Dr. Willard H. (Bill) Wattenburg


The Case for Cancer Protector Cells

The following 19 pages can be viewed as a .pdf file by clicking this link or downloaded by right clicking this link.

Additional notes, references and unpublished information follow these nineteen pages titled "UNPUBLISHED GAMMA DELTA T CELL THEORIES"






























UNPUBLISHED GAMMA DELTA T CELL THEORIES

by W. H. Wattenburg

Research Scientist

Research Foundation

California State University, Chico

October 12, 1994

Recent experimental reports (9,10,11) from other fields of immunology have added considerable support to a model for CD4+ depletion in AIDS disease proposed in 1993. This model suggests that AIDS virus infection is indirectly stimulating immunosuppressive gamma-delta T cells to suppress activated CD4+ clones in AIDS disease. The function of the mysterious gamma-delta T cells has been poorly studied. But they make up 1-15 percent of circulating T cells. They have now been shown to be the primary CD8+ T cells in the intestinal mucosa (10). They are conserved across the mammalian species. They appear early in development. Many have speculated that they perform some primitive immunoregulatory function over the much-studied alpha-beta T cells. There is increasing evidence that they play a broad immunoregulatory role with or over the alpha-beta T cells.

The model proposed has many similarities to an immunosuppression mechanism known as T Cell Vaccination (TCV) which has been shown to selectively suppress autoreactive CD4+ clones in mouse models (4). CD8+ cells of some sort are necessary for TCV. There may be an association with the highly potent gamma-delta CD8+ cytolytic cells in the mucosal tissues that have now been shown to be the effector cells in suppressing activated CD4+ cells that stimulate hypersensitive responses to recall antigens in a mouse model. AIDS disease appears to display an impaired cell-mediated immunity to recall antigens.

The now demonstrated gamma-delta T cell suppression of activated alpha-beta CD4+ cells (9) could produce broad-based suppression of CD4+ cells if the signaling pathway(s) which stimulate the gamma-delta T cells is disturbed by AIDS virus infection as proposed herein. This model is consistent with some major unexplained observations of AIDS disease.

The recent experimental reports have now verified several of the hypotheses on which the TCV-gamma-delta T cell model was based ( see October 5, update section in the draft proposal).

Some basic experiments to eliminate the possibility that this specific immunosuppression model is at work in AIDS disease can be conducted at relatively little expense using existing infected animals from failed vaccine trials before these animals are sacrificed. The virus-stimulated but ineffectual antibodies in AIDS patients must be suspected as factors that could be stimulating immunosuppression of CD4+ cells. Monoclonal antibodies that target and disable the gamma-delta T cells are available and have been used for this purpose in mouse models. These mAbs may be useful in the monkey and cat. Vaccine testing laboratories should be appraised of the analysis contained in this draft proposal because some have the means to conduct the appropriate experiments very quickly.









References:

1) E.J. Stott, NATURE 353, 393 (1991)

2) L.O. Arthur, et al, SCIENCE 258, 1935 (1992)

4) Amitabh Gaur, et al, SCIENCE 259, 91 (1993)

5) J.M. Riberdy, et al, NATURE 360, 474 (1992)

6) Peter Mombaerts, et al, NATURE 360, 225 (1992)

7) Encyclopedia of Immunology, Roitt and Delves, Ed., (1992)

8) W.H. Wattenburg, IEEE Transactions on Electronic Computers, EC-15, 378 (1966)

9) Y. Takahama, et al, NATURE 371, 67, (1994)

10) S. P. Balk, et al, SCIENCE, 265, 259 (1994)

11) C. McMenamin, SCIENCE, 265,1869 (1994)

12) Malkovsky, J. Med. Primatology,12, 113 (1992)

13) R. Gorcznski, Immunology, 81, 27 (1994)

14) Graziosi, et al, Science, 265, 248 (1994)

15) Maggi, et al, Science, 265, 244 (1994)

16) J. Cyster, et al, Nature, 371, 389 (1994)

17) De Pauli, et al, Clin Exp Immunology, 83, 187-191 (1991)

18) Cardillo, et al, Eur. J. Immunology, 23, 2597-2605 (1993)

19) Dieter Kabelitz, et al, Immunological Reviews (Munksgaard, Copenhagen), No 120, 71- 88 (1993)

20) Klaus Pechhold, et al, J Immunology, 152, 4984-4992 (1994)

21) Ibegbu, et al, Clinical Immunology and Immunopathology, 71(1), 27-32 (1994Apr)

22) Kozar, et al, J Clin Immunology, 13(3), 193-203 (1993May)

23) De Pauli, et al, Clin and Experimental Immunology, 83(2), 187-191 (1991 Feb)

24) Docke, et al, Allergie and Immunologie, 36(4),209-223 (1990, in German)

25) Margolick, et al, Clin Immunology and Immunopathology, 58(1),126-138 (1991Jan)

26) P. De Pauli, et al, Clin Exp Immunology, 83, 187-191 (1991)

27) B. Autran, et al, Clin Exp Immunology, 75, 206-210 (1989)

28) C. D. Surh,et al, Nature, 372, 100-103(1994)

29) G. Pantaleo, et al, Nature, 362, 355-358(1993)

30) J. Embretson, et al, Nature, 362, 359-362(1993)

31) Dan W. Urry, Scientific American, Jan 1994, 64-69

32) Ettaly K. Franke, et al, Nature, 372, 359-362 (1994)

33) Markus Thall, et al, Nature, 372, 363-365 (1994)



1. J.P Allison, et al, Annual Review of Immun., 1991, 9, 679-705

2. J.P Allison, et al, Current Opinion in Immunology, 1993Apr, 5, 241-246

3. J.P Allison, et al, Seminars in Immunology, 1990 Jan, 2, 59-65

4. J.P Allison, et al, J Exper. Med., 1 July 93, 178, 309-315

October 5, 1994, Update to January 20, 1993, draft:



Possibly, the most revealing evidence comes from McMenamin, et al, (11) who have recently provided strong evidence that gamma-delta CD8+ cells are the effector cells that selectively suppress delayed-type hypersensitivity to non-pathogenic (environmental and food antigens) in a mouse model. The gamma-delta cells strongly suppress alpha-beta CD4+ cell clones. They have also shown that the gamma-delta CD8+ cells are exceedingly high-potency effectors of CD4+ suppression. These results add some support to the earlier hypothesis that gamma-delta T cells are the effector cells in CD4+ cell depletion (the TCV model). McMenamin, et al, did not determine the signaling pathway that stimulates the gamma-delta T cells.

In McMenamin's studies, the gamma-delta T cells that effected suppression of the CD4+ cells were spleenocytes. Mixed spleenocytes including the gamma-delta CD8+ cells displayed a low IL-2, high IFN gamma cytokine profile similar to the cytokine profile of the seemingly anergized activated CD4+ cells in AIDS patients. This cytokine profile of the tolerized CD4+ cells was reversed in vitro when the gamma-delta CD8+ cells were eliminated.

Graziosi, et al (14), have recently reported the same cytokine profile and lack of CD4+ cell response in AIDS patients. Activated CD4+ cells from AIDS patients display a dysfunction in the in vivo environment (low IL-2 and IL-4). But when they are isolated and stimulated in vitro, they display normal cytokine profiles. Clearly, something is stomping on the activated CD4+ cells in aids patients as well as suppressing their proliferation and replenishment.



There are similarities with the demonstrated manner in which highly-potent gamma-delta T cells suppress alpha-beta CD4+ clones that stimulate hypersensitivity responses in the mouse and the selective, sequential loss of immune response to antigen groups in aids disease. In the mouse hypersensitivity studies, initial CD4+ IL-2 response to the antigen seems to be necessary to stimulate CD8+ cells. Then the gamma-deltas are stimulated to suppress the CD4+ clones that stimulate IgE against the same antigen. The gamma-deltas may be suppressing only naive CD4's that would normally proliferate to produce a hypersensitive response. Indeed, the gamma-deltas do not appear to be killing the original activated CD4+ pool because elimination of the gamma-deltas leaves CD4's that can be activated in vitro (as also seen in AIDS patients by Graziosi). Long-lived CD4's that are activated before aids virus infection and a long stimulation period for the activation of suppresor gamma-deltas could account for the latency period before full-blown Aids disease.

There is now evidence that gamma-delta T cells have a wide range of immunoregulatory functions, including even suppression of graft rejection (13). It is not out of the question that gamma-delta T cells are also involved in T Cell Vaccination suppression of autoreactive CD4+ cells.







Recent papers in diverse fields of immunology now report significant results that further support to the hypothesis that AIDS virus infection indirectly disrupts the normal controls on gamma-delta T cells and stimulates them to suppress alpha-beta CD4+ clones and/or destroy lymphoid tissue. Epithelial cells in the mucosal tissues and/or the lymphoid tissues may be in the signaling pathway to the gamma-delta T cells. Whatever the signaling pathway, if the gamma-deltas are effector cells in CD4+ cell depletion, it is possible that the gamma-deltas can be neutralized with available monoclonal antibodies administered to infected individuals.

:

9) Takahama, et al, Nature, 371, p67, 1 September 94

10) Balk, et al, Science, 265, p259, 8 July 94,

11) McMenamin, et al, Science, 265, p1869, 23 September 1994

12) Malkovsky, J. Med. Primatology,12, 113 (1992)

13) R. Gorcznski, Immunology, 81, 27 (1994)

14) Graziosi, et al, Science, 265, 248 (1994)

15) Maggi, et al, Science, 265, 244 (1994)

16) J. Cyster, et al, Nature, 371, 389 (1994)

17) De Pauli, et al, Clin Exp Immunology, 83, 187-191 ((1991)

18) Cardillo, et al, Eur. J. Immunology, 23, 2597-2605 (1993)

19) Dieter Kabelitz, et al, Immunological Reviews (Munksgaard, Copenhagen), No

120, 71-88 (1993)

20) Klaus Pechhold, et al, J Immunology, 152, 4984-4992 (1994)

  1. Ibegbu, et al, Clinical Immunology and Immunopathology, 71(1), 27-32

(1994Apr)

22) Kozar, et al, J Clin Immunology, 13(3), 193-203 (1993May)

23) De Pauli, et al, Clin and Experimental Immunology, 83(2), 187-191 (1991 Feb)

24) Docke, et al, Allergie and Immunologie, 36(4),209-223 (1990, in German)

  1. Margolick, et al, Clin Immunology and Immunopathology, 58(1),126-138

(1991Jan)

26) P. De Pauli, et al, Clin Exp Immunology, 83, 187-191 (1991)

27) B. Autran, et al, Clin Exp Immunology, 75, 206-210 (1989)



Malkovsky (12) found a large subset of MHC-unrestricted gamma-delta T cells in SIV monkeys which are cytotoxic to virus infected T cells. This subset of gamma-deltas constitute the the majority of circulating human gamma-delta T cells. His results demonstrate mechanisms that could stimulate immunosuppressive gamma-delta T cells. These gamma-delta T cells are reactive to a highly-conserved, normally intracellular protein (HSP60-groEL heat shock protein) which is abnormally expressed on the cell surface of cells infected by SIV and HIV virus. This could be a signal that is stimulating gamma-delta T cells to suppress activated CD4+ clones preferentially infected by virus-- or, possibly, attack epitheal cells in the lymph nodes that are destroyed in aids disease. Another subset of gamma-deltas is reactive to CD48 which is augmented by virus infection.

High levels of gamma-delta T cells have been found in a group of HIV infected humans (17). Malkovsky did not find elevated levels of gamma-deltas in the first few weeks after SIV infections in his monkeys. However, this is not inconsistent with the TCV model that suggests that gamma-delta T cell suppression of CD4+ clones is a selective process that is driven by subsequent antigen-group exposure after aids virus infection. The aids patients examined by De Pauli were infected for much longer periods than Malkovsky's monkeys.

Like almost all other studies of T cell interactions in aids disease, Malkovsky was investigating the role that gamma-delta T cells may play in attacking aids virus infected cells. He did not report that he was looking for possible suppression of CD4+ cells by gamma-delta T cells as an indirect result of virus infection.



Possibly, the most revealing evidence comes from McMenamin, et al, (11) who have recently provided strong evidence that gamma-delta CD8+ cells are the effector cells that selectively suppress delayed-type hypersensitivity to non-pathogenic (environmental and food antigens) in a mouse model. The gamma-delta cells strongly suppress alpha-beta CD4+ cell clones. They have also shown that the gamma-delta CD8+ cells are exceedingly high-potency effectors of CD4+ suppression. These results add some support to the earlier hypothesis that gamma-delta T cells are the effector cells in CD4+ cell depletion (the TCV model). McMenamin, et al, did not determine the signaling pathway that stimulates the gamma-delta T cells.

In McMenamin's studies, the gamma-delta T cells that effected suppression of the CD4+ cells were spleenocytes. Mixed spleenocytes including the gamma-delta CD8+ cells displayed a low IL-2, high IFN gamma cytokine profile similar to the cytokine profile of the seemingly anergized activated CD4+ cells in AIDS patients. This cytokine profile of the tolerized CD4+ cells was reversed in vitro when the gamma-delta CD8+ cells were eliminated.

Graziosi, et al (14), have recently reported the same cytokine profile and lack of CD4+ cell response in AIDS patients. Activated CD4+ cells from AIDS patients display a dysfunction in the in vivo environment (low IL-2 and IL-4). But when they are isolated and stimulated in vitro, they display normal cytokine profiles. Clearly, something is stomping on the activated CD4+ cells in aids patients as well as suppressing their proliferation and replenishment.



There are similarities with the demonstrated manner in which highly-potent gamma-delta T cells suppress alpha-beta CD4+ clones that stimulate hypersensitivity responses in the mouse and the selective, sequential loss of immune response to antigen groups in aids disease. In the mouse hypersensitivity studies, initial CD4+ IL-2 response to the antigen seems to be necessary to stimulate CD8+ cells. Then the gamma-deltas are stimulated to suppress the CD4+ clones that stimulate IgE against the same antigen. The gamma-deltas may be suppressing only naive CD4's that would normally proliferate to produce a hypersensitive response. Indeed, the gamma-deltas do not appear to be killing the original activated CD4+ pool because elimination of the gamma-deltas leaves CD4's that can be activated in vitro (as also seen in AIDS patients by Graziosi). Long-lived CD4's that are activated before aids virus infection and a long stimulation period for the activation of suppresor gamma-deltas could account for the latency period before full-blown Aids disease.

There is now evidence that gamma-delta T cells have a wide range of immunoregulatory functions, including even suppression of graft rejection (13). It is not out of the question that gamma-delta T cells are also involved in T Cell Vaccination suppression of autoreactive CD4+ cells.



Severe immunodeficiency could result from any virus infection that disrupts the controls on gamma-delta T cells and causes them to suppress all active alpha-beta CD4+ clones. It is difficult to imagine that this capability does not exist when one considers the wide range of environmental antigens for which hypersensitivity is normally suppressed. This strongly suggests that the gamma-deltas must have broad CD4+ suppression capability which is, however, normally tightly regulated by some other class of "stimulating cells" which are capable of making the distinction between harmless environmental antigens and pathogenic antigens. But what are the putative "gamma-delta stimulating cells?"

If such gamma-delta stimulating cells exist, then one line of reasoning points the finger at epithelial cells in the mucosal tissues.

The gamma-deltas are found in the mucosal tissues which are the first to encounter environmental antigens. A plausible test of whether an antigen is harmless might be whether the antigen disrupts or destroys epithelial cells in the presence of gamma-delta T cells. No damage or disruption of ep+ithelial cells in the presence of the antigen could be the signal to gamma-delta T cells that this is a harmless antigen and the gamma-deltas should suppress alpha-beta CD4+ cells that are activated against the antigen.

Several laboratories have recently reported that low HIV virus levels in the blood (viral load) of infected individuals correlates with slow progession to Aids disease (Anthony Fauci, et al, 10th International Conference on AIDS). So, by the above hypothesis, large amounts of viral antigens in contact with epithelial cells which are not infected by or harmed by the virus could be causing the epithelial cells to signal the gamma-delta T cells to suppress activated CD4+ cells that would normally stimulate immune responses against cells infected by the virus. Special attention should be given to the lymph nodes in the mucosal tissues were gamma-delta T cells and epithelial cells surround the antigen-trapping follicular cells in these lymph nodes.



Balk, et al (10), show that intestinal epitheleal cells (IEC) present MHC-like surface molecules which may be ligands for gamma-delta T cells. These ligand molecules are different than the MHC ligand molecules recognized by the alpha-beta T cells. The MHC-like molecules expresssed by intestinal epitheleal cells (IEC) do not contain the normal B2 microglobulin that presents antigens to alpha-beta T cells. It is possible that the antibody-like receptors on gamma-delta T cells are interacting with antigens from other sources in conjunction with the MHC-like ligand molecules on the epithelial cells.

(Recent papers 18-20, Oct 29, 1994. Edit paragraphs below)

Five other recent in vivo studies of gamma-delta T cells in HIV versus noninfected subjects support the hypothesis that at least one gamma-delta T cell subset, the Vd1, may be a major factor driving aids disease progression. More reason why we should try eliminating the Vd1 gamma-delta subset (TCRd1 and TCS1 mAbs ?) in infected monkeys, PDQ.

The gamma-delta Vd1 subset is strickingly increased and the Vg9 subset decreased in HIV patients, although total number of PBMC gamma-deltas does not change much in healthy controls versus HIV infected patients (19, p74; 21, 22, 23). The subset ratio in uninfected early development ( postnatal) is high Vd1, low Vg9. Uninfected adults have low Vd1, high Vg9. The stricking subset switch (5:1) in HIV adults is significant in light of the fact that no significant switch is noted in HIV infected children who already have high Vd1 (21). Hence, the differences in the rate of disease progression in children and adults correlates with the high initial Vd1 subset in children and the gradual, but eventually large increase of Vd1 in adults after HIV infection.

Kozbor, et al (22), also report much higher CD8+ Vd1 gamma-deltas in infected children than in uninfected, and this correlated with disease progression. They speculate that this profound "skewing of Vd1 ....might be involved in the HIV-induced immunodeficiency."

The compelling model that is consistent with all the significant results on gamma-deltas from diverse areas of immunology is that the Vd1 subset functions as a primary immunosurveilance mechanism that suppresses alpha-beta T cell responses in early development and then the Vd1 gamma-delta subset diminshes in number while the antigen-driven Vg9- CD4+ associated subset expands in the normal adult. The purpose of Vd1 gamma-deltas in children could be to broadly suppress developing alpha-beta CD4+ cells which would otherwise respond to maternal antibodies protecting the postnatal until the more selective alpha-beta immune system develops-- and/or the Vd1 monitor developing tissue cells for damage or abberent behavior (expression of hsp).

Hence, if improperly stimulated Vd1 gamma-deltas are suppressing CD4+ cells and/or destroying lymphoid tissue in aids disease, this model predicts that 1) the Vd1 subset would not have to expand significantly in HIV infected children, and 2) a significant expansion of Vd1 will be seen in HIV infected adults. This is precisely what has been reported (19, 21, 22, 23). Of course, the opposite hypothesis that the decreased Vg9 subset is important for virus immunity cannot be dismissed without proof.



The Vd1 appear to interact with other T cells and are suspected of being the gamma-deltas that suppress both Th1 and Th2 alpha-beta CD4+ cells in several autoimmune and hypersensitivity suppression studies.

Cardillo (18) provides more confirmation of gamma-delta T cell suppression of Th1 alpha-beta CD4+ T cells, the CD4+ subset most dysfunctional in aids disease. This selective suppression of autoimmune CD4+ cells is strongest in young animals where the gamma-delta Vd1 subset predominates (18). The early developmental role of gamma-deltas as Th1 CD4+ suppressors again suggests that they could be abberently stimulated in adult aids patients who show markedly increased Vd1 cells.

There is no good evidence on what stimulates the Vd1 subset. However, the Vd1 and Vg9 subsets appear to be mutually exclusive. The Vg9 recognize exogenous antigens and require Th1 alpha-beta CD4+ and IL-2 to proliferate (19). Gamma-delta dendritic epidermal cells also require IL-2. There is speculation that the Vg9 gamma-deltas T cells and alpha-beta Th1 CD4+ T cells may cross-regulate each other in some way (20, p4990). This is consistent with decreased numbers of both Vg9 gamma-deltas and alpha-beta Th1 CD4+ in aids disease progression.

Vg9 gamma-deltas are now being investigated as a predictive parameter for the functional capacity of Th1 Cd4+ cells in HIV infection (20, p4990, last paragraph).

Margolick, et al (25), also report low numbers of natural killer and higher numbers of CD8+ gamma-delta T cells in seropositive HIV adults.

Docke, et al (24, in German), report gamma-delta T cells responses to heat shock proteins (hsp) that may be the cause of septic disease, inflammation, and some autoimmune diseases. This is very relevant to the acute and chronic pathology seen in the gut tissues and the destruction of lymph tissue in aids disease. They suggest that the gamma-deltas are a phylogenetic old immune system with a role of immune surveilance and regulation during development of the present adult immune system in mammals. They speculate that improper signaling of gamma-deltas could cause them to attack normal cells on a broad basis.

The TCRd1 monoclonal is reported to target all of the Vd1 subset in mice and humans, hopefully monkeys as well. The papers 18-20 below list several other monoclonals for gamma-delta TCR. It was also discovered that L-leucine methyl ester (Leu-Ome) used to eliminate macrophages from PBL selectively wipes out the gamma-delta T cells in vitro (19, p82). Leu-OMe might be looked at as a possible shotgun attack on the gamma-deltas in monkeys. But, is it toxic and what will happen to the macrophages and other cells expressing the CD45RO activation antigen??



Some of the results and speculations summarized above are consistent with results and observations by J. P. Allison, et al (1,2,3,4) on gamma-delta T cell behavior in mouse models that we discussed last month (see Aidrpt11a memo references below). For instance, Allison (3) anticipated most of what Docke (24) seems to suggest about gamma-delta responses to hsp proteins and epithelial cells (if my reading of Docke in German is accurate).

The latest paper by Allison, et al (4), show an early switch in surface receptors on gamma-delta T cells during fetal development of the thymus in mice and this is the opposite compared to alpha-beta thymocytes. There may be some association here with the Vd1 subset switch that has been observed in HIV patients and the switching model proposed above.

Professor Allison said that he would be willing to send us their hampster-mouse gamma-delta monoclonal so that we can test its specificity on the monkey tissues. Chris Miller said he would contact him.

I have been reluctant to impose on Prof Allison's time to answer the many questions that came up in our earlier discussions of his papers on mouse gamma-deltas. But the associations mentioned above that are turning up in HIV and SIV studies say that it would be very valuable to have the expertise and guidance of Prof Allison, if possible, in devising the most appropriate experiments to characterize the gamma-delta functions and behavior in monkeys.



1. J.P Allison, et al, Annual Review of Immun., 1991, 9, 679-705

2. J.P Allison, et al, Current Opinion in Immunology, 1993Apr, 5, 241-246

3. J.P Allison, et al, Seminars in Immunology, 1990 Jan, 2, 59-65

4. J.P Allison, et al, J Exper. Med., 1 July 93, 178, 309-315





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NEW REFERENCES:







a) Evan Beckman, et al, Nature, 372, 691 ( 15 Dec 1994).

A new class of MHC-like molecules called CD1 present mycobacterial cell wall lipid antigens to a strange class of T cells. These antigens are highly hydrophobic.

Selected CD4- CD8- alpha-beta and gamma-delta T cells are restricted by another form of MHC-like molecule called CD1 which is not encoded in the polymorphic MHC gene region. Antigens presented by the non-polymorphic CD1 molecules may be different than those presented by MHC I and II. CD1 are still associated with Beta2-microglobulin.

They show here that CD1b presents lipid antigens, mycolic acid, from the cell wall of mycobacteria tuberculosis. Human cells do not have cell walls. Thus, lipids may be a new class of antigens. The CD1 molecules may have evolved early from the MHC genes.

CD1 recognize hydrophobic molecules resistant to protease treatment.

We should check aids virus immune complexes to see if there are any lipids in these that could stimulate the class of CD1 restricted T cells. This could trigger the anomolous immune suppression seen in leprosy and tuberculosis caused by mycobacteria infection. The responder T cells are CD4-CD8- TCR+?? They could cause suppression of CD4+ T cells responding to normal protein antigens. CD1 presenting molecules appear to be more primative. Immune responses to these lipid antigens might also take priorty over exogeneous or environemental antigens?



b) Peter Brooks, et al, Nature, 79, p1157 (30 Dec 94).

A monoclonal or cyclic peptide against an Integrin peptide halts angiogenesis in human tumors implanted on chick embryos on the chorioallantoic membrane. Tumors promote expression of the Integrin -- vascular cells enter the cell cycle. Blocking normal ligation of this Integrin with the mAb induces apoptosis in the proliferating angiogenic vascular cells and regression of the tumors, without disturbing preexisting quiescent blood vessels. The chick embryos developed normally.

The Integrins and contact with the extra-cellular matrix appear necessary for endothelial differentiation. Cells round up and die when deprived of this contact.



c) Fiona Durie, Science, 261, p1328 (3 Sept 1993).

gp39 on CD4+ cells interacts with CD40 on mature B cells to produce thymus dependent humoral immunity. Anti-gp39 antibody blocks both primary and secondary humoral responses. anti-gp39 stops arthritis induced in mice by immunization with type II collagen -- it stops the autoimmune response.

Surprisingly, anti-gp does not stop the priming of other antigen-specific T helper cells or causes the suppression of these cells -- it does not cause T helper cell tolerance.

Most significant, the mice did not develop antibodies against the anti-gp mAb raised in hamster. Activated T helper cells from anti-gp39 treated mice can be transfered to toher mice.

These results suggest that anti-gp39 selectively suppresses B cell autoimmune antibody responses, but does not suppress reponses to other antigens or cellular immunity.

If true, this could mean that anti-gp39 suppresses only the most active, on-going autoimmune response and the other activated T cells are somehow protected from suppression.

This could be a powerful tool for suppressing immune responses against monoclonal antibodies used to deplete specific cells such as the gamma-delta cells involved in aids disease, which we are now investigating, or locking up the heat shock proteins (hsp) on virus infected T cells which, hsp may be stimulating the gamma-delta T cells or other mechanisms that are suppressing CD4+ cells in aids disease.



d) Jingwu Zhang, et al, Science, 261, p1451 (10 Sept 1993).

More on T cell vaccination with human patients. Multiple sclerosis patients have T cells that attack mylelin basic protein (mbp) Myelin basic protein reactive T cells are irradiated and then used to immunize MS patients (attenuated innoculate T cells). This results in suppression of the autoreactive T cells that are activated against mbp. The suppressor CD8+ cells are stimulated by the TCR Vb chains on the innoculate T cells. Also, the suppressor T cells isolated from recipients can inhibit the T cells used for vaccination??

But the suppressor cells are specific for the disease, not other T cells activated against other antigens. Innoculation with attenuated autoreactive T cells prevents only the disease that they are able to induce. These are anti-clonotypic suppressor cells that do not react with activated T cells. They respond to the TCR of the autoreactive T cells.

Autoreactive clones against M. tuberculosis do the same ? (p1454).



e) Sergio Arruda, et al, Science, 261, p1454 ( 10 Sept 1993) next page to above.

Mycobacterium tuberculosis infects one third of all people. Asymtomatic in most. It infects macrphages and remains latent in most. It is intracellular.

(APPENDIX AND CS MODEL NOT SENT TO KOSHLAND WITH THIS VERSION. SEE AIDRPT1A)



FAX TO: DR. DANIEL KOSHLAND, JR., EDITOR-IN-CHIEF, SCIENCE

510-643-6386 October 10, 1994



Dan: Here is an updated, more accurate, and much shortened version of the original proposal I sent you. The lastest experimental reports are included. It is still much too redundant, but it contains all the relevant facts and arguments that Murray Gardner's staff and I have discussed to date. I would very much appreciate any learned comments from experts you know that could save us time on experiments and avoid chasing dead-ends. Murray wants me to meet with him and his staff this Friday. You might find it interesting to hear his comments on this --and the state of AIDS research sometime. He is very up on what is happening. I found him to be more knowlegeable on basic research results around the world than many you would think should be on top of this subject -- since they are spending great gobs of research dollars. Bill

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(CS MODEL SECTION NOT SENT TO GRADNER, BUT HAS BEEN ADDED BACK TO THIS STORED VERSION OF THE DRAFT. SEE AIDSRTP1)



FAX TO: PROFESSOR MURRAY GARDNER, UC DAVIS MEDICAL SCHOOL

1-916-752-4548 October 10, 1994



Murray: Here is the write-up for your staff and our meeting next week. I've updated the Jan 93 proposal to include the latest experimental results published that I discussed with you last week. It's still far too wordy and redundant, because I've left in all our diccussions from last year that might be of interest to your new staff.



Edit notes to incorporate in 10/12/94 draft:

10-15-94: There are similarities with the demonstrated manner in which highly-potent gamma-deltas suppress CD4+ clones that stimulate hypersensitivity responses in the mouse and the selective, sequential loss of immune response to antigen groups in aids disease. In the mouse hypersensitivity studies, initial CD4+ IL-2 response to the antigen is noted which seems to be necessary to stimulate the CD8+ gamma-delta T cells that later suppress the CD4+ clones that stimulate IgE against the same antigen. The gamma-deltas may be suppressing only naive CD4's that would normally proliferate to produce a hypersensitive response. Indeed, the gamma-deltas do not appear to be killing the original activated CD4+ pool because elimination of the gamma-deltas leaves CD4's that can be activated in vitro (see Graziosi,1994). Long-lived CD4's that are activated before aids virus infection and a long stimulation period for the activation of suppresor gamma-deltas could account for the latency period before full-blown Aids disease.



It appears that some of my speculations have been answered, and that monoclonal antibody for gamma-delta T cells in the human and rhesus monkey is available. Malkovsky at the University of Wisconsin Medical School in his paper, Journal of Medical Primatology, vol 21, p113, May 92, reports that he used a monoclonal to find monkey gamma-delta cells which are cytotoxic for SIV infected T cells. They are also reactive against a normally internal, highly-conserved peptide, human HSP58, that is abnormally expressed on the cell surface by aids virus infection, as I have speculated. This is the sort of thing that could stimulate the gamma-deltas to widely suppress activated CD4+ clones preferentially infected by virus. TCRd1 reacts with all human gamma-delta T cells (p114, top right paragraph). TCRd1 is available from T Cell Sciences, Cambridge, Mass.



Oct 18, 1994:



The model of CD4+ depletion presented herein suggests that demonstrated immunoregulatory mechanisms which can suppress hypersensitivity and autoimmunity are being disrupted by aids virus infection such that they produce immunodeficiency. This is not such a bizarre possibility when one considers the known linkages between immunodeficiency and autoimmunity. Cyster, et al (16), note the paradoxical association between immunodeficiency and auto immunity. They recently discovered a mechanism that may explain this linkage for some deficiencies in recirculating B cells. This raises the question whether there is a similar mechanism at play to explain the linkage between circulating T cell deficiencies and autoimmunity.

They have reported a mechanism for depletion of self-reacting B cells that is B-cell autonomous in that competition for niches in the primary lymphoid follicles determines which B cells will undergo apoptosis. Here is a selection mechanism dictated by the antigen-binding status of lymphoid tissues rather than genetically determined receptors on the follicular cells which were thought to induce anergy in self-reactive B cell clones independent of the circulating B cell repertoire. Their results suggest that the vast majority of recirculating B cells that hypermutate in response to antigen stimulation are excluded by this process from leaving the secondary lymphatics and re-entering the recirculating repertoire. They die within a few days. This suggests that apoptosis is still the normal fate for the majority of B cells, even though they have been selected for low self-reactivity before they leave the bone marrow. It is possible that this is also true for immature T cells and that a high level of appoptosis is the normal process that maintains tight control on the repertoire of circulating immature T cells.



Bill

--------------------------------------------------------------------------------------------------------------

This material not yet incorporated into the report 11-25-94





Dr. Bill Wattenburg

(916) 284-7066 or 894-6844

Fax: 916-284-6413

November 25, 1994

File:Aidrpt20

To: Professor Murray Gardner

and Satya Dandekar, Ph.D U.C Davis Medical School

Fax to: 916-752-4548

Subject: More on gamma-delta T cell subsets in HIV/SIV infection and experiments that we should consider.



In a previous memo (aidrpt17, October 29, 1994), I reviewed the gamma-delta subset switch seen in some HIV patients . Further review of the data in the papers that first reported this (19,26,27) and other papers (28,29,30) suggests that the experiments below could be revealing. One may be appropriate for FIV infected cats.



1. First, the most revealing information on the role of the gamma-delta subsets may come from the comparison of gamma-delta subsets in the tissues and blood of pathologic and nonpathologic animals at various stages of SIV infection. If a gamma-delta subset is involved in aids disease progression after SIV infection, there should be some differences in the activation, locations, or numbers of the subsets between SIV infected macques and SIV infected sooty mangabys that do not progress to aids disease.

It would be very significant if we happen to find that there is a subset switch between the Vg9 and Vd1 subsets in the SIV infected macaques but not in the sooty mangabys. This may be associated with the early hyperplasia in the gut epitheleal and lymphoid tissues in the SIV infected macques.



2. Elimination of gamma-delta subsets in SIV infected monkeys may be difficult with only the monoclonals we have (TCRd1 and dTCS1) and you have said these monoclonals do not seem to work in the cat. However, there was the observation by Kabelitz (20, p82) that L-leucine methyl ester (Leu-Ome) wipes out the gamma-deltas and macrophages in T cell cultures with no noticeable reduction of alpha-beta T cell function in vitro. It is possible that the same will happen in vivo if Leu-Ome is not seriously toxic in other ways.



3. We should see if IV administered Leu-Ome will selectively eliminate gamma-deltas and activated macrophages in FIV infected cats. This may be an inexpensive experiment that we could do in parallel with the monkey investigations. I understand that there are FIV infected cats in various stages of disease progression, which animals will be put down in due course. Professor James Allison may know something about Leu-Ome if it is commonly used in T cell studies.

This may not aggravate disease progression because the macrophages are suspected of being the first to be infected by aids virus and subsequently infecting CD4+ cells (30). Another recent report (28) shows that some subsets of macrophages rapidly (in hours) eliminate large numbers of apoptoptic T cells in the thymus, so quickly that it is difficult to see where they disappear without a special assay that labels DNA fragments from the dead T cells, which fragments are then found in the macrophages. Infected or improperly signaled macrophages might be involved in destruction of lymphoid tissues in aids disease.



18) Cardillo, et al, Eur. J. Immunology, 23, 2597-2605 (1993)

19) Dieter Kabelitz, et al, Immunological Reviews (Munksgaard, Copenhagen), No 120, 71-88 (1993)

20) Klaus Pechhold, et al, J Immunology, 152, 4984-4992 (1994)

21) Ibegbu, et al, Clinical Immunology and Immunopathology, 71(1), 27-32 (1994Apr)

22) Kozar, et al, J Clin Immunology, 13(3), 193-203 (1993May)

23) De Pauli, et al, Clin and Experimental Immunology, 83(2), 187-191 (1991 Feb)

24) Docke, et al, Allergie and Immunologie, 36(4),209-223 (1990, in German)

25) Margolick, et al, Clin Immunology and Immunopathology, 58(1),126-138 (1991Jan)

26) P. De Pauli, et al, Clin Exp Immunology, 83, 187-191 (1991)

27) B. Autran, et al, Clin Exp Immunology, 75, 206-210 (1989)

28) C. D. Surh,et al, Nature, 372, 100-103(1994)

29) G. Pantaleo, et al, Nature, 362, 355-358(1993)

30) J. Embretson, et al, Nature, 362, 359-362(1993)



FAX COPY TO: DR. DANIEL KOSHLAND, JR., SCIENCE, 1-510-643-6386

----------------------------------------------------------------------------------------------



FAX COPY TO: DR. DANIEL KOSHLAND, JR., SCIENCE MAGAZINE, 1-510-643-6386



Following not yet incorportated 12-27-93:

Bill Wattenburg

(916) 284-7066 or 894-6844

Fax: 916-284-6413

December 27, 1994



Revised

File:Aidrpt22

To: Murray Gardner and

Satya Dandekar U.C Davis Medical School

Fax to: 916-752-4548

Subject: Possible Role of Heat Shock Proteins (hsp) Found in Lymphoid Tissues of SIV Infected Macaques. A Clue to Aids Virus Pathogenesis.



Some recent reports suggest a possible pathogenic role for the heat shock proteins that we have recently found in the lymphoid tissues of SIV infected macaques, which hsp proteins do not seem to be present in the macaques infected with an attenuated, nonpathological cloned virus. If this differential expression of hsp proteins is confirmed by comparison with specimens from SIV infected sooty mangabys that do not suffer aids disease, we had better test the hypothesis given below as soon as possible.

These hsp proteins could be protecting virus coat proteins from recognition by immune cells and antibodies by changing the configuration of the coat proteins through folding of the proteins. This would explain one of the great mysteries of aids disease, that is, why aids virus infected T cells in the lymphoid tissues escape immune surveillance in the presence of abundant antibodies and activated cytoxic T cells present in infected individuals -- and why, however, these same antibodies and cytotoxic T cells can neutralize the virus in certain culture cells in vitro. It is quite possible that the infected culture cells do not express the hsp proteins that are protecting the antigenic determinants on the virus coat protein in vivo, gp120 in particular.

A report in the Jan 1994 Scientific Amercican by Urry (31, p38) on biopolymers has a passing reference to an intriguing possibility. He notes that heat shock proteins have the ability to fold proteins in the same fashion as the biopolymers that he has studied. I checked his published work and then found two other recent reports in Nature which show that certain cyclophilines in the cell are necessay for HIV particles to assemble properly. The cyclopilines act like heat shock proteins by folding proteins. Franke and Thall (32, 33) report that HIV is unique among the retroviruses in that it requires a certain cylcophiline not required by the others. SIV does not require the same cyclophiline as HIV, although they did not determine which cyclophiline might be required by SIV growing in monkey cells.

This evidence may be pointing to the answer to another mystery, that is, why the species specificity of the various aids viruses? If different cellular cyclophilines are required for each different virus, we might have an answer to why SIV is pathogenic in the macaque and not in the mangaby, HIV in the human and not in the chimp. Various elements of the heat shock protiens vary from one species to the next. It is possible that the African monkeys are nonpathological hosts for SIV because their immune systems have developed to recognize the virus coat proteins in all their folded configurations, whereas the macaques have not. Or, the macaques may have developed different heat shock proteins?

Satya and I explored several ways to test this hypothesis yesterday. If heat shock proteins within or on the surface of infected T cells in the lymphoid tissues are protecting these cells or virus particles from immune attack, we should be able to demonstrate this in vitro by locking up the heat shock proteins with a monoclonal or a peptide. It would seem to me that the usual neutralizing antibody tests with cultured T cells might be the place to start. If there is a significant difference in virus infectivity because we knock out the heat shock proteins, then we are on to something. Another approach might be to combine heat shock proteins with pure recombinant gp120 to see if there is a significant change in the ability of antibodies to recognize the antigenic sites on gp120. Column affinity procedures could be used here.

The most recents reports on vaccine development still show that the best polyclonal antibodies developed against pure recombinant gp120 in vitro are, nevertheless, not so effective in vivo. Some strains of the virus are not neutralized in vivo. Is is possible that proximity of heat shock proteins on, in, or near infected cells in vivo are folding or changing the configurations of virus coat proteins so as to hide the antigenic determinants that are recognized when these proteins are extended as they might be when virus particles are circulating in the blood. This could explain why infected individuals generate copious amounts of antibodies and cytotoxic T cells which, nevertheless, are unable to eradicate virus infected cells that reside in the lymphoid tissues. Chronically infected cells residing in the lymphoid tissues could lead to untold mechanisms for tissue damage and CD4 cell supression, for instance, by stimulation of the gamma delta T cell subset that we are now seeing in infected macaques.

31) Dan W. Urry, Scientific American, Jan 1995, 64-69

32) Ettaly K. Franke, et al, Nature, 372, 359-362 (1994)

33) Markus Thall, et al, Nature, 372, 363-365 (1994)



W. H. Wattenburg



Research Scientist



The University Foundation



California State University, Chico



(916) 894-6844 Fax 893-1317

File: aidsrpt1.doc

January 20, 1993

Revised October 5, 1994

Revised October 29, 1994



DRAFT



MODELS OF AIDS DISEASE DEPLETION OF CD4+ CELLS

AND APPROPRIATE EXPERIMENTS USING EXISTING

INFECTED ANIMALS



( The January 1993 proposal was a personal communication to Professor Murray Gardner, Primate Research Center, University of California Davis, and Dr. Jose Torres, U.C. Davis Medical School, extensively discussed with them in February and March 1993. This report has been updated to include recent and significant research results that support the hypotheses proposed in January 1993.)



SUMMARY:



A logical model of immunoregulation developed by the author predicted that there must be a class of non-MHC restricted immunoregulatory cells capable of monitoring and regulating antigen activated alpha-beta cells that have been positively selected for self compatibility (see appendix). These were called T' cells. The putative T' cells would also be good candidates for the effectors of CD4+ cell depletion in AIDS disease if they were abnormally stimulated as a result of virus infection of activated alpha-beta T cells.

A review of basic research reports in immunology disclosed numerous experimental observations that make the gamma-delta T cells good candidates for the putative T' cells. At least some gamma-delta T cells are not MHC restricted, and they appear to use antibody-like receptors. Many have speculated that the mysterious gamma-delta T cells perform some primative immunoregulation function because of their early appearance during development. They are conserved across the species. They constitute 1%-15% of T cells in peripheral blood. They can kill or anergize T cells with autoreactive T-cell receptors (7). However, the gamma-delta T cells are not known to be involved in, or necessary for any normal alpha-beta CD4+ or CD8+ immune responses.

In the course of this investigation, associations were found between the the receptor ligands of gamma-delta T cells and the peptides and surface molecules that stimulate an immunoregulation mechanism known as T Cell Vaccination (TCV) which can selectively suppress CD4+ clones activated against specific antigen groups in mouse models.

For instance, Guar, et al, (4) have shown that:

a) vaccination of MBP autoreactive mice with a peptide from the variable region of the B gene of the T-cell receptor can induce clonal anergy of the autoreactive CD4+ cells,

b) suppressor cells of some sort which express CD8+ are essential for mediating this vaccination induced anergy of the CD4+ clones in their mouse model,

c) only this one segment of the B gene variable region (Vb8.2) induces the specific clonal anergy of MBP responsive CD4+ clones, and

d) the CD4+ responsiveness against other antigens also associated with the Vb8.2 region is also surpressed by vaccination with the TCR Vb8.2 peptide.

TCV can also be stimulated by membranes from activated T cells that have cross-linked cell surface molecules.

The reported selectivity of the TCV immunosuppression mechanism appears similar to immunoregulation of activated alpha-beta CD4+ cells to suppress recall antigen hypersensitive responses.

T cell vaccination suppresses CD4+ responsiveness to certain antigenic groups associated with the specific TCR peptide used to induce the clonal anergy. This is consistent with the observed elimination of immune responsiveness to specific groups of antigens, the CDC groups (7), in the progression of AIDS disease in some patients.

The above observations raise the possibility that internally stimulated T Cell Vaccination has the capabilty of suppressing CD4+ cells on a broad basis, as is seen in AIDS disease, and that the gamma-delta T cells may be the effector cells carring out selective suppression of CD4+ clones.

Abundant, but ineffective anti-virus antibodies are generated in AIDS disease. Virus particles budding from infected T cells appear to carry intracellular peptides from the Ig/TCR/MHC supergene complex. It is not at all unreasonable to suspect that some of these virus infection generated peptides could stimulate immunosuppression of CD4+ cells by stimulating immunosuppressive gamma-delta T cells, and/or triggering the TCV immunosuppression of CD4+ cells on a broad basis. Hence, the models for CD4+ cell depletion presented herein. All Three possibilities demand investigation: the virus carried intracellular peptides, circulating antibodies, and the gamma-delta T cells.

Some basic elimination experiments to qualify or refute these hypotheses can be carried out in animals already fatally infected in failed vaccine trials, which animals will be sacrificed prematurely otherwise. Most of the experiments can be performed and evaluated in times much shorter than the evaluation times required for new vaccine trails.

October 5, 1994, Update to January 20, 1993, draft:



Support for the TCV Model:



Recent papers in diverse fields of immunology now report significant results that confirm some of the assumptions made in the TCV and VGP models for CD4+ cell depletion proposed in January 1993. These reports add further support to the hypothesis that AIDS virus infection indirectly disrupts the normal controls on gamma-delta T cells and stimulates them to suppress alpha-beta CD4+ clones and/or destroy lymphoid tissue. Epithelial cells in the mucosal tissues and/or the lymphoid tissues may be in the signaling pathway to the gamma-delta T cells. Whatever the signaling pathway, if the gamma-deltas are effector cells in CD4+ cell depletion, it is possible that the gamma-deltas can be neutralized with available monoclonal antibodies administered to infected individuals.

:

9) Takahama, et al, Nature, 371, p67, 1 September 94

10) Balk, et al, Science, 265, p259, 8 July 94,

11) McMenamin, et al, Science, 265, p1869, 23 September 1994

12) Malkovsky, J. Med. Primatology,12, 113 (1992)

13) R. Gorcznski, Immunology, 81, 27 (1994)

14) Graziosi, et al, Science, 265, 248 (1994)

15) Maggi, et al, Science, 265, 244 (1994)

16) J. Cyster, et al, Nature, 371, 389 (1994)

17) De Pauli, et al, Clin Exp Immunology, 83, 187-191 ((1991)

18) Cardillo, et al, Eur. J. Immunology, 23, 2597-2605 (1993)

19) Dieter Kabelitz, et al, Immunological Reviews (Munksgaard, Copenhagen), No

120, 71-88 (1993)

20) Klaus Pechhold, et al, J Immunology, 152, 4984-4992 (1994)

  1. Ibegbu, et al, Clinical Immunology and Immunopathology, 71(1), 27-32

(1994Apr)

22) Kozar, et al, J Clin Immunology, 13(3), 193-203 (1993May)

23) De Pauli, et al, Clin and Experimental Immunology, 83(2), 187-191 (1991 Feb)

24) Docke, et al, Allergie and Immunologie, 36(4),209-223 (1990, in German)

  1. Margolick, et al, Clin Immunology and Immunopathology, 58(1),126-138

(1991Jan)

26) P. De Pauli, et al, Clin Exp Immunology, 83, 187-191 (1991)

27) B. Autran, et al, Clin Exp Immunology, 75, 206-210 (1989)



Malkovsky (12) found a large subset of MHC-unrestricted gamma-delta T cells in SIV monkeys which are cytotoxic to virus infected T cells. This subset of gamma-deltas constitute the the majority of circulating human gamma-delta T cells. His results demonstrate mechanisms that could stimulate immunosuppressive gamma-delta T cells. These gamma-delta T cells are reactive to a highly-conserved, normally intracellular protein (HSP60-groEL heat shock protein) which is abnormally expressed on the cell surface of cells infected by SIV and HIV virus. This could be a signal that is stimulating gamma-delta T cells to suppress activated CD4+ clones preferentially infected by virus-- or, possibly, attack epitheal cells in the lymph nodes that are destroyed in aids disease. Another subset of gamma-deltas is reactive to CD48 which is augmented by virus infection.

High levels of gamma-delta T cells have been found in a group of HIV infected humans (17). Malkovsky did not find elevated levels of gamma-deltas in the first few weeks after SIV infections in his monkeys. However, this is not inconsistent with the TCV model that suggests that gamma-delta T cell suppression of CD4+ clones is a selective process that is driven by subsequent antigen-group exposure after aids virus infection. The aids patients examined by De Pauli were infected for much longer periods than Malkovsky's monkeys.

Like almost all other studies of T cell interactions in aids disease, Malkovsky was investigating the role that gamma-delta T cells may play in attacking aids virus infected cells. He did not report that he was looking for possible suppression of CD4+ cells by gamma-delta T cells as an indirect result of virus infection.



Possibly, the most revealing evidence comes from McMenamin, et al, (11) who have recently provided strong evidence that gamma-delta CD8+ cells are the effector cells that selectively suppress delayed-type hypersensitivity to non-pathogenic (environmental and food antigens) in a mouse model. The gamma-delta cells strongly suppress alpha-beta CD4+ cell clones. They have also shown that the gamma-delta CD8+ cells are exceedingly high-potency effectors of CD4+ suppression. These results add some support to the earlier hypothesis that gamma-delta T cells are the effector cells in CD4+ cell depletion (the TCV model). McMenamin, et al, did not determine the signaling pathway that stimulates the gamma-delta T cells.

In McMenamin's studies, the gamma-delta T cells that effected suppression of the CD4+ cells were spleenocytes. Mixed spleenocytes including the gamma-delta CD8+ cells displayed a low IL-2, high IFN gamma cytokine profile similar to the cytokine profile of the seemingly anergized activated CD4+ cells in AIDS patients. This cytokine profile of the tolerized CD4+ cells was reversed in vitro when the gamma-delta CD8+ cells were eliminated.

Graziosi, et al (14), have recently reported the same cytokine profile and lack of CD4+ cell response in AIDS patients. Activated CD4+ cells from AIDS patients display a dysfunction in the in vivo environment (low IL-2 and IL-4). But when they are isolated and stimulated in vitro, they display normal cytokine profiles. Clearly, something is stomping on the activated CD4+ cells in aids patients as well as suppressing their proliferation and replenishment.



There are similarities with the demonstrated manner in which highly-potent gamma-delta T cells suppress alpha-beta CD4+ clones that stimulate hypersensitivity responses in the mouse and the selective, sequential loss of immune response to antigen groups in aids disease. In the mouse hypersensitivity studies, initial CD4+ IL-2 response to the antigen seems to be necessary to stimulate CD8+ cells. Then the gamma-deltas are stimulated to suppress the CD4+ clones that stimulate IgE against the same antigen. The gamma-deltas may be suppressing only naive CD4's that would normally proliferate to produce a hypersensitive response. Indeed, the gamma-deltas do not appear to be killing the original activated CD4+ pool because elimination of the gamma-deltas leaves CD4's that can be activated in vitro (as also seen in AIDS patients by Graziosi). Long-lived CD4's that are activated before aids virus infection and a long stimulation period for the activation of suppresor gamma-deltas could account for the latency period before full-blown Aids disease.

There is now evidence that gamma-delta T cells have a wide range of immunoregulatory functions, including even suppression of graft rejection (13). It is not out of the question that gamma-delta T cells are also involved in T Cell Vaccination suppression of autoreactive CD4+ cells.



Severe immunodeficiency could result from any virus infection that disrupts the controls on gamma-delta T cells and causes them to suppress all active alpha-beta CD4+ clones. It is difficult to imagine that this capability does not exist when one considers the wide range of environmental antigens for which hypersensitivity is normally suppressed. This strongly suggests that the gamma-deltas must have broad CD4+ suppression capability which is, however, normally tightly regulated by some other class of "stimulating cells" which are capable of making the distinction between harmless environmental antigens and pathogenic antigens. But what are the putative "gamma-delta stimulating cells?"

If such gamma-delta stimulating cells exist, then one line of reasoning points the finger at epithelial cells in the mucosal tissues.

The gamma-deltas are found in the mucosal tissues which are the first to encounter environmental antigens. A plausible test of whether an antigen is harmless might be whether the antigen disrupts or destroys epithelial cells in the presence of gamma-delta T cells. No damage or disruption of eithelial cells in the presence of the antigen could be the signal to gamma-delta T cells that this is a harmless antigen and the gamma-deltas should suppress alpha-beta CD4+ cells that are activated against the antigen.

Several laboratories have recently reported that low HIV virus levels in the blood (viral load) of infected individuals correlates with slow progession to Aids disease (Anthony Fauci, et al, 10th International Conference on AIDS). So, by the above hypothesis, large amounts of viral antigens in contact with epithelial cells which are not infected by or harmed by the virus could be causing the epithelial cells to signal the gamma-delta T cells to suppress activated CD4+ cells that would normally stimulate immune responses against cells infected by the virus. Special attention should be given to the lymph nodes in the mucosal tissues were gamma-delta T cells and epithelial cells surround the antigen-trapping follicular cells in these lymph nodes.



Balk, et al (10), show that intestinal epitheleal cells (IEC) present MHC-like surface molecules which may be ligands for gamma-delta T cells. These ligand molecules are different than the MHC ligand molecules recognized by the alpha-beta T cells. The MHC-like molecules expresssed by intestinal epitheleal cells (IEC) do not contain the normal B2 microglobulin that presents antigens to alpha-beta T cells. It is possible that the antibody-like receptors on gamma-delta T cells are interacting with antigens from other sources in conjunction with the MHC-like ligand molecules on the epithelial cells.

(Recent papers 18-20, Oct 29, 1994. Edit paragraphs below)

Five other recent in vivo studies of gamma-delta T cells in HIV versus noninfected subjects support the hypothesis that at least one gamma-delta T cell subset, the Vd1, may be a major factor driving aids disease progression. More reason why we should try eliminating the Vd1 gamma-delta subset (TCRd1 and TCS1 mAbs ?) in infected monkeys, PDQ.

The gamma-delta Vd1 subset is strickingly increased and the Vg9 subset decreased in HIV patients, although total number of PBMC gamma-deltas does not change much in healthy controls versus HIV infected patients (19, p74; 21, 22, 23). The subset ratio in uninfected early development ( postnatal) is high Vd1, low Vg9. Uninfected adults have low Vd1, high Vg9. The stricking subset switch (5:1) in HIV adults is significant in light of the fact that no significant switch is noted in HIV infected children who already have high Vd1 (21). Hence, the differences in the rate of disease progression in children and adults correlates with the high initial Vd1 subset in children and the gradual, but eventually large increase of Vd1 in adults after HIV infection.

Kozbor, et al (22), also report much higher CD8+ Vd1 gamma-deltas in infected children than in uninfected, and this correlated with disease progression. They speculate that this profound "skewing of Vd1 ....might be involved in the HIV-induced immunodeficiency."

The compelling model that is consistent with all the significant results on gamma-deltas from diverse areas of immunology is that the Vd1 subset functions as a primary immunosurveilance mechanism that suppresses alpha-beta T cell responses in early development and then the Vd1 gamma-delta subset diminshes in number while the antigen-driven Vg9- CD4+ associated subset expands in the normal adult. The purpose of Vd1 gamma-deltas in children could be to broadly suppress developing alpha-beta CD4+ cells which would otherwise respond to maternal antibodies protecting the postnatal until the more selective alpha-beta immune system develops-- and/or the Vd1 monitor developing tissue cells for damage or abberent behavior (expression of hsp).

Hence, if improperly stimulated Vd1 gamma-deltas are suppressing CD4+ cells and/or destroying lymphoid tissue in aids disease, this model predicts that 1) the Vd1 subset would not have to expand significantly in HIV infected children, and 2) a significant expansion of Vd1 will be seen in HIV infected adults. This is precisely what has been reported (19, 21, 22, 23). Of course, the opposite hypothesis that the decreased Vg9 subset is important for virus immunity cannot be dismissed without proof.



The Vd1 appear to interact with other T cells and are suspected of being the gamma-deltas that suppress both Th1 and Th2 alpha-beta CD4+ cells in several autoimmune and hypersensitivity suppression studies.

Cardillo (18) provides more confirmation of gamma-delta T cell suppression of Th1 alpha-beta CD4+ T cells, the CD4+ subset most dysfunctional in aids disease. This selective suppression of autoimmune CD4+ cells is strongest in young animals where the gamma-delta Vd1 subset predominates (18). The early developmental role of gamma-deltas as Th1 CD4+ suppressors again suggests that they could be abberently stimulated in adult aids patients who show markedly increased Vd1 cells.

There is no good evidence on what stimulates the Vd1 subset. However, the Vd1 and Vg9 subsets appear to be mutually exclusive. The Vg9 recognize exogenous antigens and require Th1 alpha-beta CD4+ and IL-2 to proliferate (19). Gamma-delta dendritic epidermal cells also require IL-2. There is speculation that the Vg9 gamma-deltas T cells and alpha-beta Th1 CD4+ T cells may cross-regulate each other in some way (20, p4990). This is consistent with decreased numbers of both Vg9 gamma-deltas and alpha-beta Th1 CD4+ in aids disease progression.

Vg9 gamma-deltas are now being investigated as a predictive parameter for the functional capacity of Th1 Cd4+ cells in HIV infection (20, p4990, last paragraph).

Margolick, et al (25), also report low numbers of natural killer and higher numbers of CD8+ gamma-delta T cells in seropositive HIV adults.

Docke, et al (24, in German), report gamma-delta T cells reponses to heat shock proteins (hsp) that may be the cause of septic disease, inflammation, and some autoimmune diseases. This is very relevant to the acute and chronic pathology seen in the gut tissues and the destruction of lymph tissue in aids disease. They suggest that the gamma-deltas are a phylogenetic old immune system with a role of immune surveilance and regulation during development of the present adult immune system in mammals. They speculate that improper signaling of gamma-deltas could cause them to attack normal cells on a broad basis.

The TCRd1 monoclonal is reported to target all of the Vd1 subset in mice and humans, hopefully monkeys as well. The papers 18-20 below list several other monoclonals for gamma-delta TCR. It was also discovered that L-leucine methyl ester (Leu-Ome) used to eliminate macrophages from PBL selectively wipes out the gamma-delta T cells in vitro (19, p82). Leu-OMe might be looked at as a possible shotgun attack on the gamma-deltas in monkeys. But, is it toxic and what will happen to the macrophages and other cells expressing the CD45RO activation antigen??



Some of the results and speculations summarized above are consistent with results and observations by J. P. Allison, et al (1,2,3,4) on gamma-delta T cell behavior in mouse models that we discussed last month (see Aidrpt11a memo references below). For instance, Allison (3) anticipated most of what Docke (24) seems to suggest about gamma-delta responses to hsp proteins and epithelial cells (if my reading of Docke in German is accurate).

The latest paper by Allison, et al (4), show an early switch in surface receptors on gamma-delta T cells during fetal development of the thymus in mice and this is the opposite compared to alpha-beta thymocytes. There may be some association here with the Vd1 subset switch that has been observed in HIV patients and the switching model proposed above.

Professor Allison said that he would be willing to send us their hampster-mouse gamma-delta monoclonal so that we can test its specificity on the monkey tissues. Chris Miller said he would contact him.

I have been reluctant to impose on Prof Allison's time to answer the many questions that came up in our earlier discussions of his papers on mouse gamma-deltas. But the associations mentioned above that are turning up in HIV and SIV studies say that it would be very valuable to have the expertise and guidance of Prof Allison, if possible, in devising the most appropriate experiments to characterize the gamma-delta functions and behavior in monkeys.



1. J.P Allison, et al, Annual Review of Immun., 1991, 9, 679-705

2. J.P Allison, et al, Current Opinion in Immunology, 1993Apr, 5, 241-246

3. J.P Allison, et al, Seminars in Immunology, 1990 Jan, 2, 59-65

4. J.P Allison, et al, J Exper. Med., 1 July 93, 178, 309-315





---------------------------------------------------------------------------------------------------------------



The TCV model of CD4+ depletion presented herein suggests that demonstrated immunoregulatory mechanisms which can suppress hypersensitivity and autoimmunity are being disrupted by aids virus infection such that they produce immunodeficiency. This is not such a bizarre possibility when one considers the known linkages between immunodeficiency and autoimmunity. Cyster, et al (16), note the paradoxical association between immunodeficiency and auto immunity. They recently discovered a mechanism that may explain this linkage for some deficiencies in recirculating B cells. This raises the question whether there is a similar mechanism at play to explain the linkage between circulating T cell deficiencies and autoimmunity.

They have reported a mechanism for depletion of self-reacting B cells that is B-cell autonomous in that competition for niches in the primary lymphoid follicles determines which B cells will undergo apoptosis. Here is a selection mechanism dictated by the antigen-binding status of lymphoid tissues rather than genetically determined receptors on the follicular cells which were thought to induce anergy in self-reactive B cell clones independent of the circulating B cell repertoire. Their results suggest that the vast majority of recirculating B cells that hypermutate in response to antigen stimulation are excluded by this process from leaving the secondary lymphatics and re-entering the recirculating repertoire. They die within a few days. This suggests that apoptosis is still the normal fate for the majority of B cells, even though they have been selected for low self-reactivity before they leave the bone marrow. It is possible that this is also true for immature T cells and that a high level of appoptosis is the normal process that maintains tight control on the repertoire of circulating immature T cells.



Monoclonal antibodies have been used in animal models to arrest gamma-delta cell activity. Elimination of gamma-delta T cell activity in AIDS virus infected animals must be attempted to investigate the possible role of these cells in AIDS disease. The gamma-delta CD8+ cell reactivity to alpa-beta CD4+ cells in asymtomatic seropositive individuals and/or animals must be investigated and compared with the activity in others who have proceeded to AIDS disease.



Support for the VGP Model:



Mombaerts, et al, (6) earlier reported that the TCR-B gene product is essential for the maturation of CD4+CD8+ thymocytes and the expansion of CD4+CD8+ clones.

Takahama, et al, have recently shown that monoclonal antibodies which cross-link (aggregate) the T cell receptors on precursor T cells will inhibit the differentiation of CD4+CD8+ precursor cells into mature CD4+ cells in mouse models (9). However, monovalent antibody attachment to the T cell receptor stimulates matuation of CD4+ cells.

If single T cell receptor attachment by a differentiation messenger is the normal CD4+ cell maturation signal, and aggregation of T cell receptors is the suppression signal, we have here a very delicate mechanism that could easily be disturbed by abnormal multivalent antibody peptides or cell surface molecules released by AIDS virus infection of even a few activated T cells. Again, this brings into suspicion the anti-virus antibodies themselves that are ineffectual in stopping AIDS disease.

Now that it has been shown that both of the earlier postulated TCV and VGP (virus generated peptide suppression of CD4+ cell maturation, see Jan 1993 notes below) models can be triggered by cross-linked T cell receptors. It is possible that both are at play in CD4+ cell depletion. TCV could account for the selective suppression of active CD4+ clones and the loss of immunity to recall antigens (the CDC antigen groups I-IV). VGP could account for the lack of replenishment of CD4+ cells ( death of immature CD4+ cells) and the gradual reduction of CD4+ counts in peripheral blood.

The focus returns to the same possible stimulating factors: 1) abnormal antibodies, 2) abnormally expressed surface molecules on infected T cells, and 3) cross-linked T cell receptors. The reports of asymptomatic seropositive individuals who produce no anti-gp antibodies but appear to have active cell-mediated immunity points a finger at the circulating antibodies as potential stimulating factors for CD4+ cell depletion in full-blown AIDS disease.

Again, there are the curious associations with gamma-delta T cell stimulation and the T cell vaccination process which can also be stimulated by cross-linked T cell receptors on activated T cell membranes. AIDS virus preferentially infects activated T cells. Infected T cell membrane fragments are likely available, at least in the lymphoid tissues that harbor the virus. The TCV and VGP models may be one in the same.

Although a great deal of effort might be required to verify any of these possible signaling pathways at the molecular level, it may be possible to identify and/or eliminate the postulated effector cells in animal models much more easily.

EXPERIMENTS FOR THE TCV AND VGP MODELS:

1. The gamma-delta T cells should be selectively eliminated from infected animals with monoclonal antibody to see if any CD4+ responsiveness returns, as McMenamin and Graziosi (11,13) observed when anergized CD4+ cells again displayed normal cytokine patterns after they were isolated from the in vivo environment and stimulated in vitro.

2. CD4+ cells, mucosal and lymphoid epithelial cells, and gamma-delta T cells from infected animals should be tested in vitro to see if the CD4+ respond normally when stimulated with and without the presence of the other cells. Epithelial cells in the mucosal tissues and the lymphoid tissues of infected animals should be examined to see whether they express the HSP60- groEL family of intracellular proteins noted be Malkovsky as targets for gamma-delta T cells. The reactivity of gamma-delta T cells to virus infected T cells and associated epithelial cells found in the lymphoid tissues of infected animals should be studied to see if any previuosly unresponsive gamma-delta clones become reactive toward CD4+ cells as the disease progresses. Gamma-delta T cells from uninfected animals should be isolated before the animals are infected.

3 The intracellular peptides carried from infected T cells by budding virus particles should be studied to see what T cell categories may respond to these peptides, in particular, the gamma-delta T cells. Anti-gp antibodies in infected animals must be examined as well.

4. Elimination of anti-gp antibodies in early infected animals with anti-epitope antibodies would be a traumatic process, but it may be worth trying to see if this stops later CD4+ cell depletion.

5. Remove or bypass the thymus in early infected adult animals to see if the normal CD4+ clonal selection mechanisms in the thymus have been disturbed by virus infection. The thymus could be a primary site where the proposed VGP immunosuppression mechanism is being stimulated by cell to cell contact of precursor thymocytes with virus infected T cells. Thymectomized healthy adult animals appear to survive well. (However, it is likely that other lymphoid tissues and the liver that cannot be removed will also be active sites if the thymus is involved.)

6. Healthy animals not infected with the Aids virus should be vaccinated using a wide variety of peptides from the Ig/MHC/TCR gene complex of Aids virus infected T cells taken from the same animal and grown in culture (even though the yield will be small). If gamma-delta T cells become reactive to activated CD4+ cells, or if this causes CD4+ cell suppression in the animals, it would be evidence that the gamma-delta CD4+ depletion mechanism is at play and it is stimulated by one or more of these peptides.

MODELS OF CD4+ CELL DEPLETION IN AIDS DISEASE (Jan 1993 draft):

1. Two closely related models for the depletion of CD4+ cells in AIDS disease are presented herein. Both models predict that virus infection is indirectly causing CD4+ cell depletion by CD4+ cell immunosuppression mechanisms elucidated in other fields of immunology (4,9,11).. Virus infection may be releasing abnormal signal peptides or surface molecules on infected T cells or stimulating abnormal circulating antibodies which are similar to the antibody-like molecules that stimulate these specific CD4+ cell immunosuppression mechanisms.

These immunosuppressive mechanisms, herein called TCV and VGP, have the potential to selectively and sequentially suppress CD4+ cells if abnormal, antibody-like peptides are unleashed or abnormal cell surface molecules are expressed by virus infected T cells. In AIDS disease, CD4+ immunosuppression-stimulating factors could be a) embedded in the abundant but ineffectual anti-gp antibody complexes, b) intracellular peptides from the Ig/MHC supergene complex carried by virus particles budding from infected T cells, or c) abnormal receptor molecules presented on the surfaces of infected T cells.

2. The TCV model suggests that maturation and/or activation of CD4+ cells in AIDS disease is selectively suppressed by an immunosuppressive mechanism known as T cell vaccination (TCV) which suppresses specific clones of activated CD4+ cells. CD8+ cells of some sort appear to be the effector cells that produce TCV in mouse models (4).

The mysterious gamma-delta T cells are good candidates for the effector cells that carry out the selective T cell vaccination immunosuppression of activated CD4+ cells. The early appearance of gamma-delta T cells during development and their conserved nature across species suggests that they play some primary role in immunoregulation. They constitute 1%-15% of T cells in peripheral blood. They can kill cells or anergize cells with autoreactive T-cell receptors (7). They appear to use antibody-like receptors. At least some are not MHC restricted.

3. The VGP (virus generated suppressor peptides) model suggests that the normal maturation of CD4+ cells is suppressed by abnormal antibody-like peptides or T cell surface molecules produced as the result of AIDS virus infection of activated T cells. These abnormal peptides interfere with the differentiation of precursor CD4+CD8+ thymocytes into mature CD4+ cells by blocking maturation signal receptors on the precursor T cells. It is possible that direct ligation of the precursor T cell receptors by abnormal virus generated peptides or surface molecules on infected T cells is occurring.

This points a finger at the anti-gp antibodies that are ineffectual in stopping the progression of AIDS disease. These antibodies may harbor factors that actually stimulate CD4+ cell depletion. Some asymptomatic seropositive individuals do not produce anti-gp antibodies. Since high antibody titers to virus coat proteins both before and after infection have not blocked either initial virus infection or AIDS progression with wild-type virus, it is time to look at the opposite hypothosis.

4. Rapid mutation of the virus in infected individuals raises the possibility that virus replication in activated T cells is utilizing or is effected by the gene rearrangement machinery of the Ig/TCR /MCH gene complex in the infected T cells. The T cell surface receptors that signal T cell interactions and immunoregulation are coded in the Ig/TCR/MHC gene complex. Any disruption of this rearrangement machinery by virus infection could lead to expression of abnormal T cell surface molecules and/or antibody peptides that trigger TCV and/or VGP.

5. These models are at least consistent with some unexplained experimental results and clinical observations of CD4+ T cell depletion and the progression of AIDS disease in spite of high antibody titers and low-level infection of circulating CD4+ cells, progression of the disease associated with specific antigen groups (the CDC antigen groups), and asymtomatic seropositive individuals who do not produce anti-virus antibodies .

6. Some basic elimination experiments to qualify or refute these hypotheses can be carried out in animals already fatally infected in failed vaccine trials, which animals will be sacrificed prematurely otherwise. Most of the experiments can be performed and evaluated in times much shorter than the evaluation times required for new vaccine trails.

7. If experiments can verify either of these models, it may be possible to arrest depletion of CD4+ cells in AIDS disease with monoclonal antibodies to the gamma-delta T cell receptor and/or protect high-risk individuals from AIDS disease with virus antigen tolerization therapies that suppress the production of anti-virus antibodies.

References:

1) E.J. Stott, NATURE 353, 393 (1991)

2) L.O. Arthur, et al, SCIENCE 258, 1935 (1992)

4) Amitabh Gaur, et al, SCIENCE 259, 91 (1993)

5) J.M. Riberdy, et al, NATURE 360, 474 (1992)

6) Peter Mombaerts, et al, NATURE 360, 225 (1992)

7) Encyclopedia of Immunology, Roitt and Delves, Ed., (1992)

8) W.H. Wattenburg, IEEE Transactions on Electronic Computers, EC-15, 378 (1966)

9) Y. Takahama, et al, NATURE 371, 67, (1994)

10) S. P. Balk, et al, SCIENCE, 265, 259 (1994)

11) C. McMenamin, SCIENCE, 265,1869 (1994)

12) Malkovsky, J. Med. Primatology,12, 113 (1992)

13) R. Gorcznski, Immunology, 81, 27 (1994)

14) Graziosi, et al, Science, 265, 248 (1994)

15) Maggi, et al, Science, 265, 244 (1994)

16) J. Cyster, et al, Nature, 371, 389 (1994)

17) De Pauli, et al, Clin Exp Immunology, 83, 187-191 (1991)

18) Cardillo, et al, Eur. J. Immunology, 23, 2597-2605 (1993)

19) Dieter Kabelitz, et al, Immunological Reviews (Munksgaard, Copenhagen), No 120, 71- 88 (1993)

20) Klaus Pechhold, et al, J Immunology, 152, 4984-4992 (1994)

21) Ibegbu, et al, Clinical Immunology and Immunopathology, 71(1), 27-32 (1994Apr)

22) Kozar, et al, J Clin Immunology, 13(3), 193-203 (1993May)

23) De Pauli, et al, Clin and Experimental Immunology, 83(2), 187-191 (1991 Feb)

24) Docke, et al, Allergie and Immunologie, 36(4),209-223 (1990, in German)

25) Margolick, et al, Clin Immunology and Immunopathology, 58(1),126-138 (1991Jan)

26) P. De Pauli, et al, Clin Exp Immunology, 83, 187-191 (1991)

27) B. Autran, et al, Clin Exp Immunology, 75, 206-210 (1989)

28) C. D. Surh,et al, Nature, 372, 100-103(1994)

29) G. Pantaleo, et al, Nature, 362, 355-358(1993)

30) J. Embretson, et al, Nature, 362, 359-362(1993)

31) Dan W. Urry, Scientific American, Jan 1994, 64-69

32) Ettaly K. Franke, et al, Nature, 372, 359-362 (1994)

33) Markus Thall, et al, Nature, 372, 363-365 (1994)



1. J.P Allison, et al, Annual Review of Immun., 1991, 9, 679-705

2. J.P Allison, et al, Current Opinion in Immunology, 1993Apr, 5, 241-246

3. J.P Allison, et al, Seminars in Immunology, 1990 Jan, 2, 59-65

4. J.P Allison, et al, J Exper. Med., 1 July 93, 178, 309-315





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Discussion notes from the February 1993 meeting with Professor Murray Gardner and staff at UC DAvis Medical School:

TCV MODEL OF CD4+ CELL DEPLETION:

The T-cell vaccination (TCV) model for CD4+ cell depletion is consistent with clinical observations of AIDS disease in the following respects:

a) Activation of the immune system is associated with accelerated progression of AIDS disease. Early TCV experiments (7) showed that successful vaccination with isolated T-cell membranes required activated T cells with cross-linked membrane-bound receptor proteins. In some way, activation of the T cells prior to membrane separation presented the required T cell receptor (TCR) gene peptides that triggered vaccination. These TCR peptides are most likely not presented in a TCV stimulating manner by viable T cells in the body. Otherwise, TCV-induced CD4+ clonal suppression would be taking place continuously.

b) The TCV mechanism as a cause of CD4+ cell depletion correlates with the clinical observations of specific disease groups developing in some AIDS patients, the CDC groups I-IV. T-cell vaccination with a specific peptide fragment of the TCR appears to block out immune response to all antigens which associate with this region of the TCR gene, whereas TCV with other TCR variable region peptides does not diminish immune response against antigenic determinants which do not associate with these TCR gene regions (4). MHC association with specific diseases is well known.

c) The TCV model says that activated CD4+ T cell clones should be the first to be suppressed because virus infection of activated T cells presents the specific MHC/TCR peptides that characterize this activated clone. Initial infection should be followed by anergy in the T-cell clones infected. Other CD4+ clones are later suppressed as they become activated. This is not inconsistent with the CDC Group I initial infection syndrome for 10%-15% of AIDS patients.

Progression of the disease may then depend on future antigen stimulation and activation of other T cell clones. This could explain the long delays in AIDS progression which associates with different disease groupings, which in turn are associated with the specific virus-presented TCR peptides that triggered T Cell Vaccination against specific CD4+ clones.

The TCV model is supported by significant experimental results from other areas of immunology:

First are the recent studies of T Cell Vaccination (TCV) which suppresses autoreactive CD4+ clones in MBP mice. The result of TCV in these studies was to suppress autoimmune responsiveness. Gaur, et al, (4) have reported that autoreative CD4+ cells in MBP mice are suppressed when the mice are vaccinated with target antigen associated peptide from the variable region of the T cell receptor (TCR). Depleting the mice of CD8+ cells with monoclonal CD8+ antibody before vaccination with the TCR peptide blocked the suppression (anergy) of CD4+ autoreactive clones. CD8+ cells of some sort, triggered by vaccination with autoantigen- associated TCR peptide, are suppressing the autoreactive CD4+ cells. Some gamma-delta T cells also express the CD8 receptor (7).

Secondly, Arthur, et al, (2) recently demonstrated that HIV/SIV virus particles from human and monkey culture cells carry cellular MHC proteins. These virus-carried protein complexes could include peptides that initiate T cell vaccination and subsequent CD4+ clonal 3) R.C. Desrosiers, et al, SCIENCE 258, 1938 (1992)

suppression. Cross-linked, T cell membrane-bound molecules from activated T cells can trigger T cell vaccination and specific CD4+ clonal anergy (7).

Thirdly, Stott (1) has reported the surprising result that vaccination of monkeys with uninfected human culture cells alone produced immunity against the SIV virus raised in the human cells. Clearly, some protective mechanism is at play which is not dependent on the usual immunoregulatory recognition of virus antigens. The virus-carried cellular MHC peptides discovered by Arthur, et al, (2), and other culture cell peptides must be suspected.

Guar, et al, (4) have shown that:

1) vaccination of MBP autoreactive mice with a peptide from the variable region of the B gene of the T-cell receptor can induce clonal anergy of the autoreactive CD4+ cells,

2) suppressor cells of some sort which express CD8+ are essential for mediating this vaccination induced anergy of the CD4+ clones in their mouse model,

3) only this one segment of the B gene variable region (Vb8.2) induces the specific clonal anergy of MBP responsive CD4+ clones, and

4) the CD4+ responsiveness against other antigens also associated with the Vb8.2 region is also surpressed by vaccination with the TCR Vb8.2 peptide.

T cell vaccination appears to suppress CD4+ responsiveness to certain antigenic groups associated with the specific TCR peptide used to induce the clonal anergy. This is consistent with the observed elimination of immune responsiveness to specific groups of antigens, the CDC groups (7), in the progression of AIDS disease in some patients.

The mysterious gamma-delta T cells are candidates for the unknown CD4+ suppressor cells in TCV. There is also the real possibility that the ineffective anti-virus antibodies (or other peptides carried in these complexes) could be stimulating the depletion of CD4+ cells by cross-linking the TCR receptors on precursor T cells or stimulating immunosuppressor cells, such as gamma-delta CD8+ T cells, to attack CD4+ cells or their precursors.

APPENDIX:

A logical model of immunoregulation developed by the author predicted that there must be another class of immunoregulatory cells besides the alpha-beta CD4+ T cells and CD8+ T cells that have received most of the attention of immune function studies. These missing immunoregulatory cells were called T' cells to denote their regulatory role over the alpha-beta T cells. The putative T' immunoregulatory cells are necessary to a) prevent delayed-type hypersensitivity by downregulating positively selected alpha-beta T cells which become activated against harmless antigens (prevent hypersensitivity to recall antignes) and b) monitor the thymic epithelial cells that select MHC-restricted precursor alpha-beta T cells for self compatibility. Existence of the first immunoregulatory function is well established. Very little is known about regulation or monitoring of the thymic cells that select alpa-beta T cells. However, the stability of mammalian immune mechanisms is difficult to explain without assuming that there is some active surveilance of the cells that select alpha-beta T cells for activation.

Both logical and physical constraints suggest that alpha-beta T cells positively selected by thymic epitheleal cells cannot themselves perform the most important surveilance, or error checking, of the thymic cells that selected these T cells. Physical constraints futher predict that the putative T' cells would not be MHC restricted because this would conflict with the primary task of recognizing Thymic epithelial "selector" cells that present abberant or incorrect MHC receptors and thereby negatively select otherwise self-compatible T cell precursors.

A review of the literature disclosed numerous experimental observations that make the gamma-delta T cells good candidates for the putative T' cells. At least some gamma-delta T cells are not MHC restricted, and they appear to use antibody-like receptors. Many have speculated that the mysterious gamma-delta T cells perform some primative immunoregulation function because of their early appearance during development. The gamma-delta T cells (normally CD4- CD8-) display suspicious immunoregulatory functions, but are not known to be involved in, or necessary for any alpha-beta CD4+or CD8+ immune response. Some gamma-delta appear to be CD8+.

In the course of this investigation, associations were found between the the receptor ligands of gamma-delta T cells and the peptides and surface molecules that stimulate an immunoregulation mechanism known as T Cell Vaccination (TCV) which can selectively suppress CD4+ clones activated against specific antigen groups in mouse models. TCV can be stimulated by peptides from the T cell receptors of T cells activated against specific antigen groups (4).

The reported selectivity of the TCV immunosuppression mechanism correlated well with immunoregulation of activated alpha-beta CD4+ cells to suppress recall antigen hypersensitive responses. Futher investigation revealed that TCV can also be stimulated by membranes from activated T cells that that have cross-linked cell surface molecules. The cell surface molecules and the receptors used by some gamma-delta T cells also appear to be coded in the Ig/MHC supergene complex.

Reference 8:

With a team at U.C. Berkeley, 1961-1966, the author first developed and published the genetic programming rules and constraints for the construction of large self-reproducing computer programs. (Self-compiling compilers they were called. These were the first programming language translator programs written and expanded in their own "genetic" language, like DNA sequences. These translator programs then translate their own genetic description into a machine or somatic language, like proteins). The objective was to generate workable, efficient, self-reproducing translator programs, not theoretical models. Because of frequent errors in the constantly changing genetic programs and unreliability of early computer operation, the first genetic computer programs were unworkable. We had to find systematic ways to make these huge genetic programs evolve with "immune mechanisms" built into the programs. Since then, these genetic programming rules and techniques have been the basis of all the vast and sophisticated computer operating systems and programming language translators in use today.

This knowledge from the computer projects was the starting point for the models of CD4+ depletion in AIDS disease proposed above. Requiring consistency with significant experimental results and major clinical observations narrowed the logical models down to the models considered above.

In the 1966 paper (8) which described the genetic programming rules and constraints that we discovered in the construction (ontogeny) of the first workable computer genetic (self- reproducing) programs, it was noted at the end of the paper that biologists would someday discover that the cells of complex living organisms have to somehow obey similar genetic progamming rules in order to stablely evolve (stage) to more sophisticated forms (unless one assumes a supreme being is directing the process). Not enough was known about cellular programming structures at the DNA level at that time to make any direct analogies between the computer models and the immune mechanisms that must be coded in the cells of higher living organisms that develop through differentiation by sequentially expressing earlier evolutionary stages.

THE CS MODEL-- THE CLONAL SWITCH:

The CS Model is based on some first principles of genetic programming.

Positive and negative selection of T-cell clones in primary lymphoid tissues is well known. This clonal selection machinery will henceforth be called the CSM. The selection and maturation of thymocytes in the thymus is one major component of the CSM.

The CS Model predicts:

1) AIDS virus first infects cells of the CSM (primary lymphoid tissues) which are shielded from normal immune surveillance by alpha-beta T cells and antibody. This is at least consistent with the failure of neutralizing antibody to prevent infection.

2) Immature or shielded cells of the CSM which are not yet actively selecting alpha-beta T-cell clones are infected and some long period of time is required before these modified CSM cells mature and/or are active in clonal selection in the CSM. The original CSM then becomes reprogrammed to be a CSM'. ( the superscript ' indicates cells or clones with modified "self" recognition programming).

3) new CD4' clones selected by CSM' are incompatible with other cells. Signalling incompatiblility with the other MHC II immunoregulatory cells prevents expansion and normal stimulation of the CD4' clones and/or triggers apoptosis of the CD4' clones.

The major considerations that support the CS Model are:

1) Viable CD4+ cells in AIDS patients display a general unresponsiveness that would be expected if these normally "self" MHC restricted T-cells were contacting antigen presenting T-cells and B cells that carried incompatible MHC. The possiblity exists that CD4 cells in AIDS patients have been rendered MHC incompatible with other immunoregulatory cells from the standpoint of cell to cell interactions that stimulate all the normal immune responses that are absent in the CD4+ cells of AIDs patients. The MHC molecules presented could still appear normal, but something in the cellular MHC-TCR recognition machinery has been changed. For instance, virus alteration of the stroma cells in the thymus which select thymocytes could accomplish this "Clonal Switch."

2) First principles of genetic coding of self-reproducing physically realizable systems tells us that there must always be some components of any error checking system (immune system) which can not check or safeguard its own functions. (The author helped discover and develop these principles for the construction of the most sophisticated self- regenerating computer programs used extensively today (8). Without going into the logical proof of this principle, it is sufficient to say that the primary clonal selection machinery (CSM) which programs and directs the cellular error checking machinery of the body cannot use this same machinery (T-cells ) to check and protect all aspects of its own (CSM) programming.

What the above principle says is that some portion of the CSM (thymus, liver, bone marrow?) cannot necessarily protect its own cells from any and all genetic program alterations (errors) that might be normally recognized in other body cells by the immune system that is produced by the CSM. The only protection mechanism that can be used by the CSM in these cases is to isolate itself from outside influences that could modify its genetic programming, including surveilance by its own immune cells.

The likely physical consequences of this principle are:

a) Some cells that make up the clonal selection machinery (CSM) which programs the immune mechanism are shielded from immune surveilance, which means

b) Infecting viruses or other factors which can change the programming of these surveillance shielded CSM cells cannot be detected or eliminated by the normal immune mechanism.

CS MODEL EXPERIMENTS:

1) The first "shotgun" experiment to check The CS Model in early infected animals would be to remove and/or bypass the thymus and see if this has any effect on CD4 cell depletion and function.

2) Other T-cells of unknown function, such as the gamma- delta T cells, should be selectively eliminated with monoclonal antibody to see if they play any role in CD4 cell depletion and AIDS progression.

3) One manifestation of The CS Model could be the appearance of Class II MHC molecules on cells or in tissues that are not normally Class II. This could provide inappropriate signals to CD4+ cells and lead to CD4+ cell depletion.



INEFFECTIVE ANTIBODIES IN AIDS PATIENTS



The Models proposed above even beg the seemingly outrageous possibility that the apparantly ineffective neutralizing antibodies seen in infected animals and/or antibody-gp protein complexes could be functioning as signal transducers to render CD4+ clones unresponsive. (The gamma-delta T cells appear to use Ig-like receptors for target cell recognition and cell to cell interactions instead of the TCR-MHC complex.)



High antibody titers to the Aids virus in the presence of continued virus infection is still unexplained. It runs counter to all current dogma that these antibodies could in any way promote AIDS disease progression, but most of the pathogenesis of HIV/SIV is unexplained by current dogma.



EXPERIMENTS ON INFECTED ANIMALS:



Experiment: challenge AIDS-virus infected monkeys with specific antigen groups and see if CD4+ cells activated against these antigen groups are purged selectively. Many SIV infected monkeys soon to be sacrificed have not as yet developed full-blown aids disease. They still can mount an immune response against many antigens, even though they have failed to ward off aids virus infection in various vaccine trials.



1) Either eliminate the B-cell clones producing the virus antibodies, or lock up the circulating antibodies in complexes much different than normal antibody-gp protein complexes. There is the possibility that antibodies bound to virus particles or to cell membranes are the villains. In this case, elimination of reactive B-cell clones would seem to be the place to start.



2) Again, the gamma-delta T cells must be suspected. Their cell to cell interactions appear to use Ig-like combining sites rather than the conventional TCR-MHC recognition used by alpha-beta T cells. And yet, there is no evidence that the gamma-delta cells are involved in any normal immune responses to defined antigens. (7, page 1432). This begs the possibility that the gamma-delta T cell association with Ig combining sites serves some primary immunoregulatory function over alpha-beta





  1. W.H. Wattenburg, IEEE Transactions on Electronic Computers, EC-15, 378 (1966).