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Page 1 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 1 Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 Liubov Beduleva, Tatyana Khramova, Igor Menshikov, Elena Stolyarova, Svetlana Pavlova Udmurt State University, Department of Immunology and Cell Biology, 1 Universitetskaya St., Izhevsk, 426011, Russian Federation. Tel.: +73412916426. Correspondence to Liubov Beduleva; E-mail: [email protected] Running Head: RF is a cofactor of anti-CD4 autoantibodies Page 2 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 2 Abstract The development of immunodeficiency in HIV-infected patients is known to result from CD4+ lymphocyte depletion. Most CD4+ lymphocyte cells destined to die are not infected. The mechanism of HIV-uninfected cell death has not yet been fully elucidated. The aim of this study is to examine the role of anti-CD4 autoantibodies and physiological rheumatoid factor (RF) in the development of CD4+ lymphocytopenia. Immunization of Wistar rats with gp120 HIV-1 induces chronic production of anti-CD4 autoantibodies and decreases CD4+ lymphocytes in the blood. However, the anti-CD4 autoantibodies produced as part of the immune response to gp120 do not kill CD4+ cells directly. In rats producing anti-CD4 autoantibodies, a low level of peripheral CD4 lymphocytes is associated with high blood RF levels. The sera containing RF killed lymphocytes when the lymphocytes were pre-treated with sera containing anti-CD4 autoantibodies. Thus, the death of CD4+ lymphocytes in rats immunized with gp120 is a result of the combined action of anti-CD4 autoantibodies and rheumatoid factor, and the action of these factors can be separated in time. The fact that two signals are needed for CD4+ lymphocyte death in HIV gp120-immunized rats does not contradict the hypothesis of the Page 3 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 3 activation-induced death of uninfected CD4+ cells in HIV-infected humans. Keywords: gp120 HIV-I, CD4+ lymphocytopenia, anti-CD4 autoantibodies, rheumatoid factor, idiotypic regulation Introduction The development of opportunistic infections and progression to AIDS in HIV-infected patients is known to result from CD4 T lymphocyte depletion. Although a direct cytopathic effect of HIV-1 on CD4 T cells contributes to the depletion, most cells destined to die are not infected with HIV. Understanding how uninfected CD4 Т cells are eliminated is critical to the development of effective therapies. The mechanism of death of HIV-uninfected cells that results in immune deficiency has not yet been fully elucidated. Several hypotheses were offered. The role of apoptogenic HIV proteins and antibodies to gp120 that induce cross-linking of gp120 on CD4+ lymphocyte membrane has been considered.1-3 The counter-argument to hypotheses that consider viral proteins, independently or together with anti-gp120 antibodies, to be the cause of CD4 T lymphocyte death is that such theories do not explain the lack of correlation between viral load and blood levels of CD4 T cells in HIV-infected patients nor the AIDS-like symptoms Page 4 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 4 without HIV infection that are observed, for instance, in idiopathic CD4 T lymphocytopenia and mixed connective tissue disease.4-8 The hypothesis of activation-induced cell death of uninfected CD4 Т lymphocytes appears less contradictory.9 Under this hypothesis, in HIV infection, uninfected CD4 Т lymphocytes undergo a generalized activation that sensitizes the CD4 T cells to apoptosis. Subsequently, when the activated cells encounter an apoptosis inducer, they undergo apoptosis. However, neither the factor that induces this generalized activation of uninfected CD4 Т lymphocytes, nor the apoptosis inducer, i.e. the factor that kills the activated CD4 T lymphocytes, is yet known. Anti-CD4 autoantibodies can be the factor that causes the chronic polyclonal activation of uninfected CD4 cells in HIV infection and sensitizes them to apoptosis.10 The hypothesis that the autoimmune response to CD4 plays an important role in AIDS pathogenesis is not a new idea.11 Depletion of CD4+ T cells in HIV- or SIV-infected individuals was associated with plasma antibodies autoreactive with CD4+ T cells.12-15 Furthermore, Coore et al. demonstrated the presence of idiotype-anti-idiotype interactions between anti-gp120 antibodies and autoantibodies to CD4 in HIV-infected people, and the mechanism by which the autoantibodies to CD4 form during HIV Page 5 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 5 infection is clear.16 However, the autoimmune hypothesis of AIDS is not generally accepted. Perhaps the reason is that none of the studies conducted have clearly identified the mechanism by which anti-CD4 autoantibodies could mediate CD4+ T cell depletion. The aim of this study is to examine the role of anti-CD4 autoantibodies induced by gp120 HIV-1 in the development of CD4+ lymphocytopenia. The model chosen for study was immunization with purified gp120 protein from HIV, since under the autoimmune theory of AIDS the immune response to gp120 serves as the cause of anti-CD4 autoantibody induction.16 This model precludes a contribution by the virus or its apoptogenic proteins to the depletion of CD4 T lymphocytes. Humanized mice are the most appropriate for inducing an autoimmune response to CD4. However, in the pilot version we tried using Wistar rats. The basis for this attempt was the homology between rat CD4 and human CD4 in the area responsible for binding with HIV-1 gp120.17 The homology suggests that HIV-1 gp120 may be complementary to rat CD4, and consequently that the anti-CD4 lymphocytes that may be activated by anti-gp120 lymphocytes through idiotype-anti-idiotype interactions will be specific to rat CD4. Page 6 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 6 Previously we showed that resistance to experimental autoimmune diseases was associated with the production of rheumatoid factor (RF), which was detected by the method of agglutination of tanned erythrocytes loaded with homologous IgG.18 The role of RF in the formation of resistance to developing an autoimmune response to CD4 or regulating the immune response to gp120 has not been studied. Therefore, one of the aims was to investigate the role of RF in regulating the immune response to HIV gp120. Materials and methods Rats Wistar rats (8–10 weeks old) were used. They were brought from the Stolbovaya breeding facility (Russia) and housed under standard laboratory conditions with food and water at a constant temperature of 20±5 °C. Animal experiments were performed in accordance with European Commission Directive 86/609/EEC (European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes) and approved by the local animal care commission of Udmurt State University. Gp120 HIV-1 immunization and blood collection Recombinant GP120 HIV-1 glycoprotein (ACROBiosystems, Newark, USA) was emulsified with the same volume of incomplete Page 7 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 7 Freund’s adjuvant (IFA) (Sigma, St Louis, MO, USA). Rats (n=7) were injected at the tail base with gp120/adjuvant emulsion (20 μg of gp120 per rat). No booster immunization was performed. Control rats (n=8) received an injection of an emulsion containing saline solution and IFA. Blood sampling for anti-gp120 antibody, anti-CD4 autoantibody, and RF detection was carried out every week. From time to time blood samples were used to determine CD4+ cell counts. The number of exposed rats and days of CD4+ cell count determinations are presented in Fig. 2a. Determination of anti-gp120 antibodies by ELISA Plates (Corning-Costar, Acton, MA, USA) were coated overnight at 4 °C with 50 µl of gp120 HIV-1 (ACROBiosystems, Newark, USA) (10 µg/ml in 0.15 M PBS). Plates were blocked with 150 µl 5% milk powder in PBS. Serum samples were added in a 1:200 dilution with PBS/Tween-20 and incubated for 1 h at RT. The plates were then incubated for 1 h at RT with 100 µl of goat anti-rat Ig (IgG, IgM, IgA) conjugated to horseradish peroxidase (IMTEC, Moscow, Russia). Then the substrate mixture (5 ml of citrate buffered solution [pH 5.0]/3 mg ortho-phenylenediamine/15 ml 3% H2O2) was added. Absorbances were read after 15 min at 492 nm. Determination of anti-CD4 antibodies by ELISA Page 8 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 8 Plates (Corning-Costar, Acton, MA, USA) were coated overnight at 4 °C with 50 µl of recombinant rat CD4 (R&DSystems, Minneapolis, USA) (10 µg /ml in 0.15 M PBS). Plates were blocked with 150 µl 5% milk powder in PBS. Serum samples were added in a 1:100 dilution with PBS/Tween-20 and incubated for 1 h at RT. The plates were then incubated for 1 h at RT with 100 µl of goat anti-rat Ig (IgG, IgM, IgA) conjugated to horseradish peroxidase (IMTEC, Moscow, Russia). Then the substrate mixture (5 ml of citrate buffered solution (pH 5.0)/3 mg ortho-phenylenediamine/15 ml 3% H2O2) was added. Absorbances were read after 15 min at 492 nm. Measurement of RF The RF titer was determined in an agglutination test using rat-IgGloaded tanned erythrocytes. Group 0 human erythrocytes were fixed with 1% glutaric dialdehyde. The washed-off fixed erythrocytes (10% in PBS [pH 7.2]) were incubated for 10 min with the same amount of tannin solution in PBS at room temperature. The erythrocytes were then washed off. For sensitization, 4 ml of PBS (pH 6.4), 1 ml of a solution of 0.5 mg/ml normal IgG from rat serum (Equitech-bio, Kerrville, USA) in 0.9% NaCl, and 70 µl of the pelleted tanned erythrocytes were mixed. Incubation lasted for 20 min at room temperature; erythrocytes were washed with 0.9% PBS containing Page 9 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 9 0.2% bovine serum albumin. Twofold serial dilutions of serum were prepared and put into wells in the amount of 50 µl. The same amount of rat-IgG-loaded 1.5% erythrocyte suspension was then added. Agglutination results were read in 3 h. Determination of CD4+ cell counts The lymphocytes were isolated in ficoll-urografin gradient (ρ=1.08 g/cm3). Then aliquots of cells washed in PBS were stained for 1 h at 37 °C with FITC-conjugated anti-rat-CD4 (eBioscience, San Diego, USA), 0.25 mg antibody per 106 cells in a final volume of 100 µL. The lymphocytes were then washed off. After that CD4+ lymphocyte counts were determined using laser-based flow cytometry (BD FACSCanto II Flow Cytometer). Viability of lymphocytes treated with anti-rat CD4 autoantibodies and rheumatoid factor in vitro The lymphocytes were isolated in ficoll-urografin gradient (ρ=1.08 g/cm3) from intact rats. The lymphocytes were washed off in RPMI1640. After that 100 µl of serum from gp120-immunized rats containing anti-rat CD4 antibody or 100 µl of serum from IFAinjected rats containing no anti-rat CD4 antibody was added to 106 lymphocytes in RPMI-1640 in a final volume of 1 ml. The rheumatoid factor titer of the sera that were added was ≤1:4. The lymphocytes Page 10 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 10 were incubated for 24 hours in a 37 °C, 5% CO2 incubator. The lymphocytes were then washed off in RPMI-1640 and were suspended in a volume of 100 µl. Then 100 µl of serum containing rheumatoid factor (RF titer ≥1:32) and obtained from intact rats or 100 µl of serum containing no rheumatoid factor (RF titer ≤1:4) and obtained from intact rats was added. After incubation for 3 hours in a 37 °C, 5% CO2 incubator the number of cells was counted using trypan blue. Statistical analysis of the data The significance of differences was assessed by the t-test. Results Production of anti-CD4 autoantibodies and CD4+ lymphocytopenia in rats immunized with HIV 1 gp120. Immunization of Wistar rats (n=7) with HIV-1 gp120 induced the production not only of antibodies to HIV-1 gp120, but of autoantibodies to rat CD4 (Fig. 1). Anti-CD4 autoantibody production was revealed in all of the gp120-immunized rats. In rats receiving IFA (n=8), no anti-CD4 autoantibodies were found (Fig. 1). Fig. 1 shows that the production of anti-gp120 antibodies induced by a single injection of HIV-1 gp120 did not attenuate over the 4 months of observation. The production of anti-CD4 autoantibodies also was Page 11 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 11 chronic in nature; the level of anti-CD4 autoantibodies in the blood of rats immunized with gp120 remained significantly higher than that of IFA-injected rats even 4 months post immunization. In summary, immunization with HIV-1 gp120 induces chronic anti-CD4 autoantibody production in Wistar rats. The count of CD4+ cells in the blood of the gp120-immunized rats and the IFA-injected rats is shown in figure 2a. In rats that have developed an autoimmune response to CD4, the average number of CD4+ cells was significantly lower than the average number of CD4+ cells in the peripheral blood of IFA-injected rats (Fig. 2b). The lowest number of CD4+ cells induced in rats immunized with gp120 that developed an autoimmune response to CD4 was 293 cells per µl of blood (Fig. 2c, left picture). Representative flow cytometry of CD4+ cells in IFA-injected rats is shown in Fig. 2c, right picture. The decrease in CD4+ lymphocytes in rats immunized with gp120 protein was transitory in nature; a drop was followed by a compensatory rebound in CD4 lymphocyte levels. Contrary to expectations, no correlation was found between the level of anti-CD4 autoantibodies and the number of CD4+ lymphocytes in the blood (r=0.24, p=0.27). The drop in the number of CD4+ cells in the blood can be observed against a background of stable, relatively Page 12 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 12 low anti-CD4 antibodies in the blood, and vice versa - when the level of anti-CD4 antibodies is increasing rapidly, CD4 numbers can remain stable as compared to the cell numbers in IFA-injected rats. In sum, immunization of Wistar rats with HIV-1 gp120 induces chronic production of anti-CD4 autoantibodies and decreases CD4 lymphocytes in the blood. However, no correlation was found between antibodies to CD4 lymphocytes and the number of CD4 lymphocytes in the blood. Role of RF in the development of CD4+ lymphocytopenia in rats immunized with HIV-1 gp120 The time course of RF in response to HIV-1 gp120 immunization and in IFA-injected rats is shown in figure 3. RF levels were found not to increase in response to gp120 immunizations. The surges in RF production observed at 5, 9, 12, 15, and 17 weeks after gp120 immunization were delayed relative to immunization with the gp120 protein of HIV, while the kinetic curves of RF in gp120-immunized and IFA-injected rats almost entirely coincide (Fig. 3). Therefore, the observed changes in RF level in the blood of gp120-immunized rats are not the result of the immune response to gp120 nor the administration of animal IFA, but are spontaneous in nature and are natural variations in RF level. In sum, rheumatoid factor is not Page 13 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 13 involved in downregulating the immune response to gp120 HIV in rats. In an individual analysis of the parameters studied, we noticed that in the rats immunized with HIV gp120, the minimum of the blood CD4 lymphocytes corresponded with the maximum of the blood RF levels (Fig. 4a). No such association was found in the rats that had received IFA in place of gp120. We compared the level of CD4 cells at a relatively high RF level (titer >1/16) and a relatively low RF level (titer ≤1/16) in the blood of rats immunized with gp120. The same test was performed in IFAinjected rats. In the gp120-immunized rats, when RF blood levels were relatively high, the number of CD4 lymphocytes was significantly lower than in the rats with a relatively low RF level (Fig. 4b). In the IFA-injected rats, whether the blood RF was relatively high or relatively low, the blood CD4 cell levels were the same (Fig. 4b). In sum, in rats immunized with gp120 and producing anti-CD4 autoantibodies, a low level of peripheral CD4 lymphocytes is associated with high blood RF levels. In the IFA-injected rats, which have no anti-CD4 autoantibodies, the number of CD4 cells does not depend on blood RF levels. This information prompts us to suggest that the death of CD4 lymphocytes in rats immunized with gp120 is Page 14 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 14 a result of the combined action of CD4 autoantibodies and rheumatoid factor, and the action of the factors can be separated in time. Effect of serum containing anti-CD4 antibodies and serum containing RF on lymphocyte viability in vitro To test the assumption that CD4+ lymphocyte death in rats immunized with gp120 results from the combined action of anti-CD4 autoantibodies and rheumatoid factor, we performed additional in vitro studies. Lymphocytes from intact rats (n=11) were incubated with sera containing anti-CD4 antibodies and obtained from gp120immunized rats (n=11). Next, the lymphocytes were treated with serum from intact rats (n=11) that contains RF. We found that the lymphocytes pretreated with serum containing anti-CD4 antibodies died in response to the presentation of serum containing RF (Fig. 5). Lymphocytes did not die if serum without RF was added to lymphocytes incubated with serum containing antiCD4 (Fig. 5). The lymphocytes also stayed alive when the lymphocytes were incubated in serum without anti-CD4 antibodies and then presented with RF-containing serum. Thus, anti-CD4 autoantibodies and rheumatoid factor do not induce lymphocyte death on their own; the death of CD4 lymphocytes results from the Page 15 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 15 combined action of rheumatoid factor and anti-CD4 antibodies obtained from rats immunized with HIV-1 gp120. Discussion The aim of this study was to test the hypothesis that the cause of CD4 lymphocytopenia in HIV infection is an autoimmune response to CD4 induced by HIV-1 gp120. It is known that in HIV infection lymphocytes specific to CD4 are activated by lymphocytes against HIV-1 gp120 through idiotype-anti-idiotype interactions. The ability of lymphocytes against HIV gp120 to enter into idiotype-anti-idiotype interactions with lymphocytes against human CD4 is prompted by the complementarity of the antigen-recognizing receptors of these lymphocytes, which itself is a consequence of the complementarity between the gp120 CD4-binding sequence and the first domain of human CD4. In choosing rats as the subjects for testing the hypothesis, we assumed that rat lymphocytes, which can be activated by anti-gp120 lymphocytes through idiotype-anti-idiotype interactions in response to HIV-1 gp120 immunization, would not be specific to rat CD4. This doubt was supported by data on the homology of about 50% between the first domain of human CD4, which is responsible for HIV-1 gp120 binding, and the analogous section of the rat CD4 molecule. The fact that antibodies to rat CD4 Page 16 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 16 appeared in the rats in response to gp120 immunization indicated that the rat lymphocytes specific to HIV gp120 enter into idiotypeanti-idiotype interactions with autoreactive lymphocytes against rat CD4 and that immunization of rats with HIV-1 gp120 can be used to test the hypothesis. To measure anti-CD4 autoantibodies, recombinant rat CD4 was used. Some researchers are skeptical of the use of recombinant CD4 (rCD4) to measure anti-CD4 lymphocyte antibodies, because they believe that rCD4 differs from the membrane-bound molecule.19, 21 However, rCD4-specific antibodies can appear in rats in response to gp120 immunization only if the lymphocytes that produce antibodies specific to rCD4 enter into idiotype-anti-idiotype interactions with lymphocytes against gp120, and subsequently carry the internal image of gp120. Considering that gp120 interacts with membrane-bound lymphocyte CD4, we can assert that the rCD4 preparation we used also bears sections responsible for gp120 binding, and consequently in this area it is the same as membrane-bound CD4, and the auto-antibodies revealed by recombinant CD4 are specific to the available determinants on membrane-bound CD4. This assertion does not contradict the result Page 17 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 17 obtained by Corre et al. that anti-idiotypic antibodies to human antigp120 antibodies bind recombinant and cellular human CD4.16 According to the autoimmune hypothesis of AIDS, the rat anti-CD4 autoantibodies should induce death of rat CD4 T lymphocytes. In rats immunized with gp120 and producing anti-CD4 antibodies, the average levels of CD4 lymphocytes were significantly lower for the experiment than in the IFA-injected rats. However, the anti-CD4 autoantibodies produced as part of the immune response to gp120 do not kill cells directly. The lack of a correlation between anti-CD4 autoantibodies and the number of CD4 lymphocytes in the blood of gp120-immunized rats, as well as the results of in vitro experiments in which we tried to induce lymphocyte death in the presence of serum that contained CD4 autoantibodies obtained from gp120immunized mice, serve as evidence of this. The death of lymphocytes exposed to anti-CD4 autoantibodies induced by gp120 immunization commences in the presence of rheumatoid factor, both in vivo and in vitro. The fact that two signals, from anti-CD4 autoantibodies and rheumatoid factor, are needed for CD4 lymphocyte death in HIV gp120-immunized rats does not contradict the hypothesis of the activation-induced death of uninfected CD4+ cells in HIV-infected humans. This hypothesis also indicates that two Page 18 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 18 factors are needed: a factor that activates the cells and a factor that kills the activated cells, and the action of the factors can be separated in time. Furthermore, our data coincide with results obtained by Susal et al., who found the association between a high anti-Fab/anti-rCD4 ratio and a low level of CD4+ helper T cells in AIDS patients. Anti-Fab antibodies and rheumatoid factor have similar characteristics. Rheumatoid factor and anti-Fab antibodies both possess anti-Fab activity and downregulate the immune response.18,24 At the same time, our data raise new questions, particularly concerning the mechanism of action of rheumatoid factor that results in CD4 lymphocyte death. We previously conducted detailed studies of the rheumatoid factor population, which is associated with the drop in blood CD4 lymphocytes in HIV gp120-immunized rats. This RF population is found in intact animals; therefore it can be considered physiological. We showed that an increase in RF production in response to immunization with antigen-inducers of experimental autoimmune diseases prevents the development of those diseases and is also associated with termination of the normal immune response.18 A study of RF specificity found that every RF molecule has two kinds Page 19 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 19 of paratopes: a unique individual paratope and a shared paratope; the specificity of the shared paratope is the same across the different RF molecules. The individual paratopes of RFs represent internal images of the foreign antigens; therefore, the RFs are antiidiotypic antibodies, which recognize the antigen-binding sites of antibodies to foreign antigens. So the RF naturally occurring in the blood is heterogeneous in its specificity population of anti-idiotypic antibodies.18 The shared paratope recognizes the recurrent sequence that exists on antibodies of various specificities and is located in the Fab region but outside the antigen binding site.18 Furthermore, we showed that the antigenic determinants for the shared RF paratope can be created in the Fc hinge region of IgG fragments through papain cleavage of IgG.18 The specificity of the shared paratope of RF to the hinge region of IgG Fc fragments distinguishes the given RF from other known RF populations, which have determinants localized between the CH2 and CH3 domains.25 If RF is classified in accordance with the list of types of anti-idiotypic antibodies proposed by Parsons et al.,26 the properties of the individual paratope of the RF molecule enable the RF to be classed as Ab2 Beta (Ab recognizes the antigen binding site), while the properties of the shared paratope of RF resemble the properties of Page 20 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 20 the Ab2 Delta class (Ab recognizes non-binding site, non-disease specific, and outbred shared idiotope). Thus, RF is simultaneously both Ab2 Beta and Ab2 Delta. Based on this information, we proposed a hypothesis explaining how RF prevents autoimmune response.21 We think that RF normally acts as a factor depleting activated antigen-specific CD4 cells. In the course of an immune response to a foreign antigen (including antigens that induce experimental autoimmune diseases), RF is formed as anti-idiotypic antibodies; therefore, its target is only antigen-specific lymphocytes. RF does not recognize CD4 lymphocytes with other specificities. Transmission of the death signal to the activated antigen-specific CD4 lymphocytes may occur by virtue of the shared paratope on RF. The ligand for the shared paratope on the CD4 lymphocyte membrane may be an inducible molecule that is structurally similar to the Fc fragments of IgG and appears only in activated lymphocytes. Anti-CD4 autoantibodies produced in response to gp120 immunization can induce polyclonal activation of peripheral CD4 lymphocytes and the appearance thereon of the ligand for the shared paratope of RF (Fig. 6). Therefore, the RF normally in the blood gets the opportunity to bind with CD4 lymphocytes of any Page 21 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 21 specificity and exercise its ability to kill the lymphocytes (Fig. 6). In this way, we suggest that anti-CD4 autoantibodies turn CD4 lymphocytes into a target for rheumatoid factor and involve RF in a pathological process that results in immunodeficiency. Acknowledgements This study was supported by the Ministry of Education and Science of the Russian Federation, grant number 14.124.13.1159-MD. We are grateful to Jennifer Guernsey for editorial assistance. We thank Tatiana Tolstolutskaya at the Izhevsk State Medical Academy for technical assistance. Conflict of interest statement The authors declare no conflicts of interest. References 1. Alimonti J, Ball B, Fowke K: Mechanisms of CD4+ T lymphocyte cell death in human immunodeficiency virus infection and AIDS. J Gen Virol 2003;84:1649–1661. 2. Kang Y, Melo M, Scott D: An ongoing immune response to HIV envelope gp120 in human CD4-transgenic mice contributes to T cell decline upon intravenous administration of gp120. Eur J Immunol 1998;28:2253–2264. 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Susal C, Hoffman GW, Daniel V, Grant M, Opelz G: Complementarities and network interactions in AIDS. Journal of Autoimmunity 1993;6:601–610. 25. Sasso E, Barber C, Nardella F, Yount W, Mannik M: Antigenic specificities of human monoclonal and polyclonal IgM rheumatoid factors. The C gamma 2-C gamma 3 interface region contains the major determinants. J Immunol 1998;140:3098–3107. 26. Parsons MS, Muller S, Kohler H, Grant MD, Bernardab NF: On the benefits of sin: can greater understanding of the 1F7-idiotypic repertoire freeze enhance HIV vaccine development? Human Vaccines & Immunotherapeutics 2013; 9:1532–1538. AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 26 of 32 26 Correspondence to Liubov Beduleva; E-mail: [email protected] AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 27 of 32 27 Figure 1. Anti-CD4 autoantibodies production and time course of anti-gp120 antibodies in rats immunized with gp120 HIV-1. Each point is represented as mean±SD of 7 (gp120 immunized rats) and 8 (IFA injected rats). Page 28 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 28 Figure 2. CD4+ cell counts in the blood of gp120 HIV-1 immunized and IFA injected rats. (a) CD4+cell counts monitoring in the course of immune response to gp120. Mean, highest and minimum CD4+cell number in each experimental point. Numbers show how many rats were studied in each point. (b) Average number of CD4+ lymphocytes observed in blood samples obtained from gp120 immunized and IFA injected rats throughout the entire experiment (mean±SD). (с) Lowest number of CD4+cells in rats immunized with gp120 (left picture). Representative flow cytometry of CD4+cells in IFA injected rats (right picture). AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 29 of 32 29 Figure 3. The time course of rheumatoid factor in rats in response to HIV-1 gp120 immunization. Page 30 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 30 Figure 4. CD4+lymphocyte numbers in the blood of rats with high and low titer of rheumatoid factor. (a) Individual drop in CD4+ cell count coincident with high level of rheumatoid factor in gp120 immunized rats. Anti-CD4 autoantibodies level is presented as ODA of serum, predeluted 100-fold with PBS. (b) Average number of CD4+lymphocytes observed in blood samples of gp120 immunized rats and IFA injected rats with high and low RF titer (means±SD). Blood samples were separated into groups according to their RF titer. n- number of blood samples. AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 31 of 32 31 Figure 5. Death of lymphocytes after incubation with or without sera containing antiCD4 autoantibodies and treatment with RF-containing sera (means±SD). Page 32 of 32 AIDS Research and Human Retroviruses Combined action of anti-CD4 autoantibodies and rheumatoid factor in the development of CD4 lymphocytopenia in rats immunized with HIV-1 gp120 (doi: 10.1089/AID.2015.0358) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 32 Figure 6. A hypothetical scheme of death of HIV-uninfected CD4 cells in the course of HIV infection. Anti-CD4 autoantibodies produced in response to gp120 HIV-1 induce polyclonal activation of peripheral CD4 lymphocytes and the appearance thereon of the ligand (IgG Fc fragment-like molecule) for the shared paratope of rheumatoid factor. The rheumatoid factor normally in the blood gets the opportunity to bind with CD4+ lymphocytes and exercise its ability to kill the activated lymphocytes.