• Targeting of Non-Infected Cells for Destruction Due to Antibody Binding
• T4 Cell Failure Due to CD4 Crosslinking by gp120/Anti-gp120 Complexes (and Parallel to Anti-CD4 Antibody Induced T4 Cell Failure)
• Apoptosis Due to CD4 Crosslinking
• Transgenic Mice Studies

Targeting of Non-Infected Cells for Destruction Due to Antibody Binding

• Reference: Proceedings of the National Academy of Sciences USA Vol. 84, pp. 4601-4605, July 1987 Immunology

  Authors: H. Kim Lyerly, Thomas J. Matthews, Alphonose J. Langlois, Dan P. Bolognesi, and Kent J. Weinhold.

  Title: "Human T-cell lymphotropic virus IIIB glycoprotein (gp120) bound to CD4 determinants on normal lymphocytes and expressed by infected cells serves as target for immune attack."

  Abstract: "These studies demonstrate that gp120 situated on the cell surface can serve as an effective target for immune destruction by patient antibodies and effector lymphocytes.

  Introduction: "Purified gp120 readily associates with the CD4 molecules and forms a stable complex on the cell surface. This latter finding suggested the possibility that free gp120 bound to noninfected CD4-expressing lymphocytes could serve as a potential target for immune attack resulting in subsequent lympholysis.

  Results: "Since the orientation and expression of gp120 bound to noninfected lymphocytes may be different than that expressed on either virons or virus-infected cells, the ability of the anti-gp120 antisera to bind to infected cells and gp120-adsorbed cells was also examined. ... Cytoflourography revealed that all of the antibodies tested bound the virus infected cells in a manner similar to the gp120-adsorbed CD4 cells. However, mean fluorescence was higher in gp120-adsorbed CD4 cells, suggesting that antigen density was greater. ...

  "All patient sera tested were capable of directing ADCC activity against the gp120 onto CD4-positive cells consistently increases their lytic susceptibility 8- to 10-fold in the presence of patient antibodies and normal effector cells. ...

  Discussion: "While destruction of chronically infected cells may be a beneficial result of ADCC in infected patients, such antibodies could also direct the lysis of noninfected CD4-positive cells if they adsorbed gp120 in vivo and became susceptible to immune attack as demonstrated here. Such an event could represent one of many possible mechanisms of lymphocyte destruction contributing to the overwhelming lymphopenia seen in AIDS."

• Reference: The Journal of Immunology Vol. 142. 3091-3097. No. 9. May 1, 1989

  Authors: Kent J. Weinhold, H. Kim Lyerly, S. David Stanley, Arthur A. Austin, Thomas J. Matthews and Dani P. Bolognesi

  Title: "HIV-1 gp120-mediated immune suppression and lymphocyte destruction in the absence of viral infection"

  Abstract: "This "targeting" of activated lymphoblasts can occur with levels of gp120 far below that which is needed to saturate all OKT₄A-defined CD4 epitopes. Adsorbed gp120 could be demonstrated on the surface of these cells for up to 12 h, a sufficient time for interaction with host cytolytic elements.

  Results: "Thus, saturation of cell-surface CD4 determinants is not required for conferring target status on noninfected lymphocytes.

  Discussion: "Significant lysis was still apparent at approximately 60-fold less than saturating conditions (i.e., 60 ng/ml) when surface gp120 expression was barely detectable by using the gp120-specific 9284 mAb (Fig.1). This coupled with our finding that rapid dissolution of the gp120-CD4 complex does not occur, indicates that ample opportunity exists for interaction between cell surface gp120 and patient antibodies or effector cells....

  "Taken together, our data are consistent with the hypothesis that immunosuppression and cytolysis of CD4+ cells can occur in the absence of HIV-1 infection."

T₄ Cell Failure Due to CD4 Crosslinking by gp120/Anti-gp120 Complexes (and Parallel to Anti-CD4 Antibody Induced T₄ Cell Failure)

• Reference: Science, Vol. 245

  Authors: Robert S. Mittler and Michael K. Hoffmann

  Title: "Synergism Between HIV gp120 and gp120-Specific Antibody in Blocking Human T Cell Activation"

  Abstract: "Experimental findings reported here indicate that CD4-bound gp120 attracts gp120-specific antibodies derived from the blood of HIV-seropositive individuals to form a trimolecular complex with itself and CD4. Thus targeted to CD4, the gp120-specific antibody functions as an antibody to CD4; it cross-links and modulates the CD4 molecules and suppresses the activations of T cells as measured by mobilization of intracellular calcium (Cai2+).

  Body: "We speculated that the immune system may participate in the pathogenesis of AIDS and play a contributory role in its own destruction. We report experimental evidence to support this speculation.

  "Via gp120, the gp120-specific patient IgG may take the role of a CD4-reactive antibody. Cross-linkage of the CD4 molecule with CD4-specific antibody has indeed been shown by others to inhibit anti-Ti-mediated [calcium ion] mobilization.

  "We determined the sensitivity of T cells to suppression caused by gp120-anti-gp120 in terms of gp120 dose and antibody concentration. Significant inhibition of Cai2+ mobilization can be achieved with gp120 as low as 15 ng/ml in the presence of antibody to gp120. The end point dilution of antibody activity was also determined at low concentrations. Using an unfractioned patient serum, we found an end point dilution below 1:30,000.

  "It appears conceivable that gp120 disseminated by HIV-replicating cells forms blood-borne complexes with anti-gp120, thus exposing non-infected CD4-bearing cells to continuous downregulatory signals that may eventually lead to immunodeficiency of the kind seen in mice, after treatment with anti-CD4. The exquisite efficacy of gp120-anti-gp120 complexes in inhibiting T cell receptor-mediated calcium flux may also be viewed as favorable to a gp120-mediated autoimmune mechanism. It places the regulatory phenomenon in a dose range of contributory reagents (gp120 and anti-gp120) that can reasonably be expected to occur in HIV-infected individuals."

Apoptosis Due to CD4 Crosslinking

• Reference: Journal of Experimental Medicine Vol. 176

  Authors: Nirmal K. Banda, Jacques Bernier, David K. Kurahara, Roland Kurrle, Nancy Haigwood, Rafik-P. Sekaly, and Terri Helman Finkel

  Title: "Crosslinking CD4 by Human Immunodeficiency Virus gp120 Primes T Cells for Activation-induced Apoptosis"

  Abstract: "In the present study, crosslinking of bound gp120 on human CD4+ T cells followed by signaling through the T cell receptor for antigen was found to result in activation-dependant cell death by a form of cell suicide termed apoptosis, or programmed cell death.

  Results and Discussion: "There is evidence that gp120 is shed from HIV-infected cells in vitro and in vivo, and in recent work, gp120 has been measured in the blood of patients with AIDS at levels of 12-92 ng/ml. In the in vitro system reported here, as little as 10 ng/ml (10-9 M) gp120 primed CD4+ T cells for activation-induced apoptosis (data not shown.)...

  "Our observation that this priming for apoptosis requires crosslinking of bound gp120 by anti-gp120 antibody may in part explain the paradox that HIV appears to cause AIDS after the onset of antiviral immunity. Though this study does not address the question of apoptotic cell death in HIV-infected individuals, our observation that even picomolar concentrations of gp120 prime for activation-induced apoptosis suggests that this mechanism of cell death may be active in vivo.... This work suggests that deletion of CD4+ T cells upon activation may contribute to the progressive depletion of CD4+ T cells in AIDS."

• Reference: European Journal of Immunology 1995. 25: 1778-1782

  Authors: Serene Foster, Peter Beverley, and Richard Aspinall.

  Title: "gp120-induced programmed cell death in recently activated T-cells without subsequent ligation of the T cell receptor"

  Abstract: "We show that recently activated T cells are susceptible to apoptosis when exposed to HIV gp120 in the presence of anti-gp120 antibody.

  Introduction: "Functional defects in the CD4+ T cell compartment arise early in HIV-infected individuals, well before the peripheral blood CD4+ T cell count has reached the low level found at the onset of AIDS. These defects also arise when fewer than 1 in 10,000 CD4+ T cell in the peripheral blood are infected with HIV. ... Recently, Ameisen and Capron proposed that programmed cell death (PCD) could account for the qualitative and quantitative defects that arise after HIV infection. PCD is a mechanism of tissue homeostasis in which cells die by an active process often involving endonuclease activity, which degrades the DNA into fragments. ... PCD can be induced by cross-linkage of cell surface molecules in an inappropriate sequence; for example, ligation of CD4+ before the T cell receptor (TCR) complex leads to PCD in mature, resting CD4+ T cells. The effect of CD4+ monoclonal antibodies (mAb) can be mimicked by the use of gp120 cross-linked at the cell surface followed by ligation of the TCR and has been proposed as a mechanism of CD4+ depletion in HIV+ individuals.

  Results: "Ligation of CD4 with gp120 in the recently activated cell population did not increase the number of dead cells observed in the controls. However, following cross-linking of CD4 by a combination of gp120 with anti-gp120, a dramatic increase in cell death was observed (from 30% to 65%).

  "IL-2 has been shown to rescue T cells from apoptosis under particular conditions [20]. We, therefore, examined whether addition of rIL-2 would reverse the effects of cross-linking CD4 with the combination of gp120 and anti-gp120 in the recently activated cell population. ... The results reveal that the presence of rIL-2 in recently activated CD4+ cells does not rescue them from cell death...

  Discussion: "Several reports have suggested the cross-linking of CD4 by gp120 and anti-gp120 or anti-CD4 antibodies renders T cells susceptible to apoptosis upon subsequent ligation of the TCR. The results presented here support these findings, in that asynchronous ligation of the TCR and CD4 results in apoptosis, and in addition, two new observations are reported. The first is that TCR-mediated stimulation may precede separate cross-linking of CD4 and still lead to death by apoptosis. The second, that the effects of cross-linking with gp120/anti-gp120 and CD4/anti-CD4 differ. Ligation of CD4 with gp120/anti-gp120 leads to much more death by apoptosis than cross-linking by anti-CD4 antibodies. Also, addition of rIL-2 to gp120-ligated CD4 did not rescue T cells from death, while those cells treated with anti-CD4 were partially rescued.

  "Two factors would be necessary for such a mechanism to contribute to the decline in CD4+ T cells seen in HIV-infected individuals. The first is that there should be activation of lymphocytes, which has been well documented in seropositive individuals, and the second is the presence of gp120 and anti-gp120 antibodies: both have indeed been detected. Interestingly, higher levels of anti-gp120 antibodies may be associated with a poor prognosis. We conclude that this mechanism of apoptosis, induced by asynchronous ligation of the TCR and CD4, may contribute the decline in CD4 T cells in HIV-infected patients."

Transgenic Mice Studies

• Reference: European Journal of Immunology 1994. 24: 1553-1557

  Authors: Zhi-qin Wang, Thorsten Orlikowsky, Anita Dudhane, Robert Mittler, Michelle Blum, Elizabeth Lacy, Gert Riethmuller, and Michael K. Hoffmann

  Title: "Deletion of T lymphocytes in human CD4 transgenic mice induced by HIV-gp120 and gp120-specific antibodies from AIDS patients."

  Introduction: "We report here that anti-CD4hu mAb as well as gp120/anti-gp120 complexes cause deletion of mouse T cells which express the human CD4 transgene.

  Results: "Table 1 shows that complexed gp120 causes severe deletion of T cells expressing the human CD4 marker. Control treatments consisting of gp120 or anti-gp120 alone or of a combination between gp120 with normal immunoglobulin were ineffective.

  "Table 1. HIV envelope protein-containing immune complexes down-regulate HIV receptor-positive lymphocytes in the mouse.

  		OKT4+ cells (%)		Spleen cell response
  Mouse	Injections	spleen	LN	to SEB (cpm x 10-3)
  1	None	36	86	ND
  2	Normal Ig	35	76	7.9
  3	AIDS patient Ig	40	74	8.4
  4	Normal Ig + gp120	37	93	7.2
  5	AIDS patient Ig + gp120	10	17	2.6
  6	gp120	42	83	7.7

  "Line 32 CD4hu transgenic mice were injected intravenously as indicated on 3 consecutive days. Lymph node and spleen single-cell suspensions were prepared 5 days after the first injection.... The data represent the average from triplicate cultures.

  "A time course of gp120/anti-gp120-mediated deletion of CD4hu T cells in the mouse [was conducted over thirty days].... Injections were given [on days 0, 1, 2, 4, 8, 28] and blood samples were taken at [on days 0, 2, 10, 30] to test for T cells expressing the human marker. The complexed envelope protein caused a rapid initial drop of CD4hu T cells followed by a slow progression of CD4hu T cell deletion. [This drop was not seen in mice injected with gp120 only, AIDS patient Ig only, or BSS.]

  Discussion: "In summary, our experiments show that despite the fact that mouse lymphocytes which express the human CD4 receptor can not be infected with HIV-1, they become deleted in vivo when they are presented with cross-linked envelope molecules. Our findings in CD4hu transgenic mice describe therefore a mechanism of non infectious elimination of HIV-reactive T cells, reflecting perhaps a mechanism of virus-induced T cell deletion in HIV-infected persons."

• Reference: European Journal of Immunology 1997. 27: 1319-1324

  Authors: Oretta Finco, Sandra Nuti, Maria Teresa De Magistris, Lucia Mangiavacchi, Alessandro Aiuti, Pietro Forte, Antonio Fantoni, Herman van der Putten and Sergio Abrignani

  Title: "Induction of CD4+ T cell depletion in mice doubly transgenic for HIV gp120 and human CD4"

  Introduction: "To investigate the effects of cross-linking of CD4/gp120 complexes by antibodies in vivo, we have generated double-transgenic mice in which T cells express human CD4 (hCD4) and secrete HIV gp120 . Immunization of these mice with denatured gp120 results in the production of antibodies to gp120. Thus, these mice represent a mode in which the effects of continuous interaction of hCD4/gp120 and anti-gp120 antibodies can be studied in the absence of HIV infection.

  Discussion: "We demonstrated that the interaction between gp120 and hCD4 in vivo renders T cells susceptible to activation-induced apoptosis in vitro, in the presence of antibodies to gp120. Furthermore, when double-transgenic mice producing anti-gp120 antibodies were immunized with TT, it was found that both antibody and T cell responses to this antigen were much lower in animals showing high levels of anti-gp120 antibodies. These results indicate that interaction between gp120 and hCD4 per se is not sufficient to affect T cell function and that antibodies cross-linking hCD4/gp120 complexes are determinant for the outcome of the T cell response to stimuli in vitro and in vivo.

  "By monitoring T cell numbers in these mice over a 10-month period, we found that absolute numbers of T cells decreased with time, showing an inverse correlation with anti-gp120 antibody titer. Furthermore, ex vivo experiments on spleen cells demonstrated the presence of apoptotic CD4+ T cells in mice with high levels of anti-gp120 antibodies, but not in those without anti-gp120 antibodies.

  "In conclusion, while it is clear that progression from HIV infection to AIDS depends on many factors, our results support the view that interaction between CD4/gp120 complexes in the surface of CD4+ T cells and non-neutralizing anti-gp120 antibodies has an important role in the depletion of uninfected T cells in HIV pathology."

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