Rainer Zimmermann

CD4+ Lymphocyte Depletion in HIV‐Infected Patients is Associated with gp120‐Immunoglobulin‐Complement Attachment to CD4+ Cells

The mechanism of CD4+ lymphocyte depletion, which is the main immunological feature in HIV‐infected patients, is unclear. We investigated whether gp120‐immunoglobulin‐complement complexes on the surface of CD4+ cells might be involved in the elimination of CD4+ lymphocytes. The results obtained in 63 HIV‐infected patients show that gp120 is attached to a variable degree to CD4+ cells. Importantly, the percentage of CD4+gp120+ lymphocytes is inversely associated with CD4+ lymphocyte counts in the peripheral blood (p = 0.0004). CD4+gp120+ blood lymphocytes bind IgM (p = 0.0027) and IgG antibodies (p = 0.0001) and complement (p = 0.0005). These results suggest that immune complex‐mediated cell elimination is an important mechanism of CD4+ cell depletion in patients with AIDS.

Sequential occurrence of IgM, IgM/IgG, and gp120-IgM/IgG complement complexes on CD4+ lymphocytes in relation to CD4+ blood lymphocyte depletion in HIV+ hemophilia patients: results of a 10-year study.

The concept of autoimmune mechanisms playing an integral role in the pathogenesis of HIV disease is rapidly gaining ground. In this study, we determined IgM and IgG antibodies, complement fragments and gp120 on the surface of CD4+ lymphocytes using double-fluorescence flow cytometry. Sequential analysis demonstrated an inverse relationship of autoantibodies and CD4+ lymphocyte counts in the peripheral blood. HIV+ patients without autoantibodies (16/104 = 15%) had the highest CD4+ blood cell counts (324 +/- 264/microliters; mean +/- SD). CD4+ counts were successively lower in patients with complement-fixing IgM (243 +/- 240/microliter), complement-fixing IgG and IgM (139 +/- 138/microliter), or gp120-IgM/IgG complement complexes on the surface of CD4+ cells (38 +/- 45/microliter, P = 0.03). Individual patient profiles show that IgM autoantibodies typically are formed early after HIV infection and appear to deplete CD4+ lymphocytes very slowly, whereas complement-fixing IgG autoantibodies are generated at a later stage and deplete CD4+ lymphocytes more efficiently. The presence of both soluble gp120 and complement-fixing autoantibodies on CD4+ lymphocytes is associated with very low CD4+ cell counts and coincides with progression to terminal disease. Early during HIV infection autoantibody production is rather unstable, but it becomes more stable with disease progression and persists in advanced stages of the disease. These data suggest that autoantibody formation against CD4+ lymphocytes is a pathogenic mechanism for CD4+ cell depletion.

IgA-anti-Fab autoantibodies and disease progression in AIDS

There is increasing evidence that autoimmune phenomena contribute to the pathogenesis of the acquired immunodeficiency syndrome (AIDS). We investigated the relationship between IgA autoantibodies directed against the Fab part of the IgG molecule and disease progression in 87 HIV-infected hemophilia patients. AIDS patients demonstrated a significantly higher serum IgA-anti-Fab activity than HIV-positive (HIV+) patients with AIDS-related complex (ARC) (P < 0.02), HIV+ patients without AIDS/ARC (P < 0.0001), HIV negative (HIV-) patients (P = 0.0001), or healthy controls (P < 0.0001). Moreover, an inverse association was observed between serum IgA-anti-Fab activity and CD4+ cell counts (r = -0.396, P < 10(-6)). This close association was confirmed in longitudinal studies of symptomatic patients. IgA-anti-Fab antibodies are suggested to play an important role in the immunopathogenesis of AIDS, and their determination may be helpful in the monitoring of HIV-infected patients.

Reduction of viral load and immune complex load on CD4+ lymphocytes as a consequence of highly active antiretroviral treatment (HAART) in HIV-infected hemophilia patients.

BACKGROUND AND OBJECTIVES Human immunodeficiency virus (HIV)-induced immune complex load on circulating CD4+ blood lymphocytes is associated with dysfunction and depletion of CD4+ lymphocytes and with increased monocyte/macrophage function. It was investigated whether HAART reduces both the viral load in plasma and the number of immune complex-coated CD4+ lymphocytes in the blood, and whether CD4+ counts are associated with viral load and/or immune complex load. MATERIALS AND METHODS Twelve HIV+ hemophilia patients before and after conversion to HAART (group 1); eight HIV+ hemophilia patients without antiretroviral therapy (group 2). HIV-1 RNA copies in plasma using NASBA/Nuclisens kits; CD4+ lymphocytes coated in-vivo with immune complexes using flowcytometry on whole blood samples; in-vitro responses of immune complex-coated T lymphocytes in cell culture assays. RESULTS After conversion to HAART there was a significant reduction of viral load, CD4+ gp120+, CD4+ IgM+, and CD4+ IgG+ circulating blood lymphocytes and plasma neopterin, paralleled by a significant increase of CD4+ and CD8+ counts. The percentage of immune complex-coated CD4+ lymphocytes of converted patients was significantly associated with CD4+ counts, in-vitro responses to concanavalin A (Con A), pokeweed mitogen (PWM), phytohaemagglutinin (PHA), anti-CD3 and pooled allogeneic stimulator cells, and with plasma neopterin levels. CONCLUSION HAART reduces viral load and HIV-induced immune complex load on circulating CD4+ blood lymphocytes. The results of this study can be interpreted to suggest that HAART increases CD4+ lymphocyte counts in part by counteracting HIV-induced autoimmune phenomena.

Isotypes and IgG subclasses of anti-Fab antibodies in human immunodeficiency virus-infected hemophilia patients.

We reported recently that anti‐Fab autoantibodies of the IgG isotype are associated with the decrease of helper/inducer (CD4+) lymphocytes in human immunodeficiency virus‐infected (HIV+) hemophilia patients with acquired immunodeficiency syndrome (AIDS) or AIDS‐related complex (ARC). In the present study we investigated the subclass distribution of IgG‐anti‐Fab autoantibodies, and whether anti‐Fab antibodies of the IgA and IgM isotypes also are associated with the development of AIDS. Sera of HIV+ patients with AIDS had significantly higher IgA‐anti‐Fab activity than HIV+ patients with ARC (p<0.02), HIV+ patients without AIDS/ARC (p<0.0001), HIV‐negative (HIV‐) patients (p<0.001), or healthy controls (p<0.0001). An inverse association was found between IgA‐anti‐Fab activity and CD4+ cell counts (r = ‐0.396, p<10‐6). In contrast, no association of CD4+ cell counts was observed with IgM‐anti‐Fab. However, IgM‐anti‐Fab was significantly increased in patients with thrombocytopenia. We found a significant association between IgA‐anti‐Fab activity and serum neopterin concentrations (r = 0.310, p<10‐5). IgG‐anti‐Fab activity was detected mainly in the IgG3 fraction, although in HIV+ patients with AIDS/ARC various IgG subclasses were present. Affinity‐purified anti‐Fab antibodies isolated from sera of AIDS patients bound to rgp120‐preincubated CD4+ cells of a healthy individual, supporting our hypothesis that anti‐Fab antibodies and free circulating gp120 molecules are involved in the elimination of uninfected CD4+ cells. Removal of anti‐Fab autoantibodies from the circulation by immune adsorbance might be a useful approach in the treatment of AIDS.

Autoantibodies in HIV‐infected Hemophilia Patients against Different Epitopes on CD4+Lymphocytes and Recombinant CD4

We studied 684 sera obtained from 20 hemophilia patients with AIDS/AIDS‐related complex (ARC), 89 asymptomatic HIV+, 76 HIV‐ hemophilia patients and 151 healthy controls for antibodies against recombinant CD4 (rCD4). Twenty‐two percent of AIDS/ARC patients, 10% of asymptomatic HIV+ patients, 17% of HIV‐ patients, and 1% of healthy controls had anti‐rCD4 antibodies. Purified anti‐rCD4 antibodies did not react with human CD4+lymphocytes. This may explain why formation of anti‐rCD4 antibodies correlated neither with the occurrence of autoantibodies against CD4+lymphocytes nor with a decrease in CD4+cell counts. Antibodies that were eluted from CD4+lymphocytes after sequential adsorption and elution with separated CD8+and CD4+cells reacted with CD4+lymphocytes of only some healthy individuals, suggesting diversity of CD4 expression.

CD8+ lymphocyte decrease in HIV disease: association with anti-CD4+ but not with anti-CD8+ lymphocyte autoantibodies.

HIV+ patients form autoantibodies against CD4+ and CD8+ lymphocytes. It was shown that anti‐CD4+ lymphocyte autoantibodies are associated with the depletion of CD4+ cells. In the present study we analyzed the relationship of anti‐CD4+ and anti‐CDS+ autoantibodies with the CD8+ lymphocyte decrease commonly observed during HIV disease. IgM and IgG antibodies as well as complement fragments were determined on the surface of CD4+ and CD8+ lymphocytes using double fluorescence flow cytometry. Anti‐CD8+ lymphocyte autoantibodies were found more often in HIV+ hemophilia patients (75/105 = 71%) than HIV‐ hemophilia patients (13/37 = 35%; p<0.0001), patients with pharyngeal carcinoma (20/44 = 45%; p = 0.002), habitual abortions (3/13 = 23%; p = 0.0009) or healthy individuals (93/223 = 42%; p< 0.0001). Anti‐CD8+ antibodies, mostly of the IgM type, occurred significantly more frequently than anti‐CD4+ antibodies in healthy controls (p< 0.0001), patients with pharyngeal carcinoma (p = 0.0001), or HIV‐ patients (p = 0.01). In HIV+ patients, however, anti‐CD4+ autoantibodies were found more often than anti‐CD8+ antibodies (85 vs 71%; p = 0.02). 70 of 104 (67%) HIV+ patients had autoantibodies on both CD4+ and CD8+ lymphocytes and the IgG/IgM/C3d autoantibody pattern was identical in 31 (44%) of the patients. Interestingly, peripheral blood CD8+ cell counts were significantly associated with anti‐CD4+ (p = 0.01) but not with anti‐CD8+ lymphocyte autoantibodies. It is hypothesized that the inhibition and depletion of CD4+ cells by anti‐CD4+ autoantibodies is associated with a loss of regulatory functions that leads to a depletion of antiviral cytotoxic CD8+ lymphocytes.

Association of viral load in plasma samples of HIV-infected hemophilia patients with autoantibodies and gp120-containing immune complexes on CD4+ lymphocytes

OBJECTIVE We investigated whether the induction of antilymphocyte autoantibodies and immune complexes is associated with the activity of HIV replication. METHODS Viral HIV-1 RNA was measured in the plasma samples of 84 HIV+ hemophilia patients and correlated with the IgM, IgG, IgM/IgG and IgM/IgG/gp120 load of circulating CD4+ lymphocytes, CD4+ and CD8+ cell counts, plasma neopterin levels and in vitro T-cell responses to mitogens and pooled allogeneic stimulator cells. RESULTS Compared to patients with no immune complexes, on circulating CD4+ lymphocytes, viral load was increased in patients with IgM, IgM/IgG or IgM/IgG/gp120 complexes. Sequential analysis of HIV+ patients showed that peaks of retroviral activity were associated with the subsequent formation of CD4+ lymphocyte-reactive IgM and IgG autoantibodies and gp120-containing immune complexes. CONCLUSION The induction of autoantibodies and immune complexes attached to CD4+ lymphocytes is associated with periods of increased viral activity in HIV-infected patients.

Increased soluble Fas in HIV-infected hemophilia patients with CD4+ and CD8+ cell count increases and viral load and immune complex decreases.

Previous studies interpreted increases of soluble Fas (sFas) in the plasma during disease progression in HIV-infected patients as evidence of increased apoptosis of CD4(+) lymphocytes. We studied whether sFas and sFas ligand (sFasL) plasma levels are associated with CD4(+) and CD8(+) lymphocyte counts, plasma viral load, and IgM, IgG, C3d, and gp120 complexes on circulating CD4(+) blood lymphocytes in long-term surviving HIV-infected hemophilia patients, most of whom were receiving HAART. Twenty-six hemophilia patients who were infected with HIV in the early 1980s were investigated in 1997, 1998, and 1999. HAART was initiated in 1996 and 1997 in most patients. Lymphocyte subpopulations and immune complex-coated CD4(+) lymphocytes in the blood were investigated by flow cytometry, plasma viral load (HIV-1 mRNA copies/ml plasma) was tested with HIV-1 QT Nuclisens kits, sFas (ng/ml) and sFasL (ng/ml) plasma levels were measured with MBL ELISA kits, and the in vitro response of patient lymphocytes was tested in cell cultures. During the period from 1997 to 1999 we observed an increase in sFas plasma levels (p = 0.003) as well as in CD4(+) (p = 0.004) and CD8(+) (p = 0.023) cell counts; a decrease in IgG (p = 0.047), C3d (p = 0.024), and gp120 (p = 0.001)-coated CD4(+) lymphocytes in the blood; and a decrease in the number of impaired mitogen stimulation assays (p = 0.013). sFas was negatively associated with viral burden (r = -0.662, p = 0.0002) as well as with CD4(+)IgM(+) (r = -0.554, p = 0.004), CD4(+)IgG(+) (r = -0.431, p = 0.031), CD4(+)C3d(+) (r = -0.551, p = 0.041), and CD4(+)gp120(+) (r = -0.430, p = 0.041) blood lymphocytes, CD8(+)DR(+) cell counts (r = -0.700, p = 0.016), and impaired in vitro responses of patient lymphocytes to PHA (r = -0.475, p = 0.016). sFasL was negatively associated with total lymphocyte counts (r = -0.433, p = 0.027), as well as with absolute numbers of CD3(+) (r = -0.492, p = 0.011) and CD8(+) (r = -0.432, p = 0.027) cells. We conclude that, contrary to expectations, sFas plasma levels increased in long-term surviving HIV-infected hemophilia patients receiving HAART, concomitant with increases in CD4(+) and CD8(+) cell counts. Increased sFas may reflect the growing pool of T lymphocytes that recovers because of a decreasing viral burden and a decreasing immune complex load of CD4(+) lymphocytes.

Association of immune complexes and plasma viral load with CD4+ cell depletion, CD8+DR+ and CD16+ cell counts in HIV+ hemophilia patients. Implications for the immunopathogenesis of HIV-induced CD4+ lymphocyte depletion

OBJECTIVE There is evidence that HIV induces CD4+ depletion in part by the formation of immune complexes (IC) that attach to CD4+ blood lymphocytes. In the present study we examined the relationship of IC-coated CD4+ blood cells with retroviral replication in HAART-treated patients. PATIENTS AND METHODS 52 hemophilia patients were studied from 1997 to 1999. Lymphocyte subsets, IgM, IgG and gp120 on CD4+ blood cells, in vitro responses of lymphocytes to mitogens, plasma neopterin and plasma viral load were measured. RESULTS Patients with detectable viral replication and without ICs on CD4+ blood lymphocytes had a lower viral load (4100 versus 21000 HIV-1 mRNA copies/ml; P = 0.079) and higher CD4+ cell counts (310/microl versus 161/microl; P = 0.035) than patients with ICs on circulating CD4+ lymphocytes. Among patients with < 80 HIV-1 mRNA copies/ml, IC- individuals had slightly higher CD4+ lymphocyte counts than IC+ patients (384/microl versus 316/microl; n.s.). Further evidence for the clinical relevance of the ICs was obtained when 18 patients who had an undetectable viral load at previous investigations were analyzed. Among patients with a stable undetectable viral load, CD4+ counts increased in 6 of 8 IC- but in none of 2 IC+ individuals. In patients whose viral load increased during the observation period, 5 of 6 IC- but none of 2 IC+ individuals showed higher CD4+ cell counts. Impaired virus killing is suggested by lower CD16+ (35/microl versus 107/microl; P = 0.016), higher CD3+ DR+ (178/microl versus 66/microl; P = 0.006), and higher CD8+ DR+ (142/microl versus 34/microl; P = 0.017) cell counts in IC(-) patients compared to IC- patients without detectable viral load. Strong retroviral replication induced strong T cell dysfunctions. Fewer CD3+ 25+ blood lymphocytes (19/microl versus 47/microl; P = 0.006) and a lower in vitro response of T lymphocytes to the mitogens Con A (RR: 0.3 versus 1.2; P=0.023) and CD3 mab (RR: 0.5 versus 2.4; P = 0.012) was observed in IC+ patients with detectable versus undetectable viral load. CONCLUSION Our data suggest that ICs on circulating CD4+ blood lymphocytes are primarily associated with CD4+ lymphocyte depletion whereas the plasma viral load is primarily associated with decreased T lymphocyte activation, lower CD16+ counts, and higher CD8+ DR+ lymphocytes which might be the effector cells for virus elimination.