Importantly, the increase of CD4+ cells levels during 6 months of follow-up, among 39 HAART-treated patients with undetectable levels of plasma viral RNA, was inversely correlated to the proportions of CTLA-4+CD4+ (Fig. 3, r = −0.5, P = 0.003), but not to the proportions of HLA-DR+CD3+ cells (r = −0.28, P = 0.12). Accordingly, the increase in CD4+ cells during this time period was significantly higher in those patients with less than 9% CTLA-4+CD4+ cells than in those patients with more than 9% CTLA-4+CD4+ cells (Fig. 3 inset, P < 0.001). The value of 9% was chosen as a cutoff, since this value is one SD and 50% above the mean of CTLA-4+CD4+ levels found in HIV(–) individuals.
The proportion of CTLA-4+CD8+ cells was also higher in the HIV(+) group than in the HIV(–) group (1.5 ± 0.19 versus 0.96 ± 0.21%, P = 0.07), and was correlated to the proportion`of CTLA-4+CD4+ cells (r = 0.49, P < 0.05). In both HIV(+) and HIV(–) groups, however, the proportion of CTLA+CD8+ cells was significantly lower than the proportion of CTLA-4+CD4+ cells (1.5 ± 0.2 versus 10.95 ± 0.66%, P < 0.0001 and 0.96 ± 0.2 versus 6 ± 0.45%, P < 0.0001, respectively).
Interaction between CTLA-4, CD28 and CD4
Since the overall regulation of T-cell levels and response may represent the balance between positive signals through CD28 and negative signals through CTLA-4 , the ratio between CTLA-4 and CD28 expression may be more representative of the effects HIV has on the immune system. As depicted in Fig. 4a, the ratio between the percentage of CTLA-4+CD4+ cells and of CD28+CD4+ cells, increases with HIV-1 disease progression, being significantly higher in the HIV < 200 group than in HIV(–) individuals. Furthermore, this ratio was inversely correlated to the percentage of CD4 cells (r = −0.57, P < 0.0001), CD4/CD8 ratio (r = −0.49, P = 0.0001), %CD28+CD8+ cells (r = −0.47, P = 0.003) and %CD45RA+CD4+ cells (r = −0.32, P < 0.02), and positively correlated to %HLA-DR+CD3+ cells (r = 0.59, P < 0.0001) and %CD 45RO+CD4+ cells (r = 0.44, P < 0.001). These correlations were similar to those found with CTLA-4 expression itself (shown above). We then compared the relative amount of CTLA-4 and CD28 expression on CD4+ cells by triple staining (CD4, CD28 and CTLA-4) of PBMC obtained from 36 HIV(+) patients and also measured the median fluorescence intensity (MFI) of intracellular CTLA-4 and membrane CD28. As depicted in Fig. 4b, the MFI of CD28 in the CTLA-4- cells was twice as high as the MFI of CD28 on CTLA-4+ cells (140 ± 5.3 versus 70 ± 2.28, P < 0.00001). Interestingly, a very significant correlation was found between the intensity of CD28, CD4, and CTLA-4 staining for a given cell (Fig. 4c,d). Gating cells with low CD28 and CD4 revealed that the proportion of CTLA-4+ cells among this population is significantly higher than its proportion among cells expressing high CD28 and CD4 molecules (21.7 ± 8.7 versus 8.32 ± 4.9, P < 0.0001; B versus A). A very clear and strong positive correlation is seen in CD28+CD4+ cells, between CD28 and CD4 expression (Fig. 4c). The more CD28 molecules there are in a given CD4 cell, the more CD4 molecules are expressed in this cell. Notoriously, the percentage of CTLA-4+ cells is significantly higher in those cells expressing low CD4 and low CD28, than in those cells expressing high CD4 and CD28 molecules, in both HIV(+) and HIV(–) individuals.
Increased CCR5 and Ki-67 expression in CTLA-4+CD4+ cells
The two main co-receptors for HIV-1 entry into CD4+ cells are the β-chemokines receptors CCR5 and CXCR4. The proportion of cells expressing CCR5 was higher in CTLA-4+CD4+ cells than in CTLA-4-CD4+ cells, (65 ± 11.9 versus 27 ± 8.9%, P < 0.0001). Similarly, the number of CCR5 molecules/cell, as determined by the MFI of CCR5, was four-fold higher in CTLA-4+ cells, compared with CTLA-4- cells (26.1 ± 8.6 versus 6.5 ± 2.3, P < 0.0001). The percentage of CTLA-4+CD4+ and CTLA-4-CD4+ cells expressing CXCR4 was similar (~70%), although the number of CXCR4 molecules/cell was significantly higher in the CTLA-4-CD4+ compartment (MFI of 18.8 ± 4.7 versus 33.4 ± 4.6, P < 0.0001).
Ki-67, a marker for dividing cells, is expressed in cells during the late G1, M and S phases of the cell cycle . Activated cells remaining in G1 phase and not dividing may still bear this marker . This was clearly shown in a study of chronically HIV-1 infected patients, in whom 92 ± 5% of the CD4+CD45 RO+Ki67+ cells were in the G1 phase of the cell cycle . Since CTLA-4 is present in cells that are activated but arrested at the G1 stage of proliferation , the use of both markers could help distinguish between truly dividing cells and cells activated but frozen in the G1 phase. It was found that 25 ± 7.5% of the CTLA-4+CD4+ cells expressed Ki-67, whereas only 3.7 ± 2% of the CTLA-4-CD4+ cells expressed Ki-67 (P < 0.01). Similarly, the MFI of Ki-67 in CTLA-4+CD4+ cells was significantly higher than the MFI of Ki-67 in CTLA-4-CD4+ cells (23 ± 5.1 versus 8.85 ± 1.7, P < 0.0001).
Inverse correlation between proliferation of PBMC of HIV(+) individuals to anti-CD3 or HIV antigens and levels of CTLA-4+CD4+ cells
One of the main characteristics of HIV infection is anergy and lack of specific proliferative responses. Since CTLA-4 could mediate such anergy, we examined the correlation between the capacity of PBMC of HIV-1 infected patients to respond to non-specific or HIV-1 specific stimuli and the intracellular CTLA-4 levels in CD4+ cells. There was a strong inverse correlation between the %CTLA-4+CD4+ cells and the capacity of PBMC to proliferate following stimulation with anti-CD3 antibodies (r = −0.68, P = 0.0035, Fig. 5a). Similar results were obtained with PBMC taken from HIV(–) individuals (data not shown). Proliferation of the PBMC following stimulation with gp120-depleted HIV-1 antigen (Remune) or p24 (Fig. 5b,c) was also inversely correlated to CTLA-4 levels in CD4+ cells (r = −0.38, P = 0.04 and r = −0.43, P = 0.028, respectively).
Correlation between CTLA-4 and CD25 expression
As shown in Figures 6a and b, there is a clear correlation between CTLA-4 and CD25 expression for a given cell in both HIV(+) and HIV(–) individuals. Although only 4–6% of CD4+CTLA-4- cells were CD25+, 30–40% of CD4+CTLA-4+ cells were also CD25+ cells, in both HIV(+) and HIV(−) individuals (Fig. 6c,d).
The results of this study clearly indicate that CTLA-4 plays an important role during HIV-1 infection, through the following main observations: (1) the proportion of CTLA-4+CD4+ cells is significantly higher in HIV(+) individuals in comparison to HIV(-) controls; (2) intracellular CTLA-4 levels are inversely correlated to CD4+ levels and to CD4/CD8 ratio; (3) CTLA-4 levels are higher in HIV(+) patients with advanced clinical symptoms or AIDS than in asymptomatic patients; (4) CD4 cell counts increase in HAART-treated patients with undetectable viral load, and is inversely correlated to the proportions of CTLA-4+CD4+ cells; (5) CTLA-4 expression and the ratio between the proportion of CTLA-4+CD4+ cells and that of CD28+CD4+ cells, is correlated with disease stage and with immune activation; (6) CTLA-4+ cells have very low expression of the co-stimulatory molecule CD28; (7) CCR5 and Ki-67 are expressed significantly more in CTLA-4+ cells; and (8) the capacity of PBMC of HIV-1 infected patients to respond to non-specific or HIV-1-specific stimuli was inversely correlated to the levels of CTLA-4+CD4+ cells.
The upregulation and increased expression of CTLA-4 that we found in HIV(+) individuals may account for some of the major disturbances that characterize HIV infection. CTLA-4 expression plays a key role in maintenance of peripheral CD4+ and CD8+ homeostasis , CTLA-4 knockout mice have greatly increased levels of CD4 cells , and CTLA-4 mediates antigen-specific human T-cell apoptosis . CTLA-4 is important in induction of T-cell anergy [4–6], and apoptosis and anergy are highly important characteristics of advanced HIV infection and chronic immune activation [19,20]. In addition, blockage of CTLA-4 binding to its ligand B7 increases antigen-specific immune responses [8,10]. Our results, although they do not directly establish CTLA-4 expression as a mechanism of anergy, show an inverse correlation between CTLA-4 levels and proliferative responses of PBMC of HIV(+) patients stimulated with anti-CD3 Ab or HIV-1 antigens, supporting the central role of CTLA-4 in inducing such anergy in these patients. Furthermore, since 30–40% of CTLA-4+CD4+ cells are also CD25+ cells, recently shown to be suppressor/regulators cells (for example  and ), an increase in CTLA-4+CD4+ cells would mean an increase also in CD4+CD25+ suppressor/regulator cells. Thus, a small increase in the proportion of CTLA-4+CD4+, which include suppressor cells, causes anergy or suppresses immune responses in CTLA-4 negative cells as well. We are currently further investigating the role of CD25+CTLA-4+CD4+ cells in HIV-1 disease.
Cell surface CTLA-4 expression is very low, transient and rapidly cleared by endocytosis [11,12]. Surface CTLA-4 expression is therefore almost undetected by regular flow cytometry staining, and in our hands less than 1% of CD4+ cells were positive for CTLA-4 surface expression in both HIV(+) patients and HIV(–) controls (not shown). Thus, although there is an increase in the transient levels of CTLA-4 surface expression in cells with high intracellular pools of CTLA-4, this is often missed and the differences between the surface levels in cells with intermediate or high CTLA-4 levels are not easily detectable. Steiner and colleagues , by amplifying the staining signal of antibodies bound to surface CTLA-4, also found increased expression of CTLA-4 on CD4+ T-cells obtained from 27 HIV(+) patients. However, they did not find any association between CTLA-4 expression and disease stage, as we describe. The measurement by us of intracellular pools of CTLA-4, and not of the transient surface expression of CTLA-4, most probably accounts for this discrepancy.
The most likely reason for CTLA-4 upregulation in HIV infection is the immune activation caused by HIV antigens. This is supported by: (1) the proportion of CTLA-4+ cells in HIV infection is strongly correlated with other immune activation markers such as HLA-DR+CD3+ cell levels; (2) in early HIV infection, when the immune activation is low, CTLA-4 expression is low; and (3) in another chronic immune-activation state, caused by helminthic infections, we found similar increase in CTLA-4 expression together with CD4 diminution . However, in HAART-treated people, although we found significant decrease in immune activation, there was no such similar noticeable decrease in CTLA-4 expression. Furthermore, in HAART-treated individuals with no plasma viraemia, the changes of CD4+ levels were inversely correlated with CTLA-4+CD4+ levels, but not with immune activation, as determined by the levels of HLA-DR+CD3+ cells.
Increased levels of CTLA-4 result in down-regulation of ongoing T-cell responses, and in higher threshold for effective T-cell activation , both of which may contribute to the impaired ability of the host to contain the infection. Since CTLA-4 bind to B7-1 or B7-2 with 20- to 100-fold higher affinity than CD28 , and by doing so it down-regulates the immune response, the CD28/CTLA-4 ratio, may be a relevant parameter for assessment of the immune response. The importance of this ratio in making cells more susceptible to HIV infection  is supported by our findings of increased CCR5 expression in CTLA-4+ cells. The increased ratio of CTLA-4/CD28 that we have found in HIV(+) individuals is mainly due to the increased expression of CTLA-4 in CD4+ cells, and not due to the reduction of CD28 expression on CD4+ cells (data not shown).
Is this increased expression of CTLA-4 also the cause of CD4 decrease? CTLA-4 has been shown to play an important role controlling the production of CD4+ cells, as demonstrated in CTLA-4 knockout mice . Here we show interesting mutual associative relationships between CTLA-4, CD4 and CD28. The diminished expression of CD28 on CTLA-4+ cells and the clear association of CD28 with CD4 expression raises the possibility that CTLA-4 indirectly downregulates CD4 expression through downregulating CD28 expression and maybe CD4 production as well.
The observation, that CD4 counts increase significantly more among HAART-treated individuals with undetectable viral load, who have low proportions of CTLA-4+CD4+ cells, indicate that elevated CTLA-4 expression may be an important factor that impedes immune reconstitution in many HAART-treated patients. Further studies of intracellular CTLA-4 expression in HIV(+) patients, before and after HAART treatment, are required to help clarify the dissociation found in some patients on HAART, between virus suppression, and recovery or rise of CD4+ cells. Such studies may clarify the role of CTLA-4 in immune restoration following HAART, and indicate the clinical value of CTLA-4 monitoring and treatment in HIV infection.
The effect of HAART on the immune system and particularly on the rise of CD4+ T-cells has been ascribed to increased proliferation of CD4+ T-cells, as determined by increased proportion of Ki-67+CD4+ cells following HAART [27,28]. Recent studies have suggested that the rise of CD4+ T-cells following HAART is mainly due to redistribution of cells, rather than to massive proliferation of CD4+ T-cells [29,30]. Our findings that approximately 50% of the Ki-67+CD4+ cells in HIV(+) individuals are CTLA-4+ cells , and that approximately 25% of the CTLA-4+CD4+ cells are Ki-67+, and those reported by Autran and colleagues , showing that almost all CD4+CD45RO+Ki67+ cells are in the G1 phase of the cell cycle, clearly support the redistribution notion. This indicates that Ki-67+ cells are not necessarily proliferating cells [15–17], and suggests that significant proportions of activated cells during HIV infection are anergic and non-proliferating cells.
Previous studies with antibodies to CTLA-4 in other systems have shown the potential importance and implications of this approach, including the clinical use of such antibodies. In particular, the enhancement of CD4+ T-cell expansion in response to peptide antigens, superantigens and parasites , and their use in transplantation . Although the benefits of CTLA-4 blockage in HIV-1 infection and immune reconstitution in HIV(+) patients has to be studied, the clear correlation between HIV-1 disease progression and CTLA-4 expression, raise the possibility of using anti-CTLA-4 antibodies for immunotherapy during HIV infection, probably in combination with antiretroviral therapy. There are several supportive arguments for this approach. The most important one, is the potential role of anergy and CD4 cell anergy in particular, in the diminished immune response of the HIV(+) individuals. Such anergy may be responsible for the diminished specific cytotoxic function of CD8 cells during HIV infection that is dependent on CD4 help which is essential in the ability of the host to contain HIV infection. Even in successfully HAART-treated patients there seems to be insufficient immune reconstitution and the response to HIV antigens is usually missing. CD8 T-cytotoxicity against tumour cells in mice can be enhanced by blockade of CTLA-4 only in the presence of CD4 T-cells, whereas CTL activity is lost in the absence of CD4 T-cells , supporting the idea that functional CD4 T-cells are essential for CD8 CTL activity. Thus, even a small increase in dysfunctional CD4 cells, namely an increase in the proportion of CTLA-4+CD4+ cells, may have dramatic effects on other compartments of the immune system, including on the capacity of CD8 cells to specifically target HIV-infected cells.
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Keywords:© 2002 Lippincott Williams & Wilkins, Inc.
CTLA-4; AIDS; HIV-1; anergy; Ki-67; CD28; CCR5