Effects of therapy with highly active anti-retroviral therapy (HAART) and IL-2 on CD4+ and CD8+ lymphocyte apoptosis in HIV+ patients

The kinetics and effects of in vivo spontaneous apoptosis and activation-induced cell death (AICD) upon CD4+ and CD8+ lymphocyte subsets and CD4 naive cell numbers were studied in HIV+ subjects with CD4 pretreatment values > 200/mm3, who were subsequently treated for 48 weeks with HAART alone or in combination with six cycles of subcutaneous IL-2. Irrespective of the type of treatment, patients showed a statistically significant increase in CD4 cell counts after 4 weeks, although the CD4 naive subset only increased significantly in the IL-2-treated subjects at the end of treatment. The percentage of CD4 cells undergoing spontaneous apoptosis and AICD was significantly reduced in all patients after 4 weeks and this reduction was maintained until the end of therapy; however, the level always remained significantly higher in comparison with healthy subjects. A statistically significant reduction in CD8 apoptosis levels required at least 24 weeks of therapy. Together these data suggest that a reduction in the level of apoptosis may contribute to the early rise in CD4 numbers measured after HAART, but that later on HAART is unable to improve further this biological parameter. Although the use of IL-2 had no additional effects on spontaneous apoptosis and AICD, it may be beneficial by stimulating a late increase in the numbers of CD4 naive cells in HIV-treated subjects.     

Long-term CD4+ T-cell response to highly active antiretroviral therapy according to baseline CD4+ T-cell count

Current treatment guidelines for HIV infection recommend a relatively late initiation of highly active antiretroviral therapy (HAART). Nevertheless, there is still a concern that immune recovery may not be as complete once CD4+ T cells have decreased below a certain threshold. This study addressed the long-term response of CD4+ T-cell counts in patients on HAART and analyzed the influence of baseline CD4+ T-cell counts, baseline viral load, and age. An observational analysis of evolution of CD4+ T cells in 861 antiretroviral therapy-naive chronic HIV-1-infected patients who started treatment consisting of at least 3 drugs in or after 1996 was performed. Patients were classified in 4 groups according to baseline CD4+ T cells: <200 cells/mm3, 200-349 cells/mm3, 350-499 cells/mm3, and >or=500 cells/mm3. The main outcome measures were proportion of patients with CD4+ T cells <200/mm3 and >500/mm3 at last determination and rate of CD4+ T-cell recovery. Patients were followed-up for a median of 173 weeks (interquartile range [IQR], 100-234). There were no differences in follow-up between the 4 groups. CD4+ T cells increased in the whole cohort from a median of 214 cells/mm3 (IQR, 90-355) to 499 cells/mm3 (IQR, 312-733) (P<0.001). Compared with the group with a baseline CD4+ T-cell count of >or=500/mm3, the relative risk of having a last determination of CD4+ T-cell counts >200 cells/mm3 was 0.79 (95% CI, 0.75-0.83), 0.92 (95% CI, 0.89-0.96) and 1 for baseline CD4+ T cells <200 cells/mm3, 200-349 cells/mm3, and 350-499 cells/mm3, respectively. The relative risk of having a last determination of CD4+ T-cell counts >500 cells/mm3 was 0.32 (95% CI, 0.27-0.39, P<0.001), 0.69 (95% CI, 0.60-0.79, P<0.001), and 0.94 (95% CI, 0.83-1.06, P=0.38) for baseline CD4+ T-cell counts <200 cells/mm3, 200-349 cells/mm3, and 350-0499 cells/mm3, respectively, compared with a baseline CD4+ T-cell count of >or=500 cells/mm3. The increase in CD4+ T cells from baseline was statistically significant and was maintained for up to 4 years of follow-up. This increase seemed to slow down after approximately 3 years and reached a plateau after 4-5 years of follow-up even in patients who achieved and maintained viral suppression in plasma. Long-term immune recovery is possible regardless of baseline CD4+ T-cell count. However, patients who start therapy with a CD4+ T-cell count <200 cells/mm3 have poorer immunologic outcome as measured by the proportion of patients with CD4+ T cells <200/mm3 or >500/mm3 at last determination. It seems that the immune recovery slows down after approximately 3 years of HAART and reaches a plateau after 4-5 years of HAART.     

Immunological analysis of treatment interruption after early highly active antiretroviral therapy

We longitudinally evaluated HIV-specific T-cell immunity after discontinuation of highly active antiretroviral therapy (HAART). After treatment interruption (TI), some individuals could maintain a low plasma viral load (<15,000 copies/mL), whereas others could not (>50,000 copies/mL). Before HAART was initiated, plasma viral load was similar. After TI, the numbers of CD8(+) T cells increased more in individuals without viral control, whereas individuals maintaining a low viral load showed a more pronounced increase in HIV-specific CD8(+) T-cell numbers. No differences were seen in the number or percentage of cytokine-producing HIV-1-specific CD4(+) T cells, or in proliferative capacity of T cells. Four weeks after TI, the magnitude of the total HIV-1-specific CD8(+) T-cell response (IFN-γ(+) and/or IL-2(+) and/or CD107a(+)) was significantly higher in individuals maintaining viral control. Degranulation contributed more to the overall CD8(+) T-cell response than cytokine production. Whether increased T-cell functionality is a cause or consequence of low viral load remains to be elucidated.     

Functional restoration of human immunodeficiency virus and Epstein-Barr virus-specific CD8(+) T cells during highly active antiretroviral therapy is associated with an increase in CD4(+) T cells

To investigate the effect of highly active antiretroviral therapy (HAART) on HIV- and Epstein-Barr virus (EBV)-specific CD8(+) T cells, total number and function of these cells was determined in 16 HIV-infected individuals using tetrameric HLA-peptide complexes and IFN-gamma ELISPOT assays after peptide stimulation, respectively. HAART induced a significant decrease in HIV-specific tetramer(+) T cells, whereas EBV-specific tetramer(+) T cells did not change. In addition, individuals who temporarily failed on therapy showed a temporary increase in the number of HIV-specific T cells, suggesting that differences in the pool size of antigen-specific T cells was determined by the presence of antigen. Interestingly, there was an increase in the ratio of IFN-gamma-producing T cells per total number of both HIV- and EBV-specific T cells in the majority of individuals, suggesting that the function of virus-specific T cells is improved in individuals successfully treated with HAART. Despite this relative functional improvement of EBV-specific T cells, no significant changes were observed in EBV load. In four subjects who temporarily failed on HAART, the percentage of IFN-gamma-producing T cells, both for HIV and EBV, paralleled CD4(+) T cell kinetics, suggesting that function seems to be related to differences in CD4(+) T cell numbers. Overall, these data indicate that HAART improves the antigen responsiveness of both HIV- and EBV-specific T cells, which is associated with an increase in CD4(+) T cells.     

Phenotypic switch from CD45RA+ to CD45RA- by normal blood T cells is associated with increased HLA-ABC expression for CD4+ and CD8+ populations but not for the NK-associated CD4-CD8dim+ or CD4-CD8- fractions.

Using the combined techniques of immunomagnetic depletion and multiple colour flow cytometry, the expression of HLA-ABC (W6/32) by normal T-cell subpopulations, defined by 2H4 (CD45RA) expression, was examined. It is thought that a CD45RA+CD45RO- phenotype defines the 'virgin' T-cell fraction, whereas a CD45RA-CD45RO+ phenotype defines the 'primed' or memory T-cell population. In addition, an intermediate phenotype (CD45RA+CD45RO+) appears to correspond to a transitional stage of development. In this study, these three phenotypic stages were represented by distinct levels of 2H4 staining defined as 2H4+, 2H4int and 2H4-, respectively. The results of this current investigation are of importance in two main areas. Firstly, when compared to the 2H4+ component, the HLA-ABC expression of 2H4- cells was significantly higher. This was true for both CD4+CD8- and CD4-CD8+ lymphocytes, but was not the case for CD4-CD8dim+, CD3+CD4-CD8- and CD3-CD4-CD8- fractions. Additionally, when HLA-ABC expression was examined as a function of 2H4 staining intensity, it was found that, for the CD4+ fraction, the greatest increase in HLA-ABC expression occurred between the 2H4int and 2H4- stages. In contrast, the increase in HLA-ABC expression by CD8+ lymphocytes was associated with transition from 2H4+ to 2H4int status, which suggests that increased HLA-ABC expression occurs at an earlier stage in the acquisition of CD45RO in CD8+ cells than for CD4+ cells. Secondly, for each individual blood examined, a close and highly significant correlation (P = 0.002) for membrane HLA-ABC expression was found between (i) CD4+2H4+ and CD8+2H4+ and (ii) CD4+2H4- and CD8+2H4- subpopulations. This suggests that modulation of HLA-ABC expression in CD4+ and CD8+ cells is subject to common control mechanisms and remains proportionate for these lymphocyte fractions in any given individual.     

Reconstitution of CD4+ T cell responses in HIV-1 infected individuals initiating highly active antiretroviral therapy (HAART) is associated with renewed interleukin-2 production and responsiveness

Reconstitution of functional CD4(+) T cell responsiveness to in vitro stimuli is associated with continuous highly active antiretroviral therapy (HAART). Thirty-six antiretroviral naive patients received HAART over 16 weeks. Antigen-specific, mitogen and interleukin (IL)-2 induced lymphocyte proliferative responses and specific IL-2 and IL-4 production were assessed at each time-point, together with quantification of HIV-1 RNA load and lymphocyte populations. Reconstitution of recall responses was limited largely to persistent antigens such as Herpes simplex virus and Candida, rather than to HIV-1 or neo-antigens. Recall antigens, mitogens and IL-2-induced renewed responses were associated with in-vitro production of IL-2, but not IL-4. Differential responsiveness to low versus high concentration IL-2 stimulus increases in a stepwise manner, suggesting normalization of IL-2 receptor expression and improved functionality. These increases in in-vitro proliferative responses thus probably reflect short lived effector clones, driven by ongoing antigenic stimulus associated with persisting long-term organisms. In this context non-responsiveness to HIV-1 antigens suggests ongoing HIV-1 specific clonal T cell anergy.     

Low CD4+ T-cell counts in HIV patients receiving effective antiretroviral therapy are associated with CD4+ T-cell activation and senescence but not with lower effector memory T-cell function

The adverse effects of immune activation on CD4(+) T-cell recovery and the relationship between CD4(+) T-cell counts and effector T-cell function were examined in HIV-1 patients receiving long-term effective ART. Patients with nadir CD4(+) T-cell counts <100/microl, > 12 months on ART and >6 months with <50 HIV RNA copies/ml were stratified by current CD4(+) T-cell counts and patients from the lowest (n = 15) and highest (n = 12) tertiles were studied. We assessed proliferation (Ki67), activation (HLA-DR, CD38) and replicative senescence (CD57) by flow cytometry and CD4(+) T-cell responses to CMV by IFN-gamma ELISpot. Proportions of CD4(+) T-cells expressing HLA-DR or CD57 were strong univariate predictors of total (P = 0.0002 and P = 0.002) and naive (P < 0.0001 and P < 0.0001, respectively) CD4(+) T-cell counts, suggesting that CD4(+) T-cell activation drives the depletion of naive CD4(+) T-cells. This was clearest in patients with a small/undetectable thymus. IFN-gamma responses to CMV were similar in patients with low or high CD4(+) T-cell counts.     

Presence of HIV-specific CD4+ T-cell responses in HIV-infected subjects with sustained virologic control after highly active antiretroviral therapy

HIV-specific CD4+ T-helper cell responses in 40 subjects with chronic infection (CI) who had virus suppression after highly active antiretroviral therapy (HAART) were compared with those in 34 subjects treated during primary infection (PI). A CD4+ T-cell proliferative response to HIV p24 protein was present in 50% of these subjects compared with 79% of subjects treated during PI. The existence of a proliferative response in CI subjects was associated with a higher CD4+ T-cell count at initiation of HAART, a longer duration of virus suppression, and a higher CD4+ T-cell count at the time of analysis. These results show that an HIV-specific proliferative response is preferentially observed in treated CI subjects with CD4+ T-cell counts of >200/microL. However, in treated CI subjects with a significant degree of CD4+ T-cell depletion (<200/microL), it may also be observed in 35% provided that the duration of virus suppression is long enough, which may have implications for future therapeutic strategies.     

Increased lipodystrophy is associated with increased exposure to highly active antiretroviral therapy in HIV-infected children

OBJECTIVE: To assess body composition changes in HIV-infected children receiving highly active antiretroviral therapy (HAART). METHODS: Thirty-seven HIV-positive children were enrolled. Dual-energy X-ray absorptiometry (DXA) scans were performed in all HIV-infected children at baseline and after an additional 12 months of HAART and in 54 matched (for sex, age, body mass index [BMI], and pubertal stage) healthy controls. Abdominal MRI was performed in 14 of 37 HIV-positive children at baseline and in 28 of 37 HIV-positive children after additional 12 months of HAART. RESULTS: During the study period, mean HAART exposure increased from 39.3 to 50.9 months and the number of HIV-infected children with clinical lipodystrophy (LD) increased from 6 to 8, whereas mean BMI, CD4 percentage, and percentage of HIV-infected children with HIV RNA <50 copies/mL did not change. DXA scans showed an increase in lean mass, peripheral fat loss, and central fat accumulation in all HIV-infected children. As compared with controls, 70% and 84% of HIV-infected children showed DXA-detectable LD at baseline and at 12 months of follow-up, respectively. Mixed LD and central fat accumulation were the most common LD phenotype. At baseline and at 12 months of follow-up, intra-abdominal adipose tissue (IAT) was greater than in controls in 33% and 35% of HIV-infected children, and it was greater in those with LD than in those without. Peripheral fat loss and IAT content were associated with duration of HAART and were independent of immunologic stage of disease and immunologic response. CONCLUSIONS: Changes in body composition related to LD in HAART-treated children are frequent, precocious, and progressive. Duration of HAART negatively influences visceral adiposity and peripheral fat loss.     

Incomplete CD4 T cell recovery in HIV-1 infection after 12 months of highly active antiretroviral therapy is associated with ongoing increased CD4 T cell activation and turnover

To evaluate the relationship between T cell turnover, immune activation, and CD4 recovery in HIV infection, 32 antiretroviral-naive HIV-1-infected patients were studied before and after initiation of highly active antiretroviral therapy (HAART). Elevated CD4 and CD8 T cell turnover (measured by Ki67) in HIV infection decreased with HAART in blood and lymphoid tissue. Increased peripheral CD4 T cell turnover was strongly associated with immune activation even after viral suppression to less than 50 copies/mL (R = 0.8; p <.001). Increased CD4 T cell turnover correlated strongly with CD4 cell counts both before (R = -0.6; p <.001) and after (R = -0.4; p =.05) HAART. In patients with baseline CD4 cell counts of less than 350/microL, decreases in CD4 T cell turnover with HAART significantly correlated with increases in CD4 cell counts. In addition, persistently elevated levels of CD4 T cell turnover after HAART were associated with incomplete CD4 T cell recovery despite HIV RNA levels of less than 50 copies/mL. These data suggest that immune activation is central to CD4 cell depletion in HIV infection and immune reconstitution with HAART.     

Highly active antiretroviral therapy restores CD4+ Vbeta T-cell repertoire in patients with primary acute HIV infection but not in treatment-naive HIV+ patients with severe chronic infection

In drug-naive HIV+ patients, we analyzed the effects of highly active antiretroviral therapy (HAART) on the reconstitution of the T-cell receptor (TCR) repertoire. We followed 2 groups of patients for 1 year: 18 individuals who experienced acute HIV infection and 24 patients who had HIV infection for many years but never took HAART. They were compared with 10 healthy controls who were longitudinally analyzed for the same period. We performed cytofluorometric analysis of the Vbeta TCR repertoire and detected the clonality of different Vbeta families by the spectratyping method. A new statistical approach based on the use of mixed models was then employed to analyze the data. Before the beginning of therapy, the repertoire of patients with acute or chronic infection was significantly different from that of healthy controls. After therapy, patients with acute HIV infection showed an improvement of the repertoire among either CD4+ or CD8+ T lymphocytes. Conversely, patients with chronic infection were capable of changing their repertoire among CD8+ but not CD4+ T lymphocytes. Our results indicate that HAART can restore the T-cell repertoire in individuals whose immune system is not severely compromised by the infection.     

Thymic volume is associated independently with the magnitude of short- and long-term repopulation of CD4+ T cells in HIV-infected adults after highly active antiretroviral therapy (HAART)

Age is one of the main factors involved in the rapidity and the magnitude of CD4(+) T cell repopulation in human immunodeficiency virus (HIV)-infected patients on highly active antiretroviral treatment (HAART). Improved thymic function has been suggested as the main factor associated with CD4(+) T cell restoration after HAART. This work was undertaken to determine, among host factors, the predictor variable at baseline involved in the magnitude of short- and long-term recovery of CD4(+) T cells after HAART. HIV-RNA levels and CD4(+) T cell numbers were determined in 54 HIV-infected adults at baseline and at weeks 4, 12, 48 and 96 after HAART. T cell subpopulations were determined by flow cytometry, thymic volume by computed tomography, T cell receptor excision circle (TREC)-bearing cells by quantitative polymerase chian reaction (PCR) and interleukin (IL)-7 levels by enzyme linked immunosorbent assay at baseline. The phenotype of patients' isolates was determined by infecting GHOST cells expressing CCR5 and CXCR4. The possible interference of phenotype with thymic function was also analysed. Baseline thymic volume was associated independently with the magnitude of short- and long-term recovery of CD4(+) T cells after HAART, despite the patients' viral phenotype. The measurement of thymic volume before therapy may predict the magnitude of T cell increase. This result could have important clinical implications not only in HIV-infected patients, but also in other scenarios of T cell depletion such as bone marrow transplantation and chemotherapy.     

Effect of highly active anti-retroviral therapy on CD3+/CD4+/CD8+ T lymphocyte counts in HIV seropositive Kashmiri patients: a follow up study

Antiretroviral therapy has played an important role in improving the quality of life and extending the life span of HIV positive patients. In the present study 17 naive HIV positive patients out of a total of 23 positive cases from local population who had absolute CD4+ counts below 300 were given ARV therapy and followed for 1 year. The patients showed an overall improvement in CD4+T lymphocyte counts at one year survival. The values of CD3+ & CD8+ T lymphocytes also changed as expected.     

Association of CD4+ T-lymphocyte counts and new thymic emigrants in HIV-infected children during successful highly active antiretroviral therapy

BACKGROUND: In a cohort of children receiving highly active antiretroviral therapy (HAART) with sustained plasma HIV-1 RNA < 50 copies/mL, children who reached undetectable RNA after week 8 (slow responders, median: week 20) had higher HIV-1 intracellular DNA (HIV-1 DNA) and equal or greater CD4+ T-lymphocyte counts compared with children who reached undetectable plasma HIV-1 RNA by week 8 (rapid responders) throughout HAART. OBJECTIVE: To determine whether levels of T-cell receptor excision circles (TRECs) could explain the apparent inconsistency between the quantity of HIV-1 DNA and CD4+ T-lymphocyte counts in HIV-1-infected children receiving HAART with sustained virologic suppression. METHODS: T-cell receptor excision circles and HIV-1 DNA and plasma HIV-1 RNA were quantified longitudinally by PCR in 31 children (median age, 5.6 years) with sustained undetectable plasma HIV-1 RNA for >104 weeks of HAART. RESULTS: There was a positive correlation between TREC and HIV-1 DNA during HAART, notably at weeks 48 and 80 (P < .004). During the early stage of HAART, TREC levels positively correlated with CD4+ T-lymphocyte percentages (P < .02) and naive CD4+ T-lymphocyte counts (P < .001) and percentages (P = .05). Median TREC levels were consistently equal or higher in slow responders compared with rapid responders (P < .001) despite slow responders having consistently greater quantities of HIV-1 DNA. CONCLUSION: To maintain adequate levels of CD4+ T-lymphocytes, children with high HIV-1 DNA maintain high levels of TREC while receiving HAART. Thus, a thymic control mechanism is required to maintain new CD4+ T lymphocytes in the presence of persistent virus. CLINICAL IMPLICATIONS: The TREC level is a useful marker of thymic function in HIV-infected children.     

IL-18, but not IL-12, is required for optimal cytokine production by influenza virus-specific CD8+ T cells

The potent innate cytokines IL-12 and IL-18 are considered to be important antigen-independent mediators of IFN-gamma production by NK cells and T lymphocytes. The present analysis addresses the physiological role of IL-12 and IL-18 in the generation of virus-specific CD8+ T cells. Both wt C57BL/6J (B6) mice and mice with disrupted IL-12p40 (IL-12p40(-/-)) or IL-18 (IL-18(-/-)) genes were infected with an influenza A virus and the characteristics of the resultant epitope-specific CD8+ T cell responses were compared. While IL-12 appeared to have no notable effect on either virus growth or on CD8+ T cell response profiles, the absence of IL-18 was associated with delayed virus clearance from the lung and, despite normal numbers, a significantly reduced production of IFN-gamma, TNF-alpha, and IL-2 by epitope-specific CD8+ T cells. While this cytokine phenotype was broadly maintained in IL-12p40/IL-18 double-knockout mice, no evidence was seen for any additive effect. Together, our results suggest that IL-18, but not IL-12, induces optimal, antigen-specific production of key cytokines by CD8+ T cells for the efficient clearance of influenza virus from the lungs of infected mice.