Changes in thymic function in HIV-positive patients treated with highly active antiretroviral therapy and interleukin-2

Despite its potent antiviral activity, highly active antiretroviral therapy (HAART) only exerts a marginal effect on CD4⁺ T‐cell regeneration in HIV‐infected subjects. Combination therapies aimed at boosting T‐cell activity and maturation may provide an important contribution to the restoration of immune function. Here, we report the results obtained by a two‐year follow‐up of a cohort of HIV‐infected patients treated with a combination of HAART and interleukin‐2 (IL‐2). In these patients, in addition to a series of quantitative virological and immunological parameters, we investigated T‐cell regeneration by an immunophenotypic assay monitoring CD4⁺ naïve T cells, and by analysis of thymic function, through the quantification of the excision DNA products of T‐cell receptor rearrangement (TRECs) in lymphocytes. Compared with HAART alone, we found that the IL‐2 combination therapy was equally effective in reducing the levels of viremia and marginally more effective in decreasing proviral DNA load. Strikingly, the IL‐2 combination produced a marked increase in the number of CD4⁺ T cells bearing a naïve phenotype (CD45RA⁺, CD62L⁺), which was apparent for over 96 weeks after therapy. To assess whether these cells were the product of improved T‐cell generation, we exploited a competitive quantitative molecular assay to quantify TRECs in peripheral blood lymphocytes. Surprisingly, we found that the levels of these molecules were unchanged in these patients. These findings indicate that improved thymic function does not account for the early rise of CD4 naïve cells in HIV‐positive patients treated with IL‐2, and suggest that alternative mechanisms of T‐cell maturation and differentiation are responsible for this event.     

T-cell mean telomere lengths changes in treatment naïve HIV-infected patients randomized to G-CSF or placebo simultaneously with initiation of HAART

The effect of highly active antiretroviral therapy (HAART) and granulocyte colony stimulating factor (G‐CSF) on mean telomere restriction fragment (TRF) length of peripheral blood mononuclear cells (PBMC) was examined in 11 treatment naïve human immunodeficiency virus (HIV)‐infected individuals with a CD4⁺ T‐cell count < 350cells/mm³. Patients were randomized to HAART combined with G‐CSF thrice weekly for 12 weeks (n = 6) or placebo (n = 5). An increase in the mean TRF lengths was observed in PBMC of patients on HAART after 24 weeks of treatment mainly owing to increased mean CD8⁺ T‐cell TRF lengths. However, in the group of patients on HAART combined with G‐CSF no changes of PBMC mean TRF length was observed during treatment or during 12 weeks of follow‐up. The mean CD4⁺ T‐cell TRF length did not change in any of the two groups. These results confirm that HAART induces mainly the lengthening of the mean CD8⁺ T‐cell TRF length. However, G‐CSF given simultaneously with HAART induces an inhibition of the expected lengthening in mean TRF length. These results do therefore not support the use of adjuvant G‐CSF treatment simultaneously when initiating HAART and should further be evaluated before use in non‐neutropenic HIV‐infected patients.     

Effects of highly active antiretroviral therapy and its adherence on herpes zoster incidence: a longitudinal cohort study

Background

Herpes zoster (HZ) is common among HIV-infected individuals, but the impacts of highly active antiretroviral therapy (HAART) and HAART adherence on HZ risk have not been well studied.

Methods

The effects of HAART and HAART adherence on HZ incidence were evaluated by comparing HIV-infected women on HAART (HAART use group) with the HIV-infected women remaining HAART naïve (HAART naïve group) in the Women’s Interagency HIV Study (WIHS). A 1:1 matching with propensity score for predicting HAART initiation was conducted to balance background covariates at index visit, including HIV disease stage. Kaplan-Meier method was used to compare the risk of HZ development between the matched pairs. Cox proportional hazard models were used to assess the effects of HAART and HAART adherence on HZ incidence.

Results

Through propensity score matching, 389 pairs of participants were identified and they contributed 3,909 person years after matching. The background covariates were similar between the matched pairs at the index visit. The participants had a mean age around 39 years old, and about 61% of them were Black and 22% were Latina. No significant difference in HZ risk was observed between the HAART use group and the HAART naïve group during the first year of follow-up in any analyses. In the univariate analysis, the HAART use group had marginally lower HZ risk (Hazard Ratio (HR): 0.72; 95% Confidence Interval (CI): 0.48-1.1) over the entire follow-up period. However, women with a HAART adherence level of ≥95% had significantly lower HZ risk (HR: 0.54; 95% CI: 0.31, 0.94) compared to the HAART naïve women. The association remained significant after adjusting for quality of life score and acyclovir use, but it attenuated and was no longer statistically significant after adjusting for an intermediate variable, either CD4+ T cell counts or HIV viral load.

Conclusions

Among adult women, we observed a significant preventive effect of long-term HAART use on HZ incidence when a HAART adherence level of ≥95% was attained, and this effect was mediated through reduction of HIV viral load and improvement of CD4+ T cell counts.

     

Immunity in HIV-1-Infected Adults with a Previous State of Moderate-Severe Immune-Suppression and More Than 500 CD4+ T Cell After Highly Active Antiretroviral Therapy

We evaluated phenotypic and functional parameters of immune restoration of 27 HIV-infected patients on highly active antiretroviral therapy (HAART) (HIV-cases) with HIV-RNA levels below detectable limits at least during 18 months, and CD4+ cell per microliter higher than 500 at the moment of the study and lower than 300 anytime before. These patients were compared with 11 HIV-controls that never had less than 500 CD4+ cell per microliter and 20 healthy-controls (HIV seronegative subjects) in a cross-sectional study. HIV-cases had lower counts of naïve CD4+ than HIV-controls and healthy-controls. HIV-patients (both HIV-cases and HIV-controls) showed higher values of naïve and memory CD8+ counts than healthy-controls. TREC-bearing cell levels were significantly lower in HIV-cases than in healthy-controls. Peripheral blood mononuclear cells (PBMC) cultures, HIV-cases had lower values in proliferation to streptokinase (SK) and tetanus toxin (TT) than in healthy-controls. HIV-cases had lower IFN-γ and higher IL-5 production with pokeweed than healthy-controls (P < 0.01). However, IL-5 production of HIV-cases after TT stimulation was lower than in HIV-controls and healthy-controls. Total IgG and IgG1 levels were significantly higher in HIV-cases than in HIV-controls and healthy-controls. Also, IgM levels were significantly higher in HIV-cases than in healthy-controls. Nevertheless, IgG2 levels were significantly lower in HIV-cases and HIV-controls than in healthy-controls. The levels of specific Igs antipneumococcal capsular polysaccharide and TT were significantly lower in HIV-cases than in healthy-controls. HIV-patients with a previous state of severe-moderate immunosuppression normalizing their CD4+ counts have a incomplete immune reconstitution after HAART. Long-term consequences of this subclinical immune deficiency remain to be determined.     

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.     

Increased levels of regulatory T cells (Tregs) in human immunodeficiency virus‐infected patients after 5 years of highly active anti‐retroviral therapy may be due to increased thymic production of naive Tregs

This study determines levels of regulatory T cells (T_regs), naive T_regs, immune activation and cytokine patterns in 15 adult human immunodeficiency virus (HIV)‐infected patients receiving prolonged highly active anti‐retroviral therapy (HAART) who have known thymic output, and explores if naive T_regs may represent recent thymic emigrant T_regs. HIV‐infected patients treated with HAART with a median of 1 and 5 years were compared with healthy controls. Percentages of T_regs (CD3⁺CD4⁺CD25⁺CD127^low), naive T_regs (CD3⁺CD4⁺CD25⁺CD45RA⁺) and activation markers (CD38⁺human leucocyte antigen D‐related) were determined by flow cytometry. Forkhead box P3 mRNA expression and T cell receptor excision circles (T_REC) content in CD4⁺ cells were determined by polymerase chain reaction and cytokines analysed with Luminex technology. Levels of T_regs were significantly higher in HIV‐infected patients compared with controls, both after 1 and 5 years of HAART (P < 0·001), despite fully suppressed HIV‐RNA and normalization of both CD4 counts, immune activation and cytokine patterns. Furthermore, levels of naive T_regs were elevated significantly in HIV‐infected patients (P < 0·001) and were associated with thymic output measured as the T_REC frequency in CD4⁺ cells (P = 0·038). In summary, T_reg levels in HIV‐infected patients are elevated even after 5 years of HAART. Increased thymic production of naive T_regs may contribute to higher T_reg levels in HIV‐infection.     

CDC staging based on absolute CD4 count and CD4 percentage in an HIV-1-infected Indian population: treatment implications

CD4⁺ T‐cell levels are an important criterion for categorizing HIV‐related clinical conditions according to the CDC classification system and are therefore important in the management of HIV by initiating antiretroviral therapy and prophylaxis for opportunistic infections due to HIV among HIV‐infected individuals. However, it has been observed that the CD4 counts are affected by the geographical location, race, ethnic origin, age, gender and changes in total and differential leucocyte counts. In the light of this knowledge, we classified 600 HIV seropositive antiretroviral treatment (ART)‐naïve Indian individuals belonging to different CDC groups A, B and C on the basis of CDC criteria of both CD4% and CD4 counts and receiver operating characteristic (ROC) curves were generated. Importantly, CDC staging on the basis of CD4% indicated significant clinical implications, requiring an early implementation of effective antiretroviral treatment regimen in HIV‐infected individuals deprived of treatment when classified on the basis of CD4 counts.     

Occult hepatitis B in persons infected with HIV is associated with low CD4 counts and resolves during antiretroviral therapy

Occult hepatitis B virus (HBV) is defined by the presence of plasma HBV DNA in individuals with HBV core antibodies (anti‐HBc), but without HBV surface antigen (HBsAg). The prevalence of occult HBV in HIV‐infected patients remains controversial, and the risk factors, clinical significance and effect of highly active antiretroviral therapy (HAART) are unknown. The aim of this study was to determine prevalence, risk factors, and clinical significance of occult HBV in HIV‐infected patients and to evaluate the effect of HAART. Plasma HBV DNA levels were determined in 191 HIV positive, antiretroviral naïve patients, who were anti‐HBc positive and HBsAg negative. Quantitative HBV DNA was determined using a Taqman real‐time nested PCR. Additionally, plasma HIV RNA levels, CD4 cell counts, anti‐HBs‐antibodies, anti‐HCV‐antibodies, ALT, AST, and γGT were determined. Occult HBV (a plasma HBV DNA level >50 copies/ml) was detected in 9/191 (4.7%) of the patients. Among 45 anti‐HBs‐negative patients (isolated anti‐HBc positive), the prevalence was 11.1%. Patients with occult HBV had significantly lower CD4 count compared to anti‐HBc‐positive/HBsAg negative/HBV DNA‐negative patients (105 ± 157 (median ± SD) vs. 323 ± 299 cells/mm³, P = 0.019). When HAART (including lamivudine) was initiated in the patients with occult HBV, HBV DNA was no longer detectable in any of the patients during 3 years of follow‐up. In conclusion, occult HBV was associated with low CD4 counts and may be viewed as opportunistic reactivation of HBV that resolves as a consequence of HAART induced immune reconstitution and/or the effect of lamivudine. J. Med. Virol. 81:441–445, 2009. © 2009 Wiley‐Liss, Inc.     

Primary vasculitis of the central nervous system in patients infected with HIV‐1 in the HAART era

Angiitis of the central nervous system (CNS) in patients infected with HIV‐1‐is often associated with concomitant infection or lymphoproliferative disease of the CNS. Four HAART naïve patients infected with HIV‐1 with severe stroke are described. Evidence of vasculitis was found by magnetic resonance angiography. Extensive investigations excluded concomitant opportunistic, lymphoproliferative or autoimmune disorders leading to the diagnosis of primary angiitis of the CNS. Despite initiation of HAART and prolonged suppression of viral replication, these patients remained severely immunosuppressed. The addition of corticosteroids led to a significant improvement of clinical symptoms. Primary angiitis of the CNS should be considered in patients with HIV and stroke. The prognosis of these patients remain poor despite HAART. These observations suggest that the vascular inflammatory process persists despite the control of viral load under HAART in patients with persistent immunosuppression. J. Med. Virol. 81:578–581, 2009 © 2009 Wiley‐Liss, Inc.     

HIV-infected Patients with a Large Thymus Maintain Higher CD4 Counts in a 5-year Follow-up Study of Patients Treated with Highly Active Antiretroviral Therapy

CD4 recovery in HIV-infected patients treated with highly active antiretroviral therapy (HAART) is in part believed to be dependent on the degree of preserved thymic function. We investigated whether the thymus has a prolonged effect on CD4 recovery. Total and naïve CD4 counts as well as thymic output determined as the number of CD4 +  cells containing T-cell receptor-rearrangement excision DNA circles were measured prospectively in 25 HIV-infected patients with known thymic size during 5 years of HAART. Patients with larger thymic size had at all time points of follow-up significantly higher CD4 counts than patients with minimal thymic size ( P  = 0.0036). The CD4 increase from time of initiation of HAART until 6 months of follow-up differed significantly between the two thymic groups ( P  = 0.045), but did not at later time points. Thymic output remained significantly higher in patients with larger thymic size at follow-up. However, no difference in the increase in thymic output was seen between thymic groups. In conclusion, the importance of the thymus to the rate of cellular restoration seems primarily to lie within the first two years of HAART. However, patients with larger thymic size are able to maintain higher CD4 counts even after 5 years of HAART.     

HIV‐1 impairs in vitro priming of naïve T cells and gives rise to contact‐dependent suppressor T cells

Priming of T cells in lymphoid tissues of HIV‐infected individuals occurs in the presence of HIV‐1. DC in this milieu activate T cells and disseminate HIV‐1 to newly activated T cells, the outcome of which may have serious implications in the development of optimal antiviral responses. We investigated the effects of HIV‐1 on DC–naïve T‐cell interactions using an allogeneic in vitro system. Our data demonstrate a dramatic decrease in the primary expansion of naïve T cells when cultured with HIV‐1‐exposed DC. CD4⁺ and CD8⁺ T cells showed enhanced expression of PD‐1 and TRAIL, whereas CTLA‐4 expression was observed on CD4⁺ T cells. It is worth noting that T cells primed in the presence of HIV‐1 suppressed priming of other naïve T cells in a contact‐dependent manner. We identified PD‐1, CTLA‐4, and TRAIL pathways as responsible for this suppresion, as blocking these negative molecules restored T‐cell proliferation to a higher degree. In conclusion, the presence of HIV‐1 during DC priming produced cells with inhibitory effects on T‐cell activation and proliferation, i.e. suppressor T cells, a mechanism that could contribute to the enhancement of HIV‐1 pathogenesis.     

Limited Impact of the Thymus on Immunological Recovery During and After Chemotherapy in Patients with Diffuse Large B-cell Lymphoma

To investigate the impact of thymus on immunological recovery after dose-dense chemotherapy a prospective study of 17 patients diagnosed with diffuse large B-cell lymphoma (DLBCL) was conducted. Patients were monitored before, during and until 3 months after chemotherapy. The thymus was visualized using computer tomographic scans. Patients were divided into two groups according to thymic size, one group comprising of patients without detectable thymus and one group of patients with detectable thymus. Naïve CD4 and CD8 counts were measured by flow cytometry, and to measure thymic output determination of CD4+ cells containing T-cell receptor excision circles (TREC) was done. During chemotherapy, the naïve CD4 count decreased significantly as did the CD4-TREC%. Significant difference in recovery of naïve CD4 counts between patients with detectable and undetectable thymic tissue during treatment with chemotherapy was not found. CD4-TREC% was associated with lower age. It was not possible to demonstrate an association between thymic size and recovery of the naïve CD4+ cells. The study terminated 3 months after the last cycle of chemotherapy, and at that time point the naïve CD4 counts and the CD4-TREC% had not returned to pretreatment levels. However, patients with detectable thymic tissue had higher naïve CD4 counts after the first cycles of chemotherapy, suggesting that these patients may be less susceptible to infectious complications related to chemotherapy.     

Human thymus regeneration and T cell reconstitution

The thymus supports the development of T cells throughout life from hematopoietic progenitor cells migrating from the bone marrow. During the early years after birth thymic activity is highest, but progressively declines resulting in diminished naïve T cell output. Underlying causes of thymic involution may be degeneration of the stromal thymic network, providing survival and differentiation factors for developing T cells, or insufficiency of the progenitor cells to home and/or develop in the aged thymus. In young people the reduced thymic output is insignificant, since the peripheral T cell compartment is under compensatory homeostatic control. However, in more or less immunocompromised individuals, including aged people and patients depleted of T cells due to conditioning regimens before bone marrow transplantation or HIV infection, the thymus is necessary to replenish the peripheral T cell compartment. This may require rejuvenation of the thymus. Alternatively, approaches to generate mature T cells independent of the thymus have gained considerable interest.     

A new mechanism shapes the naïve CD8+ T cell repertoire: the selection for full diversity

During thymic T cell differentiation, TCR repertoires are shaped by negative, positive and agonist selection. In the thymus and in the periphery, repertoires are also shaped by strong inter-clonal and intra-clonal competition to survive death by neglect. Understanding the impact of these events on the T cell repertoire requires direct evaluation of TCR expression in peripheral naïve T cells. Several studies have evaluated TCR diversity, with contradictory results. Some of these studies had intrinsic technical limitations since they used material obtained from T cell pools, preventing the direct evaluation of clonal sizes. Indeed with these approaches, identical TCRs may correspond to different cells expressing the same receptor, or to several amplicons from the same T cell. We here overcame this limitation by evaluating TCRB expression in individual naïve CD8⁺ T cells. Of the 2269 Tcrb sequences we obtained from 13 mice, 99% were unique. Mathematical analysis of the data showed that the average number of naïve peripheral CD8⁺ T cells expressing the same TCRB is 1.1 cell. Since TCRA co-expression studies could only increase repertoire diversity, these results reveal that the number of naïve T cells with unique TCRs approaches the number of naïve cells. Since thymocytes undergo multiple rounds of divisions after TCRB rearrangement and 3–5% of thymocytes survive thymic selection events the number of cells expressing the same TCRB was expected to be much higher. Thus, these results suggest a new repertoire selection mechanism, which strongly selects for full TCRB diversity.     

Mineralocorticoid receptor signaling reduces numbers of circulating human naïve T cells and increases their CD62L,CCR7, and CXCR4 expression

The role of mineralocorticoid receptors (MRs) in human T‐cell migration is not yet understood. We have recently shown that the MR antagonist spironolactone selectively increases the numbers of circulating naïve and central memory T cells during early sleep, which is the time period in the 24 h cycle hallmarked by predominant MR activation. To investigate whether this effect is specific to spironolactone's blockade of MRs and to study the underlying molecular mechanisms, healthy humans were given the selective MR‐agonist fludrocortisone or placebo and numbers of eight T‐cell subsets and their CD62L and CXCR4 expression were analyzed. Fludrocortisone selectively reduced counts of naïve CD4⁺, central memory CD4⁺, and naïve CD8⁺ T cells and increased CXCR4 expression on the naïve subsets. In complementing in vitro studies, fludrocortisone enhanced CXCR4 and CD62L expression, which was counteracted by spironolactone. Incubation of naïve T cells with spironolactone alone reduced CD62L and CCR7 expression. Our results indicate a regulatory influence of MR signaling on human T‐cell migration and suggest a role for endogenous aldosterone in the redistribution of T‐cell subsets to lymph nodes, involving CD62L, CCR7, and CXCR4. Facilitation of T‐cell homing following sleep‐dependent aldosterone release might thus essentially contribute to sleep's well‐known role in supporting adaptive immunity.     

Plasma levels of intact and cleaved urokinase receptor decrease in HIV-1-infected patients initiating highly active antiretroviral therapy

Elevated blood levels of soluble urokinase receptor (suPAR) measured by ELISA decrease in human immunodeficiency virus‐1 (HIV‐1)‐infected patients initiating highly active antiretroviral therapy (HAART). As the suPAR ELISA measures both three‐ and two‐domain suPAR [suPAR(I–III), suPAR(II–III)] and suPAR(I–III)–ligand complexes, the amount by which the individual suPAR forms (suPAR(I–III), suPAR(II–III) and one‐domain suPAR [suPAR(I)]) decrease in plasma in HIV‐1‐infected patients initiating HAART is unknown. Consequently, the objective of this study was to investigate HAART‐induced changes in the individual plasma suPAR forms in HIV‐1‐infected patients. Plasma suPAR was measured by three time‐resolved fluorescence immunoassays detecting suPAR(I–III), suPAR(I–III) + suPAR(II–III) and suPAR(I) in 29 treatment‐naïve HIV‐1‐infected patients followed annually for 5 years after initiation of HAART and in 20 age‐ and gender‐matched healthy individuals. In addition, plasma levels of the following inflammatory markers were also investigated: soluble tumour necrosis factor receptor (sTNFr)‐II, TNF‐α, interleukins (IL)‐10, IL‐6, IL‐4, IL‐2 and interferon (IFN)‐γ. In HIV‐1‐infected patients, plasma suPAR(I–III), suPAR(II–III) and suPAR(I) decreased within the first treatment year (all P < 0.05) and suPAR(I–III) and suPAR(II–III) remained above normal throughout follow‐up (both P < 0.05). Plasma sTNFrII, IL‐6, IFN‐γ and IL‐10 also decreased during HAART (all P < 0.05). In HIV‐1‐infected patients, sTNFrII correlated with all suPAR forms before (all P < 0.01) and after 5 years HAART (all P < 0.001), whereas sTNFrII and suPAR did not correlate in healthy individuals. Intact and cleaved plasma suPAR decreased in HIV‐1‐infected patients initiating HAART but remained above normal. The positive correlation with sTNFrII suggests that the individual plasma suPAR forms are linked to immune activation in HIV‐1 infection.