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.     

New cohorts of naive T cells exacerbate ongoing allergy but can be suppressed by regulatory T cells

Although as pretreatment oral tolerance is a potent means to achieve systemic suppression, its application in ongoing disease is controversial. Here we propose that availability of naive T cells may critically determine whether immunological tolerance is achieved during ongoing antigenic reactivity. Infusion of naive antigen-specific T cells into mice directly prior to eliciting a secondary Th2 response induces these naive cells to actively engage in the antigenic response despite presence of established memory. Naive antigen-specific T-cells divided faster, produced more interleukin (IL)-2, IL-4 and IL-5 and enhanced immunoglobulin E (IgE) release during a secondary Th2 response, compared with naive T cells that were infused prior to a primary response. Despite such contribution by new cohorts of naive T cells co-infusion of mucosal Tr together with naive T cells could suppress enhanced IgE release during a secondary Th2 response. We conclude that naive T cells contribute to a secondary Th2 response and although they can still be suppressed in the presence of sufficient numbers of mucosal Tr, they may interfere with potential therapeutic application of mucosal tolerance.     

Impact of changes in antigen level on CD38/PD‐1 co‐expression on HIV‐specific CD8 T cells in chronic,untreated HIV‐1 infection

Excessive immune activation is a hallmark of chronic uncontrolled HIV infection. During the past years, growing evidence suggests that immune inhibitory signals also play an important role in progressive disease. However, the relationship between positive and negative immune signals on HIV‐specific CD8 T cells has not been studied in detail so far in chronic HIV‐1 infection. In this study, the expression of markers of positive (CD38) and negative (PD‐1) immune signals on virus‐specific CD8 T cells in chronic, untreated HIV‐1 infection was evaluated using intracellular cytokine staining. Viral escape mutations were assessed by autologous virus sequence analysis and subsequent peptide titration assays. Single‐epitope CD8 T‐cell responses toward Gag, Pol, and Nef were compared in 12 HIV‐1 controllers (viral load <5,000 cp/ml) and 12 HIV‐1 progressors (viral load >50,000 cp/ml) and a highly significant increase of CD38/PD‐1 co‐expression on virus‐specific CD8 T cells in progressors was found (P < 0.0001). The level of CD38/PD‐1 co‐expression was independent of epitope specificity. Longitudinal follow‐up revealed a clear drop in CD38/PD‐1 co‐expression on virus‐specific CD8 T cells after the suppression of antigen following either viral escape mutation or the initiation of HAART (P = 0.004). Antigen persistence with a fluctuating viral load revealed stable levels of CD38/PD‐1 co‐expression whereas significant rises in viral load were accompanied or even preceded by substantial increases in CD38/PD‐1 co‐expression. The CD38/PD‐1 phenotype clearly distinguishes HIV‐specific CD8 T‐cell responses between controllers and progressors. Whether it plays a causative role in disease progression remains debatable. J. Med. Virol. 82:358–370, 2010. © 2010 Wiley‐Liss, Inc.     

Phenotypic characterization of regulatory T cells in the human decidua

Pregnancy is a unique situation for the maternal immune system. We have studied and identified a CD4+CD25+ regulatory T (Treg) cell population isolated from the human decidua. This mucosal surface in the uterus is in direct contact with semiallogenic fetal cells. We observed that about 14% of the decidual CD4+ T cells have the CD4+CD25+ phenotype. The decidual CD4+CD25+ T cells expressed high frequency of intracellular CTLA-4 (CTLA-4i). The majority of CD4+CD25+CTLA-4i+ cells were also positive for GITR and OX40, typical markers for human Treg cells. The frequency of CD4+CD25+ T cells in the peripheral blood from pregnant women was found to be increased during the first and second trimester of gestation when compared to nonpregnant controls. Being an important molecule for Treg cells, the role of CTLA-4 in the regulation of indoleamine 2,3-dioxygenase (IDO) expression was also examined. The stimulation with CTLA-4Ig did not increase IDO mRNA expression in CD14+ cells from pregnant women, while IFN-gamma was observed to up-regulate IDO expression. The presence of Treg cells in the human decidua suggests that these cells are important in protecting the fetus from alloreactive immune responses at the maternal-fetal interface.     

CTL recognition of HIV‐1‐infected cells via cross‐recognition of multiple overlapping peptides from a single 11‐mer Pol sequence

It is known that overlapping HIV‐1 peptides of different lengths can be presented by a given HLA class I molecule. However, the role of those peptides in CD8⁺ T cells recognition of HIV‐1‐infected cells remains unclear. Here we investigated the recognition of overlapping 8‐mer to 11‐mer peptides of Pol 155–165 by HLA‐B*54:01‐restricted CD8⁺ T cells. The analysis of ex vivo T cells using ELISPOT and tetramer binding assays showed that there were different patterns of CD8⁺ T‐cell responses to these peptides among chronically HIV‐1‐infected HLA‐B*54:01⁺ individuals, though the response to the 9‐mer peptide was the strongest among them. CD8⁺ T‐cell clones with TCRs specific for the 9‐mer, 10‐mer, and/or 11‐mer peptides effectively killed HIV‐1‐infected cells. Together, these results suggest that the 9‐mer and 10‐mer peptides could be predominantly presented by HLA‐B*54:01, though it remains possible that the 11‐mer peptide was also presented by this HLA allele. The present study demonstrates effective CD8⁺ T‐cell recognition of HIV‐1‐infected cells via presentation of multiple overlapping HIV‐1 peptides and cross‐recognition by the CD8⁺ T cells.     

Comprehensive analysis of HIV Gag‐specific IFN‐γ response in HIV‐1‐ and HIV‐2‐infected asymptomatic patients from a clinical cohort in The Gambia

Majority of HIV‐2‐infected individuals meet the criteria of long‐term non‐progressors. This has been linked to superior qualitative HIV‐2‐specific cellular immune responses that correlate with viral control. However, it is unknown whether this is due to frequent targeting of immunodominant Gag epitopes in HIV‐2 than HIV‐1 infection. We describe a comprehensive comparison of the magnitude, breadth and frequency of Gag responses and the degree of cross‐recognition of frequently targeted, immunodominant Gag peptides in a cross‐sectional study of asymptomatic HIV‐1‐ and HIV‐2‐infected individuals. Fresh PBMC from 20 HIV‐1‐ and 20 HIV‐2‐infected patients with similar CD4⁺ T‐cell counts (p=0.36) were stimulated with pools of HIV‐1 and/or HIV‐2 Gag peptides in an IFN‐γ ELISPOT assay. We found no difference in the cumulative magnitude of IFN‐γ responses (p=0.75) despite significantly lower plasma viral loads in HIV‐2‐infected people (p<0.0001). However, Gag211–290 was targeted with significantly higher magnitude in HIV‐2‐infected subjects (p=0.03) although this did not correlate with viral control. There was no difference in frequently targeted Gag peptides, the breadth, immunodominance or cross‐recognition of Gag peptide pools between the two infections. This suggests that other factors may control viral replication in HIV‐2 infection.     

Human metapneumovirus keeps dendritic cells from priming antigen‐specific naive T cells

Human metapneumovirus (hMPV) is the second most common cause of acute lower respiratory tract infections in children, causing a significant public health burden worldwide. Given that hMPV can repeatedly infect the host without major antigenic changes, it has been suggested that hMPV may have evolved molecular mechanisms to impair host adaptive immunity and, more specifically, T‐cell memory. Recent studies have shown that hMPV can interfere with superantigen‐induced T‐cell activation by infecting conventional dendritic cells (DCs). Here, we show that hMPV infects mouse DCs in a restricted manner and induces moderate maturation. Nonetheless, hMPV‐infected DCs are rendered inefficient at activating naive antigen‐specific CD4⁺ T cells (OT‐II), which not only display reduced proliferation, but also show a marked reduction in surface activation markers and interleukin‐2 secretion. Decreased T‐cell activation was not mediated by interference with DC–T‐cell immunological synapse formation as recently described for the human respiratory syncytial virus (hRSV), but rather by soluble factors secreted by hMPV‐infected DCs. These data suggest that although hMPV infection is restricted within DCs, it is sufficient to interfere with their capacity to activate naive T cells. Altogether, by interfering with DC function and productive priming of antigen‐inexperienced T cells, hMPV could impair the generation of long‐term immunity.     

Plasmacytoid dendritic cell and functional HIV Gag p55‐specific T cells before treatment interruption can inform set‐point plasma HIV viral load after treatment interruption in chronically suppressed HIV‐1+ patients

The identification of immune correlates of HIV control is important for the design of immunotherapies that could support cure or antiretroviral therapy (ART) intensification-related strategies. ART interruptions may facilitate this task through exposure of an ART partially reconstituted immune system to endogenous virus. We investigated the relationship between set-point plasma HIV viral load (VL) during an ART interruption and innate/adaptive parameters before or after interruption. Dendritic cell (DC), natural killer (NK) cell and HIV Gag p55-specific T-cell functional responses were measured in paired cryopreserved peripheral blood mononuclear cells obtained at the beginning (on ART) and at set-point of an open-ended interruption from 31 ART-suppressed chronically HIV-1⁺ patients. Spearman correlation and linear regression modeling were used. Frequencies of plasmacytoid DC (pDC), and HIV Gag p55-specific CD3⁺ CD4⁻ perforin⁺ IFN-γ⁺ cells at the beginning of interruption associated negatively with set-point plasma VL. Inclusion of both variables with interaction into a model resulted in the best fit (adjusted R² = 0·6874). Frequencies of pDC or HIV Gag p55-specific CD3⁺ CD4⁻ CSFE^lo CD107a⁺ cells at set-point associated negatively with set-point plasma VL. The dual contribution of pDC and anti-HIV T-cell responses to viral control, supported by our models, suggests that these variables may serve as immune correlates of viral control and could be integrated in cure or ART-intensification strategies.     

Human leucocyte antigen‐Bw4 and Gag‐specific T cell responses are associated with slow disease progression in HIV‐1B‐infected anti‐retroviral therapy‐naive Chinese

In China, the majority of human immunodeficiency virus (HIV) infections are predominately subtype B. It is important to characterize the HIV‐1 subtype B‐specific and its T cell response within the Chinese population, with the aim of identifying protective correlates of immunity to control HIV‐1 infections. In this study, we performed a comprehensive analysis looking into the magnitude/strength of T cell responses directed at the Gag protein of the HIV‐1 subtype B, one of the most conserved HIV‐1 proteins. The study group consisted of anti‐retroviral native and chronic HIV‐1 subtype B‐infected individuals. We used enzyme‐linked immunospot (ELISPOT) assay to quantify the total T cell responses to HIV‐1 Gag at the single peptide level. Twenty‐eight (38%) peptides were recognized in 24 (82·8%) individuals. The p24 was identified as the most frequently recognized subunit protein with the greatest T cell response in the test, which correlated positively with CD4⁺ T cell count and inversely with viral load (VL). At the level of the human leucocyte antigen (HLA) supertypes, we detected the highest levels and a significant correlation with both the CD4⁺ T cell count and the VL with Gag T cell responses in Bw4/Bw4. These findings demonstrate that (i) the HIV‐1B Gag p24‐specific immune responses play an important role in controlling viral replication and slowing clinical progression; and (ii) HLA‐Bw4/Bw4 allele has stronger T cell responses, which is associated with slow clinical progression in Chinese HIV patients.     

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.     

Increased proportions of B cells with spontaneous production of interleukin‐10 in HIV‐infected individuals are normalized during combination antiretroviral therapy: a longitudinal study

Interleukin‐10 (IL‐10)‐producing B cells (B10 cells) may inhibit HIV‐specific T cells and are elevated in untreated HIV infection. We aimed to determine the effect of combination antiretroviral treatment (cART) on the proportion of B10 cells. Furthermore, we compared B10‐cell proportions in HIV‐infected progressors and viremic controllers. This was a prospective study including HIV‐infected progressors, viremic controllers and healthy controls. Progressors initiating cART were followed for 6 months. Purified B cells were stimulated with CpG, alone or in combination with HIV gp120, and the proportion of B10 cells was measured by flow cytometry. Without stimulation, the B10‐cell proportion was higher in progressors than in healthy controls, while viremic controllers and healthy controls had comparable proportions. Moreover, the proportion of CD24^hiCD38^hi transitional B cells was higher in progressors than in healthy controls. After initiation of cART, the proportion of B10 cells and transitional B cells decreased. In conclusion, progressors had elevated B10‐cell proportions, while viremic controllers displayed normal proportions. After initiation of cART, the B10‐cell proportion decreased. This could limit B10‐cell‐mediated suppression of specific CD8⁺ T‐cell responses.     

Foxp3(+) regulatory T cells, immune stimulation and host defence against infection

The immune system is intricately regulated allowing potent effectors to expand and become rapidly mobilized after infection, while simultaneously silencing potentially detrimental responses that averts immune-mediated damage to host tissues. This relies in large part on the delicate interplay between immune suppressive regulatory CD4(+) T (Treg) cells and immune effectors that without active suppression by Treg cells cause systemic and organ-specific autoimmunity. Although these beneficial roles have been classically described as counterbalanced by impaired host defence against infection, newfound protective roles for Treg cells against specific viral pathogens (e.g. herpes simplex virus 2, lymphocytic choriomeningitis virus, West Nile virus) have been uncovered using transgenic mice that allow in vivo Treg-cell ablation based on Foxp3 expression. In turn, Foxp3(+) Treg cells also provide protection against some parasitic (Plasmodium sp., Toxoplasma gondii) and fungal (Candida albicans) pathogens. By contrast, for bacterial and mycobacterial infections (e.g. Listeria monocytogenes, Salmonella enterica, Mycobacterium tuberculosis), experimental manipulation of Foxp3(+) cells continues to indicate detrimental roles for Treg cells in host defence. This variance is probably related to functional plasticity in Treg cell suppression that shifts discordantly following infection with different types of pathogens. Furthermore, the efficiency whereby Treg cells silence immune activation coupled with the plasticity in Foxp3(+) cell activity suggest that overriding Treg-mediated suppression represents a prerequisite 'signal zero' that together with other stimulation signals [T-cell receptor (signal 1), co-stimulation (signal 2), inflammatory cytokines (signal 3)] are essential for T-cell activation in vivo. Herein, the importance of Foxp3(+) Treg cells in host defence against infection, and the significance of infection-induced shifts in Treg-cell suppression are summarized.     

Association between discordant immunological response to highly active anti‐retroviral therapy,regulatory T cell percentage,immune cell activation and very low‐level viraemia in HIV‐infected patients

The mechanisms sustaining the absence of complete immune recovery in HIV‐infected patients upon long‐term effective highly active anti‐retroviral therapy (HAART) remain elusive. Immune activation, regulatory T cells (T_regs) or very low‐level viraemia (VLLV) have been alternatively suspected, but rarely investigated simultaneously. We performed a cross‐sectional study in HIV‐infected aviraemic subjects (mean duration of HAART: 12 years) to concomitantly assess parameters associated independently with inadequate immunological response. Patients were classified as complete immunological responders (cIR, n = 48) and inadequate immunological responders (iIR, n = 39), depending on the CD4⁺ T cell count (> or < 500/mm³). Clinical and virological data (including very low‐level viraemia) were collected. In parallel, immunophenotyping of CD4⁺ lymphocytes, including T_reg subsets, and CD8⁺ T cells was performed. Percentages of activated CD4⁺ T cells, T_regs, effector T_regs and terminal effector T_regs were found to be significantly elevated in iIR. Neither the percentage of activated CD8⁺ T cells nor VLLV were found to be associated with iIR. In the multivariate analysis, nadir of CD4⁺ T cell count and percentage of T_regs were the only two parameters associated independently with iIR [odds ratio (OR) = 2·339, P = 0·001, and OR = 0·803, P = 0·041]. We present here the largest study investigating simultaneously the immune response to long‐term HAART, activation of CD4⁺ and CD8⁺ T cells, T_reg percentages and very low‐level viraemia. Causative interactions between T_regs and CD4⁺ T cells should now be explored prospectively in a large patients cohort.     

The CD8+HLA‐DR+ T cells expanded in HIV‐1 infection are qualitatively identical to those from healthy controls

HIV‐induced immune activation leads to expansion of a subset of human CD8⁺ T cells expressing HLA‐DR antigens. Expansion of CD8⁺HLA‐DR⁺ T cells can be also observed in non‐HIV settings including several autoimmune diseases and aging. Although these cells are felt to represent “immune exhaustion” and/or to be anergic, their precise role in host defense has remained unclear. Here, we report that this subset of cells exhibits a restricted repertoire, shows evidence of multiple rounds of division, but lacks markers of recent TCR engagement. Detailed cell cycle analysis revealed that compared with their CD8⁺HLA‐DR^? counterpart, the CD8⁺HLA‐DR⁺ T‐cell pool contained an increased fraction of cells in S‐phase with elevated levels of the G2/M regulators: cyclin A2, CDC25C, Cdc2 (CDK1), indicating that these cells are not truly anergic but rather experiencing proliferation in vivo. Together, these data support a hypothesis that antigen stimulation leads to the initial expansion of a CD8⁺ pool of cells in vivo that undergo further expansion independent of ongoing TCR engagement. No qualitative differences were noted between CD8⁺HLA‐DR⁺ cells from HIV⁺ and HIV^? donors, indicating that the generation of CD8⁺HLA‐DR⁺ T cells is a part of normal immune regulation that is exaggerated in the setting of HIV‐1 infection.     

HIV gag‐specific immune response mediated by double negative (CD3+CD4−CD8−) T cells in HIV‐exposed seronegative individuals

Double negative (DN) T cells are CD3⁺, CD4^?, CD8^? cells with either T‐cell receptors (TCR) αβ or TCR γδ whose importance on protection against HIV infection is unknown. Since HIV‐exposed seronegative individuals correspond to an ideal group in whom correlates of protection are expected, the role of these cells was studied in 13 HIV‐serodiscordant couples in a stable relationship and reporting unprotected sexual intercourses. HIV‐specific immune responses mediated by DN T‐cells were evaluated by measuring intracellular IFNγ and MIP1β (CCL4) production in response to HIV‐Gag peptides. Thirty‐five healthy controls not exposed to HIV were tested similarly and used to define a threshold for positive responses. Interestingly, Gag‐specific DN T‐cell responses were found in 3/13 (23%) HIV‐exposed seronegative individuals (Group A), involving both DN/αβ⁺ and DN/γδ⁺ T‐cells through MIP1β and IFNγ production. 4/13 (30%) of partners infected with HIV (Group B) also showed Gag‐specific responses but were mediated exclusively by DN/γδ⁺ T‐cells, mainly through IFNγ production. DN T‐cells in Group A individuals can display differential HIV‐specific immune responses, which might contribute to the low susceptibility to infection with HIV shown by individuals in Group A. J. Med. Virol. 85:200–209, 2013. © 2012 Wiley Periodicals, Inc.     

Membrane‐bound Dickkopf‐1 in Foxp3+ regulatory T cells suppresses T‐cell‐mediated autoimmune colitis

Induction of tolerance is a key mechanism to maintain or to restore immunological homeostasis. Here we show that Foxp3⁺ regulatory T (Treg) cells use Dickkopf‐1 (DKK‐1) to regulate T‐cell‐mediated tolerance in the T‐cell‐mediated autoimmune colitis model. Treg cells from DKK‐1 hypomorphic doubleridge mice failed to control CD4⁺ T‐cell proliferation, resulting in CD4 T‐cell‐mediated autoimmune colitis. Thymus‐derived Treg cells showed a robust expression of DKK‐1 but not in naive or effector CD4 T cells. DKK‐1 expression in Foxp3⁺ Treg cells was further increased upon T‐cell receptor stimulation in vitro and in vivo. Interestingly, Foxp3⁺ Treg cells expressed DKK‐1 in the cell membrane and the functional inhibition of DKK‐1 using DKK‐1 monoclonal antibody abrogated the suppressor function of Foxp3⁺ Treg cells. DKK‐1 expression was dependent on de novo protein synthesis and regulated by the mitogen‐activated protein kinase pathway but not by the canonical Wnt pathway. Taken together, our results highlight membrane‐bound DKK‐1 as a novel Treg‐derived mediator to maintain immunological tolerance in T‐cell‐mediated autoimmune colitis.