David van Bockel

Proliferation of weakly suppressive regulatory CD4+ T cells is associated with over-active CD4+ T-cell responses in HIV-positive patients with mycobacterial immune restoration disease.

The role of Treg in patients with late‐stage HIV disease, who commence combination antiretroviral therapy (cART) and develop pathogen‐specific immunopathology manifesting as immune restoration disease (IRD) remains unclear. We hypothesised that Treg could be defective in either numbers and/or function and therefore unable to ensure the physiological equilibrium of the immune system in patients with IRD. Phenotypic and functional CD4+ T‐cell subsets of eight late‐stage HIV patients with nadir CD4 count <50 cells/μL, who developed mycobacterial IRD upon commencing cART were compared with six therapy naive HIV+ patients (nadir CD4 count <50 cells/μL), who did not develop an IRD after cART. Mycobacterium‐avium‐specific CD4+ T cells from IRD patients produced high levels of IFN‐γ and IL‐2 compared with controls (p<0.001). Surprisingly, we found a significant expansion of CD127loFoxp3+CD25+ Treg in IRD patients and a higher ratio of Treg to effector/memory subsets (p<0.001). In vitro suppression assays demonstrated reduced functional capacity of suppressor cells and diminished IL‐10 secretion in IRD patients. Plasma levels of IL‐7 were increased in patients and, interestingly, exogenous IL‐7 and other cytokines strongly inhibited Treg suppression. These data suggest that despite substantial Treg expansion in IRD, their ability to induce suppression, and thereby downregulate aberrant immune responses, is compromised.

A Molecular Basis for the Control of Preimmune Escape Variants by HIV-Specific CD8(+) T Cells.

The capacity of the immune system to adapt to rapidly evolving viruses is a primary feature of effective immunity, yet its molecular basis is unclear. Here, we investigated protective HIV-1-specific CD8+ T cell responses directed against the immunodominant p24 Gag-derived epitope KK10 (KRWIILGLNK263-272) presented by human leukocyte antigen (HLA)-B∗2705. We found that cross-reactive CD8+ T cell clonotypes were mobilized to counter the rapid emergence of HIV-1 variants that can directly affect T cell receptor (TCR) recognition. These newly recruited clonotypes expressed TCRs that engaged wild-type and mutant KK10 antigens with similar affinities and almost identical docking modes, thereby accounting for their antiviral efficacy in HLA-B∗2705+ individuals. A protective CD8+ T cell repertoire therefore encompasses the capacity to control TCR-accessible mutations, ultimately driving the development of more complex viral escape variants that disrupt antigen presentation.

TCR β-Chain Sharing in Human CD8+ T Cell Responses to Cytomegalovirus and EBV

The CD8+ TCR repertoires specific for many immunogenic epitopes of CMV and EBV are dominated by a few TCR clonotypes and involve public TCRs that are shared between many MHC-matched individuals. In previous studies, we demonstrated that the observed sharing of epitope-specific TCRβ chains between individuals is strongly associated with TCRβ production frequency, and that a process of convergent recombination facilitates the more efficient production of some TCRβ sequences. In this study, we analyzed a total of 2836 TCRβ sequences from 23 CMV-infected and 10 EBV-infected individuals to investigate the factors that influence the sharing of TCRβ sequences in the CD8+ T cell responses to two immunodominant HLA-A*0201-restricted epitopes from these viruses. The most shared TCRβ amino acid sequences were found to have two features that indicate efficient TCRβ production, as follows: 1) they required fewer nucleotide additions, and 2) they were encoded by a greater variety of nucleotide sequences. We used simulations of random V(D)J recombination to demonstrate that the in silico TCRβ production frequency was predictive of the extent to which both TCRβ nucleotide and amino acid sequences were shared in vivo. These results suggest that TCRβ production frequency plays an important role in the interindividual sharing of TCRβ sequences within CD8+ T cell responses specific for CMV and EBV.

Innate and Adaptive Immunity in Long-Term Non-Progression in HIV Disease

Long-term non-progressors (LTNP) were identified after 10–15 years of the epidemic, and have been the subject of intense investigation ever since. In a small minority of cases, infection with nef/3′LTR deleted attenuated viral strains allowed control over viral replication. A common feature of LTNP is the readily detected proliferation of CD4 T-cells in vitro, in response to p24. In some cases, the responding CD4 T-cells have cytotoxic effector function and may target conserved p24 epitopes, similar to the CD8 T-cells described below. LTNP may also carry much lower HIV DNA burden in key CD4 subsets, presumably resulting from lower viral replication during primary infection. Some studies, but not others, suggest that LTNP have CD4 T-cells that are relatively resistant to HIV infection in vitro. One possible mechanism may involve up-regulation of the cell cycle regulator p21/waf in CD4 T-cells from LTNP. Delayed progression in Caucasian LTNP is also partly associated with heterozygosity of the Δ32 CCR5 allele, probably through decreased expression of CCR5 co-receptor on CD4 T-cells. However, in approximately half of Caucasian LTNP, two host genotypes, namely HLA-B57 and HLA-B27, are associated with viral control. Immunodominant CD8 T-cells from these individuals target epitopes in p24 that are highly conserved, and escape mutations have significant fitness costs to the virus. Furthermore, recent studies have suggested that these CD8 T-cells from LTNP, but not from HLA-B27 or HLA-B57 progressors, can cross-react with intermediate escape mutations, preventing full escape via compensatory mutations. Humoral immunity appears to play little part in LTNP subjects, since broadly neutralizing antibodies are rare, even amongst slow progressors. Recent genome-wide comparisons between LTNP and progressors have confirmed the HLA-B57, HLA-B27, and delta32 CCR5 allelic associations, plus indicated a role for HLA-C/KIR interactions, but have not revealed any new genotypes so far. Nevertheless, it is hoped that studying the mechanisms of intracellular restriction factors, such as the recently identified SAMHD1, will lead to a better understanding of non-progression.

Persistent Survival of Prevalent Clonotypes within an Immunodominant HIV Gag-Specific CD8+ T Cell Response

CD8+ T cells play a significant role in the control of HIV replication, yet the associated qualitative and quantitative factors that determine the outcome of infection remain obscure. In this study, we examined Ag-specific CD8+ TCR repertoires longitudinally in a cohort of HLA-B*2705+ long-term nonprogressors with chronic HIV-1 infection using a combination of molecular clonotype analysis and polychromatic flow cytometry. In each case, CD8+ T cell populations specific for the immunodominant p24 Gag epitope KRWIILGLNK (KK10; residues 263–272) and naturally occurring variants thereof, restricted by HLA-B*2705, were studied at multiple time points; in addition, comparative data were collected for CD8+ T cell populations specific for the CMV pp65 epitope NLVPMVATV (NV9; residues 495–503), restricted by HLA-A*0201. Dominant KK10-specific clonotypes persisted for several years and exhibited greater stability than their contemporaneous NV9-specific counterparts. Furthermore, these dominant KK10-specific clonotypes exhibited cross-reactivity with antigenic variants and expressed significantly higher levels of CD127 (IL-7Rα) and Bcl-2. Of note, we also found evidence that promiscuous TCR α-chain pairing associated with alterations in fine specificity for KK10 variants could contribute to TCR β-chain prevalence. Taken together, these data suggest that an antiapoptotic phenotype and the ability to cross-recognize variant epitopes contribute to clonotype longevity and selection within the peripheral memory T cell pool in the presence of persistent infection with a genetically unstable virus.

HIV Immune Escape at an Immunodominant Epitope in HLA-B*27–Positive Individuals Predicts Viral Load Outcome

The CTL response in HLA-B*27+ HIV-infected individuals is characterized by an immunodominant response to a conserved epitope in gag p24 (aa 263–272, KRWIILGLNK; KK10). Mutations resulting in substitution of the arginine (R264) at position 2 of this epitope have been identified as escape mutations. Nineteen HLA-B*27+ long-term nonprogressors were identified from an Australian cohort with an average follow-up of 16 y following infection. Viral and host genetic factors impacting on disease progression were determined at multiple time points. Twelve of 19 had wild-type sequences at codon 264 at all time points; 7 of 19 carried CTL escape variants. Median viral load and CD4+ T cell counts were not significantly different between these groups at enrollment. Viral load, as judged by levels at their last visit (1,700 and 21,000 RNA copies/ml, respectively; p = 0.01) or by time-weighted area under the curve was higher in the escape group (p = 0.02). Escape mutants at other HLA-B*27–restricted epitopes were uncommon. Moreover, host polymorphisms, such as CCR5Δ32, CCR2-64I, and SDF1-3′A, or breadth of TCR repertoire responding to KK10 did not segregate to wild-type or escape groups. Host and viral factors were examined for a relationship to viral load. The only factor to affect viral load was the presence of the R264 escape mutations at the immunodominant epitope. CTL escape at R264 in the KK10 epitope is a major determinant of subsequent viral load in these HLA-B*27+ individuals.

Cytotoxic CD4 T cells-friend or foe during viral infection?

CD4 T cells with cytotoxic function were once thought to be an artifact due to long-term in vitro cultures but have in more recent years become accepted and reported in the literature in response to a number of viral infections. In this review, we focus on cytotoxic CD4 T cells in the context of human viral infections and in some infections that affect mice and non-human primates. We examine the effector mechanisms used by cytotoxic CD4 cells, the phenotypes that describe this population, and the transcription factors and pathways that lead to their induction following infection. We further consider the cells that are the predominant targets of this effector subset and describe the viral infections in which CD4 cytotoxic T lymphocytes have been shown to play a protective or pathologic role. Cytotoxic CD4 T cells are detected in the circulation at much higher levels than previously realized and are now recognized to have an important role in the immune response to viral infections.

Circulating gluten-specific FOXP3 + CD39 + regulatory T cells have impaired suppressive function in patients with celiac disease

Background: Celiac disease is a chronic immune‐mediated inflammatory disorder of the gut triggered by dietary gluten. Although the effector T‐cell response in patients with celiac disease has been well characterized, the role of regulatory T (Treg) cells in the loss of tolerance to gluten remains poorly understood. Objective: We sought to define whether patients with celiac disease have a dysfunction or lack of gluten‐specific forkhead box protein 3 (FOXP3)+ Treg cells. Methods: Treated patients with celiac disease underwent oral wheat challenge to stimulate recirculation of gluten‐specific T cells. Peripheral blood was collected before and after challenge. To comprehensively measure the gluten‐specific CD4+ T‐cell response, we paired traditional IFN‐&ggr; ELISpot with an assay to detect antigen‐specific CD4+ T cells that does not rely on tetramers, antigen‐stimulated cytokine production, or proliferation but rather on antigen‐induced coexpression of CD25 and OX40 (CD134). Results: Numbers of circulating gluten‐specific Treg cells and effector T cells both increased significantly after oral wheat challenge, peaking at day 6. Surprisingly, we found that approximately 80% of the ex vivo circulating gluten‐specific CD4+ T cells were FOXP3+CD39+ Treg cells, which reside within the pool of memory CD4+CD25+CD127lowCD45RO+ Treg cells. Although we observed normal suppressive function in peripheral polyclonal Treg cells from patients with celiac disease, after a short in vitro expansion, the gluten‐specific FOXP3+CD39+ Treg cells exhibited significantly reduced suppressive function compared with polyclonal Treg cells. Conclusion: This study provides the first estimation of FOXP3+CD39+ Treg cell frequency within circulating gluten‐specific CD4+ T cells after oral gluten challenge of patients with celiac disease. FOXP3+CD39+ Treg cells comprised a major proportion of all circulating gluten‐specific CD4+ T cells but had impaired suppressive function, indicating that Treg cell dysfunction might be a key contributor to disease pathogenesis.

T-cell responses in primary HIV-1 infection.

Purpose of reviewImportant immunological events, especially involving T cells, occur during primary HIV-1 infection. The qualitative nature of the primary immune response to the virus may determine long-term outcome. Whereas CD4 T cells are being rapidly depleted, CD8 T cells play an important role in the initial control of viral replication. There is significant individual variability in the extent of viral control. Understanding the mechanisms underlying these differences and the causes of the development of dysfunctional T-cell responses will allow the identification of opportunities for therapeutic intervention that might change the long-term outcome. Recent findingsRecent studies have revealed early dysfunction of T cells demonstrating increased expression of PD-1, CTLA-4 and reduced expression of CD127. Those studies suggest disruption of the interaction between CD4 and CD8 T cells. In addition, a few regions, mainly within the Gag protein, have been highlighted as potentially important targets for effective immune responses inducing viral control. SummaryDespite recent studies emphasizing the critical nature of acute HIV-1 infection, current intervention strategies have failed to influence disease progression. Recent findings have indicated potential new strategies to re-enable functional properties of T cells and direct these responses towards critical regions of the virus.

The primary immune response to Vaccinia virus vaccination includes cells with a distinct cytotoxic effector CD4 T-cell phenotype

BACKGROUND Smallpox was eradicated by a global program of inoculation with Vaccinia virus (VV). Robust VV-specific CD4 T-cell responses during primary infection are likely essential to controlling VV replication. Although there is increasing interest in cytolytic CD4 T-cells across many viral infections, the importance of these cells during acute VV infection is unclear. METHODS We undertook a detailed functional and genetic characterization of CD4 T-cells during acute VV-infection of humans. VV-specific T-cells were identified by up-regulation of activation markers directly ex vivo and through cytokine and co-stimulatory molecule expression. At day-13-post primary inoculation with VV, CD38highCD45RO+ CD4 T-cells were purified by cell sorting, RNA isolated and analysed by microarray. Differential expression of up-regulated genes in activated CD4 T-cells was confirmed at the mRNA and protein levels. We compared analyses of VV-specific CD4 T-cells to studies on 12 subjects with primary HIV infection (PHI). VV-specific T-cells lines were established from PBMCs collected post vaccination and checked for cytotoxicity potential. RESULTS A median 11.9% CD4 T-cells were CD38highCD45RO+ at day-13 post-VV inoculation, compared to 3.0% prior and 10.4% during PHI. Activated CD4 T-cells had an up-regulation of genes related to cytolytic function, including granzymes K and A, perforin, granulysin, TIA-1, and Rab27a. No difference was seen between CD4 T-cell expression of perforin or TIA-1 to VV and PHI, however granzyme k was more dominant in the VV response. At 25:1 effector to target ratio, two VV-specific T-cell lines exhibited 62% and 30% cytotoxicity respectively and CD107a degranulation. CONCLUSIONS We show for the first time that CD4 CTL are prominent in the early response to VV. Understanding the role of CD4 CTL in the generation of an effective anti-viral memory may help develop more effective vaccines for diseases such as HIV.