Extensive major histocompatibility complex class I binding promiscuity for Mycobacterium tuberculosis TB10.4 peptides and immune dominance of human leucocyte antigen (HLA)‐B*0702 and HLA‐B*0801 alleles in TB10.4 CD8+ T‐cell responses

The molecular definition of major histocompatibility complex (MHC) class I‐presented CD8⁺ T‐cell epitopes from clinically relevant Mycobacterium tuberculosis (Mtb) target proteins will aid in the rational design of T‐cell‐based diagnostics of tuberculosis (TB) and the measurement of TB vaccine‐take. We used an epitope discovery system, based on recombinant MHC class I molecules that cover the most frequent Caucasian alleles [human leucocyte antigen (HLA)‐A*0101, A*0201, A*0301, A*1101, A*2402, B*0702, B*0801 and B*1501], to identify MHC class I‐binding peptides from overlapping 9‐mer peptides representing the Mtb protein TB10.4. A total of 33 MHC class I‐binding epitopes were identified, spread across the entire amino acid sequence, with some clustering at the N‐ and C‐termini of the protein. Binding of individual peptides or closely related peptide species to different MHC class I alleles was frequently observed. For instance, the common motif of xIMYNYPAMx bound to six of eight alleles. Affinity (50% effective dose) and off‐rate (half life) analysis of candidate Mtb peptides will help to define the conditions for CD8⁺ T‐cell interaction with their nominal MHC class I‐peptide ligands. Subsequent construction of tetramers allowed us to confirm the recognition of some of the epitopes by CD8⁺ T cells from patients with active pulmonary TB. HLA‐B alleles served as the dominant MHC class I restricting molecules for anti‐Mtb TB10.4‐specific CD8⁺ T‐cell responses measured in CD8⁺ T cells from patients with pulmonary TB.     

A novel T‐cell receptor mimic defines dendritic cells that present an immunodominant West Nile virus epitope in mice

We used a newly generated T‐cell receptor mimic monoclonal antibody (TCRm MAb) that recognizes a known nonself immunodominant peptide epitope from West Nile virus (WNV) NS4B protein to investigate epitope presentation after virus infection in C57BL/6 mice. Previous studies suggested that peptides of different length, either SSVWNATTAI (10‐mer) or SSVWNATTA (9‐mer) in complex with class I MHC antigen H‐2D^b, were immunodominant after WNV infection. Our data establish that both peptides are presented on the cell surface after WNV infection and that CD8⁺ T cells can detect 10‐ and 9‐mer length variants similarly. This result varies from the idea that a given T‐cell receptor (TCR) prefers a single peptide length bound to its cognate class I MHC. In separate WNV infection studies with the TCRm MAb, we show that in vivo the 10‐mer was presented on the surface of uninfected and infected CD8α⁺CD11c⁺ dendritic cells, which suggests the use of direct and cross‐presentation pathways. In contrast, CD11b⁺CD11c^? cells bound the TCRm MAb only when they were infected. Our study demonstrates that TCR recognition of peptides is not limited to certain peptide lengths and that TCRm MAbs can be used to dissect the cell‐type specific mechanisms of antigen presentation in vivo.     

Human herpesvirus 6B immediate‐early I protein contains functional HLA‐A*02, HLA‐A*03, and HLA‐B*07 class I restricted CD8+ T‐cell epitopes

Human herpesvirus 6B (HHV‐6B) is a ubiquitous pathogen with frequent reactivation observed in immunocompromised patients such as BM transplant (BMT) recipients. Adoptive immunotherapy is a promising therapeutic avenue for the treatment of opportunistic infections, including herpesviruses. While T‐cell immunotherapy can successfully control CMV and EBV reactivations in BMT recipients, such therapy is not available for HHV‐6 infections, in part due to a lack of identified protective CD8⁺ T‐cell epitopes. Our goal was to identify CD8⁺ T‐cell viral epitopes derived from the HHV‐6B immediate‐early protein I and presented by common human leukocyte Ag (HLA) class I alleles including HLA‐A*02, HLA‐A*03, and HLA‐B*07. These epitopes were functionally tested for their ability to induce CD8⁺ T‐cell expansion and kill HHV‐6‐infected autologous cells. Cross‐reactivity of specific HHV‐6B‐expanded T cells against HHV‐6A‐infected cells was also confirmed for a conserved epitope presented by HLA‐A*02 molecule. Our findings will help push forward the field of adoptive immunotherapy for the treatment and/or the prevention of HHV‐6 reactivation in BMT recipients.     

Interleukin‐10‐secreting T cells define a suppressive subset within the HIV‐1‐specific T‐cell population

Recent studies have indicated that Treg contribute to the HIV type 1 (HIV‐1)‐related immune pathogenesis. However, it is not clear whether T cells with suppressive properties reside within the HIV‐1‐specific T‐cell population. Here, PBMC from HIV‐1‐infected individuals were stimulated with a 15‐mer Gag peptide pool, and HIV‐1‐specific T cells were enriched by virtue of their secretion of IL‐10 or IFN‐γ using immunomagnetic cell‐sorting. Neither the IL‐10‐secreting cells nor the IFN‐γ‐secreting cells expressed the Treg marker FOXP3, yet the IL‐10‐secreting cells potently suppressed anti‐CD3/CD28‐induced CD4⁺ as well as CD8⁺ T‐cell proliferative responses. As shown by intracellular cytokine staining, IL‐10‐ and IFN‐γ‐producing T cells represent distinct subsets of the HIV‐1‐specific T cells. Our data collectively suggest that functionally defined HIV‐1‐specific T‐cell subsets harbor potent immunoregulatory properties that may contribute to HIV‐1‐associated T‐cell dysfunction.     

High‐affinity human leucocyte antigen class I binding variola‐derived peptides induce CD4+ T cell responses more than 30 years post‐vaccinia virus vaccination

Interferon‐γ secreting T lymphocytes against pox virus‐derived synthetic 9‐mer peptides were tested by enzyme‐linked immunospot in peripheral blood of individuals vaccinated with vaccinia virus more than 30 years ago. The peptides were characterized biochemically as high‐affinity human leucocyte antigen (HLA) class I binders (K_D ≤ 5 nM). However, five of the individuals tested did not show typical CD8⁺ T cell‐mediated HLA class I‐restricted responses. Instead, these donors showed CD4⁺ T cell‐dependent responses against four of a total of eight antigenic 9‐mer peptides discovered recently by our group. These latter responses were blocked specifically in the presence of anti‐HLA class II antibody. We conclude that long‐lived memory responses against pox virus‐derived 9‐mer peptides, with high binding affinity for HLA class I molecules, are mediated in some cases by CD4⁺ T cells and apparently restricted by HLA class II molecules.     

Defining the HLA class I‐associated viral antigen repertoire from HIV‐1‐infected human cells

Recognition and eradication of infected cells by cytotoxic T lymphocytes is a key defense mechanism against intracellular pathogens. High‐throughput definition of HLA class I‐associated immunopeptidomes by mass spectrometry is an increasingly important analytical tool to advance our understanding of the induction of T‐cell responses against pathogens such as HIV‐1. We utilized a liquid chromatography tandem mass spectrometry workflow including de novo‐assisted database searching to define the HLA class I‐associated immunopeptidome of HIV‐1‐infected human cells. We here report for the first time the identification of 75 HIV‐1‐derived peptides bound to HLA class I complexes that were purified directly from HIV‐1‐infected human primary CD4⁺ T cells and the C8166 human T‐cell line. Importantly, one‐third of eluted HIV‐1 peptides had not been previously known to be presented by HLA class I. Over 82% of the identified sequences originated from viral protein regions for which T‐cell responses have previously been reported but for which the precise HLA class I‐binding sequences have not yet been defined. These results validate and expand the current knowledge of virus‐specific antigenic peptide presentation during HIV‐1 infection and provide novel targets for T‐cell vaccine development.     

Dimorphic HLA‐B signal peptides differentially influence HLA‐E‐ and natural killer cell‐mediated cytolysis of HIV‐1‐infected target cells

As a mechanism of self‐protection, signal peptides cleaved from human leukocyte antigen (HLA) class I products bind to HLA‐E before the complex interacts with the natural killer (NK) cell receptor CD94/NKG2A to inhibit NK‐mediated cell lysis. Two types of the signal peptides differ in their position 2 (P2) anchor residue, with P2‐methionine (P2‐M) having higher HLA‐E binding affinity than P2‐threonine (P2‐T). All HLA‐A and HLA‐C molecules carry P2‐M, whereas HLA‐B products have either P2‐M or P2‐T. Epidemiological evidence suggests that P2‐M is unfavourable in the context of HIV‐1 infection, being associated with accelerated acquisition of HIV‐1 infection in two African cohorts. To begin elucidating the functional mechanism, we studied NK‐mediated killing of CD4⁺ T cells and monocyte‐derived macrophages infected with two laboratory‐adapted HIV‐1 strains and two transmitted/founder (T/F) viruses. In the presence of target cells derived from individuals with the three HLA‐B P2 genotypes (M/M, M/T and T/T), NK‐mediated cytolysis was elevated consistently for P2‐T in a dose‐dependent manner for all cell and virus combinations tested (P = 0·008–0·03). Treatment of target cells with an anti‐HLA‐E monoclonal antibody restored NK‐mediated cytolysis of cells expressing P2‐M. Observations on cell lysis were also substantiated by measurements of HIV‐1 p24 antigen in the culture supernatants. Overall, our experiments indicate that the anti‐HIV‐1 function mediated by NK cells is compromised by P2‐M, corroborating the association of HLA‐B genotype encoding P2‐M with accelerated HIV‐1 acquisition.     

Identification of a dengue virus‐specific HLA‐A*0201‐restricted CD8+ T cell epitope

In this study, a combination of epitope‐prediction programs and in vitro assays was used to identify dengue virus (DENV)‐specific CD8⁺ T cell epitopes. Peripheral blood mononuclear cells (PBMCs) isolated from patients who recovered from dengue fever were stimulated with candidate epitope peptides derived from DENV, which were predicted by using SYFPEITHI and RANKpep epitope‐prediction programs. The IFN‐γ ELISpot results and the results of intracellular staining of IFN‐γ showed that peptides NS4b_40 (TLYAVATTI), E_256 (QEGAMHTAL), NS3_205 (LPAIVREAI), NS5_210 (SRNSTHEMY), and NS3_207 (AIVREAIKR) could induce the recall response of CD8⁺ T cells. Furthermore, the results of the MHC–peptide complex stabilization assay revealed that peptide NS4b_40 (TLYAVATTI) has a high affinity for HLA‐A*0201 molecules. The IFN‐γ ELISpot results and staining of intracellular IFN‐γ confirmed that this peptide could induce high‐level CD8⁺ T cell response in HLA‐A*0201 positive PBMCs. Peptide NS4b_40 (TLYAVATTI) was identified as a novel DENV‐specific HLA‐A*0201‐restricted CD8⁺ T cell epitope. J. Med. Virol. 82:642–648, 2010. © 2010 Wiley‐Liss, Inc.     

Analysis of nucleophosmin–anaplastic lymphoma kinase (NPM‐ALK)‐reactive CD8+ T cell responses in children with NPM‐ALK+ anaplastic large cell lymphoma

Cellular immune responses against the oncoantigen anaplastic lymphoma kinase (ALK) in patients with ALK‐positive anaplastic large cell lymphoma (ALCL) have been detected using peptide‐based approaches in individuals preselected for human leucocyte antigen (HLA)‐A*02:01. In this study, we aimed to evaluate nucleophosmin (NPM)‐ALK‐specific CD8⁺ T cell responses in ALCL patients ensuring endogenous peptide processing of ALK antigens and avoiding HLA preselection. We also examined the HLA class I restriction of ALK‐specific CD8⁺ T cells. Autologous dendritic cells (DCs) transfected with in‐vitro‐transcribed RNA (IVT‐RNA) encoding NPM–ALK were used as antigen‐presenting cells for T cell stimulation. Responder T lymphocytes were tested in interferon‐gamma enzyme‐linked immunospot (ELISPOT) assays with NPM–ALK‐transfected autologous DCs as well as CV‐1 in Origin with SV40 genes (COS‐7) cells co‐transfected with genes encoding the patients’ HLA class I alleles and with NPM–ALK encoding cDNA to verify responses and define the HLA restrictions of specific T cell responses. NPM–ALK‐specific CD8⁺ T cell responses were detected in three of five ALK‐positive ALCL patients tested between 1 and 13 years after diagnosis. The three patients had also maintained anti‐ALK antibody responses. No reactivity was detected in samples from five healthy donors. The NPM–ALK‐specific CD8⁺ T cell responses were restricted by HLA‐C‐alleles (C*06:02 and C*12:02) in all three cases. This approach allowed for the detection of NPM–ALK‐reactive T cells, irrespective of the individual HLA status, up to 9 years after ALCL diagnosis.     

The impact of HLA class I and EBV latency‐II antigen‐specific CD8+ T cells on the pathogenesis of EBV+ Hodgkin lymphoma

In 40% of cases of classical Hodgkin lymphoma (cHL), Epstein–Barr virus (EBV) latency‐II antigens [EBV nuclear antigen 1 (EBNA1)/latent membrane protein (LMP)1/LMP2A] are present (EBV⁺cHL) in the malignant cells and antigen presentation is intact. Previous studies have shown consistently that HLA‐A*02 is protective in EBV⁺cHL, yet its role in disease pathogenesis is unknown. To explore the basis for this observation, gene expression was assessed in 33 cHL nodes. Interestingly, CD8 and LMP2A expression were correlated strongly and, for a given LMP2A level, CD8 was elevated markedly in HLA‐A*02^– versus HLA‐A*02⁺ EBV⁺cHL patients, suggesting that LMP2A‐specific CD8⁺ T cell anti‐tumoral immunity may be relatively ineffective in HLA‐A*02^– EBV⁺cHL. To ascertain the impact of HLA class I on EBV latency antigen‐specific immunodominance, we used a stepwise functional T cell approach. In newly diagnosed EBV⁺cHL, the magnitude of ex‐vivo LMP1/2A‐specific CD8⁺ T cell responses was elevated in HLA‐A*02⁺ patients. Furthermore, in a controlled in‐vitro assay, LMP2A‐specific CD8⁺ T cells from healthy HLA‐A*02 heterozygotes expanded to a greater extent with HLA‐A*02‐restricted compared to non‐HLA‐A*02‐restricted cell lines. In an extensive analysis of HLA class I‐restricted immunity, immunodominant EBNA3A/3B/3C‐specific CD8⁺ T cell responses were stimulated by numerous HLA class I molecules, whereas the subdominant LMP1/2A‐specific responses were confined largely to HLA‐A*02. Our results demonstrate that HLA‐A*02 mediates a modest, but none the less stronger, EBV‐specific CD8⁺ T cell response than non‐HLA‐A*02 alleles, an effect confined to EBV latency‐II antigens. Thus, the protective effect of HLA‐A*02 against EBV⁺cHL is not a surrogate association, but reflects the impact of HLA class I on EBV latency‐II antigen‐specific CD8⁺ T cell hierarchies.     

Invariant chain as a vehicle to load antigenic peptides on human MHC class I for cytotoxic T‐cell activation

Protective T‐cell responses depend on efficient presentation of antigen (Ag) in the context of major histocompatibility complex class I (MHCI) and class II (MHCII) molecules. Invariant chain (Ii) serves as a chaperone for MHCII molecules and mediates trafficking to the endosomal pathway. The genetic exchange of the class II‐associated Ii peptide (CLIP) with antigenic peptides has proven efficient for loading of MHCII and activation of specific CD4⁺ T cells. Here, we investigated if Ii could similarly activate human CD8⁺ T cells when used as a vehicle for cytotoxic T‐cell (CTL) epitopes. The results show that wild type Ii, and Ii in which CLIP was replaced by known CTL epitopes from the cancer targets MART‐1 or CD20, coprecipitated with HLA‐A*02:01 and mediated colocalization in the endosomal pathway. Furthermore, HLA‐A*02:01‐positive cells expressing CLIP‐replaced Ii efficiently activated Ag‐specific CD8⁺ T cells in a TAP‐ and proteasome‐independent manner. Finally, dendritic cells transfected with mRNA encoding IiMART‐1 or IiCD20 primed naïve CD8⁺ T cells. The results show that Ii carrying antigenic peptides in the CLIP region can promote efficient presentation of the epitopes to CTLs independently of the classical MHCI peptide loading machinery, facilitating novel vaccination strategies against cancer.     

Specific CD8+ T cells recognize human herpesvirus 6B

The importance of human herpesvirus 6 (HHV‐6) species as human pathogens is increasingly appreciated. However, we do not understand how infection is controlled in healthy virus carriers, and why control fails in patients with disease. Other persistent viruses are under continuous surveillance by antigen‐specific T cells, and specific T‐cell repertoires have been well characterized for some of them. In contrast, knowledge on HHV‐6‐specific T‐cell responses is limited, and missing for CD8⁺ T cells. Here we identify CD8⁺ T‐cell responses to HHV‐6B, the most widespread HHV‐6 species, in healthy virus carriers. HHV‐6B‐specific CD8⁺ T‐cell lines and clones recognized HLA‐A2‐restricted peptides from the viral structural proteins U54 and U11, and displayed various antigen‐specific antiviral effector functions. These CD8⁺ T cells specifically recognized HHV‐6B‐infected primary CD4⁺ T cells in an HLA‐restricted manner, produced antiviral cytokines, and killed infected cells, whereas HHV‐6A‐infected cells were not recognized. Thus, HHV‐6B‐specific CD8⁺ T cells are likely to contribute to control of infection, overcoming the immunomodulatory effects exerted by the virus. Potentially, HHV‐6‐associated disease could be addressed by active or passive immunotherapy that reconstitutes virus‐specific CD8⁺ T‐cell responses.     

High proportion of PD‐1‐expressing CD4+ T cells in adipose tissue constitutes an immunomodulatory microenvironment that may support HIV persistence

We and others have demonstrated that adipose tissue is a reservoir for HIV. Evaluation of the mechanisms responsible for viral persistence may lead to ways of reducing these reservoirs. Here, we evaluated the immune characteristics of adipose tissue in HIV‐infected patients receiving antiretroviral therapy (ART) and in non‐HIV‐infected patients. We notably sought to determine whether adipose tissue's intrinsic properties and/or HIV induced alteration of the tissue environment may favour viral persistence. ART‐controlled HIV infection was associated with a difference in the CD4/CD8 T‐cell ratio and an elevated proportion of Treg cells in subcutaneous adipose tissue. No changes in Th1, Th2 and Th17 cell proportions or activation markers expression on T cell (Ki‐67, HLA‐DR) could be detected, and the percentage of CD69‐expressing resident memory CD4⁺ T cells was not affected. Overall, our results indicate that adipose‐tissue‐resident CD4⁺ T cells are not extensively activated during HIV infection. PD‐1 was expressed by a high proportion of tissue‐resident memory CD4⁺ T cells in both HIV‐infected patients and non‐HIV‐infected patients. Our findings suggest that adipose tissue's intrinsic immunomodulatory properties may limit immune activation and thus may strongly contribute to viral persistence.     

Highly avid,oligoclonal, early‐differentiated antigen‐specific CD8+ T cells in chronic HIV‐2 infection

HIV‐1‐specific CD8⁺ T cells are present in most HIV‐1‐infected people and play an important role in controlling viral replication, but the characteristics of an effective HIV‐specific T‐cell response are largely unknown. The majority of HIV‐2‐infected people behave as long‐term non‐progressors while those who progress to AIDS do so in a manner indistinguishable from HIV‐1. A detailed study of HIV‐2 infection may identify protective immune responses. Robust gag p26‐specific T‐cell responses are elicited during HIV‐2 infection and correlate with control of viremia. In this study, we analyzed features of an HLA‐B^*3501‐restricted T‐cell response to HIV‐2 p26 that may contribute to virus control. In contrast to HIV‐1, HIV‐2‐specific T cells are at an early stage of differentiation (CD27⁺CD28⁺), a finding that relates directly to CD4⁺ T‐cell levels and inversely to immune activation. The cells demonstrate IFN‐γ secretion, oligoclonal T‐cell receptor Vβ gene segment usage, exceptional avidity and secretion of pro‐inflammatory cytokines. Despite the potentially strong selection pressure imposed on the virus by these cells, there was no evidence of HIV‐2 sequence evolution. We propose that in chronic HIV‐2 infection, the maintenance of early‐differentiated, highly avid CD8⁺ T cells could account for the non‐progressive course of disease. Such responses may be desirable from an HIV vaccine.     

Naturally processed HLA‐DR3‐restricted HHV‐6B peptides are recognized broadly with polyfunctional and cytotoxic CD4 T‐cell responses

Human herpes virus 6B (HHV‐6B) is a widespread virus that infects most people early in infancy and establishes a chronic life‐long infection with periodic reactivation. CD4 T cells have been implicated in control of HHV‐6B, but antigenic targets and functional characteristics of the CD4 T‐cell response are poorly understood. We identified 25 naturally processed MHC‐II peptides, derived from six different HHV‐6B proteins, and showed that they were recognized by CD4 T‐cell responses in HLA‐matched donors. The peptides were identified by mass spectrometry after elution from HLA‐DR molecules isolated from HHV‐6B‐infected T cells. The peptides showed strong binding to matched HLA alleles and elicited recall T‐cell responses in vitro. T‐cell lines expanded in vitro were used for functional characterization of the response. Responding cells were mainly CD3⁺CD4⁺, produced IFN‐γ, TNF‐α, and low levels of IL‐2, alone or in combination, highlighting the presence of polyfunctional T cells in the overall response. Many of the responding cells mobilized CD107a, stored granzyme B, and mediated specific killing of peptide‐pulsed target cells. These results highlight a potential role for polyfunctional cytotoxic CD4 T cells in the long‐term control of HHV‐6B infection.     

Distinct activation phenotype of a highly conserved novel HLA‐B57‐restricted epitope during dengue virus infection

Variation in the sequence of T‐cell epitopes between dengue virus (DENV) serotypes is believed to alter memory T‐cell responses during second heterologous infections. We identified a highly conserved, novel, HLA‐B57‐restricted epitope on the DENV NS1 protein. We predicted higher frequencies of B57‐NS1_26–34‐specific CD8⁺ T cells in peripheral blood mononuclear cells from individuals undergoing secondary rather than primary DENV infection. However, high tetramer‐positive T‐cell frequencies during acute infection were seen in only one of nine subjects with secondary infection. B57‐NS1_26–34‐specific and other DENV epitope‐specific CD8⁺ T cells, as well as total CD8⁺ T cells, expressed an activated phenotype (CD69⁺ and/or CD38⁺) during acute infection. In contrast, expression of CD71 was largely limited to DENV epitope‐specific CD8⁺ T cells. In vitro stimulation of cell lines indicated that CD71 expression was differentially sensitive to stimulation by homologous and heterologous variant peptides. CD71 may represent a useful marker of antigen‐specific T‐cell activation.     

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 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.     

Co‐expression of HLA‐B7 and HLA‐B27 alleles is associated with B7‐restricted immunodominant responses following influenza infection

It is recognized that host response following viral infection is characterized by immunodominance, but deciphering the different factors contributing to immunodominance has proved a challenge due to concurrent expression of multiple MHC class I alleles. To address this, we generated H2‐K^?/?/D^?/? double‐knockout transgenic mice expressing either one or two human MHC‐I alleles. We hypothesized that co‐expression of different allele combinations figures critically in immunodominance and examined this in influenza‐infected, double Tg MHC‐I mice. In A2/B7 or A2/B27 mice, using ELISpot assays with the A2‐restricted matrix I.58–66, the B7‐restricted NP418–426 or the B27‐restricted NP383–391 influenza A (flu) epitopes, we observed the expected recognition of both peptides for both alleles. In contrast, in flu‐infected B7/B27 mice, a significantly reduced level of B27/NP383‐restricted CTL response was detected while there was no change in the B7/NP418‐restricted CTL response. Flu‐specific tetramer studies revealed a partial deletion of Vβ8.1⁺ NP383/B27‐restricted CD8⁺ T cells, and a diminished Vβ12⁺ CD8⁺ T‐cell expansion in B7/B27 Tg mice. Using HLA Tg chimeric mice, we confirmed these findings. These findings shed light on the immune consequences of co‐dominant expression of MHC‐I alleles for host immune response to pathogens.     

Inhibition of R5‐tropic HIV type‐1 replication in CD4+ natural killer T cells by γδ T lymphocytes

After the development of highly active anti‐retroviral therapy, it became clear that the majority of emergent HIV‐1 is macrophage‐tropic and infects CD4⁺, CCR5‐expressing cells (R5‐tropic). There are three distinct cell populations, R5‐tropic, HIV‐1‐susceptible CD4⁺ cells: (i) natural killer T (NKT) cells, (ii) dendritic cells and macrophages, and (iii) tissue‐associated T cells residing primarily at mucosal surfaces. We have confirmed that CD4⁺ NKT cells derived from peripheral blood mononuclear cells (PBMCs) predominantly express CCR5 rather than CXCR4, whereas the reverse is true for CD4⁺ T cells derived from circulating PBMCs, and that R5‐tropic HIV‐1 expands efficiently in the CD4⁺ NKT cells. Moreover, when PBMCs depleted of CD8α⁺ cells were stimulated in the presence of α‐galactosylceramide (α‐GalCer) and R5‐tropic HIV‐1 [NL(AD8)], the production of HIV‐1 virions was not suppressed, whereas, similar to the untreated PBMCs, depletion of CD8β⁺ cells from PBMCs significantly inhibited virion production. These findings suggest that CD8αα⁺ but not CD8αβ⁺ cells may have the ability to inhibit R5‐tropic HIV‐1 replication in CD4⁺ NKT cells. Here, we show that co‐culturing R5‐tropic HIV‐1‐infected CD4⁺ NKT cells with CD8αα⁺ γδ T cells, in particular Vγ1Vδ1 cells, but not with CD8αα⁺ NKT cells or CD8αα⁺ dendritic cells, inhibits HIV‐1 replication mainly by secreting chemokines, such as macrophage inflammatory proteins 1α and 1β and RANTES. Collectively, these results indicate the importance of CD8αα⁺ γδ T cells in the control of R5‐tropic HIV‐1 replication and persistence in CD4⁺ NKT cells.