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.     

Interaction of a dengue virus NS1‐derived peptide with the inhibitory receptor KIR3DL1 on natural killer cells

Killer immunoglobulin‐like receptors (KIRs) interact with human leucocyte antigen (HLA) class I ligands and play a key role in the regulation and activation of NK cells. The functional importance of KIR–HLA interactions has been demonstrated for a number of chronic viral infections, but to date only a few studies have been performed in the context of acute self‐limited viral infections. During our investigation of CD8⁺ T cell responses to a conserved HLA‐B57‐restricted epitope derived from dengue virus (DENV) non‐structural protein‐1 (NS1), we observed substantial binding of the tetrameric complex to non‐T/non‐B lymphocytes in peripheral blood mononuclear cells (PBMC) from a long‐standing clinical cohort in Thailand. We confirmed binding of the NS1 tetramer to CD56^dim NK cells, which are known to express KIRs. Using depletion studies and KIR‐transfected cell lines, we demonstrated further that the NS1 tetramer bound the inhibitory receptor KIR3DL1. Phenotypical analysis of PBMC from HLA‐B57⁺ subjects with acute DENV infection revealed marked activation of NS1 tetramer‐binding natural killer (NK) cells around the time of defervescence in subjects with severe dengue disease. Collectively, our findings indicate that subsets of NK cells are activated relatively late in the course of acute DENV illness and reveal a possible role for specific KIR–HLA interactions in the modulation of disease outcomes.     

Altered effector functions of virus‐specific and virus cross‐reactive CD8+ T cells in mice immunized with related flaviviruses

Memory cross‐reactive CD8⁺ T‐cell responses may induce protection or immunopathology upon secondary viral challenge. To elucidate the potential role of T cells in sequential flavivirus infection, we characterized cross‐reactive CD4⁺ and CD8⁺ T‐cell responses between attenuated and pathogenic Japanese encephalitis virus (JEV) and pathogenic West Nile virus (WNV). A previously reported WNV NS4b CD8⁺ T‐cell epitope and its JEV variant elicited CD8⁺ T‐cell responses in both JEV‐ and WNV‐infected mice. The peptide variant homologous to the immunizing virus induced greater cytokine secretion and activated higher frequencies of epitope‐specific CD8⁺ T cells. However, there was a virus‐dependent, peptide variant‐independent pattern of cytokine secretion; the IFNγ⁺‐to‐IFNγ⁺TNFα⁺ CD8⁺ T‐cell ratio was greater in JEV‐ than in WNV‐infected mice. Despite similarities in viral burden for pathogenic WNV and JEV viruses, CD8⁺ T cells from pathogenic JEV‐immunized mice exhibited functional and phenotypic profiles similar to those seen for the attenuated JEV strain. Patterns of killer cell lectin‐like receptor G1 (KLRG1) and CD127 expression differed by virus type, with a rapid expansion and contraction of short‐lived effector cells in JEV infection and persistence of high levels of short‐lived effector cells in WNV infection. Such cross‐reactive T‐cell responses to primary infection may affect the outcomes of sequential flavivirus infections.     

Epitope‐specific CD4+, but not CD8+, T‐cell responses induced by recombinant influenza A viruses protect against Mycobacterium tuberculosis infection

Tuberculosis remains a global health problem, in part due to failure of the currently available vaccine, BCG, to protect adults against pulmonary forms of the disease. We explored the impact of pulmonary delivery of recombinant influenza A viruses (rIAVs) on the induction of Mycobacterium tuberculosis (M. tuberculosis)‐specific CD4⁺ and CD8⁺ T‐cell responses and the resultant protection against M. tuberculosis infection in C57BL/6 mice. Intranasal infection with rIAVs expressing a CD4⁺ T‐cell epitope from the Ag85B protein (PR8.p25) or CD8⁺ T‐cell epitope from the TB10.4 protein (PR8.TB10.4) generated strong T‐cell responses to the M. tuberculosis‐specific epitopes in the lung that persisted long after the rIAVs were cleared. Infection with PR8.p25 conferred protection against subsequent M. tuberculosis challenge in the lung, and this was associated with increased levels of poly‐functional CD4⁺ T cells at the time of challenge. By contrast, infection with PR8.TB10.4 did not induce protection despite the presence of IFN‐γ‐producing M. tuberculosis‐specific CD8⁺ T cells in the lung at the time of challenge and during infection. Therefore, the induction of pulmonary M. tuberculosis epitope‐specific CD4⁺, but not CD8⁺ T cells, is essential for protection against acute M. tuberculosis infection in the lung.     

Increasing inflationary T‐cell responses following transient depletion of MCMV‐specific memory T cells

Murine CMV (MCMV) infection induces effector CD8⁺ T cells that continue to increase in frequency after acute infection (“inflation”) and are stably maintained at a high frequency, with up to 20% of the CD8⁺ T‐cell compartment being specific for one epitope, although the flexibility and turnover of these populations is not fully defined. Here we report that effector/memory CD8⁺ T cells induced by MCMV can be paradoxically boosted following transient depletion of epitope specific CD8⁺ T cells. Treatment of MCMV‐infected mice with MHC‐Class I‐saporin tetramers led to partial (80–90%) depletion of epitope‐specific CD8⁺ T cells—rapidly followed by a rebound, leading to expansion and maintenance of up to 40% of total CD8⁺ T cells, with minimal changes in response to a control epitope (M45). These data indicate the tight balance between host and virus during persistent infection and the functional flexibility of the “inflated” CD8⁺ T cell responses during persistent infection.     

Emergence of a distinct HIV‐specific IL‐10‐producing CD8+ T‐cell subset with immunomodulatory functions during chronic HIV‐1 infection

Interleukin‐10 (IL‐10) plays a key role in regulating proinflammatory immune responses to infection but can interfere with pathogen clearance. Although IL‐10 is upregulated throughout HIV‐1 infection in multiple cell subsets, whether this is a viral immune evasion strategy or an appropriate response to immune activation is unresolved. Analysis of IL‐10 production at the single cell level in 51 chronically infected subjects (31 antiretroviral (ART) naïve and 20 ART treated) showed that a subset of CD8⁺ T cells with a CD25^neg FoxP3^neg phenotype contributes substantially to IL‐10 production in response to HIV‐1 gag stimulation. The frequencies of gag‐specific IL‐10‐ and IFN‐γ‐producing T cells in ART‐naïve subjects were strongly correlated and the majority of these IL‐10⁺ CD8⁺ T cells co‐produced IFN‐γ; however, patients with a predominant IL‐10⁺/IFN‐γ^neg profile showed better control of viraemia. Depletion of HIV‐specific CD8⁺ IL‐10⁺ cells from PBMCs led to upregulation of CD38 on CD14⁺ monocytes together with increased IL‐6 production, in response to gag stimulation. Increased CD38 expression was positively correlated with the frequency of the IL‐10⁺ population and was also induced by exposure of monocytes to HIV‐1 in vitro. Production of IL‐10 by HIV‐specific CD8⁺ T cells may represent an adaptive regulatory response to monocyte activation during chronic infection.     

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.     

Epitope specificity of memory CD8+ T cells dictates vaccination-induced mortality in LCMV-infected perforin-deficient mice

Perforin‐deficient (PKO) mice serve as models for familial hemophagocytic lympho‐histiocytosis, a uniformly fatal disease associated with viral infection of perforin‐deficient humans. Naïve perforin‐deficient BALB/c mice survive while vaccinated PKO mice containing virus‐specific memory CD8 ⁺ T cells rapidly succumb to lymphocytic choriomeningitis virus (LCMV) infection. Thus, vaccination converts a nonlethal persistent infection into a fatal disease mediated by virus‐specific memory CD8 ⁺ T cells. Here, we determine the extent to which vaccination‐induced mortality in PKO mice following LCMV challenge is due to differences in vaccine modalities, the quantity or epitope specificity of memory CD8⁺ T cells. We show that LCMV‐induced mortality in immune PKO mice is independent of vaccine modalities and that the starting number of memory CD8 ⁺ T cells specific to the immunodominant epitope NP_118‐126 dictates the magnitude of secondary CD8 ⁺ T‐cell expansion, the inability to regulate production of CD8⁺ T‐cell‐derived IFN‐γ, and mortality in the vaccinated PKO mice. Importantly, mortality is determined by the epitope specificity of memory CD8 ⁺ T cells and the associated degree of functional exhaustion and cytokine dysregulation but not the absolute magnitude of CD8 ⁺ T‐cell expansion. These data suggest that deeper understanding of the parameters that influence the outcome of vaccine‐induced diseases would aid rational vaccine design to minimize adverse outcomes after infection.     

Regulatory T‐cells in acute dengue viral infection

Although regulatory T‐cells (T_regs) have been shown to be expanded in acute dengue, their role in pathogenesis and their relationship to clinical disease severity and extent of viraemia have not been fully evaluated. The frequency of T_regs was assessed in 56 adult patients with acute dengue by determining the proportion of forkhead box protein 3 (FoxP3) expressing CD4⁺ CD25⁺T‐cells (FoxP3⁺ cells). Dengue virus (DENV) viral loads were measured by quantitative real‐time polymerase chain reaction (PCR) and DENV‐specific T‐cell responses were measured by ex‐vivo interferon (IFN)‐γ enzyme‐linked immunospot (ELISPOT) assays to overlapping peptide pools of DENV‐NS3, NS1 and NS5. CD45RA and CCR4 were used to phenotype different subsets of T‐cells and their suppressive potential was assessed by their expression of cytotoxic T lymphocyte‐antigen 4 (CTLA‐4) and Fas. While the frequency of FoxP3⁺ cells in patients was significantly higher (P < 0·0001) when compared to healthy individuals, they did not show any relationship with clinical disease severity or the degree of viraemia. The frequency of FoxP3⁺ cells did not correlate with either ex‐vivo IFN‐γ DENV‐NS3‐, NS5‐ or NS1‐specific T‐cell responses. FoxP3⁺ cells of patients with acute dengue were predominantly CD45RA⁺ FoxP3^low, followed by CD45RA‐FoxP3^low, with only a small proportion of FoxP3⁺ cells being of the highly suppressive effector Treg subtype. Expression of CCR4 was also low in the majority of T‐cells, with only CCR4 only being expressed at high levels in the effector T_reg population. Therefore, although FoxP3⁺ cells are expanded in acute dengue, they predominantly consist of naive T_regs, with poor suppressive capacity.     

CD70 and IFN‐1 selectively induce eomesodermin or T‐bet and synergize to promote CD8+ T‐cell responses

CD70‐mediated stimulation of CD27 is an important cofactor of CD4⁺ T‐cell licensed dendritic cells (DCs). However, it is unclear how CD70‐mediated stimulation of T cells is integrated with signals that emanate from signal 3 pathways, such as type‐1 interferon (IFN‐1) and IL‐12. We find that while stimulation of CD27 in isolation drives weak Eomesodermin^hiT‐bet^lo CD8⁺ T‐cell responses to OVA immunization, profound synergistic expansion is achieved by cotargeting TLR. This cooperativity can substantially boost antiviral CD8⁺ T‐cell responses during acute infection. Concomitant stimulation of TLR significantly increases per cell IFN‐γ production and the proportion of the population with characteristics of short‐lived effector cells, yet also promotes the ability to form long‐lived memory. Notably, while IFN‐1 contributes to the expression of CD70 on DCs, the synergy between CD27 and TLR stimulation is dependent upon IFN‐1's effect directly on CD8⁺ T cells, and is associated with the increased expression of T‐bet in T cells. Surprisingly, we find that IL‐12 fails to synergize with CD27 stimulation to promote CD8⁺ T‐cell expansion, despite its capacity to drive effector CD8⁺ T‐cell differentiation. Together, these data identify complex interactions between signal 3 and costimulatory pathways, and identify opportunities to influence the differentiation of CD8⁺ T‐cell responses.     

High‐frequency vaccine‐induced CD8+ T cells specific for an epitope naturally processed during infection with Mycobacterium tuberculosis do not confer protection

Relatively few MHC class I epitopes have been identified from Mycobacterium tuberculosis, but during the late stage of infection, CD8⁺ T‐cell responses to these epitopes are often primed at an extraordinary high frequency. Although clearly available for recognition during infection, their role in resistance to mycobacterial infections still remain unclear. As an alternative to DNA and viral vaccination platforms, we have exploited a novel CD8⁺ T‐cell‐inducing adjuvant, cationic adjuvant formulation 05 (dimethyldioctadecylammonium/trehalose dibehenate/poly (inositic:cytidylic) acid), to prime high‐frequency CD8 responses to the immunodominant H2‐K^b‐restricted IMYNYPAM epitope contained in the vaccine Ag tuberculosis (TB)10.4/Rv0288/ESX‐H (where ESX is mycobacterial type VII secretion system). We report that the amino acid C‐terminal to this minimal epitope plays a decisive role in proteasomal cleavage and epitope priming. The primary structure of TB10.4 is suboptimal for proteasomal processing of the epitope and amino acid substitutions in the flanking region markedly increased epitope‐specific CD8⁺ T‐cell responses. One of the optimized sequences was contained in the closely related TB10.3/Rv3019c/ESX‐R Ag and when recombinantly expressed and administered in the cationic adjuvant formulation 05 adjuvant, this Ag promoted very high CD8⁺ T‐cell responses. This abundant T‐cell response was functionally active but provided no protection against challenge, suggesting that CD8⁺ T cells play a limited role in protection against M. tuberculosis in the mouse model.     

CD4+ and CD8+ T‐cell immunity to Dengue – lessons for the study of Zika virus

Dengue virus (DENV) and Zika virus (ZIKV) are rapidly emerging mosquito‐borne flaviviruses that represent a public health concern. Understanding host protective immunity to these viruses is critical for the design of optimal vaccines. Over a decade of research has highlighted a significant contribution of the T‐cell response to both protection and/or disease enhancement during DENV infection, the latter being mainly associated with sub‐optimal cross‐reactive T‐cell responses during secondary infections. Phase IIb/III clinical trials of the first licensed tetravalent dengue vaccine highlight increased vaccine efficacy in dengue‐immune as opposed to dengue‐naive vaccinees, suggesting a possible immunoprotective role of pre‐existing DENV‐specific T cells that are boosted upon vaccination. No vaccine is available for ZIKV and little is known about the T‐cell response to this virus. ZIKV and DENV are closely related viruses with a sequence identity ranging from 44% and 56% for the structural proteins capsid and envelope to 68% for the more conserved non‐structural proteins NS3/NS5, which represent the main targets of the CD4⁺ and CD8⁺ T‐cell response to DENV, respectively. In this review we discuss our current knowledge of T‐cell immunity to DENV and what it can teach us for the study of ZIKV. The extent of T‐cell cross‐reactivity towards ZIKV of pre‐existing DENV‐specific memory T cells and its potential impact on protective immunity and/or immunopathology will also be discussed.     

Clonotype‐specific avidity influences the dynamics and hierarchy of virus‐specific regulatory and effector CD4+ T‐cell responses

A key component of immunity against viruses, CD4⁺ T cells expand and differentiate into functional subsets upon primary infection, where effector (Teff) cells facilitate infection control and regulatory (Treg) cells mitigate immunopathology. After secondary infection, Teff cells mount a robust response from the memory pool. Here, we show that Treg‐cell responses are diminished upon secondary infection, and Treg‐cell response dynamics are associated more with T‐cell receptors (TCRs) repertoire and avidity than with epitope specificity. In the murine model, the IA^bM₂₀₉ epitope of respiratory syncytial virus is recognized by both CD4⁺ Treg and Teff cells, while the IA^bM2₂₆ epitope is recognized almost exclusively by CD4⁺ Teff cells expressing high avidity TCR Vβ8.1/8.2 and dominating the CD4⁺ T‐cell response during primary and secondary infections. IA^bM₂₀₉‐Teff cells express relatively low avidity TCRs during early primary infection, but high avidity TCR Vβ7‐expressing IA^bM₂₀₉‐Teff cells emerge during the late phase, and become dominant after secondary infection. The emerging high avidity IA^bM₂₀₉‐Teff cells outcompete IA^bM₂₀₉‐Treg cells that share the same epitope, but have low avidity and are restricted to TCR Vβ2 and Vβ6 subpopulations. These data indicate that MHC‐peptide‐TCR interactions can produce different kinetic and functional profiles in CD4⁺ T‐cell populations even when responding to the same epitope.     

T‐cell help dependence of memory CD8+ T‐cell expansion upon vaccinia virus challenge relies on CD40 signaling

Due to their capacity to differentiate into long‐lived memory cells, CD8⁺ T cells are able to resolve subsequent infections faster than during the primary response. Among other factors, CD4⁺ T cells play a crucial role during primary and secondary CD8⁺ T‐cell responses. However, the timing and mechanisms by which they influence CD8⁺ T cells may differ in primary and secondary responses. Here, we demonstrate that during both primary and secondary vaccinia virus infection, CD4⁺ T cells are necessary to promote CD8⁺ T‐cell responses. While CD4⁺ T cells contributed to memory CD8⁺ T‐cell development, they were even more important during memory recall responses during challenge, as absence of CD4⁺ T cells during challenge resulted in markedly decreased proliferation and increased apoptosis. T‐cell help during primary and secondary responses was mediated via CD40 signaling, with DCs being an integral part of that pathway. As opposed to primary CD8⁺ T‐cell responses where only a combination of agonistic CD40 signaling and provision of IL‐2 could substitute for T‐cell help, agonistic CD40 triggering alone was sufficient to rescue memory CD8⁺ T‐cell responses in absence of T‐cell help in the context of vaccinia virus infection.     

Mycobacterium tuberculosis‐specific CD8+ T cells are functionally and phenotypically different between latent infection and active disease

Protective immunity to Mycobacterium tuberculosis (Mtb) remains poorly understood and the role of Mtb‐specific CD8⁺ T cells is controversial. Here we performed a broad phenotypic and functional characterization of Mtb‐specific CD8⁺ T cells in 326 subjects with latent Mtb infection (LTBI) or active TB disease (TB). Mtb‐specific CD8⁺ T cells were detected in most (60%) TB patients and few (15%) LTBI subjects but were of similar magnitude. Mtb‐specific CD8⁺ T cells in LTBI subjects were mostly T_EMRA cells (CD45RA⁺CCR7^?), coexpressing 2B4 and CD160, and in TB patients were mostly T_EM cells (CD45RA^?CCR7^?), expressing 2B4 but lacking PD‐1 and CD160. The cytokine profile was not significantly different in both groups. Furthermore, Mtb‐specific CD8⁺ T cells expressed low levels of perforin and granulysin but contained granzymes A and B. However, in vitro‐expanded Mtb‐specific CD8⁺ T cells expressed perforin and granulysin. Finally, Mtb‐specific CD8⁺ T‐cell responses were less frequently detected in extrapulmonary TB compared with pulmonary TB patients. Mtb‐specific CD8⁺ T‐cell proliferation was also greater in patients with extrapulmonary compared with pulmonary TB. Thus, the activity of Mtb infection and clinical presentation are associated with distinct profiles of Mtb‐specific CD8⁺ T‐cell responses. These results provide new insights in the interaction between Mtb and the host immune response.     

HIV–TB coinfection impairs CD8+ T‐cell differentiation and function while dehydroepiandrosterone improves cytotoxic antitubercular immune responses

Tuberculosis (TB) is the leading cause of death among HIV‐positive patients. The decreasing frequencies of terminal effector (T_TE) CD8⁺T cells may increase reactivation risk in persons latently infected with Mycobacterium tuberculosis (Mtb). We have previously shown that dehydroepiandrosterone (DHEA) increases the protective antitubercular immune responses in HIV–TB patients. Here, we aimed to study Mtb‐specific cytotoxicity, IFN‐γ secretion, memory status of CD8⁺T cells, and their modulation by DHEA during HIV–TB coinfection. CD8⁺T cells from HIV–TB patients showed a more differentiated phenotype with diminished naïve and higher effector memory and T_TE T‐cell frequencies compared to healthy donors both in total and Mtb‐specific CD8⁺T cells. Notably, CD8⁺T cells from HIV–TB patients displayed higher Terminal Effector (T_TE) CD45RA^dim proportions with lower CD45RA expression levels, suggesting a not fully differentiated phenotype. Also, PD‐1 expression levels on CD8⁺T cells from HIV–TB patients increased although restricted to the CD27⁺ population. Interestingly, DHEA plasma levels positively correlated with T_TE in CD8⁺T cells and in vitro DHEA treatment enhanced Mtb‐specific cytotoxic responses and terminal differentiation in CD8⁺T cells from HIV–TB patients. Our data suggest that HIV–TB coinfection promotes a deficient CD8⁺ T‐cell differentiation, whereas DHEA may contribute to improving antitubercular immunity by enhancing CD8⁺T‐cell functions during HIV–TB coinfection.     

IL‐4 and IL‐15 promotion of virtual memory CD8+ T cells is determined by genetic background

Virtual memory (VM) CD8⁺ T cells are present in unimmunized mice, yet possess T‐cell receptors specific for foreign antigens. To date, VM cells have only been characterized in C57BL/6 mice. Here, we assessed the cytokine requirements for VM cells in C57BL/6 and BALB/c mice. As reported previously, VM cells in C57BL/6 mice rely mostly on IL‐15 and marginally on IL‐4. In stark contrast, VM cells in BALB/c mice rely substantially on IL‐4 and marginally on IL‐15. Further, NKT cells are the likely source of IL‐4, because CD1d‐deficient mice on a BALB/c background have significantly fewer VM cells. Notably, this NKT/IL‐4 axis contributes to appropriate effector and memory T‐cell responses to infection in BALB/c mice, but not in C57BL/6 mice. However, the effects of IL‐4 are manifest prior to, rather than during, infection. Thus, cytokine‐mediated control of the precursor population affects the development of virus‐specific CD8⁺ T‐cell memory. Depending upon the genetic background, different cytokines encountered before infection may influence the subsequent ability to mount primary and memory anti‐viral CD8⁺ T‐cell responses.     

HCV‐specific T cells in HCV/HIV co‐infection show elevated frequencies of dual Tim‐3/PD‐1 expression that correlate with liver disease progression

Co‐infection of HCV with HIV has been associated with more rapid progression of HCV‐related disease. HCV‐specific T‐cell immune responses, which are essential for disease control, are attenuated in co‐infection with HIV. T‐cell exhaustion has recently been implicated in the deficient control of chronic viral infections. In the current study, we investigated the role of programmed death‐1 (PD‐1) and T‐cell immunoglobulin and mucin domain‐containing molecule‐3 (Tim‐3) expression in T‐cell exhaustion during HCV/HIV co‐infection. We show that in HCV/HIV co‐infection, both total and HCV‐specific T cells co‐express Tim‐3 and PD‐1 in significantly higher frequencies, compared with HCV mono‐infection. Co‐expression of these two markers on HCV‐specific CD8⁺ T cells positively correlated with a clinical parameter of liver disease progression. HCV‐specific CD8⁺ T cells showed greater frequencies of Tim‐3/PD‐1 co‐expression than HIV‐specific CD8⁺ T cells, which may indicate a greater degree of exhaustion in the former. Blocking Tim‐3 or PD‐1 pathways restored both HIV‐ and HCV‐specific CD8⁺ T‐cell expansion in the blood of co‐infected individuals. These data demonstrate that co‐expression of Tim‐3 and PD‐1 may play a significant role in HCV‐specific T‐cell dysfunction, especially in the setting of HIV co‐infection.