Involvement of CD91 and scavenger receptors in Hsp70‐facilitated activation of human antigen‐specific CD4+ memory T cells

Hsp70 plays several roles in the adaptive immune response. Based on the ability to interact with diverse peptides, extracellular Hsp70:peptide complexes exert profound effects both in autoimmunity and in tumor rejection by evoking potent T cell responses to the chaperoned peptide. The interaction with receptors on APC represents the basis for the immunological functions of Hsp70 and a critical point where the immune response can be regulated. Various surface proteins (e.g. CD91, scavenger receptors (SR)) have been implicated in binding of Hsp70. In this study, antigenic peptides from tetanus toxin and influenza hemagglutinin complexed to human stress‐inducible Hsp70 were found to enhance the proliferation and cytokine production of human antigen‐specific CD4⁺ T cells. This was demonstrated in proliferation experiments using human monocytes as APC. Proliferated antigen‐specific cells were detected combining HLA‐DRB1^*0401 or HLA‐DRB1^*1101 tetramer and CFSE staining. Treating monocytes with CD91 siRNA diminished these effects. Additional blocking of SR by the SR ligand fucoidan completely abolished enhanced proliferation and production of Th1 and Th2 cytokines. Taken together, our data indicate that in the human system, CD91 and members of the SR family efficiently direct Hsp70:peptide complexes into the MHC class II presentation pathway and thus enhance antigen‐specific CD4⁺ T cell responses.     

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.     

T‐cell receptors: Tugging on the anchor for a tighter hold on the tumor‐associated peptide

Although it has been shown that human tumor‐associated, HLA anchor residue modified “heteroclitic” peptides may induce stronger immune responses than wild‐type peptides in cancer vaccine trials, it has also been shown that some T cells primed with these heteroclitic peptides subsequently fail to recognize the natural, tumor‐expressed peptide efficiently. This may provide a molecular reason for why clinical trials of these peptides have been thus far unsuccessful. In this issue of the European Journal of Immunology, Madura et al. [Eur. J. Immunol. 2015. 45: 584–591] highlight a novel twist on T‐cell receptor (TCR) recognition of HLA–peptide complexes. Tumor‐associated peptides often lack canonical anchor residues, which can be substituted for the optimal residue to improve their antigenicity. T‐cell cross‐reactivity between the natural and modified (heteroclitic) peptides is essential for this approach to work and depends on whether the anchor residue substitution influences peptide conformation. The Melan‐A/MART‐1_26‐35 peptide epitope is an example where T cells can make this distinction, with the natural peptide stimulating higher affinity CD8⁺ T cells than the heteroclitic peptide, despite the heteroclitic peptide's more stable association with HLA‐A2. The molecular basis for peptide discrimination is identified through the structure of the TCR bound to the natural peptide; TCR engagement of the natural peptide “lifts” its amino‐terminus partly away from the HLA peptide binding groove, forming a higher affinity interface with the TCR than is formed with the anchor residue “optimized” heteroclitic peptide, which cannot be “pulled” from the HLA groove.     

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.     

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.     

Allergen‐specific naïve and memory CD4+ T cells exhibit functional and phenotypic differences between individuals with or without allergy

Although allergen‐specific CD4⁺ T cells are detectable in the peripheral blood of both individuals with or without allergy, their frequencies and phenotypes within the memory as well as naïve repertoires are incompletely known. Here, we analyzed the DRB1*0401‐restricted responses of peripheral blood‐derived memory (CD4⁺CD45RO⁺) and naïve (CD4⁺CD45RA⁺) T cells from subjects with or without allergy against the immunodominant epitope of the major cow dander allergen Bos d 2 by HLA class II tetramers in vitro. The frequency of Bos d 2_127–142‐specific memory T cells in the peripheral blood‐derived cultures appeared to be higher in subjects with allergy than those without, whereas naïve Bos d 2_127–142‐specific T cells were detectable in the cultures of both groups at nearly the same frequency. Surprisingly, the TCR avidity of Bos d 2_127–142‐specific T cells of naïve origin, as assessed by the intensity of HLA class II tetramer staining, was found to be higher in individuals with allergy. Upon restimulation, long‐term Bos d 2_127–142‐specific T‐cell lines generated from both memory and naïve T‐cell pools from individuals with allergy proliferated more strongly, produced more IL‐4 and IL‐10, and expressed higher levels of CD25 but lower levels of CXCR3 than the T‐cell lines from individuals without allergy, demonstrating differences also at the functional level. Collectively, our current results suggest that not only the memory but also the naïve allergen‐specific T‐cell repertoires differ between individuals with or without allergy.     

Direct cloning and tetramer staining to measure the frequency of intestinal gluten‐reactive T cells in celiac disease

Knowledge of the frequency of disease‐driving CD4⁺ T cells in lesions of chronic human inflammatory diseases is limited. In celiac disease (CD), intestinal gluten‐reactive CD4⁺ T cells, which recognize gluten peptides only in the context of the disease‐associated HLA‐DQ molecules, are key pathogenic players. By cloning CD4⁺ T cells directly from intestinal biopsies of CD patients, we found that 0.5–1.8% of CD4⁺ T cells were gluten reactive. About half of the gluten‐reactive T cells were specific for either the immuno‐dominant DQ2.5‐glia‐α1a or DQ2.5‐glia‐α2 epitopes, suggesting that direct visualization of gluten‐specific T cells could be possible. Assessed by flow cytometry, tetramer‐positive T cells were present in 10/10 untreated CD patients with a frequency of 0.1–1.2% of CD4⁺ T cells. Gluten‐specific T cells were also detectable in most treated CD patients (7/10). Moreover, the frequency of gluten‐specific T cells correlated with the degree of histological damage in the gut mucosa as scored by Marsh‐grading, and also with serum IgA anti‐transglutaminase 2 antibody levels. Tetramer staining of gluten‐reactive T cells in biopsy material is a useful tool for future studies of such cells in CD and could also potentially serve as a diagnostic supplement in selected cases.     

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.     

Characterization of the human CD4+ T‐cell repertoire specific for major histocompatibility class I‐restricted antigens

While CD4⁺ T lymphocytes usually recognize antigens in the context of major histocompatibility (MHC) class II alleles, occurrence of MHC class‐I restricted CD4⁺ T cells has been reported sporadically. Taking advantage of a highly sensitive MHC tetramer‐based enrichment approach allowing detection and isolation of scarce Ag‐specific T cells, we performed a systematic comparative analysis of HLA‐A*0201‐restricted CD4⁺ and CD8⁺ T‐cell lines directed against several immunodominant viral or tumoral antigens. CD4⁺ T cells directed against every peptide‐MHC class I complexes tested were detected in all donors. These cells yielded strong cytotoxic and T helper 1 cytokine responses when incubated with HLA‐A2⁺ target cells carrying the relevant epitopes. HLA‐A2‐restricted CD4⁺ T cells were seldom expanded in immune HLA‐A2⁺ donors, suggesting that they are not usually engaged in in vivo immune responses against the corresponding peptide‐MHC class I complexes. However, these T cells expressed TCR of very high affinity and were expanded following ex vivo stimulation by relevant tumor cells. Therefore, we describe a versatile and efficient strategy for generation of MHC class‐I restricted T helper cells and high affinity TCR that could be used for adoptive T‐cell transfer‐ or TCR gene transfer‐based immunotherapies.     

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.     

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.     

Novel HIV‐1 clade B candidate vaccines designed for HLA‐B*5101+ patients protected mice against chimaeric ecotropic HIV‐1 challenge

Novel candidate HIV‐1 vaccines have been constructed, which are tailor‐designed for HLA‐B^*5101⁺ patients infected with HIV‐1 clade B. These vaccines employ novel immunogen HIVB‐B^*5101 derived from consensus HIV‐1 clade B Gag p17 and p24 regions coupled to two Pol‐derived B^*5101‐restricted epitopes, which are together with a third B^*5101 epitope in Gag dominant in HIV‐1‐infected long‐term non‐progressing patients. Both plasmid DNA and modified vaccinia virus Ankara (MVA) vectors supported high expression levels of the HIVB‐B^*5101 immunogen in cultured cells. Heterologous DNA prime‐recombinant MVA boost regimen induced efficiently HIV‐1‐specific CD8⁺ T‐cell responses in BALB/c mice. These vaccine‐elicited T cells were multifunctional, killed efficiently target cells in vivo, and protected mice against challenge with ecotropic HIV‐1/NL4‐3 and ecotropic HIV‐1/NDK chimaeric viruses with HIV‐1 clade B or D backbones, respectively, and ecotropic murine leukemia virus gp80 envelope, and therefore did so in the absence of anti‐HIV‐1 gp120 antibodies. These results support further development of HIVB‐B^*5101 vaccines in combined heterologous‐modality regimens. The use of allele‐specific vaccines in humans is discussed in the context of other developments in the HIV‐1 field.     

Functional islet‐specific Treg can be generated from CD4+CD25− T cells of healthy and type 1 diabetic subjects

CD4⁺CD25⁺FOXP3⁺ Treg cells require TCR engagement for suppressive function, thus ensuring that suppression occurs only in the presence of specific antigens; however, to date no studies have addressed the function of self‐antigen‐specific Treg in humans. These studies were designed to determine whether peripheral generation and function of islet antigen‐specific adaptive Treg are defective in human subjects with type 1 diabetes (T1D). Islet antigen‐specific adaptive Treg were induced in vitro by activation of CD4⁺FOXP3^? T cells with glutamic acid decarboxylase and islet‐specific glucose‐6‐phosphate catalytic subunit‐related protein peptides in the context of T1D‐associated HLA‐DRβ alleles. Antigen‐specific Treg were characterized using flow cytometry for FOXP3 and class II tetramer and assessed for the ability to inhibit proliferation. These adaptive Treg were then compared with influenza‐specific Treg from the same study population. The function of tetramer⁺ cells that expressed FOXP3 was similar for both influenza and islet antigens generated from control and T1D subjects. In fact, the potency of suppression correlated with FOXP3 expression, not antigen specificity. Thus, these data suggest that development of functional adaptive Treg can occur in response to islet antigens and activation of islet‐specific Treg may potentially be used as a targeted immunotherapy in T1D.     

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.     

Differential CD4+ T‐cell responses of allergic and non‐allergic subjects to the immunodominant epitope region of the horse major allergen Equ c 1

The responses of allergen‐specific CD4⁺ T cells of allergic and healthy individuals are still incompletely understood. Our objective was to investigate the functional and phenotypic properties of CD4⁺ T cells of horse‐allergic and healthy subjects specific to the immunodominant epitope region of the major horse allergen Equ c 1. Specific T‐cell lines (TCLs) and clones were generated from peripheral blood mononuclear cells with Equ c 1_143–160, the peptide containing the immunodominant epitope region of Equ c 1. The frequency, proliferative response, cytokine production and HLA restriction of the cells were examined. The frequency of Equ c 1‐specific CD4⁺ T cells was low (approximately 1 per 10⁶ CD4⁺ T cells) in both allergic and non‐allergic subjects. The cells of allergic subjects had a stronger proliferative capacity than those of non‐allergic subjects, and they predominantly emerged from the memory T‐cell pool and expressed the T helper type 2 cytokine profile, whereas the cells of non‐allergic subjects emerged from the naive T‐cell pool and produced low levels of interferon‐γ and interleukin‐10. T‐cell response to Equ c 1_143–160 was restricted by several common HLA class II molecules from both DQ and DR loci. As the phenotypic and functional properties of Equ c 1‐specific CD4⁺ T cells differ between allergic and non‐allergic subjects, allergen‐specific T cells appear to be tightly implicated in the development of diseased or healthy outcome. Restriction of the specific CD4⁺ T‐cell response by multiple HLA alleles suggests that Equ c 1_143–160 is a promising candidate for peptide‐based immunotherapy.     

Heterologous vaccination against human tuberculosis modulates antigen‐specific CD4+ T‐cell function

Heterologous prime‐boost strategies hold promise for vaccination against tuberculosis. However, the T‐cell characteristics required for protection are not known. We proposed that boost vaccines should induce long‐lived functional and phenotypic changes to T cells primed by Bacille Calmette Guerin (BCG) and/or natural exposure to mycobacteria. We characterized changes among specific CD4⁺ T cells after vaccination with the MVA85A vaccine in adults, adolescents, and children. CD4⁺ T cells identified with Ag85A peptide‐bearing HLA class II tetramers were characterized by flow cytometry. We also measured proliferative potential and cytokine expression of Ag85A‐specific CD4⁺ T cells. During the effector phase, MVA85A‐induced specific CD4⁺ T cells coexpressed IFN‐γ and IL‐2, skin homing integrins, and the activation marker CD38. This was followed by contraction and a transition to predominantly IL‐2‐expressing, CD45RA^?CCR7⁺CD27⁺ or CD45RA⁺CCR7⁺CD27⁺ specific CD4⁺ T cells. These surface phenotypes were similar to Ag85A‐specific T cells prior to MVA85A. However, functional differences were observed postvaccination: specific proliferative capacity was markedly higher after 6–12 months than before vaccination. Our data suggest that MVA85A vaccination may modulate Ag85A‐specific CD4⁺ T‐cell function, resulting in greater recall potential. Importantly, surface phenotypes commonly used as proxies for memory T‐cell function did not associate with functional effects of vaccination.     

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.