Influenza A antigen exposure selects dominant Vbeta17+ TCR in human CD8+ cytotoxic T cell responses.

During acute human viral infections, such as influenza A, specific cytotoxic T lymphocytes (CTL) are generated which aid virus clearance. We have observed that in HLA-A*0201+ subjects, CTL expressing Vbeta17+ TCR and recognizing a peptide from the influenza A matrix protein (M1(58-66)) dominate this response. In experimental models of infection such dominance can be due to inheritance of a restricted T cell repertoire or acquired consequent on expansion of CTL bearing an optimum TCR conformation against the MHC-peptide complex. To examine how influenza A infection might influence the development of TCR Vbeta17 expansion, we studied influenza A-specific CTL in a cross-sectional study of 82 HLA-A*0201+ individuals from birth (cord blood) to adulthood. Primary M1(58-66) -specific CTL were detected in cord blood, but their TCR were diverse and depletion of Vbeta17+ cells did not abrogate specific cytotoxicity. In contrast following natural influenza A infection, TCR Vbeta17+ CTL dominated to the extent that only one of nine adult CTL lines retained any functional activity after in vitro depletion of Vbeta17+ CTL. These results suggest that the dominance of Vbeta17+ TCR among adult M1(58-66)-specific CTL results from maturation and focussing of the response driven by exposure to influenza, and have implications for optimum immunization strategies.     

Chimeric immune receptors (CIRs) specific to JC virus for immunotherapy in progressive multifocal leukoencephalopathy (PML)

Progressive multifocal leukoencephalopathy (PML) is a deadly brain disease caused by the polyomavirus JC (JCV). The aim of this study is to develop 'designer T cells' armed with anti-JCV TCR-based chimeric immune receptors (CIRs) by gene modification for PML immunotherapy. Two T cell lines specific to two dominant CTL epitopes derived from JCV VP1 protein (termed p36 and p100) from an HLA-A0201+ PML survivor were generated for TCR cloning. Two distinct dominant TCR alpha chains (Valpha6 and Valpha12) and a unique TCR beta chain (Vbeta5.1) were cloned from the p36-specific cell line, while only one alpha (Valpha8.6) and one beta (Vbeta2) chains were dominant in the p100-specific line. Retroviral constructs encoding CIRs were created with the extracellular domains of TCR alpha and beta chains fused to the transmembrane and cytoplasmic portions of CD3zeta (ValphaCalphaCD3zeta or VbetaCbetaCD3zeta). Cellular expression and screening for binding specific peptide-HLA-A0201 tetramer confirmed the reactivity of the p100 TCRalphabeta and of one of the two pairs of p36 TCRalphabeta (Valpha12 and Vbeta5.1). Functional tests confirmed CIR-expressing T cells secreted cytokines and expressed potent cytotoxicity on contact with A0201+ B-lymphoblastoid line loaded with peptides and/or with HLA-A0201+ cells expressing native JCV VP1 protein. In conclusion, anti-JCV designer T cells were generated.     

Programmed death-1 (PD-1)/PD-1 ligand pathway-mediated immune responses against human T-lymphotropic virus type 1 (HTLV-1) in HTLV-1-associated myelopathy/tropical spastic paraparesis and carriers with autoimmune disorders

Human T-lymphotropic virus-1 (HTLV-1) causes HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia-lymphoma in individuals with dysfunctional immune responses. In this study, to characterize the HTLV-1-specific cytotoxic T lymphocyte (CTL) populations in asymptomatic HTLV-1 carriers (ACs), HAM/TSP patients, and carriers with autoimmune disorders (CAIDs), we examined the role of programmed death-1 and its ligand (PD-1/PD-L1) in HTLV-1-specific CTL functions using an HTLV-1 Tax/HLA-A*0201 tetramer and an HTLV-1 Tax/HLA-A*2402 tetramer. Interestingly, the percentage of HTLV-1 Tax301-309/HLA-A*2402 tetramer(+)CD8(+) cells expressing PD-1 in ACs was significantly higher than the percentage of HTLV-1 Tax11-19/HLA-A*0201 tetramer(+)CD8(+) cells expressing PD-1. PD-1 expression was significantly downregulated on HTLV-1-specific CTLs in HAM/TSP compared with ACs. PD-L1 expression was observed in a small proportion of unstimulated lymphocytes from ACs and was greater in ACs than in HAM/TSP and CAIDs after short-term culture. Furthermore, CTL degranulation was impaired in HAM/TSP, whereas anti-PD-L1 blockade significantly increased CTL function in ACs. Downregulation of PD-1 on HTLV-1-specific CTLs and loss of PD-L1 expression in HAM/TSP and CAIDs, along with impaired function of HTLV-1-specific CTLs in HAM/TSP, may underlie the apparently dysfunctional immune response against HTLV-1.     

Functional profile of human influenza virus-specific cytotoxic T lymphocyte activity is influenced by interleukin-2 concentration and epitope specificity

The ability of influenza A virus-specific cytotoxic T lymphocytes (CTL) to degranulate and produce cytokines upon antigenic restimulation was studied in four HLA-A*0101 and HLA-A*0201 positive subjects. Peripheral blood mononuclear cells of these subjects were stimulated with influenza A virus in the presence of high or low interleukin (IL)-2 concentrations. CD8(+) T cell populations specific for the HLA-A*0101 restricted epitope NP(44-52) and the HLA-A*0201 restricted epitope M1(58-66) were identified by positive staining with tetramers of peptide major histocompatibility complexes (MHC) (NP-Tm and M1-Tm, respectively). Within these populations, the proportion of cells mobilizing CD107a, or expressing interferon (IFN)-gamma and tumour necrosis factor-(TNF)-alpha upon short-term peptide restimulation was determined by flow cytometry. Independent of IL-2 concentrations, large subject-dependent differences in the mobilization of CD107a and expression of IFN-gamma and TNF-alpha by both NP- and M1-specific T cells were observed. In two of the four subjects, the functional profile of NP-Tm(+) and M1-Tm(+) cells differed considerably. Overall, no difference in the proportion of NP-Tm(+) or M1-Tm(+) cells expressing CD107a was observed. The proportion of M1-Tm(+) cells that produced IFN-gamma (P < 0.05) was larger than for NP-Tm(+) cells, independent of IL-2 concentration. When cultured under IL-2(hi) concentrations higher TNF-alpha expression was also observed in M1-Tm(+) cells (P < 0.05). The IL-2 concentration during expansion of virus-specific cells had a profound effect on the functionality of both M1-Tm(+) and NP-Tm(+) cells.     

The structural dynamics and energetics of an immunodominant T cell receptor are programmed by its Vbeta domain

Immunodominant and public T cell receptor (TCR) usage is relatively common in many viral diseases yet surprising in the context of the large naive TCR repertoire. We examined the highly conserved Vbeta17:Valpha10.2 JM22 T cell response to the influenza matrix peptide (58-66)-HLA-A*0201 (HLA-A2-flu) through extensive kinetic, thermodynamic, and structural analyses. We found several conformational adjustments that accompany JM22-HLA-A2-flu binding and identified a binding "hotspot" within the Vbeta domain of the TCR. Within this hotspot, key germline-encoded CDR1 and CDR2 loop residues and a crucial but commonly coded residue in the hypervariable region of CDR3 provide the basis for the substantial bias in the selection of the germline-encoded Vbeta17 domain. The chances of having a substantial number of T cells in the naive repertoire that have HLA-A2-flu-specific Vbeta17 receptors may consequently be relatively high, thus explaining the immunodominant usage of this clonotype.     

Cytotoxic T cell recognition of allelic variants of HLA B35 bound to an Epstein-Barr virus epitope: influence of peptide conformation and TCR-peptide interaction.

Fine specificity analysis of HLA B35-restricted Epstein-Barr virus (EBV)-specific cytotoxic T lymphocyte (CTL) clones revealed a unique heterogeneity whereby one group of these clones cross-recognized an EBV epitope (YPLHEQHGM) on virus-infected cells expressing either HLA B*3501 or HLA B*3503, while another group cross-recognized this epitope in association with either HLA B*3502 or HLA B*3503. Peptide binding and titration studies ruled out the possibility that these differences were due to variation in the efficiency of peptide presentation by the HLA B35 alleles. Sequence analysis of the TCR genetic elements showed that these clonotypes either expressed BV12/AV3 or BV14/ADV17S1 heterodimers. Interestingly, CTL analysis with monosubstituted alanine mutants of the YPLHEQHGM epitope indicated that the BV12/AV3+ clones preferentially recognized residues towards the C terminus of the peptide, while the BV14/ADV17S1+ clones interacted with residues towards N terminus of the peptide. Molecular modelling of the MHC-peptide complexes suggests that the differences in two floor positions (114 and 116) of the HLA B35 alleles dictate different conformations of the peptide residues L3 and/or H7 and directly contribute in the discerning allele-specific immune recognition by the CTL clonotypes. These results provide evidence for a critical role for the selective interaction of the TCR with specific residues within the peptide epitope in the fine specificity of CTL recognition of allelic variants of an HLA molecule.     

骨髓瘤抗原Sp17 HLA-A*0201限制性CTL表位预测及初步鉴定

目的鉴定骨髓瘤抗原Sp17的HLA-A＊0201限制性CTL表位。方法采用超基序和量化基序法预测Sp17的HLA-A＊0201限制性CTL表位；用T2细胞亲和力实验和稳定性实验对该表位进行初步鉴定。结果超基序和量化基序法预测得出Sp17抗原的4个CTL表位（LLEGLTREI（19-27）、ILREQPDNI（27-35）、SLLEKREKT（45-53）、KEKEEVAAV（111-119）），亲和力实验结果表明LLEGLTREI（19-27）、ILREQPDNI（27-35）、SLLEKREKT（45-53）有较高亲和力，而KEKEEVAAV（111-119）肽亲和力较低；稳定性实验表明ILREQPDNI（27-35）、SLLEKREKT（45-53）与HLA-A＊0201分子结合稳定性较好。结论ILREQPDNI（27-35）、SLLEKREKT（45-53）有可能是骨髓瘤抗原Sa17 HLA-A＊0201限制件CTL表位。     

The effect of epitope variation on the profile of cytotoxic T lymphocyte responses to the HIV envelope glycoprotein [In Process Citation]

To address the relationship between viral and host factors during HIV infection, we analyzed the effect of viral mutations on T cell responses in seropositive, asymptomatic HLA-A2+ individuals using four envelope (env)-specific peptides with the HLA-A*0201 binding motif. We showed that the natural sequence variation was frequent within epitopes located in the C-terminal region of the env glycoprotein and was largely responsible for a lower env-specific cytotoxic T lymphocyte (CTL) activity in the peptide-stimulated cultures. The highest CTL responses in vitro were induced with conserved epitopes D1 and 4.3 that mapped to the N-terminal region of the env glycoprotein. These peptides exhibited high binding affinity for HLA-A*0201 molecules and stimulated CD8+ T cells of relatively limited TCR Vbeta chain repertoire. Decreased CTL activities to the D1 epitope were observed in the absence of any detectable viral mutation, and were associated with lower proliferative responses and expression of the CD28 antigen. Results of this study demonstrate that the degree of sequence variation within a stimulatory epitope of the viral quasispecies, as well as proliferative potential of the effector cells, are among the factors underlying decreased CTL activity in HIV-infected patients. These experiments also provide evidence that the D1 peptide might be useful for the development of vaccines and immune-based therapy.      

Cytomegalovirus immune reconstitution occurs in recipients of allogeneic hematopoietic cell transplants irrespective of detectable cytomegalovirus infection.

The question of when immune reconstitution of cytomegalovirus (CMV)-specific CD8 T cells occurs after hematopoietic cell transplantation and, more specifically, to which CMV targets this immunity is likely to be directed remains poorly understood. The dependence of immune reconstitution on CMV reactivation is even less clear. To better understand these events, 44 CMV-seropositive HLA-A*0201 subjects were followed up at approximately days 40, 90, 120, 150, 180, and 360 after hematopoietic cell transplantation for CMV immunity as measured by 2 types of assays: (1) an HLA-A*0201 tetramer-binding assay for both CMV pp65 (pp65) and immediate-early 1 (IE-1) or (2) intracellular cytokine interferon gamma responses induced by pp65 or IE-1-derived peptides. To verify the reliability of IE-1-specific assays relative to the pp65-based assays, a pilot study first compared the development of IE-1-specific immunity in a subgroup by using multiple HLA-A*0201-restricted peptides, and then these recipients were followed up for 1 year for immunologic function and for CMV infection. The IE-1-specific response occurred to each of the 3 HLA-A*0201-restricted peptides studied (IE-1-256, -297, and -316), and there was no predominant IE peptide response. However, the immunodominant HLA-A*0201-restricted pp65 peptide was recognized significantly more frequently than these IE-1 peptides. When this was compared with the occurrence of CMV infection, the overall immune reactivity, as measured by the mean or median number of CD8+ T cells reactive to either pp65 or IE-1 peptides by intracellular cytokine or tetramer binding assay, was not significantly different in those with and without CMV infection. For patients who demonstrated reconstituted immunity to CMV at 1 year, all were reconstituted by 6 months, and the timing of the first observed immune reactivity to either of the pp65 or the IE peptides was not different in those with and without detectable CMV infection.     

SARS冠状病毒M蛋白HLA-A*0201限制性CTL表位的预测

目的 预测SAR冠状病霉M蛋白的HLA-A*0201限制性CTL表位。方法 联合应用简单基序法和延展基序法。结果 预的测出4个九肽CTL表位。结论通过对SARS冠状病毒M蛋白抗原CTL表位进行预测，从而为SARS冠状病毒M蛋白CTL表位的实验探测和鉴定提供了线索，为认识CTL介导的细胞免疫机制以及基于CTL表位的疫苗研制提供了基础资料。     

Functional supertype of HLA-A2 in the presentation of Flu matrix p58-66 to induce CD8+ T-cell response in a Northern Chinese population

The functional supertype of HLA-A2 was investigated in the presentation of the A*0201-restricted Flu matrix p58-66 peptide to activate recall CD8+ T-cell response. In healthy Northern Chinese, the HLA-A2 supertype was mainly composed of the six alleles, A*0201 (26.4%), A*0206 (12.7%), A*0203 (8.2%), A*0207 (7.3%), A*0210 (1.8%) and A*0205 (0.9%), as analyzed by PCR using sequence specific primer (PCR-SSP) and sequence based typing (SBT). The IFN-gamma release Elispot assay was employed to assess effector CD8+ T cells. In A*0201-bearing individuals, the CD8+ T-cell response was potent when stimulated with autologous CD8- PBMCs. The frequency of the effector CD8+ T cells was 96.6% with the magnitude of effector CD8+ T cells of 225 SFC/5 x 104 CD8+ T cells and the RI of 25.7. In non-A*0201 individuals, the effector CD8+ T cells were minimally detectable while the peptide was presented by the autologous CD8- PBMCs. However, the induction of the response of CD8+ T cells obtained from non-A*0201 individuals was remarkably improved when the peptide was presented by autologous dendritic cells instead of CD8- PBMCs. The HLA-A2 alleles possessing cross-reactivity in the peptide presentation were mainly of A*0206 and non-A*0201 heterozygotes of A*0206 and A*0210. Moreover, A*0206 as the HLA-A2 functional supertype was further confirmed by tetramer assay. In two A*0206+ donors with CD8+ T-cell response to the peptide, the CD8+ T-cell frequency assessed by specific binding of peptide HLA-A*0201 tetramer was 4.62% and 1.66%, respectively. Thus, our results have substantiated the immunological relevance of the HLA-A2 supertype, which may benefit the design of peptide vaccines with the potential to be applicable in broader populations.     

Identification of an HLA-A*0201-restricted T-cell epitope on the MPT51 protein, a major secreted protein derived from Mycobacterium tuberculosis, by MPT51 overlapping peptide screening

CD8+ T cells play a pivotal role in protection against Mycobacterium tuberculosis infection. We identified a novel HLA-A*0201-restricted CD8+ T-cell epitope on a dominant secreted antigen of M. tuberculosis, MPT51, in HLA-A*0201 transgenic HHD mice. HHD mice were immunized with plasmid DNA encoding MPT51 with gene gun bombardment, and gamma interferon (IFN-gamma) production by the immune splenocytes was analyzed. In response to overlapping synthetic peptides covering the mature MPT51 sequence, the splenocytes were stimulated to produce IFN-gamma by only one peptide, p51-70. Three-color flow cytometric analysis of intracellular IFN-gamma and cell surface CD4 and CD8 staining revealed that the MPT51 p51-70 peptide contains an immunodominant CD8+ T-cell epitope. Further analysis using computer algorithms permitted identification of a bona fide T-cell epitope, p53-62. A major histocompatibility complex class I stabilization assay using T2 cells confirmed that this epitope binds to HLA-A*0201. The T cells were capable of lysing MPT51 p53-62 peptide-pulsed T2 cells. In addition, MPT51 p53-62-specific memory CD8+ T cells were found in tuberculin skin test-positive HLA-A*0201+ healthy individuals. Use of this HLA-A*0201-restricted CD8+ T-cell epitope for analysis of the role of MPT51-specific T cells in M. tuberculosis infection and for design of vaccines against tuberculosis is feasible.     

氨基酸非键作用指数用于HLA-A*0201限制性CTL表位定量预测研究

目的 预测SSX-1和SSX-4抗原HLA-A*0201限制性表位.方法 从分子的三维空间结构出发,基于静电、立体、疏水这三类与生物活性直接相关的非键作用方式,经主成分分析技术(PCA)得到了一种新的分子结构表达方法--氨基酸非键作用指数.利用该指数结合偏最小二乘(PLS)算法对152个HLA-A*0201限制性CTL表位进行了定量构效关系(QSAR)建模,再使用该模型对SSX-1和SSX-4抗原HLA.A*0201限制性表位进行预测.结果 预测出8个活性值大于7的九肽表位.结论 通过对SSX-1和SSX-4抗原HLA-A*0201限制性表位的预测,从而为SSX-1和SSD-4抗原HLA-A*0201限制性表位的实验探测和鉴定打下了基础.     

Analysis of HLA-A24-restricted CMVpp65 peptide-specific CTL with HLA-A*2402-CMVpp65 tetramer

Developing precise and efficient methods of monitoring immune responses against cytomegalovirus (CMV) infection in immunocompromised transplantation patients is important. With the aim of optimizing the monitoring strategy, an HLA-A24-CMVpp65 tetramer-based analysis of CMVpp65 peptide-specific CTL lines was performed. Previously, the HLA-A24-restricted CTL epitope of CMVpp65 matrix protein was identified (QYDPVAALF aa 341-349). In the present study, CMVpp65 (aa 341-349) peptide-specific CTL lines were obtained from PBLs of 12 HLA-A24+ healthy donors by two stimulations with peptide-pulsed dendritic cells (DC). Among 12 CTL lines, 9 showed HLA-A*2402-CMVpp65 tetramer staining, which was found to be strongly co-related to the amount of IFN-gamma produced by CMVpp65 peptide-restimulated CTL lines (r=0.943, P<0.001). These results suggested that HLA-A*2402-CMVpp65 tetramer staining was an efficient way to monitor immune responses against CMV infection in HLA-A24+ immunocompromised hosts.     

肿瘤抗原TRAG-3 HLA-A2.1限制性CTL表位的鉴定

目的寻找并鉴定TRAG-3来源的HLA-A2．1限制性CTL表位，为临床开展基于TRAG-3表位的特异性免疫治疗奠定基础。方法应用超基序和量化基序方案，预测TRAG-3 HLA-A2．1限制性CTL表位。用计算机分子模拟的方法，对预测表位进行分子模拟。用流式细胞仪分析测定各肽与HLA-A2．1分子的结合力。最后，应用HLA-A2．1阳性健康志愿者外周血单个核细胞(PBMC)对其体外诱导的CTL效应进行鉴定。结果应用超基序和量化基序方案，预测出4个候选表位肽，用计算机分子模拟发现其中只有TRAG-3（37-45)不符合HLA-A2．1限制性CTL表位的特点。在上述4个九肽中，TRAG-3(58-66)与HLA-A2．1分子的结合力最高。在进一步的CTL诱导实验中，发现TRAG-3(58-66)能够诱导健康志愿者PBMC产生特异性CTL。结论表位预测、计算机分子模拟、结合力分析和体外CTL诱导实验的一致性较好。4种方法一致认为，TRAG-3（58-66）(ILLRDA-GLV)为HLA-A2．1限制性CTL表位。该表位肽可望用于基于TRAG-3抗原多肽疫苗的临床实验。     

Identification of a new HLA-A*0201-restricted CD8+ T cell epitope from hepatocellular carcinoma-associated antigen HCA587

For the development of peptide-based cancer immunotherapies, we aimed to identify specific HLA-A*0201-restricted CTL epitopes in hepatocellular carcinoma (HCC) associated antigen HCA587, which has been identified as a member of the cancer/testis (CT) antigens highly expressed in HCC. We first combined the use of an HLA-A*0201/peptide binding algorithm and T2 binding assays with the induction of specific CD8(+) T cell lines from normal donors by in vitro priming with high-affinity peptides, then IFN-gamma release and cytotoxicity assays were employed to identify the specific HLA-A*0201 CD8(+) T cell epitope using peptide-loaded T2 cells or the HCA587 protein(+) HCC cell line HepG2. In the six candidate synthesized peptides, two peptides showed higher binding ability in T2 binding assays. No. 2 peptide, encompassing amino acid residues FLAKLNNTV (HCA587(317-325)), was able to activate a HCA587-specific CD8(+) T-cell response in human lymphocyte cultures from two normal donors and two HCC patients, and these HCA587-specific CD8(+) T cells recognized peptide-pulsed T2 cells as well as the HCA587 protein(+) HCC cell line HepG2 in IFN-gamma release and cytotoxicity assays. The results indicate that no. 2 peptide is a new HLA-A*0201-restricted CTL epitope capable of inducing HCA587-specific CTLs. Our data suggest that identification of this new HCA587/HLA-A*0201 peptide FLAKLNNTV may facilitate the design of peptide-based immunotherapies for the treatment of HCA587-bearing HCC patients.     

慢性白血病抗原CML28 HLA-A*0201限制性CTL表位预测

目的鉴定慢性白血病肿瘤抗原CML28的HIA-A*0201限制性CTL表位。方法 采用超基序和量化基序结合的方法初步预测CM128的HLA-A*0201限制性CTL表位；利用T2细胞亲合力实验初步验证预测结果。结果 在预测的4个候选CTL表位中，ALVDAGVPM和ALDSDGTLV有较高的亲和力，ALFCGVACA和SLLACCLNA仅有低度亲和力。结论ALVDAGVPM与ALDSDGTLV最有可能是慢性白血病肿瘤抗原CML28的HLA-A*0201限制性CTL表位。     

Prediction of HLA class I-restricted T-cell epitopes of islet autoantigen combined with binding and dissociation assays

Identification of cognate peptides recognized by human leucocyte antigen (HLA)/T cell receptor (TCR) complex provides insight into the pathogenic process of type 1 diabetes (T1D). We hypothesize that HLA-binding assays alone are inadequate metrics for the affinity of peptides. Zinc transporter-8 (ZnT8) has emerged in recent years as a novel, major, human autoantigen. Therefore, we aim to identify the HLA-A2-restricted ZnT8 epitopes using both binding and dissociation assays. HLA class I peptide affinity algorithms were used to predict candidate ZnT8 peptides that bind to HLA-A2. We analyzed 15 reported epitopes of seven β-cell candidate autoantigens and eight predicted candidate ZnT8 peptides using binding and dissociation assays. Using IFN-γ ELISpot assay, we tested peripheral blood mononuclear cells (PBMCs) from recent-onset T1D patients and healthy controls for reactivity to seven reported epitopes and eight candidate ZnT8 peptides directly ex vivo. We found five of seven recently reported epitopes in Chinese T1D patients. Of the eight predicted ZnT8 peptides, ZnT8(153-161) had a strong binding affinity and the lowest dissociation rate to HLA-A*0201. We identified it as a novel HLA-A*0201-restricted T-cell epitope in three of eight T1D patients. We conclude that ZnT8(153-161) is a novel HLA-A*0201-restricted T-cell epitope. We did not observe a significant correlation (P = 0.3, R = - 0.5) between cytotoxic T cell (CTL) response and peptide/HLA*0201 complex stability. However, selection of peptides based on affinity and their dissociation rate may be helpful for the identification of candidate CTL epitopes. Thus, we can minimize the number of experiments for the identification of T-cell epitopes from interesting antigens.     

Induction of CTL response by a minimal epitope vaccine in HLA A*0201/DR1 transgenic mice: dependence on HLA class II restricted T(H) response

CTL play a pivotal role in the immune response during viral infections. In this study, the HLA class II restricted T(H) requirement for optimal in vivo induction of HLA class I restricted CTL responses has been investigated. Towards this goal, transgenic mice expressing both HLA class I (A*0201 or A2.1) and class II (DRB1*0101 or DR1) molecules have been derived. Immunization of these mice with an HLA A*0201-restricted and CMV-specific CTL epitope (pp65(495-503)), and either of three different tetanus toxin-derived MHC class II-binding T(H) epitopes, resulted in a vigorous CTL response. CTL specific for the pp65(495-503) epitope were dramatically enhanced in mice expressing both the HLA-DR1 and HLA-A*0201 transgenes. Notably, preinjection of three TT peptides (TT(639-652), TT(830-843), and TT(947-967)) increased the capability of HLA A*0201/DR1 Tg mice to respond to subsequent immunization with the T(H) + CTL peptide mixture. These results indicate that the use of HLA A*0201/DR1 Tg mice constitute a versatile model system (in lieu of immunizing humans) for the study of both HLA class I and class II restricted T-cell responses. These studies provide a rational model for the design and assessment of new minimal-epitope vaccines based on their in vivo induction of a pathogen-specific CTL response.