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Immunity to pulmonary infection typically requires elicitation of lung-resident T cells that subsequently confer protection against secondary infection. The presence of tissue-resident T cells in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) convalescent patients is unknown. Using a sublethal mouse model of coronavirus disease 2019, we determined if SARS-CoV-2 infection potentiated Ag-specific pulmonary resident CD4+ and CD8+ T cell responses and if these cells mediated protection against secondary infection. S protein–specific T cells were present in resident and circulating populations. However, M and N protein–specific T cells were detected only in the resident T cell pool. Using an adoptive transfer strategy, we found that T cells from SARS-CoV-2 immune animals did not protect naive mice. These data indicate that resident T cells are elicited by SARS-CoV-2 infection but are not sufficient for protective immunity.

Flagellin is an immunodominant Ag in Crohn disease, with many patients showing anti-flagellin Abs. To study the clonality of flagellin-reactive CD4 cells in Crohn patients, we used a common CD154-based enrichment method following short-term Ag exposure to identify Ag-reactive CD4 cells. CD154 expression and cytokine production following Ag exposure compared with negative control responses (no Ag exposure) revealed that only a small fraction of CD154-enriched cells could be defined by Ag-reactive cytokine responses. This was especially true for low-frequency flagellin-reactive CD4 cells compared with polyclonal stimulation or Candida albicans Ag exposure. Moreover, we found that culture conditions used for the assay contributed to background CD40L (CD154) expression in the CD154-enriched CD4 cells. Using a cut-off rule based on flow cytometry results of the negative control CD154-enriched CD4 cells, we could reliably find the fraction of Ag-reactive cells in the CD154-enriched population. Ag-reactive CD4 cytokine production was restricted to CD4 cells with an effector memory phenotype and the highest levels of induced CD154 expression. This has important implications for identifying Ag-specific T cells of interest for single cell cloning, phenotyping, and transcriptomics.

Invariant NKT (iNKT) cells are an innate-like population characterized by their recognition of glycolipid Ags and rapid cytokine production upon activation. Unlike conventional T cells, which require TCR ligation, iNKT cells can also be stimulated independently of their TCR. This feature allows iNKT cells to respond even in the absence of glycolipid Ags, for example, during viral infections. Although the TCR-dependent and -independent activation of iNKT cells have been relatively well established, the exact contributions of IL-12, IL-18, and TLRs remain unclear for these two activation pathways. To definitively investigate how these components affect the direct and indirect stimulation of iNKT cells, we used mice deficient for either MyD88 or the IL-12Rβ2 in the T cell lineage. Using these tools, we demonstrate that IL-12, IL-18, and TLRs are completely dispensable for the TCR activation pathway when a strong agonist is used. In contrast, during murine CMV infection, when the TCR is not engaged, IL-12 signaling is essential, and TLR signaling is expendable. Importantly, to our knowledge, we discovered an intrinsic requirement for IL-18 signaling by splenic iNKT cells but not liver iNKT cells, suggesting that there might be diversity, even within the NKT1 population.

The availability of MHC-binding prediction tools has been useful in guiding studies aimed at identifying candidate target Ags to generate reactive T cells and to characterize viral and tumor-reactive T cells. Nevertheless, prediction algorithms appear to function poorly for epitopes containing cysteine (Cys) residues, which can oxidize and form disulfide bonds with other Cys residues under oxidizing conditions, thus potentially interfering with their ability to bind to MHC molecules. Analysis of the results of HLA-A*02:01 class I binding assays carried out in the presence and absence of the reducing agent 2-ME indicated that the predicted affinity for 25% of Cys-containing epitopes was underestimated by a factor of 3 or more. Additional analyses were undertaken to evaluate the responses of human CD8+ tumor-reactive T cells against 10 Cys-containing HLA class I–restricted minimal determinants containing substitutions of α-aminobutyric acid (AABA), a cysteine analogue containing a methyl group in place of the sulfhydryl group present in Cys, for the native Cys residues. Substitutions of AABA for Cys at putative MHC anchor positions often significantly enhanced T cell recognition, whereas substitutions at non-MHC anchor positions were neutral, except for one epitope where this modification abolished T cell recognition. These findings demonstrate the need to evaluate MHC binding and T cell recognition of Cys-containing peptides under conditions that prevent Cys oxidation, and to adjust current prediction binding algorithms for HLA-A*02:01 and potentially additional class I alleles to more accurately rank peptides containing Cys anchor residues.

Tau protein is found to be aggregated and hyperphosphorylated (p-tau) in many neurologic disorders, including Parkinson disease (PD) and related parkinsonisms, Alzheimer disease, traumatic brain injury, and even in normal aging. Although not known to produce autoimmune responses, we hypothesized that the appearance of aggregated tau and p-tau with disease could activate the immune system. We thus compared T cell responses to tau and p-tau–derived peptides between PD patients, age-matched healthy controls, and young healthy controls (

Adoptive immunotherapy based on chimeric antigen receptor–modified T (CAR-T) cells has been demonstrated as one of the most promising therapeutic strategies in the treatment of malignancies. However, CAR-T cell therapy has shown limited efficacy for the treatment of solid tumors. This is, in part, because of tumor heterogeneity and a hostile tumor microenvironment, which could suppress adoptively transferred T cell activity. In this study, we, respectively, engineered human- or murine-derived–armored glypican-3 (GPC3)–specific CAR-T cells capable of inducibly expressing IL-12 (GPC3-28Z-NFAT-IL-12) T cells. The results showed that GPC3-28Z-NFAT-IL-12 T cells could lyse GPC3+ tumor cells specifically and increase cytokine secretion compared with GPC3-28Z T cells in vitro. In vivo, GPC3-28Z-NFAT-IL-12 T cells augmented the antitumor effect when encountering GPC3+ large tumor burdens, which could be attributed to IL-12 increasing IFN-γ production, favoring T cells infiltration and persistence. Furthermore, in immunocompetent hosts, low doses of GPC3-m28Z-mNFAT-mIL-12 T cells exerted superior antitumor efficacy without prior conditioning in comparison with GPC3-m28Z T cells. Also, mIL-12 secretion decreased regulatory T cell infiltration in established tumors. In conclusion, these findings demonstrated that the inducible expression of IL-12 could boost CAR-T function with less potential side effects, both in immunodeficient and immunocompetent hosts. The inducibly expressed IL-12–armored GPC3–CAR-T cells could broaden the application of CAR-T–based immunotherapy to patients intolerant of lymphodepletion chemotherapy and might provide an alternative therapeutic strategy for patients with GPC3+ cancers.

Antitumor T cell responses involve CD8+ T cells with high affinity for mutated self-antigen and low affinity for nonmutated tumor-associated Ag. Because of the highly individual nature of nonsynonymous somatic mutations in tumors, however, immunotherapy relies often on an effective engagement of low-affinity T cells. In this study, we studied the role of T cell affinity during peripheral priming with single-peptide vaccines and during the effector phase in the tumor. To that end, we compared the antitumor responses after OVA257–264 (N4) peptide vaccination of CD8+ T cells carrying TCRs with high (OT-1) and low (OT-3) avidity for the N4 peptide in B16.N4 tumor-bearing C57BL/6 mice. Additionally, we assessed the response of OT-1 cells to either high-affinity (B16.N4) or low-affinity (B16.T4) Ag-expressing tumors after high-affinity (N4) or low-affinity (T4) peptide vaccination. We noticed that although low-affinity tumor-specific T cells expand less than high-affinity T cells, they express lower levels of inhibitory receptors and produce more cytokines. Interestingly, tumor-infiltrating CD8+ T cells show similar in vivo re-expansion capacity to their counterparts in secondary lymphoid organs when transferred to tumor-free hosts, suggesting that T cells in tumors may be rekindled upon relief of tumor immunosuppression. Moreover, our results show that αPD-1 treatment enhances tumor control of high- and low-affinity ligand-expressing tumors, suggesting that combination of high-affinity peripheral priming by altered peptide ligands and checkpoint blockade may enable tumor control upon low-affinity Ag recognition in the tumor.

The successes of antitumor immuno-based therapies and the application of next-generation sequencing to mutation profiling have produced insights into the specific targets of antitumor T cells. Mutated proteins have tremendous potential as targets for interventions using autologous T cells or engineered cell therapies and may serve as important correlates of efficacy for immunoregulatory interventions including immune checkpoint blockade. As mutated self, tumors present an exceptional case for host immunity, which has primarily evolved in response to foreign pathogens. Tumor Ags’ resemblance to self may limit immune recognition, but key features appear to be the same between antipathogen and antitumor responses. Determining which targets will make efficacious Ags and which responses might be elicited therapeutically are key questions for the field. Here we discuss current knowledge on antitumor specificity, the mutations that provide immunogenic targets, and how cross-reactivity and immunodominance may contribute to variation in immune responses among tumor types.

Cyclin B1 (CCNB1) is considered as a potential target for a cancer vaccine, as it is overexpressed in many malignant cells, while being transiently expressed in normal cells. To evaluate the CD4 T cell response to CCNB1, we derived T cell lines by multiple weekly rounds of stimulation with recombinant CCNB1 of T cells collected in healthy donors (long-term T cell assays). T cell lines were specific for 15 immunodominant peptides and derived preferentially from naive T cells. From 74 overlapping peptides, 20 peptides were selected for their broad specificity of binding to HLA class II molecules and included most of the immunodominant epitopes. They primed in vitro a large number of specific CD4 T cell lines in all the donors. Immunodominant epitopes were the most efficacious in long-term T cell assays, both in terms of number of specific T cell lines and number of responding donors. The 20 peptides were also submitted to short-term T cell assays using cells collected in healthy and cancer patients with the aim to evaluate the memory response. The recognized peptides differed from the immunodominant peptides and were part of the best promiscuous peptides. We also observed pre-existing CCNB1-specifc IgG Abs in both healthy and cancer donors. Long- and short-term T cell assays revealed that CCNB1 contained many CD4 T cell epitopes, which are differentially recognized by pre-existing naive and memory CD4 T cells. These observations are of value for the design of cancer vaccines.

Foxp3+ regulatory T cell (Treg)-based immunotherapy holds promise for autoimmune diseases. However, this effort has been hampered by major caveats, including the low frequency of autoantigen-specific Foxp3+ Tregs and lack of understanding of their molecular and cellular targets, in an unmanipulated wild-type (WT) immune repertoire. In this study, we demonstrate that infusion of myelin in WT mice results in the de novo induction of myelin-specific Foxp3+ Tregs in WT mice and amelioration of experimental autoimmune encephalomyelitis. Myelin-specific Foxp3+ Tregs exerted their effect both by diminishing Ag-bearing inflammatory dendritic cell (iDC) recruitment to lymph nodes and by impairing their function. Transcriptome analysis of ex vivo–isolated Treg-exposed iDCs showed significant enrichment of transcripts involved in functional properties of iDCs, including chemotaxis-related genes. To this end, CCR7 expression by iDCs was significantly downregulated in tolerant mice and this was tightly regulated by the presence of IL-10. Collectively, our data demonstrate a novel model for deciphering the Ag-specific Foxp3+ Treg-mediated mechanisms of tolerance and delineate iDCs as a Foxp3+ Treg cellular target in unmanipulated mice.

The systematic assessment of the human immune system bears huge potential to guide rational development of novel immunotherapies and clinical decision making. Multiple assays to monitor the quantity, phenotype, and function of Ag-specific T cells are commonly used to unravel patients’ immune signatures in various disease settings and during therapeutic interventions. When compared with tests measuring soluble analytes, cellular immune assays have a higher variation, which is a major technical factor limiting their broad adoption in clinical immunology. The key solution may arise from continuous control of assay performance using TCR-engineered reference samples. We developed a simple, stable, robust, and scalable technology to generate reference samples that contain defined numbers of functional Ag-specific T cells. First, we show that RNA-engineered lymphocytes, equipped with selected TCRs, can repetitively deliver functional readouts of a controlled size across multiple assay platforms. We further describe a concept for the application of TCR-engineered reference samples to keep assay performance within or across institutions under tight control. Finally, we provide evidence that these novel control reagents can sensitively detect assay variation resulting from typical sources of error, such as low cell quality, loss of reagent stability, suboptimal hardware settings, or inaccurate gating.

Identification of the specific HLA locus and allele presenting an epitope for recognition by specific TCRs (HLA restriction) is necessary to fully characterize the immune response to Ags. Experimental determination of HLA restriction is complex and technically challenging. As an alternative, the restricting HLA locus and allele can be inferred by genetic association, using response data in an HLA-typed population. However, simple odds ratio (OR) calculations can be problematic when dealing with large numbers of subjects and Ags, and because the same epitope can be presented by multiple alleles (epitope promiscuity). In this study, we develop a tool, denominated Restrictor Analysis Tool for Epitopes, to extract inferred restriction from HLA class II–typed epitope responses. This automated method infers HLA class II restriction from large datasets of T cell responses in HLA class II–typed subjects by calculating ORs and relative frequencies from simple data tables. The program is validated by: 1) analyzing data of previously determined HLA restrictions; 2) experimentally determining in selected individuals new HLA restrictions using HLA-transfected cell lines; and 3) predicting HLA restriction of particular peptides and showing that corresponding HLA class II tetramers efficiently bind to epitope-specific T cells. We further design a specific iterative algorithm to account for promiscuous recognition by calculation of OR values for combinations of different HLA molecules while incorporating predicted HLA binding affinity. The Restrictor Analysis Tool for Epitopes program streamlines the prediction of HLA class II restriction across multiple T cell epitopes and HLA types.

The characteristics of the TCR repertoire expressed by epitope-specific CD8+ T cells can be an important determinant of the quality of immune protection against virus infection. Most studies of epitope-specific TCR repertoires focus solely on an analysis of TCR β-chains, rather than the combined TCRαβ heterodimers that confer specificity. Hence, the importance of complementary α- and β-chain pairing in determining TCR specificity and T cell function is not well understood. Our earlier study of influenza-specific TCR repertoires in a C57BL/6J mouse model described a structural basis for preferred TCRαβ pairing that determined exquisite specificity for the DbPA224 epitope from influenza A virus. We have now extended this analysis using retrogenic mice engineered to express single TCR α- or β-chains specific for the DbNP366 or DbPA224 epitopes derived from influenza A virus. We found that particular TCRαβ combinations were selected for recognition of these epitopes following infection, indicating that pairing of certain α- and β-chain sequences is key for determining TCR specificity. Furthermore, we demonstrated that some TCRαβ heterodimers were preferentially expanded from the naive repertoire in response to virus infection, suggesting that appropriate αβ pairing confers optimal T cell responsiveness to Ag.

Burkholderia pseudomallei is the causative agent of melioidosis characterized by pneumonia and fatal septicemia and prevalent in Southeast Asia. Related Burkholderia species are strong risk factors of mortality in cystic fibrosis (CF). The B. pseudomallei flagellar protein FliC is strongly seroreactive and vaccination protects challenged mice. We assessed B. pseudomallei FliC peptide binding affinity to multiple HLA class II alleles and then assessed CD4 T cell immunity in HLA class II transgenic mice and in seropositive individuals in Thailand. T cell hybridomas were generated to investigate cross-reactivity between B. pseudomallei and the related Burkholderia species associated with Cepacia Complex CF. B. pseudomallei FliC contained several peptide sequences with ability to bind multiple HLA class II alleles. Several peptides were shown to encompass strong CD4 T cell epitopes in B. pseudomallei–exposed individuals and in HLA transgenic mice. In particular, the p38 epitope is robustly recognized by CD4 T cells of seropositive donors across diverse HLA haplotypes. T cell hybridomas against an immunogenic B. pseudomallei FliC epitope also cross-reacted with orthologous FliC sequences from Burkholderia multivorans and Burkholderia cenocepacia, important pathogens in CF. Epitopes within FliC were accessible for processing and presentation from live or heat-killed bacteria, demonstrating that flagellin enters the HLA class II Ag presentation pathway during infection of macrophages with B. cenocepacia. Collectively, the data support the possibility of incorporating FliC T cell epitopes into vaccination programs targeting both at-risk individuals in B. pseudomallei endemic regions as well as CF patients.

αβ and γδ T cells are thought to arise from a common precursor in the thymus but play distinct roles in pathogen resistance. Although conventional αβ T cells exit the thymus in a naive state and acquire effector function in the periphery, the effector fate of many γδ T cells is specified in the thymus and exhibits limited plasticity thereafter. This review describes the current models that have been proposed to explain the acquisition of effector fate by γδ T cells, as well as the apparent linkage to Vγ gene usage. The two predominant models are the predetermination model, which suggests that effector fate is determined prior to TCR expression, perhaps in association with the developmental timing of Vγ rearrangement, and the TCR-dependence model, which proposes that the nature of the TCR signal, particularly its intensity or duration, plays an important role in influencing effector fate.

Extensive diversity in the human repertoire of TCRs for Ag is both a cornerstone of effective adaptive immunity that enables host protection against a multiplicity of pathogens and a weakness that gives rise to potential pathological self-reactivity. The complexity arising from diversity makes detection and tracking of single Ag-specific CD4 T cells (ASTs) involved in these immune responses challenging. We report a tandem, multistep process to quantify rare TCRβ-chain variable sequences of ASTs in large polyclonal populations. The approach combines deep high-throughput sequencing (HTS) within functional CD4 T cell compartments, such as naive/memory cells, with shallow, multiple identifier–based HTS of ASTs identified by activation marker upregulation after short-term Ag stimulation in vitro. We find that clonotypes recognizing HLA class II–restricted epitopes of both pathogen-derived Ags and self-Ags are oligoclonal and typically private. Clonotype tracking within an individual reveals private AST clonotypes resident in the memory population, as would be expected, representing clonal expansions (identical nucleotide sequence; “ultraprivate”). Other AST clonotypes share CDR3β amino acid sequences through convergent recombination and are found in memory populations of multiple individuals. Tandem HTS-based clonotyping will facilitate studying AST dynamics, epitope spreading, and repertoire changes that arise postvaccination and following Ag-specific immunotherapies for cancer and autoimmune disease.

Human CD1a mediates foreign Ag recognition by a T cell clone, but the nature of possible TCR interactions with CD1a/lipid are unknown. After incubating CD1a with a mycobacterial lipopeptide Ag, dideoxymycobactin (DDM), we identified and measured binding to a recombinant TCR (TRAV3/ TRBV3-1, KD of ≈100 μM). Detection of ternary CD1a/lipid/TCR interactions enabled development of CD1a tetramers and CD1a multimers with carbohydrate backbones (dextramers), which specifically stained T cells using a mechanism that was dependent on the precise stereochemistry of the peptide backbone and was blocked with a soluble TCR. Furthermore, sorting of human T cells from unrelated tuberculosis patients for bright DDM-dextramer staining allowed recovery of T cells that were activated by CD1a and DDM. These studies demonstrate that the mechanism of T cell activation by lipopeptides occurs via ternary interactions of CD1a/Ag/TCR. Furthermore, these studies demonstrate the existence of lipopeptide-specific T cells in humans ex vivo.

Cigarette smoke (CS) exposure is the primary risk factor for the development of chronic obstructive pulmonary disease (COPD). COPD is characterized by chronic peribronchial, perivascular, and alveolar inflammation. The inflammatory cells consist primarily of macrophage, neutrophils, and lymphocytes. Although myeloid cells are well studied, the role of lymphocyte populations in pathogenesis of COPD remains unclear. Using a mouse model of CS-induced emphysema, our laboratory has previously demonstrated that CS exposure causes changes in the TCR repertoire suggestive of an Ag-specific response and triggers a pathogenic T cell response sufficient to cause alveolar destruction and inflammation. We extend these findings to demonstrate that T cells from CS-exposed mice of the BALB/cJ or C57B6 strain are sufficient to transfer pulmonary pathology to CS-naive, immunosufficient mice. CS exposure causes a proinflammatory phenotype among pulmonary T cells consistent with those from COPD patients. We provide evidence that donor T cells from CS-exposed mice depend on Ag recognition to transfer alveolar destruction using MHC class I–deficient recipient mice. Neither CD4+ nor CD8+ T cells from donor mice exposed to CS alone are sufficient to cause inflammation or pathology in recipient mice. We found no evidence of impaired suppression of T cell proliferation among regulatory T cells from CS-exposed mice. These results suggest that CS exposure initiates an Ag-specific response that leads to pulmonary destruction and inflammation that involves both CD8+ and CD4+ T cells. These results are direct evidence for an autoimmune response initiated by CS exposure.

We re-examined the observation that γδ T cells, when transferred from mice tolerized to an inhaled conventional Ag, suppress the allergic IgE response to this Ag specifically. Using OVA and hen egg lysozyme in crisscross fashion, we confirmed the Ag-specific IgE-regulatory effect of the γδ T cells. Although only Vγ4+ γδ T cells are regulators, the Ag specificity does not stem from specificity of their γδ TCRs. Instead, the Vγ4+ γδ T cells failed to respond to either Ag, but rapidly acquired Ag-specific regulatory function in vivo following i.v. injection of non-T cells derived from the spleen of Ag-tolerized mice. This correlated with their in vivo Ag acquisition from i.v. injected Ag-loaded splenic non-T cells, and in vivo transfer of membrane label provided evidence for direct contact between the injected splenic non-T cells and the Vγ4+ γδ T cells. Together, our data suggest that Ag itself, when acquired by γδ T cells, directs the specificity of their IgE suppression.

Memory T cells form a highly specific defense layer against reinfection with previously encountered pathogens. In addition, memory T cells provide protection against pathogens that are similar, but not identical to the original infectious agent. This is because each T cell response harbors multiple clones with slightly different affinities, thereby creating T cell memory with a certain degree of diversity. Currently, the mechanisms that control size, diversity, and cross-reactivity of the memory T cell pool are incompletely defined. Previously, we established a role for apoptosis, mediated by the BH3-only protein Noxa, in controlling diversity of the effector T cell population. This function might positively or negatively impact T cell memory in terms of function, pool size, and cross-reactivity during recall responses. Therefore, we investigated the role of Noxa in T cell memory during acute and chronic infections. Upon influenza infection, Noxa−/− mice generate a memory compartment of increased size and clonal diversity. Reinfection resulted in an increased recall response, whereas cross-reactive responses were impaired. Chronic infection of Noxa−/− mice with mouse CMV resulted in enhanced memory cell inflation, but no obvious pathology. In contrast, in a model of continuous, high-level T cell activation, reduced apoptosis of activated T cells rapidly led to severe organ pathology and premature death in Noxa-deficient mice. These results establish Noxa as an important regulator of the number of memory cells formed during infection. Chronic immune activation in the absence of Noxa leads to excessive accumulation of primed cells, which may result in severe pathology.

A small number of HIV-infected individuals known as elite controllers experience low levels of chronic phase viral replication and delayed progression to AIDS. Specific HLA class I alleles are associated with elite control, implicating CD8+ T lymphocytes in the establishment of these low levels of viral replication. Most HIV-infected individuals that express protective HLA class I alleles, however, do not control viral replication. Approximately 50% of Mamu-B*00801+ Indian rhesus macaques control SIVmac239 replication in the chronic phase in a manner that resembles elite control in humans. We followed both the immune response and viral evolution in SIV-infected Mamu-B*00801+ animals to better understand the role of CD8+ T lymphocytes during the acute phase of viral infection, when viral control status is determined. The virus escaped from immunodominant Vif and Nef Mamu-B*00801–restricted CD8+ T lymphocyte responses during the critical early weeks of acute infection only in progressor animals that did not control viral replication. Thus, early CD8+ T lymphocyte escape is a hallmark of Mamu-B*00801+ macaques who do not control viral replication. By contrast, virus in elite controller macaques showed little evidence of variation in epitopes recognized by immunodominant CD8+ T lymphocytes, implying that these cells play a role in viral control.

Vaccine strategies aimed at generating CD8+ T cell memory responses are likely to show augmented efficacy against chronic challenges like tumor. The abundance in variety of memory CD8+ T cells behooves development of vaccine strategies that generate distinct memory responses and evaluate them for tumor efficacy. In this study, we demonstrate the ability of a variety of rapamycin treatment regimens to regulate virus vaccination-induced CD8+ T cell memory responses and tumor efficacy. Strikingly, a short course of high-dose, but not low-dose, rapamycin treatment transiently blocks viral vaccination-induced mammalian target of rapamycin activity in CD8+ T cells favoring persistence and Ag-recall responses over type 1 effector maturation; however, prolonged high-dose rapamycin administration abrogated memory responses. Furthermore, a short course of high-dose rapamycin treatment generated CD8+ T cell memory responses that were independent of IL-15 and IL-7 and were programmed early for sustenance and greater tumor efficacy. These results demonstrate the impact a regimen of rapamycin treatment has on vaccine-induced CD8+ T cell responses and indicates that judicious application of rapamycin can augment vaccine efficacy for chronic challenges.

Minimization of immunosuppression and donor-specific tolerance to MHC-mismatched organ grafts are important clinical goals. The therapeutic potential of regulatory T cells (Tregs) has been demonstrated, but conditions for optimizing their in vivo function posttransplant in nonlymphocyte-depleted hosts remain undefined. In this study, we address mechanisms through which inhibition of the mammalian target of rapamycin (Rapa) synergizes with alloantigen-specific Treg (AAsTreg) to permit long-term, donor-specific heart graft survival in immunocompetent hosts. Crucially, immature allogeneic dendritic cells allowed AAsTreg selection in vitro, with minimal expansion of unwanted (Th17) cells. The rendered Treg potently inhibited T cell proliferation in an Ag-specific manner. However, these AAsTreg remained unable to control T cells stimulated by allogeneic mature dendritic cells, a phenomenon dependent on the release of proinflammatory cytokines. In vivo, Rapa administration reduced danger-associated IL-6 production, T cell proliferation, and graft infiltration. Based on these observations, AAsTreg were administered posttransplant (day 7) in combination with a short course of Rapa and rendered >80% long-term (>150 d) graft survival, a result superior to that achieved with polyclonal Treg. Moreover, graft protection was alloantigen-specific. Significantly, long-term graft survival was associated with alloreactive T cell anergy. These findings delineate combination of transient mammalian target of Rapa inhibition with appropriate AAsTreg selection as an effective approach to promote long-term organ graft survival.

Although recent studies have focused on CD4+ regulatory T cells (Treg), CD8+ Treg have also been reported to play important roles in the induction and maintenance of immune tolerance. Adoptive transfer of CD8+ Treg in rodents or induction of CD8+ Treg in humans can prevent or treat allograft rejection and autoimmune diseases. However, no approaches have been reported for the generation of human Ag-specific CD8+ Treg at a practical scale for clinical use. Here, we found that two novel CD8+ T cell subsets with different levels of CD8 surface expression, CD8high and CD8low, could be induced from naive CD8+ precursors in vitro by allogeneic CD40-activated B cells, whereas only CD8high T cells were alloantigen-specific Treg with relatively poor alloantigen-specific cytotoxicity. Importantly, alloantigen-specific CD8high Treg could be induced and expanded from naive CD8+CD25− T cells at a large scale after 3 wk of culture without exogenous cytokines. These induced alloantigen-specific Treg were CD45RO+ and CCR7− memory cells, and they expressed Foxp3, CD25, CD27, CD28, and CD62L. The induction and expansion of CD8high Treg by CD40-activated B cells were dependent on endogenously expressed IFN-γ, IL-2, IL-4, and CTLA-4. This approach may facilitate the clinical application of CD8+ Treg-based immunotherapy in transplantation and autoimmune diseases.

Alloreactivity is the response of T cells to MHC molecules not encountered during thymic development. A small population (1–8%) of peripheral T cells in mice and humans express two TCRs due to incomplete allelic exclusion of TCRα, and we hypothesized they are highly alloreactive. FACS analysis of mouse T cell MLR revealed increased dual TCR T cells among alloreactive cells. Quantitative assessment of the alloreactive repertoire demonstrated a nearly 50% reduction in alloreactive T cell frequency among T cells incapable of expressing a secondary TCR. We directly demonstrated expansion of the alloreactive T cell repertoire at the single cell level by identifying a dual TCR T cell with distinct alloreactivities for each TCR. The importance of dual TCR T cells is clearly demonstrated in a parent-into-F1 model of graft-vs-host disease, where dual TCR T cells comprised up to 60% of peripheral activated T cells, demonstrating a disproportionate contribution to disease.

Genes of the MHC show the strongest genetic association with multiple sclerosis (MS), but the underlying mechanisms have remained unresolved. In this study, we asked whether the MS-associated MHC class II molecules, HLA-DRB1*1501, HLA-DRB5*0101, and HLA-DRB1*0401, contribute to autoimmune CNS demyelination by promoting pathogenic T cell responses to human myelin basic protein (hMBP), using three transgenic (Tg) mouse lines expressing these MHC molecules. Unexpectedly, profound T cell tolerance to the high-affinity MHC-binding hMBP82-100 epitope was observed in all Tg mouse lines. T cell tolerance to hMBP82-100 was abolished upon back-crossing the HLA-DR Tg mice to MBP-deficient mice. In contrast, T cell tolerance was incomplete for low-affinity MHC-binding hMBP epitopes. Furthermore, hMBP82-100-specific type B T cells escaped tolerance in HLA-DRB5*0101 Tg mice. Importantly, T cells specific for low-affinity MHC-binding hMBP epitopes and hMBP82-100-specific type B T cells were highly encephalitogenic. Collectively, the results show that MS-associated MHC class II molecules are highly efficient at inducing T cell tolerance to high-affinity MHC-binding epitope, whereas autoreactive T cells specific for the low-affinity MHC-binding epitopes and type B T cells can escape the induction of T cell tolerance and may promote MS.

Lymphocyte differentiation from naive CD4+ T cells into mature Th1, Th2, Th17, or T regulatory cell (Treg) phenotypes has been considered end stage in character. In this study, we demonstrate that dendritic cells (DCs) activated with a novel immune modulator B7-DC XAb (DCXAb) can reprogram Tregs into T effector cells. Down-regulation of FoxP3 expression after either in vitro or in vivo Treg-DCXAb interaction is Ag-specific, IL-6-dependent, and results in the functional reprogramming of the mature T cell phenotype. The reprogrammed Tregs cease to express IL-10 and TGFβ, fail to suppress T cell responses, and gain the ability to produce IFN-γ, IL-17, and TNF-α. The ability of IL-6+ DCXAb and the inability of IL-6−/− DCXAb vaccines to protect animals from lethal melanoma suggest that exogenously modulated DC can reprogram host Tregs. In support of this hypothesis and as a test for Ag specificity, transfer of DCXAb into RIP-OVA mice causes a break in immune tolerance, inducing diabetes. Conversely, adoptive transfer of reprogrammed Tregs but not similarly treated CD25− T cells into naive RIP-OVA mice is also sufficient to cause autoimmune diabetes. Yet, treatment of normal mice with B7-DC XAb fails to elicit generalized autoimmunity. The finding that mature Tregs can be reprogrammed into competent effector cells provides new insights into the plasticity of T cell lineage, underscores the importance of DC-T cell interaction in balancing immunity with tolerance, points to Tregs as a reservoir of autoimmune effectors, and defines a new approach for breaking tolerance to self Ags as a strategy for cancer immunotherapy.

The manner in which cells die is believed to have a major impact on the nature of immune responses to their released Ags. In this study, we present the first direct analysis of tumor-specific immune responses to in vivo occurring tumor cell death through apoptosis or necrosis. Mice bearing thymidine kinase-transfected tumors were treated either with ganciclovir to induce tumor cell apoptosis in vivo or a vascular targeting agent, ZD6126, to induce tumor cell necrosis in vivo. In contrast to tumor apoptosis, induction of necrosis reduced the frequency and impaired the function of tumor-specific CD8+ T cells. Adoptive transfer of lymphocytes from mice with apoptotic tumors into tumor-challenged mice resulted in a significant tumor protection, which was absent when splenocytes were transferred from mice with necrotic tumors. Anti-CD40 treatment reversed impaired Ag-specific CD8+ T cell responses in these mice. These observations have not only fundamental importance for the development of immunotherapy protocols but also help to understand the underlying mechanism of in vivo immune responses to tumor cell death.

A major issue regarding T cell responses in autoimmunity is how the repertoire compares between the periphery and target organ. In type 1 diabetes, the status of at-risk or diabetic individuals can be monitored by measuring β cell-specific T cells isolated from PBL, but whether these T cells accurately reflect the repertoire residing in the pancreatic islets is unclear. The TCR repertoire of disease-relevant, tetramer-sorted CD8+ T cells was examined at the single-cell level in PBL, pancreatic lymph nodes (PLN), and the islets of individual NOD mice. CDR3α and CDR3β sequences demonstrated that the same repertoire of T cells in PBL was detected in the islets and PLN, although the frequency of specific clonotypes varied. Albeit infrequent, clonotypes that were prevalent in the islets but not found in PBL were also detected. β cell Ag immunization expanded immunodominant PBL clonotypes present in the islets and PLN. These results show that insight into repertoire profiles of islet-infiltrating T cells can be obtained from PBL.

Influenza A virus infection of C57BL/6 (B6) mice is characterized by prominent CD8+ T cell responses to H2Db complexed with peptides from the viral nucleoprotein (NP366, ASNENMETM) and acid polymerase (PA224, SSLENFRAYV). An in vivo cytotoxicity assay that depends on the adoptive transfer of peptide-pulsed, syngeneic targets was used in this study to quantitate the cytotoxic potential of DbNP366- and DbPA224-specific acute and memory CD8+ T cells following primary or secondary virus challenge. Both T cell populations displayed equivalent levels of in vivo effector function when comparable numbers were transferred into naive B6 hosts. Cytotoxic activity following primary infection clearly correlated with the frequency of tetramer-stained CD8+ T cells. This relationship looked, however, to be less direct following secondary exposure, partly because the numbers of CD8+DbNP366+ T cells were greatly in excess. However, calculating the in vivo E:T ratios indicated that in vivo lysis, like many other biological functions, is threshold dependent. Furthermore, the capacity to eliminate peptide-pulsed targets was independent of the differentiation state (i.e., primary or secondary effectors) and was comparable for the two T cell specificities that were analyzed. These experiments provide insights that may be of value for adoptive immunotherapy, where careful consideration of both the activation state and the number of effector cells is required.

In this report, we studied T cell responses to a prostate cancer Ag by adoptively transferring tumor Ag-specific T cells into prostate tumor-bearing mice. Our findings demonstrate that CD8+ T cells initially encountered tumor Ag in the lymph node and underwent an abortive proliferative response. Upon isolation from the tumor, the residual tumor-specific T cells were functionally tolerant of tumor Ag as measured by their inability to degranulate and secrete IFN-γ and granzyme B. We next sought to determine whether providing an ex vivo-matured, peptide-pulsed dendritic cell (DC) vaccine could overcome the tolerizing mechanisms of tumor-bearing transgenic adenocarcinoma of the mouse prostate model mice. We demonstrate that tumor Ag-specific T cells were protected from tolerance following provision of the DC vaccine. Concurrently, there was a reduction in prostate tumor size. However, even when activated DCs initially present tumor Ag, T cells persisting within the tolerogenic tumor environment gradually lost Ag reactivity. These results suggest that even though a productive antitumor response can be initiated by a DC vaccine, the tolerizing environment created by the tumor still exerts suppressive effects on the T cells. Furthermore, our results demonstrate that when trying to elicit an effective antitumor immune response, two obstacles must be considered: to maintain tumor Ag responsiveness, T cells must be efficiently primed to overcome tumor Ag presented in a tolerizing manner and protected from the suppressive mechanisms of the tumor microenvironment.

Plasmacytoid dendritic cells (pDC) are capable of producing high levels of type I IFNs upon viral stimulation, and play a central role in modulating innate and adaptive immunity against viral infections. Whereas many studies have assessed myeloid dendritic cells (mDC) in the induction of antitumor immune responses, the role of pDC in antitumor immunity has not been addressed. Moreover, the interaction of pDC with other dendritic cell subsets has not been evaluated. In this study, we analyzed the capacity of pDC in stimulating an Ag-specific T cell response. Immunization of mice with Ag-pulsed, activated pDC significantly augmented Ag-specific CD8+ CTL responses, and protected mice from a subsequent tumor challenge. Immunization with a mixture of activated pDC plus mDC resulted in increased levels of Ag-specific CD8+ T cells and an enhanced antitumor response compared with immunization with either dendritic cell subset alone. Synergy between pDC and mDC in their ability to activate T cells was dependent on MHC I expression by mDC, but not pDC, suggesting that pDC enhanced the ability of mDC to present Ag to T cells. Our results demonstrate that pDC and mDC can interact synergistically to induce an Ag-specific antitumor immune response in vivo.

Superantigens activate large fractions of T cells through unconventional interactions with both TCR β-chain V domains (Vβs) and MHC class II molecules. The bacterial superantigen streptococcal pyrogenic exotoxin C (SpeC) primarily stimulates human Vβ2+ T cells. Herein, we have analyzed the SpeC-Vβ2.1 interaction by mutating all SpeC residues that make contact with Vβ2.1 and have determined the energetic and functional consequences of these mutations. Our comprehensive approach, including mutagenesis, functional readouts from both bulk T cell populations, and an engineered Vβ2.1+ Jurkat T cell, as well as surface plasmon resonance binding analysis, has defined the SpeC “functional epitope” for TCR engagement. Although only two SpeC residues (Tyr15 and Arg181) are critical for activation of virtually all human CD3+ T cells, a larger cluster of four hot spot residues are required for interaction with Vβ2.1. Three of these residues (Tyr15, Phe75, and Arg181) concentrate their binding energy on the CDR2 loop residue Ser52a, a noncanonical residue insertion found only in Vβ2 and Vβ4 chains. Plasticity of this loop is important for recognition by SpeC. Although SpeC interacts with the Vβ2.1 hypervariable CDR3 loop, our data indicate these contacts have little to no influence on the functional interaction with Vβ2.1. These studies also provide a molecular basis for selectivity and cross-reactivity of SpeC-TCR recognition and reveal a degree of fine specificity in these interactions, whereby certain SpeC mutants are capable of distinguishing between different alleles of the same Vβ domain subfamily.

Human adenovirus (HAdV) infection is a frequent and potentially severe complication following allogeneic stem cell transplantation in children. Because treatment with antiviral drugs is often ineffective, adoptive transfer of donor-derived HAdV-specific T cells able to control viral replication of HAdV of multiple serotypes may be an option for therapy. In healthy donors, predominantly HAdV-specific T cells expressing CD4 are detected. In this study, a preclinical in vitro model was used to measure the antiviral effect of HAdV-specific CD4+ T cells. CD4+ HAdV-specific T cell clones restricted by HLA class II molecules were generated and most of these clones recognized conserved peptides derived from the hexon protein. These cross-reactive T cell clones were able to control viral replication of multiple serotypes of HAdV in EBV-transformed B cells (B-LCL), melanoma cells (MJS) and primary bronchial epithelial cells through cognate interaction. The HAdV-specific CD4+ T cell clones were able to specifically lyse infected target cells using a perforin-dependent mechanism. Antigenic peptides were also presented to the CD4+ T cell clones when derived from endogenously produced hexon protein. Together, these results show that cross-reactive HAdV-specific CD4+ T cells can control replication of HAdV in vitro and provide a rationale for the use of HAdV-specific T cells in adoptive immunotherapy protocols for control of life-threatening HAdV-infections in immunocompromised patients.

TLR are evolutionarily conserved molecules that play a key role in the initiation of innate antimicrobial immune responses. Through their influence on dendritic cell maturation, these receptors are also thought to indirectly shape the adaptive immune response. However, no data are currently available regarding both TLR expression and function in human CD8+ T cell subsets. We report that a subpopulation of CD8+ T cells, i.e., effector, but neither naive nor central memory cells, constitutively expresses TLR3. Moreover, the ligation of the receptor by a specific agonist in TLR3-expressing CD8+ T cells increased IFN-γ secretion induced by TCR-dependent and -independent stimulation, without affecting proliferation or specific cytolytic activity. These results thereby suggest that TLR3 ligands can not only indirectly influence the adaptive immune response through modulation of dendritic cell activation, but also directly increase IFN-γ production by Ag-specific CD8+ T cells. Altogether, the present work might open new perspectives for the use of TLR ligands as adjuvants for immunotherapy.

Immune elimination of tumor cells requires the close cooperation between CD8+ CTL and CD4+ Th cells. We circumvent MHC class II-restriction of CD4+ T cells by expression of a recombinant immunoreceptor with an Ab-derived binding domain redirecting specificity. Human CD4+ T cells grafted with an immunoreceptor specific for carcinoembryonic Ag (CEA) are activated to proliferate and secrete cytokines upon binding to CEA+ target cells. Notably, redirected CD4+ T cells mediate cytolysis of CEA+ tumor cells with high efficiencies. Lysis by redirected CD4+ T cells is independent of death receptor signaling via TNF-α or Fas, but mediated by perforin and granzyme because cytolysis is inhibited by blocking the release of cytotoxic granules, but not by blocking of Fas ligand or TNF-α. CD4+ T cells redirected by Ab-derived immunoreceptors in a MHC class II-independent fashion substantially extend the power of an adoptive, Ag-triggered immunotherapy not only by CD4+ T cell help, but also by cytolytic effector functions. Because cytolysis is predominantly mediated via granzyme/perforin, target cells that are resistant to death receptor signaling become sensitive to a cytolytic attack by engineered CD4+ T cells.

Human T cell lymphotropic virus type 1 (HTLV-1)-specific CTL are thought to be immune effectors that reduce the risk of adult T cell leukemia (ATL). However, in vivo conditions of anti-HTLV-1 CTL before and after ATL development have yet to be determined. To characterize anti-HTLV-1 CTL in asymptomatic HTLV-1 carriers (AC) and ATL patients, we analyzed the frequency and diversity of HTLV-1-specific CD8+ T cells in PBMC of 35 AC and 32 ATL patients using 16 distinct epitopes of HTLV-1 Tax or Env/HLA tetramers along with intracellular cytolytic effector molecules (IFN-γ, perforin, and granzyme B). Overall frequency of subjects possessing Tax-specific CD8+ T cells was significantly lower in ATL than AC (53 vs 90%; p = 0.001), whereas the difference in Env-specific CD8+ T cells was not statistically significant. AC possessed Tax11–19/HLA-A*0201-specific tetramer+ cells by 90% and Tax301–309/HLA-A*2402-specific tetramer+ cells by 92%. Some AC recognized more than one epitope. In contrast, ATL recognized only Tax11–19 with HLA-A*0201 and Tax301–309 with HLA-A*2402 at frequencies of 30 and 55%. There were also significant differences in percentage of cells binding Tax11–19/HLA-A*0201 and Tax301–309/HLA-A*2402 tetramers between AC and ATL. Anti-HTLV-1 Tax CD8+ T cells in AC and ATL produced IFN-γ in response to Tax. In contrast, perforin and granzyme B expression in anti-HTLV-1 CD8+ T cells of ATL was significant lower than that of AC. Frequency of Tax-specific CD8+ T cells in AC was related to proviral load in HLA-A*0201. These results suggest that decreased frequency, diversity, and function of anti-HTLV-1 Tax CD8+ T cell clones may be one of the risks of ATL development.

H2-M3-restricted CD8+ T cells provide early protection against bacterial infections. In this study, we demonstrate that activated H2-M3-restricted T cells provide early signals for efficient CD4+ T cell priming. C57BL/6 mice immunized with dendritic cells coated with the MHC class II-restricted listeriolysin O peptide LLO190–201 (LLO) generated CD4+ T cells capable of responding to Listeria monocytogenes (LM) infection. Inclusion of a H2-M3-restricted formylated peptide fMIGWII (fMIG), but not MHC class Ia-restricted peptides, during immunization with LLO significantly increased IFN-γ-producing CD4+ T cell numbers, which was associated with increased protection against LM infection. Studies with a CD4+ T cell-depleting mAb indicate that the reduction in bacterial load in fMIG plus LLO immunized mice is likely due to augmented numbers of LLO-specific CD4+ T cells, generated with the help of H2-M3-restricted CD8+ T cells. We also found that augmentation of LLO-specific CD4+ T lymphocytes with H2-M3-restricted T cells requires presentation of LLO and fMIG by the same dendritic cells. Interestingly, the augmented CD4+ T cell response generated with fMIG also increased primary LM-specific responses by MHC class Ia-restricted CD8 T cells. Coimmunization with LLO and fMIG also increases the number of memory Ag-specific CD4+ T cells. We also demonstrate that CD8 T cells restricted to another MHC class Ib molecule, Qa-1, whose human equivalent is HLA-E, are also able to enhance Ag-specific CD4+ T cell responses. These results reveal a novel function for H2-M3- and Qa-1-restricted T cells; provision of help to CD4+ Th cells during the primary response.

Many recombinant poxviral vaccines are currently in clinical trials for cancer and infectious diseases. However, these agents have failed to generate T cell responses specific for recombinant gene products at levels comparable with T cell responses associated with natural viral infections. The recent identification of vaccinia-encoded CTL epitopes, including a new epitope described in this study, allows the simultaneous comparison of CTL responses specific for poxviral and recombinant epitopes. We performed detailed kinetic analyses of CTL responses in HLA-A*0201 patients receiving repeated injections of recombinant modified vaccinia Ankara encoding a string of melanoma tumor Ag epitopes. The vaccine-driven CTL hierarchy was dominated by modified vaccinia Ankara epitope-specific responses, even in patients who had not received previous smallpox vaccination. The only recombinant epitope that was able to impact on the CTL hierarchy was the melan-A26–35 analog epitope, whereas responses specific for the weaker affinity epitope NY-ESO-1157–165 failed to be expanded above the level detected in prevaccination samples. Our results demonstrate that immunodominant vaccinia-specific CTL responses limit the effectiveness of poxviruses in recombinant vaccination strategies and that more powerful priming strategies are required to overcome immunodominance of poxvirus-specific T cell responses.

HIV-1 replication is associated with reduced or absent HIV-1-specific CD4+ T cell proliferation and skewing of HIV-1-specific CD4+ T cells toward an IFN-γ-producing, CCR7− phenotype. The CCR7− T cell population is heterogeneous and can be subdivided based on the expression of CD57. Although CD57 expression on CD8+ T cells is associated with proliferation incompetence and replicative senescence, less is known about the function of CD57-expressing CD4+ T cells. In this study, the frequency, phenotype, and function of CD57+CD4+ T cells were evaluated in 25 HIV-1-infected subjects and 10 seronegative controls. CD57+CD4+ T cells were found to be proliferation incompetent, even after strong mitogen stimulation. Percentages of CD4+ T cells that expressed CD57 were significantly higher in untreated HIV-1-infected subjects than in HIV-1-seronegative donors, and CD57 expression did not normalize in subjects receiving at least 6 mo of effective antiretroviral therapy. CD57 was predominately expressed on the CCR7− fraction of the CD4+ T cell compartment and accounted for the majority of cells in the CCR7−CD45RA+ population from untreated HIV-1-infected subjects. HIV-1-specific CD4+ T cells producing only IFN-γ had the highest expression of CD57, whereas few cells producing IL-2 alone expressed CD57. These findings further define a novel population of proliferation-incompetent CD4+ T cells that are generated in the presence of chronic Ag exposure. A better understanding of the generation and persistence of CD57+ T cells in HIV-1 infection could provide important insights into the immunopathogenesis of this disease.

A vigorous hepatitis C virus (HCV)-specific Th cell response is regarded as essential to the immunological control of HCV viremia. The aim of this study was to comprehensively define the breadth and specificity of dominant HCV-specific CD4+ T cell epitopes in large cohorts of subjects with chronic and spontaneously resolved HCV viremia. Following in vitro stimulation of PBMC, HCV-specific cell cultures from each subject were screened with an overlapping panel of synthetic 20-mer peptides spanning the entire HCV polyprotein. Of 22 subjects who spontaneously controlled HCV viremia, all recognized at least one of a group of six epitopes situated within the nonstructural (NS) proteins NS3, NS4, and NS5, each of which was detected by >30% of subjects, but most subjects recognized additional, more heterogeneous specificities. In contrast, none of the most frequently targeted epitopes was detected by >5% of persons with chronic infection. The most frequently recognized peptides showed promiscuous binding to multiple HLA-DR molecules in in vitro binding assays and were restricted by different HLA-DR molecules in functional assays in different persons. These data demonstrate that predominant CD4+ T cell epitopes in persons with resolved HCV infection are preferentially located in the nonstructural proteins and are immunogenic in the context of multiple class II molecules. This comprehensive characterization of CD4+ T cell epitopes in resolved HCV infection provides important information to facilitate studies of immunopathogenesis and HCV vaccine design and evaluation.

cDNAs encoding TCR α- and β-chains specific for HLA-A2-restricted cancer-testis Ag NY-ESO-1 were cloned using a 5′RACE method from RNA isolated from a CTL generated by in vitro stimulation of PBMC with modified NY-ESO-1-specific peptide (p157–165, 9V). Functionality of the cloned TCR was confirmed by RNA electroporation of primary PBL. cDNA for these α- and β-chains were used to construct a murine stem cell virus-based retroviral vector, and high titer packaging cell lines were generated. Gene transfer efficiency in primary T lymphocytes of up to 60% was obtained without selection using a method of precoating retroviral vectors onto culture plates. Both CD4+ and CD8+ T cells could be transduced at the same efficiency. High avidity Ag recognition was demonstrated by coculture of transduced lymphocytes with target cells pulsed with low levels of peptide (

The members of the lymphotoxin (LT) family of molecules play a critical role in lymphoid organogenesis. Whereas LTα-deficient mice lack all lymph nodes and Peyer’s patches, mice deficient in LTβ retain mesenteric lymph nodes and cervical lymph nodes, suggesting that an LTβ-independent pathway exists for the generation of mucosal lymph nodes. In this study, we describe the presence of a lymph node in LTβ-deficient mice responsible for draining the genital mucosa. In the majority of LTβ-deficient mice, a lymph node was found near the iliac artery, slightly misplaced from the site of the sacral lymph node in wild-type mice. The sacral lymph node of the LTβ-deficient mice, as well as that of the wild-type mice, expressed the mucosal addressin cell adhesion molecule-1 similar to the mesenteric lymph node. Following intravaginal infection with HSV type 2, activated dendritic cells capable of stimulating a Th1 response were found in this sacral lymph node. Furthermore, normal HSV-2-specific IgG responses were generated in the LTβ-deficient mice following intravaginal HSV-2 infection even in the absence of the spleen. Therefore, an LTβ-independent pathway exists for the development of a lymph node associated with the genital mucosa, and such a lymph node serves to generate potent immune responses against viral challenge.

Recognition by CD8+ T lymphocytes (CTL) of epitopes that are derived from conserved gene products, such as Gag and Pol, is well documented and conceptually supports the development of epitope-based vaccines for use against diverse HIV-1 subtypes. However, many CTL epitopes from highly conserved regions within the HIV-1 genome are highly variable, when assessed by comparison of amino acid sequences. The TCR is somewhat promiscuous with respect to peptide binding, and, as such, CTL can often recognize related epitopes. In these studies, we evaluated CTL recognition of five sets of variant HIV-1 epitopes restricted to HLA-A*0201 and HLA-A*1101 using HLA transgenic mice. We found that numerous different amino acid substitutions can be introduced into epitopes without abrogating their recognition by CTL. Based on our findings, we constructed an algorithm to predict those CTL epitopes capable of inducing responses in the HLA transgenic mice to the greatest numbers of variant epitopes. Similarity of CTL specificity for variant epitopes was demonstrated for humans using PBMC from HIV-1-infected individuals and CTL lines produced in vitro using PBMC from HIV-1-uninfected donors. We believe the ability to predict CTL epitope immunogenicity and recognition patterns of variant epitopes can be useful for designing vaccines against multiple subtypes and circulating recombinant forms of HIV-1.

Formation of an immunological synapse (IS) between APCs and T CD4+ lymphocytes is a key event in the initiation and the termination of the cognate immune response. We have analyzed the contribution of the APC to IS formation and report the implication of the actin cytoskeleton, the signaling proteins and the lipid rafts of B lymphocytes. Recruitment of MHC class II molecules to the IS is concomitant with actin cytoskeleton-dependent B cell raft recruitment. B cell actin cytoskeleton disruption abrogates both IS formation and T cell activation, whereas protein kinase C inhibition only impairs T cell activation. Pharmacological B cell lipid raft disruption inhibited peptide-dependent T lymphocyte activation and induced peptide-independent but HLA-DR-restricted APC-T cell conjugate formation. Such peptide-independent conjugates did not retain the ability to activate T cells. Thus, B cell lipid rafts are bifunctional by regulating T cell activation and imposing peptide stringency.

Two papers highlighted here as Pillars of Immunology led to the discovery, more than two decades ago, of expanded T lymphocyte populations with a limited TCR α-chain diversity ([1][1], [2][2]). This would be a mere factoid if these specialized cell populations were not highly conserved, suggestive