TCRBV Gene Research Articles

Profiling of T Cell Receptor β-Chain Complimentary Determining Regions 3 Repertoire in Subarachnoid Hemorrhage Patients Using High-Throughput Sequencing.

ObjectiveThe adaptive immune response following subarachnoid hemorrhage (SAH) is not well understood. We evaluated and compared the T cell receptor (TCR) immune repertoire of good-grade and poor-grade SAH patients to elucidate the T cell immunology after ictus. MethodsPeripheral blood from six SAH patients was collected at two different times, admission and at the 7-day follow-up. Composition and variation of the TCR β-chain (TCRB) complimentary determining regions (CDR) 3 repertoire was examined using high-throughput sequencing; the analysis was based on sampling time and disease severity (good vs. poor-grade SAH). ResultsClonality at admission and follow-up were 0.059 (0.037–0.038) and 0.027 (0.014–0.082) (median, 25th–75th percentile). Poor-grade SAH (0.025 [0.011–0.038]) was associated with significantly lower clonality than good-grade SAH (0.095 [0.079–0.101]). Poor-grade SAH patients had higher diversity scores than good-grade SAH patients. CDR length was shorter in good-grade SAH vs. poor-grade SAH. Differences in clonotype distribution were more prominent in TCRBV gene segments than TCRBJ segments. TCRBV19-01/TCRBJ02-04 and TCRBV28-01/TCRBJ02-04 were the most increased and the most decreased V-J pairs in the 7-day follow-up compared to admission in good-grade SAH. The most increased and decreased V-J pairs in poor-grade SAH patients were TCRBV28-01/TCRBJ02-06 and TCRBV30-01/TCRBJ02-04, respectively. ConclusionThe TCRB repertoire is dynamic in nature following SAH. TCRB repertoire may facilitate our understanding of adaptive immune response according to SAH severity.

Clonal Kinetics and Single Cell Transcriptional Profiling of Adoptively Transferred CD19 CAR-T Cells

INTRODUCTION:When introduced into polyclonal T cells, chimeric antigen receptors (CAR) redirect specificity of the engineered T cells to an antigen recognized by the CAR. We conducted a phase I/II clinical trial of treatment of relapsed and refractory CD19-positive B cell malignancies using a defined formulation of CD4+ and CD8+ CD19-specific CAR-T cells (NCT01865617). Little is known about the transcriptional heterogeneity of CAR-T cells in the infused product and their clonal kinetics after adoptive transfer.METHODS:To understand the factors that impact clonal CAR-T cell behavior in vivo, we performed TCRBV sequencing and single cell transcriptional profiling (10X Genomics) on CD8+ CAR-T cells isolated from infused products and the blood of treated patients. TCRBV sequencing was performed on 0.8 to 1.5 million cells from the infused product and 700-65,000 CAR-T cells from blood after CAR-T infusion. For single-cell RNA sequencing (scRNAseq), we obtained paired 5' gene expression and V(D)J data from individual CAR-T cells isolated from infused products and from the blood at the peak of in vivo expansion, after contraction, and at a late time point.RESULTS:High-throughput sequencing of the TCRBV genes revealed that CAR-T cells were polyclonal in the infused products, and during in vivo expansion and contraction, and at late times (≥ 3 months) after adoptive transfer. We evaluated the diversity of the TCRBV repertoire using the Shannon entropy index, and found that clonal diversity was highest in the infused product and declined at later time points after adoptive transfer. Loss of diversity after adoptive transfer was due to both expansion of higher frequency CAR-T cell clones and loss of low-frequency clones. We identified distinct CAR-T cell clones in the infused product and in blood at multiple time points after infusion that exhibited different kinetics of expansion and contraction. To examine the transcriptional programs that regulate the fate of CAR-T cells after infusion, we performed scRNAseq on CD8+ CAR-T cells, and found transcriptional heterogeneity in the infused products, which declined in CD8+ CAR-T cells isolated from patient blood after adoptive transfer. Gene set enrichment analysis showed that the infused products expressed higher levels of genes associated with hypoxia, glycolysis, and proliferation, and lower levels of genes associated with cytotoxicity compared to CAR-T cells isolated after adoptive transfer. In the infused product, genes associated with cytotoxicity were expressed at higher levels in CAR-T cells harboring clonotypes that were subsequently represented at relatively higher levels in vivo after adoptive transfer.CONCLUSIONS:There is transcriptional heterogeneity in the infused product and distinct CAR-T cell clones exhibit different kinetics of expansion and contraction after infusion. A better understanding of the kinetics of clonal expansion of CAR-T cells after adoptive transfer may provide insight into strategies to improve CAR-T cell immunotherapy. DisclosuresTurtle:Nektar Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Caribou Biosciences: Membership on an entity's Board of Directors or advisory committees; Juno/Celgene: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Structural and Mechanistic Implications of Rearrangement Frequencies within Human TCRBV Genes.

The T cell repertoire is a function of thymic V(D)J rearrangement and of peripheral selection. The mature repertoire embodies TCR sequences that are important for survival and can identify important structural aspects of the TCR. Analysis of the circulating TCRBV19 CD8 T cell repertoire showed that a majority of NDN-encoded CDR3 amino acid motifs start at CDR3 position four, well within the V region. Rearrangement at this position indicates that the DNA hairpin loop is not opened at the position adjacent to the recombination signal sequence, but rather is trimmed back three or more bases. In this article, we show that the rearrangement frequency distribution within the V region reveals selection on CDR3 position four. The selection is already established in single-positive CD8 thymocytes. Crystal structures reveal a possible basis for this selection due to the location of this residue in a bend that positions the remaining portion of CDR3 to interact with the peptide and MHC. Examination of other TCRBV families also shows selection for rearrangement within the V region of a number of genes and for CD8 and CD4 cells. The exact profile of rearrangement within the V region appears to be V gene specific. The frequent observation of side chains associated with turn motifs at CDR3 positions three and four fits with the structural need for a bend. The data are discussed in terms of the generation of a structural turn motif, the rearrangement mechanism, and selection of the repertoire on the peptide and MHC.

Abstract P4-04-10: Early-stage breast carcinomas are infiltrated by oligoclonal T cell populations highly enriched relative to the blood

Abstract BACKGROUD: The immunogenicity of some human tumors towards T lymphocytes is well established. Recently, encouraging results have been obtained with immunotherapies inhibiting immune checkpoints in cancers such as melanoma, NSCLC and bladder cancer. Fewer studies explored these treatments in breast cancer (BC) as these tumors are often considered to be poorly immunogenic. METHODS: We analysed the T cell receptor β-chains variable genes (TCRBV) repertoires of tumor-infiltrating T cells in 17 early BC. We looked for clonally amplified T cells as their presence is an expected consequence of tumor immunogenicity. RNA was extracted and reverse-transcribed from formalin-fixed, paraffin-embedded tumor tissues. A short random sequence was added to the cDNA and used as a unique molecular identifier (UMI) for each cDNA molecule. cDNA encoding TCRBV genes was then amplified and sequenced using high throughput sequencing. Usage of UMIs during this procedure strongly improved the accuracy of the analysis by avoiding amplification biases inherent to the construction of the TCRBV library and by allowing an absolute quantification of TCRBV mRNA molecules normalized with the RPP30 housekeeping gene. TCRBV sequences were aligned using IMGT/HighV-QUEST. The Simpson's index was used to evaluate TCRBV repertoires diversity (ranging from 0 = infinite diversity to 1 = no diversity). For 3 patients, the same procedure was applied on blood T cells collected a few days before tumor resection and the analysis was also carried out on 3 normal tissues obtained from breast reduction surgery. RESULTS: T cell infiltration varied strongly from one tumor to another ranging from 5 to 2498 TCRBV/103 RPP30 mRNA molecules. TCRBV repertoires analysis indicated that infiltrated T cells corresponded to oligoclonal populations. We observed 3 clonotypes in the smaller repertoire and 74 in the largest one and the Simpson's index ranged from 0.01 to 0.65. Most tumors (16/17) contained at least one clonotype that made up ≥10% of the infiltrating T cells, with the highest observed proportions reaching 80%. Normal breast samples were infiltrated by a more diverse repertoire: 130 to 368 clonotypes were identified in those tissues and Simpson's index ranged from 0.002 to 0.008. Highest observed frequency among those clonotypes was 2%. For 3 BC patients, the frequencies of the most prevalent clonotypes in the tumor were compared to those of the same clonotypes in blood prior to surgery. These T cell clones were 250 to >34000 times more frequent in the tumor than in the blood. CONCLUSIONS: Some early BC are infiltrated by oligoclonal T cell populations that are highly enriched relative to the blood. Quantitative T cell repertoire analysis allows to distinguish 3 types of BC: (1) tumors without T cell infiltration, (2) tumors with a high T cell infiltration and a small T cell repertoire, and (3) tumors with a high T cell infiltration and a large repertoire. Our observations suggest that anti-tumor T cell responses are ongoing in some early BC and this warrants boosting such responses with immune checkpoint inhibitors in selected patients. T cell repertoire evaluation could be used as a predictive biomarker to identify patients who will benefit from this treatment. Citation Format: Carrasco J, Schröder D, Coulie PG, Godelaine D, Berlière M, Theate I, Delrée P, Vannuffel P, Galant C, Duhoux FP, Machiels J-P, Canon J-L. Early-stage breast carcinomas are infiltrated by oligoclonal T cell populations highly enriched relative to the blood. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-10.

The Ankylosing Spondylitis-associated HLA-B*2705 presents a B*0702-restricted EBV epitope and sustains the clonal amplification of cytotoxic T cells in patients.

HLA-B*27 is strongly associated with an inflammatory autoimmune disorder, the Ankylosing Spondylitis (AS) and plays a protective role in viral infections. The two aspects might be linked. In this work, we compared in B*2705/B*07 positive patients with AS, the CD8+ T cell responses to two immunodominant EBV-derived epitopes restricted for either the HLA-B*27 (pEBNA3C) or the HLA-B*07 (pEBNA3A). We have unexpectedly found that the HLA-B*07-restricted EBNA3A peptide is presented by both the B*0702 and the B*2705 but not by the non AS-associated B*2709, that differs from the AS-associated B*2705 for a single amino acid in the peptide-binding groove (His116Asp). We then analysed 38 B*2705-positive/B*07-negative (31 AS-patients and 7 healthy donors) and 8 B*2709-positive/B*07-negative subjects. EBNA3A-specific CD8+ T lymphocytes were present in 55.3% of the HLA-B*2705 but in none of the B*2709 donors (p=0.0049). TCR β-chain analysis identified common TCRBV and TCRBJ gene segments and shared CDR3β sequences in pEBNA3A-responsive CTLs of B*2705 carriers, suggesting the existence of a shared TCR repertoire for recognition of the uncanonical B*2705/pEBNA3A complex. These data highlight the plasticity of the AS-associated HLA-B*2705, which presents peptides with suboptimal binding motifs, possibly contributing both to its enhanced capacity to protect against pathogens and to predispose to autoimmunity.

Factors Influencing Reconstitution of Mucosal-Associated Invariant T Cells Following Allogeneic Hematopoietic Stem Cell Transplantation

Background: Mucosal-associated invariant T (MAIT) cells are innate-like T cells characterized by high expression of CD161 and a semi-invariant T cell receptor (TCR) comprised of a Vα7.2-Jα33 alpha chain and a limited Vβ repertoire that enables their activation by riboflavin metabolites produced by distinct bacterial and fungal species. MAIT cells are infrequent in cord blood, but undergo TCR-dependent accumulation in neonates in response to gastrointestinal (GI) commensal colonization to comprise approximately 10% of T cells in adult blood. The GI localization of MAIT cells, their capacity to secrete IL-17, and their activation by microbial metabolites suggests a role in mucosal immunity that may be particularly important after allogeneic hematopoietic stem cell transplantation (HCT) when the GI mucosal barrier is compromised and adaptive immunity is impaired. After HCT, the composition of the GI microbiota may be modified by antibiotics, mucositis and immunosuppression, yet its impact on MAIT cell reconstitution, function and post-transplant immunity remain unknown.Aims: To characterize and identify factors influencing MAIT cell reconstitution and function after HCT.Methods: Blood and stool samples were collected from healthy donors and HCT patients prior to and at distinct times after HCT. Absolute counts of MAIT cells, identified as CD3+/CD161hi/Vα7.2+events, were determined in peripheral blood using flow cytometry performed in conjunction with a complete blood count. The bacterial composition of stool was characterized using bacterial 16S rRNA gene PCR with high throughput sequencing and phylogenetic assignment of the amplified fragments. TCR signaling pathway activation in MAIT cells and conventional T cells was evaluated using flow cytometry analysis of phosphoprotein expression after stimulation through the TCR-CD3 complex with anti-CD3/anti-CD28 monoclonal antibodies. TCR Vβ repertoire assessment was performed using high throughput TCRBV gene sequencing.Results: High throughput TCRBV gene sequencing showed that MAIT cells from different donors (n = 3) shared TCRBV sequences, consistent with their capacity to be activated by common GI microbial TCR ligands. Despite GI microbial colonization, MAIT cells from adult donor blood were quiescent and did not proliferate to TCR stimulation. Phosphoprotein flow cytometry established that phosphorylation of proximal TCR signaling pathway molecules (CD3ζ, Lck, and ZAP-70) was diminished and responsible for impaired TCR signaling in adult MAIT cells compared to conventional αβ T cells. MAIT cell proliferation was restored by TCR stimulation in the presence of IL-1β, IL-12, IL-18 and IL-23, raising the possibility that the post-HCT inflammatory environment might be permissive for MAIT cell proliferation driven by GI microbial TCR ligands. We examined the kinetics of MAIT cell reconstitution in HCT patients (n = 163). MAIT cell numbers were lower in patients before conditioning compared to healthy individuals, and were further depleted on the day of stem cell infusion; however, they proliferated in the post-HCT environment in association with induction of Ki67 expression and reached a plateau after day 30 post-HCT (healthy, 56.8/μL; day 30, 6.7/μL). MAIT cell reconstitution after peripheral blood stem cell (PBSC) transplantation was similar comparing myeloablative (MA) and reduced intensity conditioning (RIC) regimens and related compared to unrelated donors, but was highly variable between individuals. Short tandem repeat PCR chimerism studies showed that MAIT cells were of donor origin early after MA and RIC PBSC transplantation. MAIT cell reconstitution was markedly impaired in recipients of cord blood, which contains few MAIT cells, compared to those receiving PBSC, in which MAIT cells are plentiful, suggesting that early MAIT cell reconstitution is primarily derived from mature cells transferred with the HCT graft. Analysis of stool samples from HCT recipients (n = 17) has shown that the relative abundance of distinct gut bacterial species is highly variable between recipients and changed during the course of HCT. Analyses of the relationship between the microbiota and MAIT cell reconstitution will be presented.Conclusions: MAIT cell recovery following HCT varies between different types of transplants and may be influenced by the transferred graft source, the post-HCT environment, and the gut microbiome. DisclosuresNo relevant conflicts of interest to declare.

Therapy of B Cell Malignancies with CD19-Specific Chimeric Antigen Receptor-Modified T Cells of Defined Subset Composition

BACKGROUND:The adoptive transfer of CD19-specific chimeric antigen receptor-modified (CD19 CAR) T cells is a promising strategy for treating patients with CD19+ B cell acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphoma (NHL). Dramatic responses have been observed in a subset of patients receiving CD19 CAR T cell therapy, and prior studies suggest that persistence of transferred T cells may correlate with the extent of tumor regression. The use of unselected T cells to prepare CAR T cells results in variation in the phenotypic composition of the infused product in individual patients, making it difficult to determine whether particular T cell subsets contribute to efficacy and/or toxicity. Studies in our lab demonstrated that genetically modified effector T cells derived from purified T cell subsets differ in the capacity to persist in vivo after adoptive transfer, and that a combination of CAR-modified CD8+ central memory (TCM) and CD4+ T cells provides optimal antitumor activity in tumor xenograft models. Based on these data, we designed the first clinical trial in which patients with CD19+ B cell malignancies receive CD19 CAR T cells comprised of a defined composition of CD8+ TCM and CD4+T cells engineered to express a CD19 CAR.METHODS:Patients with relapsed or refractory CD19+ ALL, CLL or NHL are eligible for this phase I/II study. CD8+ TCM and CD4+ T cells were separately enriched by immunomagnetic selection from a leukapheresis product from each patient, and cryopreserved. The CD8+ TCM and CD4+ T cells were stimulated in independent cultures with anti-CD3/anti-CD28 paramagnetic beads, and transduced with a lentivirus encoding the murine FMC63 anti-CD19 scFv, 4-1BB and CD3 zeta signaling domains. After in vitro expansion, the cell product for infusion was formulated in a 1:1 ratio of CD4+:CD8+ CAR+ T cells. A truncated non-functional human epidermal growth factor receptor (EGFRt) encoded in the transgene cassette allowed identification of transgene-expressing T cells by flow cytometry. Lymphodepleting chemotherapy was administered followed by infusion of EGFRt+ CAR T cells at one of three dose levels (2 x 105 EGFRt+ cells/kg, 2 x 106 EGFRt+ cells/kg, 2 x 107 EGFRt+cells/kg).RESULTS:Twenty patients with relapsed or refractory ALL (n = 9), NHL (n = 10) or CLL (n = 1), including those who failed prior autologous (n = 4) or allogeneic (n = 4) stem cell transplant have been treated on the trial. Fifteen of 20 treated patients received a product that conformed to the prescribed CD8+ T­CM:CD4 composition. Five patients received a product manufactured using a modified strategy either due to low blood lymphocyte counts (n = 3) or due to failure to propagate T cells in culture (n = 2). CD8+ TCM and CD4+ T cells have been isolated from 12 additional patients and cryopreserved for therapy. Patients have been treated at all three dose levels without acute infusional toxicity. Severe cytokine release syndrome (sCRS) consisting of fever, hypotension, and reversible neurotoxicity associated with elevated serum IFN-γ and IL-6 was only observed in ALL patients with a high tumor burden. One ALL patient treated at the highest cell dose died of complications associated with sCRS. None of the NHL patients had sCRS. Of patients who are >6 weeks after CD19 CAR T cell therapy, best responses included complete (n=1) or partial (n=5) remission in 6/9 patients with NHL and complete remission in 5/7 patients with ALL. Both CD4+ and CD8+ CAR-T cells expanded in vivo and could be detected in blood, marrow and CSF. The peak level and duration of persistence of both CD4+ and CD8+ EGFRt+ T cells were associated with clinical response. TCRBV gene sequencing of flow sorted CD4+ and CD8+ EGFRt+CAR T cells from 2 patients showed that proliferating CAR T cells were polyclonal. A subset of NHL patients in whom CAR T cells became undetectable developed a T cell immune response to sequences in the murine CD19-specific scFv component of the CAR transgene.CONCLUSION:Adoptive immunotherapy with CD19 CAR T cells of defined subset composition is feasible and safe in a majority of heavily pretreated patients with refractory B cell malignancies and has potent anti-tumor activity. Persistence of CAR-T cells may be limited in some patients by transgene product immunogenicity. Data from this ongoing clinical trial will be updated at the meeting. DisclosuresTurtle:Juno Therapeutics: Research Funding. Berger:Juno Therapeutics: Patents & Royalties. Hudecek:Juno Therapeutics: Patents & Royalties. Jensen:Juno: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding. Riddell:Juno Therapeutics: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding. Maloney:Juno Therapeutics: Research Funding.

Comparing TCR beta chain variable gene profile skewness between children with tuberculosis and BCG-vaccinated children.

We compared the T cell antigen receptor (TCR-BV) gene families of peripheral blood mononuclear cells (PMBC) between children with tuberculosis (TB) and those inoculated with the Bacille Calmette Guerin (BCG) vaccine. The total RNA was extracted from PMBC of 15 TB children, 15 BCG-vaccinated children and 15 healthy controls. The RNAs were reverse-transcribed and amplified by polymerase chain reaction (PCR). PCR products were separated on 1.5% agarose gel and analyzed with the Genescan technique. Some TCR-BV gene families in TB children and BCG-vaccinated children exhibited a blur band in the predicted position on 1.5% agarose gel, some showed a distinct or fainted band. In general, many shared predominant clonal TCR-BV gene families (Vβ2, Vβ16, Vβ21, Vβ22) and the restricted-expression families (Vβ14 and Vβ17). All the gene families of the control children only exhibited blur bands and polyclonal. The skewed profile of TCR-BV gene families in TB children and BCG-vaccinated children are similar, which may probably explain the protective effects of BCG-vaccine against TB in children.

Biased usage of T cell receptor β‐chain variable region genes of Wilms’ tumor gene (WT1)‐specific CD8+ T cells in patients with solid tumors and healthy donors

Wilms' tumor gene 1 (WT1) protein is a promising tumor-associated antigen. In patients with WT1-expressing malignancies, WT1-specific CTLs are spontaneously induced as a result of an immune response to the WT1 protein. In the present study, we performed single cell-level comparative analysis of T cell receptor β-chain variable region (TCR-BV) gene families of a total of 750 spontaneously induced WT1(126) peptide (amino acids 126-134, WT1(126))-specific CTLs in both HLA-A*0201(+) patients with solid tumors and healthy donors (HDs). This is the first report of direct usage analysis of 24 kinds of TCR-BV gene families of WT1(126)-specific CTLs at the single cell level. Usage analysis with single-cell RT-PCR of TCR-BV gene families of individual FACS-sorted WT1(126) tetramer(+) CD8(+) T cells showed, for the first time, that: (i) BVs 3, 6, 7, 20, 27, and 28 were commonly biased in patients and HDs; (ii) BVs 2, 11, and 15 were biased only in patients; and (iii) BVs 4, 5, 9, and 19 were biased only in HDs. However, statistical analysis of similarity of individual usage frequencies of 24 kinds of TCR-BV gene families between patients and HDs indicated that the usage frequencies of TCR-BV gene families in patients reflected those in HDs. These results should provide us with a novel insight for a better understanding of WT1-specific immune responses.

Extending T-cell transfer therapy to melanoma and oral squamous carcinoma.

e13071 Background: T-cell based immune therapy in melanoma has shown markedly objective clinical responses in a single-center study at the NIH. In order to extend the treatment to other centers and investigate application to other histologies, we compare the characteristics of tumor infiltrating lymphocytes (TIL) expanded from two groups of cancer patients: malignant melanoma (MM) and squamous cell carcinoma (SCC) of the oral cavity. Methods: Expanded TIL cultures were examined for T-cell phenotype subsets using FACS and clonality by TCRBV gene segment semi quantification with PCR. Tumor reactivity was assessed by INFγ Elispot, CD107a expression by FACS and Cr51 release assay. To identify possible Tumor Associated Antigen (TAA) specific populations TIL were screened for a panel of epitopes from the known TAA Survivin, Telomerase, Bcl-x, Bcl-2, Ny-ESO 1, MART-1, and IDO. Results: Preclinical part: In both patient cohorts TIL were expanded within 5 weeks in 80% of the patients. Cultures comprised an oligoclonal repertoire of T effector memory cells (TEM). Tumor activity against HLA-A matched cell lines showed varying responses regarding frequency and magnitude; activity directed against autologous tumor cells were characterized by a strikingly high frequency of INFg producing cells and/or distinct CD107a+ populations with tumorlytic capacity. Peptide screening revealed that specific TIL cultures were not correlated with tumor activity although the TAA mRNA were identified in the tumor cell lines. Clinical part: TIL were expanded in 5 of 5 (100%) MM patients included. Kinetics, phenotypes, and preliminary activity data resemble preclinical results. Three patients have completed treatment and two have been evaluated by CT scan (1 SD and 1 PR). Immune monitoring is pending. Conclusions: Results on ex vivo expanding TEM from both MM and SCC show comparable outcome and demonstrates shared activity against several common TAA. A clinical pilot trial is ongoing in MM patients with metastatic disease and a pilot trial in patients with primary SCC stage T3/T4 will start in 2010. No significant financial relationships to disclose.

Public TCR use by herpes simplex virus-2-specific human CD8 CTLs.

Recombination of germline TCR alpha and beta genes generates polypeptide receptors for MHC peptide. Ag exposure during long-term herpes simplex infections may shape the T cell repertoire over time. We investigated the CD8 T cell response to HSV-2 in chronically infected individuals by sequencing the hypervariable regions encoding TCR alpha and beta polypeptides from T cell clones recognizing virion protein 22 aa 49-57, an immunodominant epitope. The most commonly detected TCRBV gene segment, found in four of five subjects and in 12 of 50 independently derived T cell clones, was TCRBV12-4. Nineteen to seventy-two percent of tetramer-binding cells in PBMCs were stained ex vivo with a TCRBV12 mAb. Three alpha-chain and three beta-chain public TCR sequences were shared between individuals. Public heterodimers were also detected. Promiscuous pairing of a specific TCRVA1-1 sequence with several different TCRB polypeptides was observed, implying a dominant structural role for the TCRA chain for these clonotypes. Functional avidity for cytotoxicity and IFN-gamma release was relatively invariant, except for one subject with both high avidity and unique TCR sequences and lower HSV-2 shedding. These data indicate that the CD8 response to a dominant alpha-herpesvirus epitope converges on preferred TCR sequences with relatively constant functional avidity.

Biased usage of BV gene families of T‐cell receptors of WT1 (Wilms’ tumor gene)‐specific CD8+ T cells in patients with myeloid malignancies

WT1 (Wilms' tumor gene 1) protein is a potent pan-tumor-associated antigen (TAA) and WT1-specific cytotoxic T lymphocytes (WT1 tetramer(+) CD8(+) T cells) are spontaneously induced in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). We conducted a single-cell level comparative analysis of T-cell receptor beta-chain variable region (TCR-BV) gene families of a total of 1242 spontaneously induced WT1 tetramer(+) CD8(+) T cells in HLA-A*2402(+) patients with AML or MDS and those in healthy donors (HDs). This is the first report of direct usage analysis of TCR-BV gene families of individual TAA-specific CD8(+) T cells at single-cell level. Usage analysis using single-cell RT-PCR of TCR-BV gene families of individual FACS-sorted WT1 tetramer(+) CD8(+) T cells showed for the first time (i) that BVs 5, 6, 20, and 27 were commonly biased in both HDs and patients; (ii) that BV4 was commonly biased in HDs and MDS patients; (iii) that BV19 was commonly biased in the patients; and (iv) that BVs 7 and 28, BVs 9 and 15, and BVs 12 and 29 were specifically biased in HDs, AML, and MDS patients, respectively. However, statistical analysis of similarity among HD, AML, and MDS of individual usage frequencies of 24 kinds of TCR-BV gene families indicated that the usage frequencies of TCR-BV gene families in AML and MDS patients reflect those in HDs. These findings represent a novel insight for a better understanding of WT1-specific immune response.

Minor viral and host genetic polymorphisms can dramatically impact the biologic outcome of an epitope-specific CD8 T-cell response

AbstractHuman immunodeficiency virus-1 subtypes A and C differ in the highly conserved Gag-TL9 epitope at a single amino acid position. Similarly, the TL9 presenting human leukocyte antigen (HLA) class I molecules B42 and B81 differ only at 6 amino acid positions. Here, we addressed the influence of such minor viral and host genetic variation on the TL9-specific CD8 T-cell response. The clonotypic characteristics of CD8 T-cell populations elicited by subtype A or subtype C were distinct, and these responses differed substantially with respect to the recognition and selection of TL9 variants. Irrespective of the presenting HLA class I molecule, CD8 T-cell responses elicited by subtype C exhibited largely comparable TL9 variant cross-recognition properties, expressed T-cell receptors that used almost exclusively the TRBV 12-3 gene, and selected for predictable patterns of viral variation within TL9. In contrast, subtype A elicited TL9-specific CD8 T-cell populations with completely different, more diverse TCRBV genes and did not select for viral variants. Moreover, TL9 variant cross-recognition properties were extensive in B81+ subjects but limited in B42+ subjects. Thus, minor viral and host genetic polymorphisms can dramatically alter the immunologic and virologic outcome of an epitope-specific CD8 T-cell response.

Changes in autoreactive T cell avidity during type 1 diabetes development

The activation threshold for antigen-specific T cell responses is dependent on the avidity of the trimolecular interaction between TCR, antigen, and MHC. We compared CD4+ T cell avidities for the diabetes-associated autoantigen glutamic acid decarboxylase 555-567 (GAD 555) among serial samples from autoantibody-positive subjects at high risk of progression to type 1 diabetes (T1D). T cells from three at-risk subjects demonstrated significant avidity increases (p<0.05 by F test) over time. This avidity shift correlated with the outgrowth of T cells expressing TCR BV 9, 15, 17 or 20 that demonstrated higher GAD 555 tetramer-binding levels compared to cells expressing other TCR BV genes. Similar analysis of autoantibody-negative, first-degree relatives and T1D patients did not demonstrate similar changes in avidity. These data implicate the outgrowth of T cells expressing higher affinity TCR in a process of antigen-specific T cell avidity maturation during the pre-clinical stage of T1D.

Association of T-cell receptor Vβ haplotypes with dry skin in DS-Nhmice

Although dry skin and T cell-dependent disease exacerbation are characteristic features of atopic dermatitis (AD), the involvement of T cells in the development of dry skin remains unclear. We aimed to elucidate the role of T cells in the development of dry skin in DS-Nh mice as a model for AD, and to evaluate this skin condition pharmacologically. We prepared DS-Nh mice harbouring a T-cell receptor (TCR)Vbeta(a) haplotype with a central deletion in the TCRBV gene segments, and mice harbouring a TCRVbeta(b) haplotype without any deletion. We analysed the TCRVbeta chain usage and cytokine response to antimouse CD3 monoclonal antibodies in the splenocytes from the two mouse substrains. Transepidermal water loss (TEWL) was measured, and histochemical examination of these mice was carried out. Finally, a pharmacological analysis using loratadine was also performed to evaluate the features of spontaneous dry skin in DS-Nh mice as a model of AD. Although the deletion of TCRBV gene segments in the TCRVbeta(a) haplotype yielded different representations of each TCRVbeta mRNA, this deletion did not evoke distinct cytokine profiles in the splenocytes compared with those of mice with the TCRVbeta(b) haplotype. Furthermore, our results indicated that the onset of dry skin occurred earlier in mice with TCRVbeta(b) than in those with TCRVbeta(a). Pharmacologically, AD-like dry skin in DS-Nh with TCRVbeta(b) mice is susceptible to an H1 blocker. A specific lymphocyte subpopulation bearing T-cell receptors may be responsible for loratadine-responsive dermatitis in DS-Nh mice.

Natural Naive CD4+CD25+CD127low Regulatory T Cell (Treg) Development and Function Are Disturbed in Multiple Sclerosis Patients: Recovery of Memory Treg Homeostasis during Disease Progression

Patients with relapsing-remitting multiple sclerosis (RR-MS) show a suboptimal CD4(+)CD25(+) regulatory T cell (Treg) function, whereas no Treg alterations are observed in secondary progressive MS (SP-MS) patients. To clarify the difference in Treg activity between early and chronic disease stages in MS, we analyzed the functional capacity and homeostatic parameters of naive CD4(+)CD25(+)CD127(low)CD45RA(+) Tregs (nTregs) and their memory counterparts CD4(+)CD25(+)CD127(low)CD45RO(+) Tregs (mTregs) in untreated MS patients and healthy controls. Interestingly, whereas the suppressive capacity of FACS-sorted nTregs was impaired in both early and chronic MS patients, only the latter group showed a restored mTreg function. Consistent with this observation, chronic MS patients had increased numbers of mTregs as compared with age-matched early MS patients, whereas nTreg frequencies did not differ significantly. TCR excision circle numbers were reduced in nTregs of early MS patients, suggestive of a diminished nTreg thymic output. Moreover, a decreased number of CD31(+) mTregs were observed in early vs chronic MS patients, indicating that inflammatory processes drive the homeostatic turnover of mTregs during the early disease stage. Additionally, early MS patients showed a more restricted nTreg and mTreg TCR BV gene profile as compared with healthy controls and chronic MS patients. Finally, analysis of IFN-beta and glatiramer acetate-treated MS patients showed that these immunomodulatory drugs modify nTreg homeostasis. Taken together, this study provides strong evidence for a disturbed thymic nTreg development and function in MS patients. Moreover, memory Treg but not naive Treg homeostasis recovers during disease progression.

The Context of Epitope Presentation Can Influence Functional Quality of Recalled Influenza A Virus-Specific Memory CD8+ T Cells

Lipopeptide constructs offer a novel strategy for eliciting effective cellular and humoral immunity by directly targeting the vaccine Ag to dendritic cells. Importantly, it is not known how closely immunity generated after lipopeptide vaccination mimics that generated after natural infection. We have used a novel lipopeptide vaccine strategy to analyze both the quantity and quality of CD8(+) T cell immunity to an influenza A virus epitope derived from the acidic polymerase protein (PA(224)) in B6 mice. Vaccination with the PA(224) lipopeptide resulted in accelerated viral clearance after subsequent influenza virus infection. The lipopeptide was also effective at recalling secondary D(b)PA(224) responses in the lung. Lipopeptide recalled D(b)PA(224)-specific CTL produced lower levels of IFN-gamma and TNF-alpha, but produced similar levels of IL-2 when compared with D(b)PA(224)-specific CTL recalled after virus infection. Furthermore, lipopeptide- and virus-recalled CTL demonstrated similar TCR avidity. Interestingly, lipopeptide administration resulted in expansion of D(b)PA(224)-specific CTL using a normally subdominant TCRBV gene segment. Overall, these results demonstrate that protective CTL responses elicited by lipopeptide vaccines can be correlated with TCR avidity, IL-2 production, and broad TCR repertoire diversity. Furthermore, factors that impact the quality of immunity are discussed. These factors are important considerations when evaluating the efficacy of novel vaccine strategies that target dendritic cells for eliciting cellular immunity.

Discrete T Cell Populations with Specificity for a Neo-Self-Antigen Bear Distinct Imprints of Tolerance

Mice expressing the Torpedo acetylcholine receptor alpha-chain as a neo-self-Ag exhibit a reduced frequency of T cells responding to the immunodominant epitope Talpha146-162 indicating a degree of tolerance. We characterized tolerance induction in these animals by analyzing the residual Talpha146-162-responsive T cell population and comparing it to that of nontransgenic littermates. Using CD4(high) sorting, we isolated the vast majority of Ag-reactive T cells from both strains of mice. Quantitative studies of the CD4(high) populations in transgenic mice following immunization with Talpha146-162 revealed a diminished expansion of cells expressing the canonical TCRBV6 but not other TCRBV gene segments when compared with nontransgenic littermates. In addition, CD4(high) cells from transgenic mice were functionally hyporesponsive to Talpha146-162 in terms of proliferation and cytokine secretion regardless of TCRBV gene segment use. TCR sequence analysis of transgenic Vbeta6(+)CD4(high) cells revealed a reduced frequency of cells expressing a conserved motif within the TCRbeta CDR3. Thus, the canonical Talpha146-162 responsive, Vbeta6(+) population demonstrates both quantitative and qualitative deficits that correlate with an altered TCR repertoire whereas the non-Vbeta6 population in transgenic mice exhibits only a reduction in peptide responsiveness, a qualitative defect. These data demonstrate that discrete autoreactive T cell populations with identical peptide/MHC specificity in Torpedo acetylcholine receptor-alpha-transgenic animals bear distinct tolerance imprints.

Impact of T‐cell receptor Vβ haplotypes on the development of dermatitis in DS‐Nh mice: synergistic production of interleukin‐13 caused by staphylococcal enterotoxin C and peptide glycans from Staphylococcus aureus

Although the pathogenic role of interleukin-13 (IL-13) is a key for atopic dermatitis (AD), the mechanism of IL-13 production in AD remains unclear. To investigate the role of the T-cell receptor Vbeta (TCR Vbeta) haplotype in the development of dermatitis and the production of IL-13 in the naturally occurring dermatitis model by staphylococcal enterotoxin C (SEC)-producing Staphylococcus aureus, we raised DS-Nh mice harbouring the TCR Vbeta(a) haplotype with a central deletion in the TCRBV gene segments, including TCR Vbeta8S2. Observation and histopathological analysis of the two mouse substrains with spontaneous dermatitis indicated that later onset and weaker severity of AD-like dermatitis were identified in mice with TCR Vbeta(a) compared to those with TCR Vbeta(b). Immunohistochemical examination revealed the infiltration of a large number of CD4-bearing T cells in the skin lesions in mice with TCR Vbeta(b) but not in those with TCR Vbeta(a). Interestingly, much lower levels of serum IL-13 were detected in mice with the TCR Vbeta(a) than in those with the TCR Vbeta(b) haplotype. In vitro, synthetic ligands (Pam(2)CSK4) of toll-like receptor 2 (TLR2) synergistically produced IL-13 with SEC in splenocytes of mice with TCR Vbeta(b) but not of those with TCR Vbeta(a), and natural killer T cells were essential for this synergism. Our findings suggested that this TCR Vbeta-haplotype-dependent synergism with TLR2 plays an important role in the development of AD-like dermatitis in DS-Nh mice.

Identical TCR β-Chain Rearrangements in Streptococcal Angina and Skin Lesions of Patients with Psoriasis Vulgaris

Tonsillar infection with Streptococcus pyogenes may induce several nonsuppurative autoimmune sequelae. The precise pathogenetic mechanisms behind this clinically well-established association are still unresolved. Using TCR analysis, we sought to identify a link between streptococcal tonsillitis and the T cell-mediated autoimmune response in psoriasis. Three patients with streptococcal-induced psoriasis underwent tonsillectomy. Using size spectratyping and sequencing of TCR beta-chain variable region gene (TCRBV) rearrangements, we compared the TCR usage of psoriatic skin lesions, blood, tonsils, and tonsillar T cells fractionated according to the expression of the skin address in "cutaneous lymphocyte-associated Ag" (CLA). TCRBV-size spectratype analysis of the blood lymphocytes, tonsils, and the CLA-negative tonsillar T cells revealed largely unselected T cell populations. Instead, TCRBV gene families of the psoriatic lesions and skin-homing CLA-positive tonsillar T cells displayed highly restricted spectratypes. Sequencing of TCRBV cDNA identified various clonal TCRBV rearrangements within the psoriatic lesions that indicated Ag-driven T cell expansion. Several of these clonotypes were also detected within the tonsils and, in one of the patients, within the small subset of CLA-positive tonsillar T cells, suggesting that T cells from the same T cell clones were simultaneously present within skin and tonsillar tissue. Because after tonsillectomy psoriasis cleared in all three patients our observations indicate that T cells may connect psoriatic inflammation to streptococcal angina. They suggest that the chronic streptococcal immune stimulus within the tonsils could act as a source for pathogenic T cells in poststreptococcal disorders, and they may help to explain why eliminating this source with tonsillectomy may improve streptococcal-induced sequelae.