Cell Receptor Beta Chain Research Articles

Analysis of the Expression of the TRBC1 in T lymphocyte tumors

T cell therapy represents a new class of immunotherapies garnering considerable attention. T cell receptor beta chain constant region 1 (TRBC1) is partially expressed in subsets of normal T cells. However, the immunotherapy of T lymphocyte tumors is rarely validated in clinical trials. Here, we aim to explore whether TRBC1 is a promising target for the immunotherapy of T lymphocyte tumors. This study examined TRBC1 expression in 25 healthy bone marrow samples, 39 patients with T-lineage acute lymphocytic leukemia (T-ALL), 4 patients with mature T cell neoplasms, and 5 patients suspected with mature T cell neoplasms with evidence of T cell neoplasia. Moreover, the expression of TRBC1 was evaluated by flow cytometry and through PCR detection of TCR gene rearrangements. The expression of monophasic TRBC1 was identified in all 25 normal bone marrows (23.83% ± 2.74% positive rate). The expression of TRBC1 was positive in 5 patients (12.8%) among the 39T-ALL patients. TRBC1 was partially expressed in 1 patient (25%) with T cell non-Hodgkin's lymphoma (T-NHL) and in 1 patient (20%) suspected to have T-NHL. Healthy donors showed a pattern of partial expression and patients with T-lymphocyte tumors showed a polytypic TRBC1 expression pattern. Thus, TRBC1 may be a diagnostic and therapeutic marker for T lymphocyte tumors.

Single-cell sequencing reveals clonal expansions of pro-inflammatory synovial CD8 T cells expressing tissue-homing receptors in psoriatic arthritis

Psoriatic arthritis (PsA) is a debilitating immune-mediated inflammatory arthritis of unknown pathogenesis commonly affecting patients with skin psoriasis. Here we use complementary single-cell approaches to study leukocytes from PsA joints. Mass cytometry demonstrates a 3-fold expansion of memory CD8 T cells in the joints of PsA patients compared to peripheral blood. Meanwhile, droplet-based and plate-based single-cell RNA sequencing of paired T cell receptor alpha and beta chain sequences show pronounced CD8 T cell clonal expansions within the joints. Transcriptome analyses find these expanded synovial CD8 T cells to express cycling, activation, tissue-homing and tissue residency markers. T cell receptor sequence comparison between patients identifies clonal convergence. Finally, chemokine receptor CXCR3 is upregulated in the expanded synovial CD8 T cells, while two CXCR3 ligands, CXCL9 and CXCL10, are elevated in PsA synovial fluid. Our data thus provide a quantitative molecular insight into the cellular immune landscape of psoriatic arthritis.

Preimmune Control of the Variance of TCR CDR-B3: Insights Gained From Germline Replacement of a TCR Dβ Gene Segment With an Ig D H Gene Segment.

We have previously shown that the sequence of the immunoglobulin diversity gene segment (DH) helps dictate the structure and composition of complementarity determining region 3 of the immunoglobulin heavy chain (CDR-H3). In order to test the role of germline D sequence on the diversity of the preimmune TCRβ repertoire of T cells, we generated a mouse with a mutant TCRβ DJC locus wherein the Dβ2-Jβ2 gene segment cluster was deleted and the remaining diversity gene segment, Dβ1 (IMGT:TRDB1), was replaced with DSP2.3 (IMGT:IGHD2-02), a commonly used B cell immunoglobulin DH gene segment. Crystallographic studies have shown that the length and thus structure of TCR CDR-B3 places amino acids at the tip of CDR-B3 in a position to directly interact with peptide bound to an MHC molecule. The length distribution of complementarity determining region 3 of the T cell receptor beta chain (CDR-B3) has been proposed to be restricted largely by MHC-specific selection, disfavoring CDR-B3 that are too long or too short. Here we show that the mechanism of control of CDR-B3 length depends on the Dβ sequence, which in turn dictates exonucleolytic nibbling. By contrast, the extent of N addition and the variance of created CDR3 lengths are regulated by the cell of origin, the thymocyte. We found that the sequence of the D and control of N addition collaborate to bias the distribution of CDR-B3 lengths in the pre-immune TCR repertoire and to focus the diversity provided by N addition and the sequence of the D on that portion of CDR-B3 that is most likely to interact with the peptide that is bound to the presenting MHC.

1063P Profiling of peripheral T cell receptor beta chain repertoire in non-small cell lung cancer (NSCLC) patients treated with anti-PD1

PD-L1 expression is the most implemented biomarker in clinical practice for immunotherapy in NSCLC; however, not all patients benefit from this treatment and PD-L1 testing alone is not yet an adequate biomarker for patient selection. Consequently, new approach is necessary to identify effective biomarkers. The aim of the project is to characterize T cell receptor β chain (TCRβ) repertoire in a cohort of advanced NSCLC patients treated with anti-PD1.

Abstract 3189: Dynamics characteristics and prognostic significance of profiling the peripheral blood T-cell receptor repertoire during immune checkpoint blockade in cancer

Abstract Introduction: Immune checkpoint blockade (ICB) targeted to CTLA-4, PD-1, and PD-L1 can promote anti-tumor T cell immunity and clinical responses. Tracking the dynamic changes of the T Cell Repertoire (TCR) can reflect the evolution of the immune system in tumor immunotherapy patients, predict the immune response to ICB, and provide high-value information for clinical monitoring of immune function status of cancer patients to make effective clinical decisions. The purpose of this study was to explore the correlation between the peripheral blood TCR repertoire dynamic changes and the immune response of patients with anti-PD-1/PD-L1, and to accurately guide the individualized treatment of immunotherapy. Methods: To comprehensively profile the TCR repertoire, high-throughput sequencing was used to identify hypervariable rearrangements of complementarity determining region 3 (CDR3) of the TCR β chain in peripheral blood samples from 31 advanced solid tumors (most were non-small cell lung cancer) patients treated with immune checkpoint blockade. The blood samples were obtained at baseline of treatment with PD-1/PD-L1 antibody, one to two additional blood samples were obtained during treatment to assess dynamic changes. Shannon-Wiener index was used to identify the diversity of TCR receptor. D50 index was used to account for diversity from the cumulative 50% of the total CDR3s counted in the sample. Results:Shannon-Wiener index at baseline was higher in durable clinical benefit (CB) group than non-clinical benefit (NCB) group (p value = 0.0040) treated with anti-PD1/PD-L1, and significantly increased in CB group ( p value = 0.031). There is a significant correlation between D50 index and clinical benefit during treatment (p value = 0.025). The higher the D50 index, the more likely it is to have durable clinical benefit. Significant difference in progression-free survival (PFS) rate was identified between the D50 high and D50 low groups (p value = 0.037). We validated this result in an independent cohort of NSCLC patients treated with anti-PD1/PD-L1(N=12). Conclusions:This study revealed the dynamic changes of peripheral blood T cell receptor beta-chain repertoire in patients with solid tumors before and during anti-PD1/PD-L1 treatment. This study found that baseline TCR diversity was associated with clinical efficacy. Patients with durable clinical benefit had an increased overall diversity of TCR. The D50 index of the sample during treatment can reflect whether the patient could benefit from anti-PD1/PD-L1 treatment or not, and was a significant prognostic factor for progression-free (PFS). These analyses provide that the peripheral blood TCR repertoire parameters may be useful for prognosis prediction and rapid determination of therapeutic outcomes for patients treated with immune checkpoint inhibitors. Citation Format: Ye Li, Jiaqian Wang, Shengjie Sun, Liangliang Wu, YunXiao Zhang, Guoqing Zhang, Xiaoting Li, Shunchang Jiao. Dynamics characteristics and prognostic significance of profiling the peripheral blood T-cell receptor repertoire during immune checkpoint blockade in cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3189.

Profiling the pattern of human TRB/IGH-CDR3 repertoire in liver transplantation patients via high-throughput sequencing analysis.

Immune processes in liver transplantation remain poorly understood. Acute allograft rejection in liver transplantation is a kind of T cell-mediated inflammatory disease accompaniedby inflammatory cell infiltration. However, the effect of acute allograft rejection on the immunological characteristics of TCRs in peripheralbloodmononuclearcell is unknown. In this study, we characterized the pattern of the human T cell receptor beta chain (TRB) and immunoglobulin heavy chain (IGH) complementarity-determining region 3 (CDR3) repertoires via high-throughput sequencing in 11 acute allograft rejection (AG) cases, 23 patients with stable allograft liver function (ST) who had liver transplantation performed and 20 healthy controls (HC). The diversity of TRB-CDR3 was significantly reduced in the AG group compared with the ST group and healthy controls (HC). The CDR3 and N-addition length distribution were not significantly different between the AG and ST groups. However, N-addition length distribution was significantly changed compared to HC. It seemed that AG used more short N-additions and healthy people used more long N-additions in TRB-CDR3 repertoire. Our findings suggested that the TRB-CDR3 region of AG had distinctive V gene use compared with that of HC. The characteristics of ST seemed to be in between those of AG and HC although the difference is not significant. Cluster analysis showed that the TRB repertoire could not effectively distinguish AG from ST. This research might give to a better understanding of the immune process of liver transplantation.

Enrichment of melanoma-associated T cells in 6-thioguanine-resistant T cells from metastatic melanoma patients.

This study examines whether 6-thioguanine resistant T cells (mutant) from metastatic melanoma patients are enriched for melanoma-associated T cells compared to T cells obtained analogously without thioguanine selection (wild-type). Melanoma-associated antigen pentamer staining was performed on 5 tumour and 9 peripheral blood samples from metastatic melanoma patients. T cell receptor beta chain repertoire was examined via Sanger sequencing of mutant and wild-type in blood and tumour from metastatic melanoma patients at times of tumour progression (n = 8) and via Illumina sequencing in tumour derived T cells and in uncultured T cells (uncultured), wild-type and mutant from blood before and after immune checkpoint blockade (n = 1). Mutant from tumour (3 of 5; P < 0.001), but not blood (0 of 9), were enriched compared to wild-type for binding melanoma-associated antigen pentamers. T cell receptor beta analysis in patients with tumour progression (n = 8) detected increased melanoma associated T cells in mutant compared to wild-type from blood (Monte Carlo P = 10). Comparison of blood samples before and after immune checkpoint blockade with prior tumor from one metastatic melanoma patient detected increased T cell receptor beta sharing between tumour and mutant compared to tumour and wild-type or tumour and uncultured: 11.0% (72/656), 1.5% (206/13 639) and 1.3% (381/29 807), respectively (Monte Carlo P = 10 for mutant versus wild-type and mutant versus uncultured). These data demonstrate that mutant in metastatic melanoma patients are enriched for melanoma-associated T cells and are candidate probes to study in vivo melanoma-reactive T cells.

Molecular analysis of primary melanoma T cells identifies patients at risk for metastatic recurrence.

Primary melanomas >1 mm thickness are potentially curable by resection, but can recur metastatically. We assessed the prognostic value of T cell fraction (TCFr) and repertoire T cell clonality, measured by high-throughput-sequencing of the T cell receptor beta chain (TRB) in T2-T4 primary melanomas (n=199). TCFr accurately predicted progression-free survival (PFS) and was independent of thickness, ulceration, mitotic rate, or age. TCFr was second only to tumor thickness in its predictive value, using a gradient boosted model. For accurate PFS prediction, adding TCFr to tumor thickness was superior to adding any other histopathological variable. Furthermore, a TCFr >20% was protective regardless of tumor ulceration status, mitotic rate or presence of nodal disease. TCFr is a quantitative molecular assessment that predicts metastatic recurrence in primary melanoma patients whose disease has been resected surgically. This study suggests that a successful T cell-mediated antitumor response can be present in primary melanomas.

Analysis of the TCR Repertoire in HIV-Exposed but Uninfected Infants

Maternal human immunodeficiency virus (HIV) infection has been shown to leave profound and lasting impacts on the HIV-exposed uninfected (HEU) infant, including increased mortality and morbidity, immunological changes, and developmental delays compared to their HIV-unexposed (HU) counterparts. Exposure to HIV or antiretroviral therapy may influence immune development, which could increase morbidity and mortality. However, a direct link between the increased mortality and morbidity and the infant’s immune system has not been identified. To provide a global picture of the neonatal T cell repertoire in HEU versus HU infants, the diversity of the T cell receptor beta chain (TRB) expressed in cord blood samples from HEU infants was determined using next-generation sequencing and compared to healthy (HU) infants collected from the same community. While the TRB repertoire of HU infants was broadly diverse, in line with the expected idea of a naïve T cell repertoire, samples of HEU infants showed a significantly reduced TRB diversity. This study is the first to demonstrate differences in TRB diversity between HEU and HU cord blood samples and provides evidence that maternal HIV, in the absence of transmission, influences the adaptive immune system of the unborn child.

Somatic hypermutation of T cell receptor gamma, delta, and beta chains in nurse sharks

Abstract Many γδ T cells typically bind antigen in a manner similar to that of immunoglobulins, recognizing and directly binding small molecules and intact proteins without presentation by classical MHC:Ag complexes. In this way, γδ T cells combine an innate-like immune response with an adaptive recognition strategy, providing both an immediate response to pathogen invasion and an ongoing, adaptive response to inflammation. However, γδ T cells often recombine tissue-specific, restricted sets of genes that have limited junctional diversity. Thus, somatic hypermutation (SHM) presents a useful solution for diversifying these antibody-like T cell receptors (TCR) by fine-tuning ligand recognition or permitting changes within the loci that allow receptors to evolve more rapidly to changing ligand environments. In fact, recent studies in sandbar shark have confirmed definitively that the γ chain locus of γδ T cells employs SHM as a mechanism to generate more diverse γδ TCR, and follow-up studies in dromedary camel indicated that loci of both γ and δ chains use SHM to diversify γδ TCR. In nurse sharks, we found evidence that SHM acts in the thymus, altering the α chain locus of αβ T cells to broaden the primary αβ T cell repertoire. Further, early results suggest that SHM modifies both γ and δ chain loci (but not β chain locus of αβ T cells) and this alteration may occur in the thymus, indicating that this receptor fine-tuning may precede ligand recognition.

Deep targeted sequencing reveals the diversity of TRB-CDR3 repertoire in patients with preeclampsia

Preeclampsia (PE) is one of the major causes of maternal and perinatal mortality worldwide. This study aimed to determine the immunological characteristics of PE patients and normal pregnancy at the T cell receptor beta-chain (TRB) level by using high-throughput sequencing. High-throughput sequencing was performed to analyze the expression of TRB-CDR3 in circulating T cells. T cells were isolated from 36 healthy pregnant women, 24 patients with severe PE, and 18 patients with moderate PE. Rearranged mRNA sequences were assigned to their germline V, D, and J counterparts, and translated into proper amino acids by the IMGT database. In general, PE samples had more TRB-CDR3 reads and types than those of normal pregnant woman in the circulation, but the mean number of TRB-CDR3 reads and unique TRB-CDR3 reads in severe group was lower than that in the moderate group. In PE patients, the V7_9 and V20_1 gene loci were more prevalent than in healthy pregnant women. In addition, 4 kinds of TRB-CDR3 peptides were found to be highly relevant to the pathogenesis of PE. Of them, peptides matched to herpes simplex virus antigen-specific T cells were much lower in PE samples.

Isolation and Validation of the First Functional HLA-a*01:01 Restricted EBV-LMP2 Specific T Cells for Treatment of EBV Associated Type II/III Lymphomas

Epstein Barr virus (EBV) is associated with the development of a broad range of malignancies, including Burkitt's lymphoma, Hodgkin and non-Hodgkin lymphomas, post-transplant lymphoproliferative disorder (PTLD), nasopharyngeal carcinoma and gastric carcinoma. Differential expression of immunogenic antigens (e.g. EBV Nuclear Antigen (EBNA2-6) and Latent membrane proteins (LMPs)) is seen at the different latent phases of the virus. Although many EBV associated lymphomas only express weakly immunogenic EBV antigens (e.g. EBNA1 and BARF1), lymphomas with type II or III latency express LMP1 and LMP2. Different strategies have been developed to manufacture EBV LMP1/2 specific T cell products for clinical application. Surprisingly, to date, no EBV specific T cells recognizing a peptide in the common HLA allele A*01:01 have been found. Furthermore, an HLA-A*01:01 associated risk for EBV+ Hodgkin lymphomas and infectious mononucleosis has been reported. A need thus exists for HLA-A*01:01 restricted EBV specific T cell products, especially EBV-LMP1/2 specific T cells.Based on MHC class I peptide predictions, HLA-A*01:01 binding peptides derived from different immunogenic EBV antigens were identified, and HLA-A*01:01/peptide tetramer complexes were synthesized (EBNA3A-YTDHQTTPT, EBNA3A-FLQRTDLSY, BZLF1-FTPDPYQVPF, LMP2-ESEERPPTPY, LMP2-LTEWGSGNRTY). For these 5 specificities, tetramer positive CD8 T cells were sorted by flow cytometry from peripheral blood mononuclear cells (PBMC) of 6 HLA-A*01:01 positive healthy donors and subsequently cultured with 1µM of specific peptide. Specificity of expanded T cells was confirmed by tetramer staining. Functional avidity was assessed using TAP2 deficient T2 cells transduced with HLA-A*01:01 and exogenously loaded with 10-12M to 10-6M of the respective specific peptide. The recognition of endogenously processed and presented antigen was analyzed using K562 cells transduced with HLA-A*01:01 and a retroviral vector encoding the full protein sequence of LMP2, HLA-A*01:01 positive EBV-LCLs and IFNy ELISA as read-out.HLA-A*01:01 restricted EBV specific T cells were present at low frequencies in total PBMCs from all 6 donors (EBNA3A-YTD 0.008%; range 0.001-0.02%, EBNA3A-FLQ 0.01%; range 0.0016-0.012%, BZLF1-FTP 0.0033%; range 0.0025-0.0075%, LMP2-ESE 0.0049%; range 0.0015-0.15% and LMP2-LTE 0.003%; range 0.002-0.025%). After flow cytometric cell sorting, only EBV-LMP2-ESE specific T cells of 5/6 donors expanded, resulting in pure tetramer+ CD8 T cell lines. Analysis of the T cell receptor beta chain (TCR-Vβ) usage showed that these EBV-LMP2-ESE specific T cell populations were oligoclonal. To further analyze their functional avidity, EBV-LMP2-ESE specific T cells were tested against HLA-A*01:01 transduced TAP2 deficient T2 cells exogenously loaded with peptide. Four out of 5 isolated T cell populations recognized T2 cells loaded with 1-103nM peptide, indicating intermediate to high avidity. To investigate whether this specific LMP2-derived peptide can be functionally processed and presented and whether these T cells were able to recognize endogenously processed and presented antigen, EBV-LMP2-ESE specific T cells were tested against HLA-A*01:01/LMP2 transduced K562 cells. All functional EBV-LMP2-ESE specific T cell populations could recognize these transduced K562 cell lines, indicating that this LMP2-ESE peptide is successfully processed, presented and recognized by EBV-LMP2-ESE specific T cells. Recognition of EBV-LCLs illustrated the potential of the EBV-LMP2-ESE specific T cells to recognize endogenous LMP2.We describe the isolation and validation of the first functional HLA-A*01:01 restricted EBV-LMP2 specific T-cell populations, which can be used for adoptive transfer to treat EBV associated type II/III lymphomas, malignancies of epithelial origin and PTLDs. Despite the very low frequency of these T cells, we were able to obtain these T cells from 5 out of 6 donors and showed their capacity to recognize both exogenously loaded as endogenously processed and presented EBV-LMP2-ESE peptide. DisclosuresNo relevant conflicts of interest to declare.

Structure Guided Engineering of Highly Specific Chimeric Antigen Receptors for the Treatment of T Cell Lymphomas

IntroductionMature T cell lymphomas are aggressive, treatment resistant cancers that are associated with poor prognosis. Clinical application of immunotherapeutic approaches has been limited by a lack of target antigens that discriminate malignant from healthy T cells. Unlike B cell depletion, pan-T cell aplasia is prohibitively toxic. Previously we reported a targeting strategy based on the mutually exclusive expression of T cell receptor beta-chain constant domains 1 and 2 (TRBC1 and TRBC2). We identified an antibody with unique TRBC1 specificity and demonstrated that anti TRBC1 chimeric antigen receptor (CAR) T cells can ablate cells expressing TRBC1 TCRs while sparing those expressing TRBC2 TCRs. A phase I clinical study investigating the efficacy of our TRBC1 CAR is ongoing.T cell malignancies are clonal, and the ratio of TRBC2 to TRBC1 expressing lymphoma cases is predicted to be approximately 2:1. To treat all cases of T cell lymphoma, a CAR that targets TRBC2 is needed. TRBC1 and 2 are highly homologous. Structural studies suggest that amino acid inversions at positions 4 and 5 of the constant beta chain provide an accessible discriminating portion between the two proteins.Given the structural similarities between TRBC1 and TRBC2 and our characterized binder against TRBC1, we explored generating antibodies with specificity towards TRBC2 via a structure guided computational biology approach; engineering the previously identified TRBC1 antibody and reversing its specificity such that it recognised TRBC2.ResultsThe crystal structure of the TRBC1 specific monoclonal antibody was solved in complex with a TRBC1-TCR to 2.4Å, Figure 1. Through computational biology and protein engineering we rationally designed a mutant version of TRBC1 binder that was specific for TRBC2 and had a 1000 fold decreased affinity towards TRBC1. Flow cytometry analysis of the TRBC2 specific antibody demonstrated the ability to bind to T-cells expressing TRBC2 TCRs. We further showed that the engineered antibody retained favourable biophysical characteristics with high stability (Fab Tm > 65oC) and low aggregation propensity (>99% monomer).We used the engineered monoclonal antibody to generate a 2nd generation anti-TRBC2 CAR. We demonstrated that our anti-TRBC2 CAR showed specificity, cytokine release and cytotoxicity in 72hr co-cultures against TRBC2+ cell lines but not TRBC1+ cell lines or cell lines that did not express TCR on the surface. Anti-TRBC2 CAR T-cells also demonstrated proliferative capacity in long-term co-culture assays.ConclusionsWe have utilised structural biology and rational protein design to generate CAR-T cells capable of specifically targeting TRBC2. The combination of TRBC1 and 2 targeting CAR-T cell products with a patient stratification companion diagnostic assay offers a therapeutic strategy for the treatment of a wide range of, otherwise untreatable, T-cell lymphomas.Figure 1. A. Structural interface between TCR Beta and Fab fragment of TRBC1 specific antibody. B and C. CDR fold of TRBC1 binder and 90o rotation. [Display omitted] DisclosuresOnuoha:Autolus Ltd: Employment, Equity Ownership, Patents & Royalties. Ferrari:Autolus Ltd: Employment, Equity Ownership. Bulek:Autolus Ltd: Employment, Equity Ownership, Patents & Royalties. Bughda:Autolus Ltd: Employment, Equity Ownership, Patents & Royalties. Manzoor:Autolus Ltd: Employment, Equity Ownership, Patents & Royalties. Srivastava:Autolus Ltd: Employment, Equity Ownership. Ma:Autolus Ltd: Employment. Karattil:Autolus Ltd: Employment, Equity Ownership, Patents & Royalties. Kinna:Autolus Ltd: Employment, Equity Ownership. Thomas:Autolus Ltd: Employment, Equity Ownership, Patents & Royalties. Cordoba:Autolus Ltd: Employment; Autolus Ltd: Patents & Royalties; Autolus Ltd: Equity Ownership. Maciocia:Autolus: Equity Ownership, Patents & Royalties: UCLB. Pule:Autolus Ltd: Employment, Equity Ownership, Other: Salary contribution paid for by Autolus, Research Funding; University College London: Patents & Royalties: Patent with rights to Royalty share through UCL.

The landscape and diagnostic potential of T and B cell repertoire in Immunoglobulin A Nephropathy

Immunoglobulin A Nephropathy (IgAN) is the most common glomerulonephritis worldwide. The pathologic hallmark of IgAN is immune complex deposited in glomerular mesangium, which induces inflammation and affects the kidney's normal functions. The exact pathogenesis of IgAN, however, remains obscure. Further, in current clinical practice, the diagnosis relies on needle biopsy of renal tissue. Therefore, a non-invasive method for diagnosis and prognosis surveillance of the disease is highly desirable. To this end, we investigated the T cell receptor beta chain (TCRB) and immunoglobulin heavy chain (IGH) repertoire in circulating lymphocytes and compared them with kidney infiltrating lymphocytes using immune repertoire high throughput sequencing. We found that some features of TCRB and IGH in renal tissues were remarkably different from that in the blood, including decreased repertoire diversity, increased IgA and IgG frequency, and more antigen-experienced B cells. The complementarity-determining region 3 (CDR3) length of circulating TCRB and IGH in IgAN patients was significantly shorter than that in healthy controls, which is the result of both VDJ rearrangement and clonal selection. The IgA1 frequency in the blood of IgAN patients is significantly higher than that in other Nephropathy (NIgAN) patients and healthy control. Importantly we identified a set of TCRB and IGH clones, which can be used to distinguish IgAN from NIgAN and healthy controls with high accuracy. These results indicated that the TCRB and IGH repertoire can potentially serve as non-invasive biomarkers for the diagnosis of IgAN. The characteristics of the kidney infiltrating and circulating lymphocytes repertoires shed light on IgAN detection, treatment and surveillance.

Abstract 987: Organoid-based characterization of patient tumors and microenvironments at single cell resolution

Abstract The advent of microenvironment directed cancer treatments such as antiangiogenic agents and recent immunotherapies have produced a pressing need for robust and systematic characterization of patient tumors and corresponding stromal populations. However, in vitro cultures of tumor biopsies do not typically preserve both the tumor epithelium and tumor microenvironment as an intact syngeneic unit. To address these limitations, we developed organoid cultures of surgically resected patient cancer biopsies that intrinsically retain diverse tumor microenvironmental cellular components without requiring reconstitution. We compared transcriptome profiles of fresh tumor cell populations to organoid cultures by droplet based single cell 5' RNA sequencing (scRNA-seq). Analysis of &amp;gt;50k single cell transcriptome profiles revealed tumor and stromal cell populations including tumor epithelia, fibroblasts and immune cells in fresh tumors. scRNA-seq of organoid cultures revealed a similar composition of native cancer cells and stromal components in both immune and non immune populations. In particular, 5' scRNA-seq enabled simultaneous characterization of paired T cell receptor alpha and beta chains as well as generalized gene expression in individual cells. We observed clonotype expansion of cytotoxic T cells in both fresh tumors and organoid cultures. The faithful recapitulation of tumor microenvironmental diversity within human organoid cultures should facilitate the in vitro exploration of immunotherapeutic agents and modeling of associated patient-specific responses. Citation Format: Ameen A. Salahudeen, Junjie Zhu, Jihang Ju, Arpit Batish, Ken Sutha, James T. Neal, Valeria Giangarra, Luz Montesclaros, Jerald Sapida, Osman Sharifi, Josephine Lee, Grace X. Zheng, Dhananjay A. Wagh, John A. Coller, Joel W. Neal, Sukhmani K. Padda, Chiara Sabatti, Calvin J. Kuo. Organoid-based characterization of patient tumors and microenvironments at single cell resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 987.

TCR Vβ Usage of Peripheral Blood and Liver Infiltrating Lymphocytes in Patients with Chronic Hepatitis B

Introduction. Chronic hepatitis B (CHB) is still a public health problem and its mechanism remains unclear. In this study, we detect the skewness of T cell receptor beta chain variable gene (TCR Vβ) in peripheral blood lymphocytes (PBL) and the liver infiltrating lymphocytes (LIL) of patients with CHB; and hope to provide information for further research on the pathogenic mechanism of CHB.Material and methods. Fifteen patients with CHB, ten healthy volunteers and three patients with liver cysts were recruited as the subjects. The usage of TCR Vβ of PBL and LIL were measured and compared; the associations of the TCR Vβ usage of PBL with some hematological indices, including human leukocyte antigen (HLA) alleles, percents of CD4+ and CD8+ T cells, sera levels of HBV-DNA and IFN-γ, were analyzed.Results. In PBL, Vβ12 and Vβ13.1 were the highest predominant usage genes which usage frequencies were all 46.7%; Vβ23 was the key limited usage gene (40.0%). In LIL, the mainly predominant and limited usage gene was Vβ13.1 (73.3%) and Vβ23 (46.7%), respectively. About half of the patients with CHB with HLA-DR9 or HLA-DR12 showed the predominant usage of Vβ5.2 or Vβ13.2. In patients with CHB, the percentage of CD4+ T cells was 33.41 ± 5.39 %, that of CD8+ T cells was 28.67 ± 6.77 %; the concentration of IFN-γ was 182.52 ± 44.16 pg/mL. Compared to the healthy controls, there were significant differences for these data (P < 0.05). Neither ALT nor HBV-DNA was relative to the usage of TCR Vβ.Conclusions. PBL and LIL share the common sknewness of TCR Vβ genes which probably relates to some hematological indices. However, the roles of such similarities and associations in the development of CHB need further study.

Analysis of T cell receptor repertoire in monozygotic twins concordant and discordant for chronic hepatitis B infection

Due to their identical inheritance and shared surroundings, identical twins have been the recommended group for studying the susceptibility and prognosis of diseases. Here, CD8+ T cell receptor beta (TCRβ) chains were analyzed by high-throughput sequencing in three pairs of healthy identical twins and chronic hepatitis B patients. The data showed a high level of similarity in the TCR repertoire of each pair in terms of average TCR Vβ segment expression and frequency of the complementary determining region 3 (CDR3) pattern and skewed or oligoclonal clonotypes. Notably, the level of similarity in TCR Vβ expression between the twins appeared to be independent of the consistency or inconsistency of chronic HBV infection, although the detailed CDR3 pattern and frequency were related to disease prognosis. There were more immunodominant clonotypes in patients with HBV antigen seroconversion, which showed an increased abundance. These immunodominant clonotypes may be used as favorable prognostic biomarkers and potential targets for immunotherapy. Thus, delineating the CD8+ T cell repertoire of identical twins with concordant chronic viral infections provides a promising means to screen protective TCR genes for immunotherapy.

Perturbation of the T cell receptor repertoire occurs with increasing age in dogs

Immunosenescence is the gradual deterioration in immune system function associated with ageing. This decline is partly due to involution of the thymus, which leads to a reduction in the output of naive T cells into the circulating lymphocyte pool. Expansion of existing naive and memory T cell populations, to compensate for the reduction in thymic output, can lead to reduced diversity in the T cell repertoire with increasing age, resulting in impairment of immune responses to novel antigenic challenges, such as during infection and vaccination. Since associations between T cell repertoire and age have only been examined in a limited number of species, to gain further insights into this relationship, we have investigated age-related changes in the canine T cell receptor (TCR) repertoire.Blood samples were obtained from Labrador retriever dogs of varying ages and variation in the complementary determining region 3 (CDR3) of the T cell receptor beta (TCRB) chain was investigated. CDR3 size spectratyping was employed to evaluate clonal expansion/deletion in the T cell repertoire, allowing identification of profiles within individual variable (V) region families that skewed away from a Gaussian distribution. Older dogs (10–13 years) were found to have an increased number of TCRB V gene spectratypes that demonstrated a skewed distribution, compared with young dogs (≤3 years). Additionally, there was a reduction in the number of clonal peaks present in the spectratypes of old dogs, compared with those of young dogs. The study findings suggest that there is an age-associated disturbance in the diversity of the T cell receptor repertoire in dogs.

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs.

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have been conducive to downstream in vivo functional analysis of TCR heterodimers. We have developed an improved protocol based on a two-step multiplex-nested PCR, which results in a PCR product that spans entire variable regions of a human TCR alpha and beta chains. By identifying unique restriction sites and incorporating them into the PCR primers, we have made the PCR product compatible with direct sub-cloning into the template retroviral vector. The resulting retroviral construct encodes a chimeric human/mouse TCR with a mouse intracellular domain, which is functional in mouse cells or in in vivo mouse models. Overall, the protocol described here combines human single cell paired TCR alpha and beta chain identification with streamlined generation of retroviral vectors readily adaptable for in vitro and in vivo TCR expression. The video and the accompanying material are designed to give a highly detailed description of the single cell PCR, so that the critical steps can be followed and potential pitfalls avoided. Additionally, we provide a detailed description of the cloning steps necessary to generate the expression vector. Once mastered, the whole procedure from single cell sorting to TCR expression could be performed in a short two-week period.

Alanine Mutagenesis in the Complementarity Determining Region 3 of the MTB and HIV-1 Peptide-Bispecific T Cell Receptor Beta Chain Affects Ligand Recognition.

Mycobacterium tuberculosis/human immunodeficiency virus (MTB/HIV) coinfection presents a special challenge to the prevention and treatment of tuberculosis and HIV/AIDS. Adoptive transfer of high-affinity T cell receptor (TCR) gene-modified T cells against MTB and HIV antigens is a promising approach to treating MTB/HIV coinfected patients whose cellular immunity is obviously disordered. We have previously successfully identified that a bispecific TCR screened out from peripheral blood mononuclear cells of a HLA-A*0201+ healthy individual using the complementarity determining region 3 (CDR3) spectratype analysis recognizes both MTB Ag85B199–207 and HIV-1 Env120–128 peptide. However, it has not been known how residues on CDR3 loops, which have been shown to play a leading role in antigen binding and specificity contribute to the bispecific TCR contact with the peptide–major histocompatibility complex (MHC) complexes. In this study, we provided an extensive investigation of residues in the predicted CDR3 of the bispecific TCR beta (β) chain using alanine scanning mutagenesis. Our data showed that three of the five substituted residues (G115A, T116A, A117G) in CDR3β of the bispecific TCR caused a significantly diminished T cell response to antigen, whereas the remaining two substituted residues (D114A, S118A) resulted in completely eliminated response, thus identifying the two residues that were particularly critical for the recognition of peptide–MHC in the bispecific TCR. These findings will provide an imperative foundation for generating an improved high-affinity bispecific TCR for use in T cell adoptive immunotherapy for MTB/HIV coinfected individuals.