TCRβ Rearrangements Research Articles

Abstract LB074: Preclinical development of API-192: An engineered CAR-NKT allogeneic cell therapy targeting CD19 and CD20 for the treatment of B-cell malignancies

Abstract Autologous chimeric antigen receptor (CAR)-T therapies have elicited high rates of durable complete responses in B cell lymphomas. Unfortunately, disease progression, manufacturing failures, and prohibitive costs highlight the need for an alternative source of cells that would provide a consistent and readily available therapy for patients. The “Appia Cells Utilized for Allogeneic” (ACUA) platform is an innovative process that differentiates cord blood-derived CD34+ hematopoietic stem and progenitor cells into large numbers of natural killer T (NKT) cells to support off-the-shelf treatment. NKT cells express an invariant T cell receptor (iTCR) and are naturally non-alloreactive, making them an ideal cell type for an allogeneic product. The ACUA platform circumvents the challenge of expanding NKT cells from the periphery where the cells comprise less than 1% of T cells. API-192 is an ACUA-derived, NKT cell product engineered to express dual CARs targeting CD19 and CD20 to prevent tumor antigen escape. At research scale, API-192 is a highly pure product (>90% iTCR+ 19CAR+ 20CAR+) that secretes soluble interleukin-15 for improved expansion and persistence. The transgenic iTCR is not expressed above physiologic levels and upon engagement, API-192 proliferates in response to and is cytotoxic against CD1d+ tumor cells in a ligand-dependent manner. In response to Raji and Nalm6 tumor cells, API-192 secretes pro-inflammatory cytokines, proliferates, and is cytotoxic in a dose- and CAR-dependent manner. API-192 can also control multiple rounds of tumor cell rechallenge in vitro. Using human tumor xenograft mouse models, we show that API-192 expands, controls tumor growth, and prolongs survival. Furthermore, API-192 persists in the periphery, enabling control of a tumor rechallenge at 70 days after initial dosing of effector cells. Indicative of a favorable immunogenic profile, API-192 expresses lower levels of MHC I and MHC II molecules relative to conventional CAR-T cells and elicits less interferon (IFN)-γ release by NK and T cells in a mixed lymphocyte reaction (MLR) assay. To address a theoretical concern that endogenous TCRαβ rearrangement could occur during the ACUA process, we performed genomic DNA based TCR sequencing to quantify the level of TCRα or TCRβ rearrangement and preliminary data suggested negligible levels (<0.2%). Using a MLR assay, we demonstrate that compared to conventional CAR-T cells, API-192 secretes minimal levels of IFN-γ in response to co-culture with healthy donor peripheral blood mononuclear cells, consistent with a low graft vs host risk. In an autonomous growth assay, API-192 expands at a low level and subsequently contracts, suggesting a lack of dysregulated growth. Collectively, these results demonstrate the favorable manufacturability, potency, and safety of API-192 and its potential as a novel allogeneic therapy. Citation Format: Lanny Gov, Jiaji Yu, Maha Qubain, Saige L. Pompura, Sean D. Allen, Michael A. Christopher, Jeff Wiezorek. Preclinical development of API-192: An engineered CAR-NKT allogeneic cell therapy targeting CD19 and CD20 for the treatment of B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB074.

Polycomb Group Protein Yin Yang 1 Controls DNA Methylation in Murine T Lymphocyte Development

Transcription factors and epigenetic regulators play key functions in T lymphocyte lineage commitment and development. Mammalian PcG proteins are major negative regulators of gene expression that typically bind as large complexes (such as Polycomb Repressive Complex 1 (PRC1) and PRC2) to gene regulatory regions selectively targeted by an as yet unknown mechanism. While nearly all mammalian PcG proteins are lack of specific DNA binding capacities, transcription factor Yin Yang 1 (YY1) can bind at the DNA sites specifically and recruit other PcG proteins to specific DNA sequences. A small 25 amino acid YY1 domain (the REPO domain) is necessary and sufficient for the recruitment of PcG proteins, PcG-mediated stable transcriptional repression and histone modification. While YY1ΔREPO is competent for DNA binding, transcriptional activation, transient transcriptional repression and interaction with transcriptional coregulators such as HDACs, YY1ΔREPO is defective in all YY1 PcG functions. Our prior study demonstrated that deletion of YY1 in the hematopoietic system leads to T cell development blockage at the double-negative 3 (DN3) stage. Interestingly, ectopic expression of YY1∆REPO in YY1 null background by retroviral bone marrow transplantation could not rescue YY1 deficiency mediated T cell survival defect. This suggests that the YY1 REPO domain/PcG function plays a vital role in T cell development. Herein, we generated the YY1 REPO domain conditional knockout mouse model ( Yy1 f/∆REPO Vav-Cre) by CRISPR/Cas9 to further dissect the YY1 REPO domain/PcG function in T cell development. Yy1 f/∆REPO Vav-Cre ( Yy1 -/∆REPO) mice have reduced thymus weight and cellularity with medulla aplasia. Compared with wild-type (WT) and YY1 heterozygous ( Yy1 +/-) mice, Yy1 -/∆REPO mice have an increased percentage of DN3 cells with decreased DN4 percentage supporting a developmental blockage at the DN3 stage. Yy1 -/∆REPO DN T cells fail to go through TCRβ rearrangement with increased apoptosis and reduction of TCRβ + cells in the thymus compared with WT and Yy1 +/-cells. Interestingly, RNA-seq analysis in sorted primary DN3 cells revealed the genetic network governing DNA methylation, histone methylation, chromosome maintenance, and translation initiation complex formation are deregulated in Yy1 -/∆REPO mice compared to WT and Yy1 +/-mice. DN T cell development requires precise and dynamic regulation of histone and DNA methylation and demethylation. Interestingly, the DNA demethylase enzymes ten-eleven translocation (TET) family proteins Tet1 and Tet2 are downregulated in Yy1 -/∆REPO DN3 thymocytes compared with WT and Yy1 +/- DN3 cells. Our data show that the YY1 REPO domain/PcG function is required for DN3-DN4 transition via regulating TCRβ rearrangement and the YY1 REPO domain/PcG function plays a critical role in proper DNA methylation status in early T cell development. As PcG proteins modify histone via trimethylation of histone H3 on lysine 27, we will further assess the impact of YY1 REPO domain/PcG function on histone methylation in T cell development. By utilizing a powerful mouse model for identifying YY1 functions as PcG domain-dependent or PcG domain-independent, our study has generated fundamental new insights into the epigenetic landscape in T cell development.

Deficiency of salt-inducible kinase 2 (SIK2) promotes immune injury by inhibiting the maturation of lymphocytes.

Salt-inducible kinase 2 (SIK2) belongs to the serine/threonine protein kinases of the AMPK/SNF1 family, which has important roles in cell cycle, tumor, melanogenesis, neuronal damage repair and apoptosis. Recent studies showed that SIK2 regulates the macrophage polarization to make a balance between inflammation and macrophage. Macrophage is critical to initiate immune regulation, however, whether SIK2 can be involved in immune regulation is not still well understood. Here, we revealed that the protein of SIK2 was highly expressed in thymus, spleen, lung, and brain. And SIK2 protein content increased in RAW264.7 and AHH1 cells with a time and dose-dependent after-ionizing radiation (IR). Inhibition of SIK2 could promote AHH1 cells apoptosis Moreover, we used the Cre-LoxP system to construct the SIK2+/- mice, and the research on function suggested that the deficiency of SIK2 could promote the sensitivity of IR. The deficiency of SIK2 promoted the immune injury via inhibiting the maturation of T cells and B cells. Furthermore, the TCRβ rearrangement was inhibited by the deficiency of SIK2. Collectively, this study demonstrated that SIK2 provides an essential function of regulating immune injury, which will provide new ideas for the treatment of immune injury-related diseases.

Distinguishing between monozygotic twins' blood samples through immune repertoire sequencing.

Monozygotic (MZ) twins with highly similar genomic DNA sequences can not be distinguished by conventional forensic DNA testing. The immune repertoire (IR) reflects an individual's immune history, which is unique between individuals, has been applied to individualized treatment in precision medicine. However, the application of IR in forensic genetics has not been reported to date. In this study, the diversity in the complementary determining region 3 (CDR3) of both the T-cell receptor β chain (TCRβ) and B-cell receptor heavy chain (also known as immunoglobulin heavy chain, IGH) in four pairs of MZ twins were analyzed. The results showed that the amino acid sequences length distribution frequency of TCRβ CDR3 had 4-10 differences, and the nucleic acid sequences length distribution frequency of TCRβ CDR3 had 2-7 differences between MZ twins. The shared difference of four pairs of MZ twins focused on the length distribution frequency of 34bp nucleotide sequences in TCRβ. By analyzing the usage frequency of V and J genes in TCRβ and IGH CDR3 DNA sequence rearrangements, we also found that there were biases between each pair of MZ twins, and the usage frequency of TRBJ2-3 showed common differences between each pair of MZ twins. Furthermore, each pair of MZ twins had its own unique V-J genes combination mode in TCRβ and IGH CDR3 DNA sequences. This study, for the first time, suggested that IR can be used as a potential biological marker to distinguish MZ twins.

Abstract NG14: Dissecting immune microenvironment of T-cell acute lymphoblastic leukemia

Abstract NG14: Dissecting immune microenvironment of T-cell acute lymphoblastic leukemia

The case of T-ALL presenting with NK phenotype after COVID-19 vaccination

NK-lymphoblastic leukemia/lymphoma (NK-LL) is an extremely rare hematopoietic tumor consisting of natural killer (NK) precursor cells, and their lineage overlaps with T-cells, making it challenging to diagnose. COVID-19 vaccination is recommended for people with a risk of aggravation such as cancer-bearing patients, including hematopoietic tumors. We present a 55-year-old man who had cervical lymph node swelling post vaccination for COVID-19. Hematological malignancy was suspected due to the presence of atypical lymphoid cells with an elevated IL-2R in laboratory data. Tumor cells were positive for CD7, CD56, cyCD3, and terminal deoxynucleotidyl transferase (TdT) evidenced through flow cytometry of the bone marrow and the lymph node. The histopathological findings showed monotonous tumor cell proliferation, the cells being positive for CD3 and TdT in the bone marrow and they were positive for CD3, TdT, and CD56 in lymph node. Even though these findings suggested NK-LL, clonal T-cell receptor (TCR) β gene rearrangement by Southern blot hybridization was observed in the bone marrow. TCRβ rearrangement led to the final diagnosis of T-cell lymphoblastic leukemia (T-ALL). The causal relationship between COVID-19 vaccination and carcinogenesis is not clear, and more cases need to be studied in order to elucidate the relationship between the two factors.

A Transcriptional Classifier Identifies Pediatric T-Cell Acute Lymphoblastic Leukemias at High Risk for End of Induction Minimal Residual Disease

Upfront risk stratification with incorporation of risk-adapted therapy continues to be a pressing need for pediatric T-ALL. However, robust molecular or cytogenetic biomarkers are currently lacking. Instead, initial response to therapy, measured as end of induction minimal residual disease (MRD), is the only prognostic factor used in most cooperative groups. This inability to stratify patients at diagnosis limits the early introduction of rationally-designed therapies that may better achieve long-term remissions. To address this, we asked whether gene expression profiling performed at diagnosis may identify patients at high risk for future MRD+, enabling earlier therapeutic intervention. To test this, we used a training dataset of RNA-seq data from diagnostic samples from patients enrolled on the Children's Oncology Group (COG) AALL0434 trial. Using leave-one-out cross-validation, we defined a risk score (RS) for MRD+ based on the expression of 119 genes. This RS indicates the probability of being MRD+ for a given gene expression pattern. Gene ontology analysis of the genes in our model demonstrated enrichment for genes involved in signal transduction, lymphocyte differentiation and activation, and cell death. Using this model, we found that early T-cell precursor (ETP) samples (n=19), of which 79% were MRD+, had uniformly high RS, with an average of 78 (SD=17). In contrast, non-ETP samples (n=146), of which 26% were MRD+, had an average RS of 26 (SD=21), with MRD+ patients having a significantly higher RS relative to MRD- patients (p<0.0001). When we analyzed the expression pattern of the 119 genes, we found that ETP samples had a relatively uniform pattern, consistent with their high rate of MRD+. Non-ETP samples with RS<50 had a pattern that was inverted relative to the ETP samples, while those with RS>50 closely mirrored the ETP samples. Using a RS cutoff of 50 to identify patients at high risk for MRD+ gives a 97% specificity in this training cohort. Given the similar gene expression pattern between ETPs and RS>50 non-ETPs, we hypothesized that this RS classification may relate to differences in developmental stage. We found that ETP and RS>50 non-ETP samples were significantly enriched for the LMO2/LYL1, HOXA, and TLX3 subgroups of T-ALL, with the subgroup distribution in the RS>50 non-ETP samples differing significantly from the RS<50 non-ETP samples (p=0.001). Furthermore, we found that the RS>50 non-ETP samples, relative to the RS<50 samples, were enriched for lacking a TCRb rearrangement (p<0.0001), indicative of an earlier developmental stage. To determine the generalizability of our gene expression classifier, we applied our fixed model to an independent validation cohort of 826 non-ETP samples from the same COG AALL0434 trial. Here, we again found that RS>50 non-ETP samples (n=130) were significantly enriched for MRD+ relative to RS<50 samples (n=696; p<0.0001). Next, we applied our classifier model to a pilot cohort of samples from patients enrolled on COG AALL1231, which used dexamethasone rather than prednisone as the induction steroid. In this group of 31 diagnostic non-ETP samples, RS>50 samples were similarly enriched for MRD+ (p=0.05), suggesting that this classifier may retain its prognostic value across multiple induction steroids. Finally, to facilitate clinical implementation of this prognostic tool, we transitioned to the NanoString nCounter platform, a more rapid and cost-effective means of performing targeted gene expression analysis. Using 364 samples from COG AALL0434 for which we had corresponding RNA-seq data, we assessed the performance of our custom NanoString assay and found a strong correlation between the RS calculated using each of the two platforms (concordance correlation coefficient = 0.98), suggesting that the prognostic value of the classifier is retained on the NanoString assay. In conclusion, while ETP samples are commonly MRD+ and are readily identifiable by their unique cell surface immunophenotype, non-ETP samples lack a surface immunophenotype that correlates with MRD+. Here, we present a targeted gene expression classifier, validated on a clinically-tractable platform, that identifies at diagnosis a subset of non-ETP T-ALLs with a gene expression pattern that resembles that of ETP T-ALLs and that are at high risk for future MRD+.

Predictive value of intra‐tumoural TCRβ rearrangements in precisely selecting adjuvant therapy for EGFR‐mutant non‐small‐cell lung cancer

AbstractIn this commentary, we discussed the important role of intratumoural specific T‐cell receptor (TCR) β chain rearrangements on tumour‐infiltrating lymphocytes in predicting clinical outcomes of adjuvant tyrosine kinase inhibitor therapy or chemotherapy in patients with non‐small‐cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutation from two aspects. The in‐depth study of the local immune microenvironment may help to discover possible biomarkers for predicting the overall survival and disease‐free survival and may further direct to select of the appropriate adjuvant therapy in EGFR‐mutant NSCLC patients according to the special TCR rearrangements.

Notch1 deficiency alters the migratory behavior of developing T cells and calcium signaling in the thymus of medaka.

The differentiation of T cells from lymphoid progenitors in the thymus follows sequential developmental stages that constantly require interaction with thymic epithelial cells. Several distinct aspects of early T cell development depend on the activation of Notch receptors on thymocytes, while the selection of thymocytes at later stages are believed to be Notch independent. Using reverse genetic approaches and whole-thymus live imaging in an in vivo teleost model, the medaka, we report that Notch1 signals is required for proliferation and specification of developing T cells as well as involved in their selection in the thymus. We reveal that Notch1 controls the migratory behavior of thymocytes through controlling the chemokine receptor Ccr9b and thereby influence the T cell receptor (TCR) activation. Hence, we propose that, in lower vertebrates, the function of Notch signaling extends to all stages of T cell development, except when thymocytes undergo TCRβ rearrangement.

Validation of Next Generation Sequencing-Based Clonality Assays for Diagnosis and Minimal Residual Disease Tracking in Lymphoid Malignancies

Introduction:B or T cell receptor (BCR/TCR) clonal rearrangements have served as important diagnostic markers and minimal residual disease (MRD) tracking markers to guide treatment decisions in lymphoid malignancies. Next generation sequencing (NGS)-based BCR/TCR clonality assessment allows easier sample preparation, higher sensitivity and simpler standardization than flow cytometry and polymerase chain reaction (PCR)-based assays. Here, we developed NGS-based BCR/TCR clonality assays to identify and track disease-associated clonotypes of IGH, IGK, IGL, TCRB and TCRG rearrangements and BCL1/2-IGH translocations in lymphoid malignant cells. Our studies validated the analytical performance of the assays using genomic DNA (gDNA) from cell lines and patient samples diagnosed with acute lymphoblastic leukemia (ALL), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), lymphoma and lymphoid blast phase chronic myeloid leukemia (BP-CML), as well as peripheral blood gDNA and circulating tumor DNA (ctDNA) from lymphoma patients.Methods:Our BCR/TCR clonality assays were based on two rounds of multiplex PCR followed by NGS. In the first-round PCR, the sequences of complementary determining region 3 (CDR3) in rearranged immune receptor genes were amplified by multiplex primers, and sample-specific index and NGS adapters were then added in the second-round PCR. Sequencing was performed on NovaSeq 6000 System and processed with customized bioinformatics pipelines. BCR clonality assays could identify IGH (V H-D H-J H or D H-J H), IGK (V κ-J κ, V κ-Kde and intronRSS-Kde) and IGL (V λ-J λ) rearrangements, as well as BCL1-IGH and BCL2-IGH translocations. TCR clonality assays could detect TCRB (V β-D β-J β) and TCRG (V γ-J γ) rearrangements. To evaluate the performance of our assays, we detected BCR or TCR clonality in gDNA from 4 cell lines and 40 clinical samples with ALL, MM, CLL and lymphoma, as well as paired chronic- and blast-phase samples of CML. Limit-of-detection (LOD) was estimated by clinical samples and cell lines with a background of peripheral blood gDNA from healthy donors. Linearity of detection was established with gDNA of cell lines spiked into normal gDNA to generate across orders of magnitude of clonal frequencies. To ensure consistent performance of the assays, we tested separate reactions and a single mixed reaction for IGH VDJ, IGH DJ, IGK and IGL. In addition, peripheral blood gDNA matching aforementioned lymphoma cases and ctDNA samples were also tested by our assays.Results:Both BCR clonality of B-cell lymphoid malignancies (B-ALL, MM, CLL and lymphoma) and TCR clonality of T-cell lymphoid malignancies (T-ALL and lymphoma) showed above 90% positive detection rate and mostly positive in more than one receptor gene. Chronic and blast phase samples from the same CML patient showed an identical dominant clonotype. The assays had high sensitivity, with LoD defined between 1 to 2 malignant cells in both BCR and TCR clonality assessment. Linearity was observed with clonal frequencies from 1 to 10 -6, which indicated consistence between observed and expected frequencies. The sequencing results with a single adjusted mixed reaction for IGH VDJ, IGH DJ, IGK and IGL were comparable to that with separate reactions, suitable for both diagnosed samples and MRD samples, suggesting the robustness of the assays. Our testing results also showed that peripheral blood gDNA of lymphoma patients carried identical clonotypes found in malignant tissues and ctDNA.Conclusion:We characterized and validated the performance of our NGS -based BCR/TCR clonality assays. We also demonstrated its potential application as a highly sensitive tool for diagnosis and MRD tracking for lymphoid malignancies, including ALL, MM, CLL, lymphoma and even CML at risk of lymphoid blast transformation. DisclosuresNo relevant conflicts of interest to declare.

Rapid and Sustained Reduction of Immunosuppressive T-Cells and Focusing of the T-Cell Repertoire in t(11;14) Relapsed/Refractory Multiple Myeloma Patients Treated with Venetoclax in Combination with Daratumumab and Dexamethasone

Rapid and Sustained Reduction of Immunosuppressive T-Cells and Focusing of the T-Cell Repertoire in t(11;14) Relapsed/Refractory Multiple Myeloma Patients Treated with Venetoclax in Combination with Daratumumab and Dexamethasone

TCRβ rearrangements without a D segment are common, abundant, and public

Significance The human body detects foreign pathogens by T cells with specific receptors. These are not directly encoded in the genome but generated in a random process that combines small gene segments into functional subunits of the receptor. The β -chain of the T cell receptor is normally composed of three such gene segments. Here we identify a group of T cells that lack the middle segment in their receptor sequence. We find that such sequences are mostly generated before birth, persist over a human lifetime, and, as a result, are excessively shared between individuals.

Genome-Wide Analysis Identifies Rag1 and Rag2 as Novel Notch1 Transcriptional Targets in Thymocytes.

Recombination activating genes 1 (Rag1) and Rag2 are expressed in immature lymphocytes and essential for generating the vast repertoire of antigen receptors. Yet, the mechanisms governing the transcription of Rag1 and Rag2 remain to be fully determined, particularly in thymocytes. Combining cDNA microarray and ChIP-seq analysis, we identify Rag1 and Rag2 as novel Notch1 transcriptional targets in acute T-cell lymphoblastic leukemia (T-ALL) cells. We further demonstrate that Notch1 transcriptional complexes directly bind the Rag1 and Rag2 locus in not only T-ALL but also primary double negative (DN) T-cell progenitors. Specifically, dimeric Notch1 transcriptional complexes activate Rag1 and Rag2 through a novel cis-element bearing a sequence-paired site (SPS). In T-ALL and DN cells, dimerization-defective Notch1 causes compromised Rag1 and Rag2 expression; conversely, dimerization-competent Notch1 achieves optimal upregulation of both. Collectively, these results reveal Notch1 dimerization-mediated transcription as one of the mechanisms for activating Rag1 and Rag2 expression in both primary and transformed thymocytes. Our data suggest a new role of Notch1 dimerization in compelling efficient TCRβ rearrangements in DN progenitors during T-cell development.

Bim Regulates the Survival and Suppressive Capability of CD8+ Foxp3+ Regulatory T Cells during Graft Versus Host Disease

Graft versus host disease (GVHD) is a proinflammatory syndrome that is the major complication of allogeneic hematopoietic stem cell transplantation and can lead to significant pathological damage. The failure to reconstitute an effective regulatory T cell compartment is a defining characteristic of this disease and has been the genesis for strategies designed to augment reconstitution of regulatory T cells (Tregs) by either in vivo expansion or adoptive transfer of this population. While CD4+ Foxp3+ T cells have been the most carefully examined Treg population, CD8+ Foxp3+ Tregs also emerge early during GVHD, have suppressive function, and can attenuate disease severity. The precise role of these cells and the extent to which CD8+ Tregs possess functional and ontological similarities to CD4+ Tregs, however, have not been well delineated. Using established murine models of GVHD, we observed that adoptively transferred alloantigen-specific CD8+ Tregs had reduced in vivo survival in all target organs and were significantly less potent than CD4+ Tregs for the prevention of lethal GVHD. To define the mechanistic basis for their reduced efficacy, we first examined the T cell receptor (TCR) repertoire of in vivo-derived CD4+ and CD8+ Tregs to determine whether a restricted repertoire was a potential explanation for the diminished ability of CD8+ Tregs to prevent GVHD. We employed GVHD models in which both polyclonal CD8+ [B6→Balb/c] and alloantigen-specific [B6→Balb.B] CD8+ H60+ Treg populations could be assessed and used the Shannon entropy index to define TCR repertoire diversity which is critical for the maintenance of self-tolerance by Tregs. Amplicon sequencing of TCR-β rearrangements to analyze the TCR repertoires of in vivo-induced CD8+ and CD4+ Tregs, along with their conventional CD8+ and CD4+ T cell counterparts, revealed that polyclonal CD8+ and alloantigen-specific CD8+ H60+ Tregs had repertoire diversity that was similar to that of conventional CD8+ T cells, indicating that a more restricted repertoire was not the proximate cause of decreased suppressive capability. Subsequent transcriptional profiling and principal component analysis revealed that CD8+ Tregs arising during GVHD possessed a canonical Treg transcriptional signature that was similar to that observed in CD4+ Tregs, yet distinct from conventional CD8+ T cells. Gene set enrichment and Ingenuity pathway analyses, however, both demonstrated that CD8+ Tregs had differential gene expression in pathways involved in cell death and survival when compared to CD4+ Tregs. This was further confirmed by gene expression analysis and protein expression studies which revealed that CD8+ Tregs had increased expression of the pro-apoptotic proteins Bim, cleaved caspase 3, and cleaved caspase 7, as well as a reduction in the anti-apoptotic protein, Mcl-1. To determine whether Bim was functionally important for CD8+ Treg survival, we comparatively examined in vivo-derived CD4+ and CD8+ Treg induction using wild type Foxp3EGFP and Bim-/-Foxp3EGFP animals as donors in transplantation studies. The absence of Bim in the CD8+ Treg compartment resulted in a significant increase in absolute CD8+ Treg numbers in the liver, lung, and colon when examined on days 7, 14, and 21 post transplantation. Given that the absence of Bim augmented in vivo-generated CD8+ Treg survival, we then examined whether Bim also regulated the survival of adoptively transferred, alloantigen-specific CD8+ Tregs which we had observed were inferior to CD4+ Tregs for the suppression of GVHD. We noted an increased percentage and absolute number of adoptively transferred CD8+ Foxp3+ Bim-/- Tregs relative to wild type CD8+ Tregs in all tissue sites, indicating that the absence of Bim resulted in enhanced stabilization of Foxp3 expression and augmented in vivo survival. Finally, animals that were transplanted with in vitro-derived, alloantigen-specific CD8+ Tregs from Bim-/- Foxp3EGFP mice had significantly prolonged survival relative to mice reconstituted with wild type CD8+ Foxp3EGFP Tregs, demonstrating that the prolonged in vivo survival conferred by the absence of Bim resulted in an increased ability to prevent GVHD. Collectively, these studies demonstrate that Bim plays a critical role in the regulation of CD8+ Treg survival which may have clinical implications for the use of regulatory T cell therapy in the treatment and prevention of GVHD. DisclosuresVincent:Merck: Research Funding; Pharmacyclics: Research Funding; Nanostring: Membership on an entity's Board of Directors or advisory committees. Serody:Merck: Research Funding.

Cutting Edge: TCR-β Selection Is Required at the CD4+CD8+ Stage of Human T Cell Development.

T cell development is predicated on the successful rearrangement of the TCR gene loci, which encode for Ag-specific receptors. Recombination-activating gene (RAG) 2 is required for TCR gene rearrangements, which occur during specific stages of T cell development. In this study, we differentiated human pluripotent stem cells with a CRISPR/Cas9-directed deletion of the RAG2 gene (RAG2-KO) to elucidate the requirement for the TCR β-chain in mediating β-selection during human T cell development. In stark contrast to mice, human RAG2-KO T lineage progenitors progressed to the CD4+CD8+ double-positive (DP) stage in the absence of TCRβ rearrangements. Nonetheless, RAG2-KO DPs retrovirally transduced to express a rearranged TCR β-chain showed increased survival and proliferation as compared with control-transduced RAG2-KO DPs. Furthermore, transcriptomic analysis showed that TCRβ- and control-transduced RAG2-KO DPs differed in gene pathways related to survival and proliferation. Our results provide important insights as to the distinct requirement for the TCR β-chain during human T cell development.

CALM-AF10 is a common fusion transcript in T-ALL and is specific to the TCRγδ lineage

AbstractThe t(10;11)(p13-14;q14-21) associated with CALM-AF10 is considered to be rare and associated with a variety of acute lymphoid and myeloid leukemias. Twelve (9%) of 131 unselected T-cell acute lymphoid leukemias (T-ALLs) expressed CALM-AF10 by reverse transcription–polymerase chain reaction or fluorescence in situ hybridization (or both), including 8% of children and 10% of adults, of whom only half demonstrated a t(10;11) by classical cytogenetics. CALM-AF10 was not found in T-cell–receptor αβ (TCRαβ) lineage T-ALLs, as defined by expression of TCRαβ, cytoplasmic TCRβ, or TCRβVDJ rearrangement in immature cytoplasmic TCRβ- cases, compared with 19% of TCRγδ T-ALLs and 33% of immature δ/γ T-ALLs. The latter differed from their CALM-AF10- immature counterparts by a CD5+/CD2-phenotype, as found in TCRγδ but not TCRαβ T-ALLs and in their TCRγ and TCRδ configurations, altogether suggesting that CALM-AF10+ immature δ/γT-ALLs are TCRγδ precursors and that, within T-ALL, CALM-AF10 is specific for this lineage. Nine of 12 immature CALM-AF10 T-ALLs demonstrated 3′ fusion transcripts, whereas 6 of 7 TCRγδ T-ALLs demonstrated 5′ fusion transcripts. The latter retain the AF10 extended LAP/PHD domain necessary for homo-oligomerization. All 8 patients with CALM-AF10+TCRγδ T-ALLs are alive, compared with only 3 of 12 with immature CALM-AF10+ T-ALLs. Six CALM-AF10+ non-T acute leukemias all expressed CD7 and demonstrated T-restricted TCRδ rearrangements, suggesting that they may also be related to the TCRγδ lineage. CALM-AF10 is therefore the most common fusion protein in T-ALL. It requires molecular and immunophenotypic characterization for appropriate prognostic evaluation and should be included in diagnostic screening panels of T-ALL and immature acute leukemias. Analysis of immature CALM-AF10+ leukemias will also facilitate analysis of the early stages of development of the TCRγδ lineage.

Profiling T-Cell Receptor Diversity and Dynamics during Lymphoma Immunotherapy Using Cell-Free DNA (cfDNA)

Profiling T-Cell Receptor Diversity and Dynamics during Lymphoma Immunotherapy Using Cell-Free DNA (cfDNA)

Impact of T Cell Repertoire Diversity on Mortality Following Cord Blood Transplantation.

IntroductionCord blood transplantation (CBT) recipients are at increased risk of mortality due to delayed immune recovery (IR). Prior studies in CBT patients have shown that recovery of absolute lymphocyte count is predictive of survival after transplant. However, there are no data on the association of T-cell receptor (TCR) and clinical outcomes after CBT. Here we retrospectively performed TCR beta chain sequencing on peripheral blood (PB) samples of 34 CBT patients.MethodsAll patients received a total body irradiation based conditioning regimen and cyclosporine and MMF were used for graft versus host disease (GvHD) prophylaxis. PB was collected pretransplant on days 28, 56, 80, 180, and 1-year posttransplant for retrospective analysis of IR utilizing high-throughput sequencing of TCRβ rearrangements from genomic DNA extracted from PB mononuclear cells. To test the association between TCR repertoire diversity and patient outcomes, we conducted a permutation test on median TCR repertoire diversity for patients who died within the first year posttransplant versus those who survived.ResultsMedian age was 27 (range 1–58 years) and most of the patients (n = 27) had acute leukemias. There were 15 deaths occurring between 34 to 335 days after transplant. Seven deaths were due to relapse. Rapid turnover of T cell clones was observed at each time point, with TCR repertoires stabilizing by 1-year posttransplant. TCR diversity values at day 100 for patients who died between 100 and 365 days posttransplant were significantly lower than those of the surviving patients (p = 0.01).ConclusionsUsing a fast high-throughput TCR sequencing assay we have demonstrated that high TCR diversity is associated with better patient outcomes following CBT. Importantly, this assay is easily performed on posttransplant PB samples, even as early as day 28 posttransplant, making it an excellent candidate for early identification of patients at high risk of death.

Phosphate Transporter Profiles in Murine and Human Thymi Identify Thymocytes at Distinct Stages of Differentiation

Thymocyte differentiation is dependent on the availability and transport of metabolites in the thymus niche. As expression of metabolite transporters is a rate-limiting step in nutrient utilization, cell surface transporter levels generally reflect the cell's metabolic state. The GLUT1 glucose transporter is upregulated on actively dividing thymocytes, identifying thymocytes with an increased metabolism. However, it is not clear whether transporters of essential elements such as phosphate are modulated during thymocyte differentiation. While PiT1 and PiT2 are both phosphate transporters in the SLC20 family, we show here that they exhibit distinct expression profiles on both murine and human thymocytes. PiT2 expression distinguishes thymocytes with high metabolic activity, identifying immature murine double negative (CD4−CD8−) DN3b and DN4 thymocyte blasts as well as immature single positive (ISP) CD8 thymocytes. Notably, the absence of PiT2 expression on RAG2-deficient thymocytes, blocked at the DN3a stage, strongly suggests that high PiT2 expression is restricted to thymocytes having undergone a productive TCRβ rearrangement at the DN3a/DN3b transition. Similarly, in the human thymus, PiT2 was upregulated on early post-β selection CD4+ISP and TCRαβ−CD4hiDP thymocytes co-expressing the CD71 transferrin receptor, a marker of metabolic activity. In marked contrast, expression of the PiT1 phosphate importer was detected on mature CD3+ murine and human thymocytes. Notably, PiT1 expression on CD3+DN thymocytes was identified as a biomarker of an aging thymus, increasing from 8.4 ± 1.5% to 42.4 ± 9.4% by 1 year of age (p < 0.0001). We identified these cells as TCRγδ and, most significantly, NKT, representing 77 ± 9% of PiT1+DN thymocytes by 1 year of age (p < 0.001). Thus, metabolic activity and thymic aging are associated with distinct expression profiles of the PiT1 and PiT2 phosphate transporters.

PD-1/PD-L1 signaling regulates the thymic niche size of agonistly selected T cells

Abstract Effective peripheral T cell responses rely upon a diverse T cell repertoire, generated during thymic selection. Signaling through the TCR establishes thresholds for thymocyte selection in order to create this repertoire. Because co-stimulation modifies TCR signaling, it may contribute to the regulation of T cell development by determining signal strength thresholds that direct thymocytes into specific lineages. Programmed death-1 (PD-1), a member of the B7/CD28 family of costimulatory/inhibitory receptors, has been implicated in the development of thymocytes because it is expressed in CD4− CD8− double-negative thymocytes during TCRb rearrangement. Further, the ligands for PD-1, PD-L1 and PD-L2, are expressed on thymic epithelial cells. PD-L2 is restricted to the medulla whereas PD-L1 is expressed throughout the thymus. Here, we show that PD-1 modulates agonist selection, a process in which some high-affinity thymocytes will mature into unconventional T cell lineages (e.g. regulatory T cells, NKT cells). Transgenic mice lacking either PD-L1 or PD-1 have increased numbers of agonistly selected T cells compared to wildtype animals. Interestingly, loss of PD-L1 increases the selection of conventional and agonistly selected T cells whereas loss of PD-1 limits the phenotype to agonist lineages. These data demonstrate that PD-1, PD-L1, and possibly PD-L2 interactions, potentially within the thymic microenvironment in which they are expressed, play a role in determining T cell fate and shaping the final T cell repertoire.