TCRβ Repertoire Research Articles

Longitudinal analysis reveals age-related changes in the T cell receptor repertoire of human T cell subsets.

A diverse T cell receptor (TCR) repertoire is essential for protection against a variety of pathogens, and TCR repertoire size is believed to decline with age. However, the precise size of human TCR repertoires, in both total and subsets of T cells, as well as their changes with age, are not fully characterized. We conducted a longitudinal analysis of the human blood TCRα and TCRβ repertoire of CD4+ and CD8+ T cell subsets using a unique molecular identifier–based (UMI-based) RNA-seq method. Thorough analysis of 1.9 × 108 T cells yielded the lower estimate of TCR repertoire richness in an adult at 3.8 × 108. Alterations of the TCR repertoire with age were observed in all 4 subsets of T cells. The greatest reduction was observed in naive CD8+ T cells, while the greatest clonal expansion was in memory CD8+ T cells, and the highest increased retention of TCR sequences was in memory CD8+ T cells. Our results demonstrated that age-related TCR repertoire attrition is subset specific and more profound for CD8+ than CD4+ T cells, suggesting that aging has a more profound effect on cytotoxic as opposed to helper T cell functions. This may explain the increased susceptibility of older adults to novel infections.

Chronic Hypergravity Induces a Modification of Histone H3 Lysine 27 Trimethylation at TCRβ Locus in Murine Thymocytes.

Gravity changes are major stressors encountered during spaceflight that affect the immune system. We previously evidenced that hypergravity exposure during gestation affects the TCRβ repertoire of newborn pups. To identify the mechanisms underlying this observation, we studied post-translational histone modifications. We first showed that among the four studied post-translational histone H3 modifications, only lysine 27 trimethylation (H3K27me3) is downregulated in the thymus of mice exposed to 2× g for 21 days. We then asked whether the TCRβ locus chromatin structure is altered by hypergravity exposure. ChIP studies performed on four Vβ segments of the murine double-negative SCIET27 thymic cell line, which corresponds to the last maturation stage before V(D)J recombination, revealed increases in H3K27me3 after 2× g exposure. Finally, we evaluated the implication for the EZH2 methyltransferase in the regulation of the H3K27me3 level at these Vβ segments by treating SCIET27 cells with the GSK126-specific inhibitor. These experiments showed that the downregulation of H3K27me3 contributes to the regulation of the Vβ germline transcript expression that precedes V(D)J recombination. These data show that modifications of H3K27me3 at the TCRβ locus likely contribute to an explanation of why the TCR repertoire is affected by gravity changes and imply, for the first time, EZH2 in the regulation of the TCRβ locus chromatin structure.

Attenuated T cell activation and rearrangement of T cell receptor β repertoire in silica nanoparticle-induced pulmonary fibrosis of mice

Silica nanoparticles (SiNPs) cause pulmonary fibrosis through a complex immune response, but the underlying mechanisms by which SiNPs interact with T cells and affect their functions remain unclear. The T cell receptor (TCR) repertoire is closely related to T cell activation and proliferation and mediates innate and adaptive immunity. High-throughput sequencing of the TCR enables comprehensive monitoring of the immune microenvironment. Here, the role of the TCRβ repertoire was explored using a mouse model of SiNP-induced pulmonary fibrosis and a co-culture of RAW264.7 and CD4+ T cells. Our results demonstrated increased TCRβ expression and decreased CD25 and CD69 expression in CD4+ T cells from peripheral blood and lung collected 14 days after the induction of pulmonary fibrosis by SiNPs. Simultaneously, SiNPs significantly decreased CD25 and CD69 expression in CD4+ T cells in vitro via RAW264.7 cell presentation. Mechanistically, pLCK and pZap70 expression, involved in mediating T cell activation, were also decreased in the lung of mice with SiNP-induced pulmonary fibrosis. Furthermore, the profile of the TCRβ repertoire in mice with SiNP-induced pulmonary fibrosis showed that SiNPs markedly altered the usage of V genes, VJ gene combinations, and CDR3 amino acids in lung tissue. Collectively, our data suggested that SiNPs could interfere with T cell activation by macrophage presentation via the LCK/Zap70 pathway and rearrange the TCRβ repertoire for adaptive immunity and the pulmonary microenvironment.

A set point in the selection of the αβTCR T cell repertoire imposed by pre-TCR signaling strength

Signaling via the T cell receptor (TCR) is critical during the development, maintenance, and activation of T cells. Quantitative aspects of TCR signaling have an important role during positive and negative selection, lineage choice, and ability to respond to small amounts of antigen. By using a mutant mouse line expressing a hypomorphic allele of the CD3ζ chain, we show here that the strength of pre-TCR–mediated signaling during T cell development determines the diversity of the TCRβ repertoire available for positive and negative selection, and hence of the final αβTCR repertoire. This finding uncovers an unexpected, pre-TCR signaling–dependent and repertoire–shaping role for β-selection beyond selection of in-frame rearranged TCRβ chains. Our data furthermore support a model of pre-TCR signaling in which the arrangement of this receptor in stable nanoclusters determines its quantitative signaling capacity.

DNA-PK inhibition plus immune adjuvants promotes CD8 TIL infiltration, neoantigen presentation, and diversifies the tumor-reactive TCRβ repertoire in B16 melanoma

Abstract Despite significant improvements in cancer immunotherapies, enhancing immunogenicity of non-responsive tumors warrants further investigation. A prior drug screen of ~3,000 compounds identified NU7441, a DNA PK inhibitor, as an effective compound that promotes immunogenicity of various melanoma lines in vitro. In this study, we hypothesized that in vivo combination therapy NU7441, STING-L, and CD40 agonist will enhance tumor immunogenicity resulting in both expansion and increased cytotoxic activity of CD8+TCRβ tumor-reactive TILs in B16 melanoma models. Results obtained by flow cytometry demonstrated that combination treatment 1) significantly increased the ratio of CD8/CD4 TILs, 2) expanded several tumor-reactive TCRβ clones, 3) increased granzyme B production and expression of 4-1BB and PD-1, 4) increased ratio of DC/MDSC infiltration, and 5) potentially identified a novel cytotoxic CD8+CD11c+GR-1+ population. Additionally, the expansion of tumor-reactive TCRs was attributed to DNA-PKi’s ability to both expand and diversify the number of neoantigen transcripts resulting in a broader neoantigen expression profile. RNA-seq identified 27 unique neoantigens as potential novel targets in melanoma immunotherapy. TILs isolated from B16 tumors were co-cultured with dendritic cells transfected with tandem-mini genes, each encoding ~10 neoantigens, and T cells were analyzed by flow cytometry to quantify the TCRβ repertoire and functional response to neoantigens. We demonstrate combination treatment with NU7441, STING-L and CD40 agonist enhance antitumor responses through increased myeloid cell infiltration and sensitization of tumor cells to T cell-mediated killing from an expanded CD8+TCRβ repertoire. Supported by R01CA207913 NCI BX004935-01, VA, Merit Award P30CA046934, NCI Leukemia and Lymphoma Society Department of Defense Gates Grubstake University of Colorado Anschutz Medical Campus Cancer League of Colorado

SARS-CoV-2 epitope-specific CD8+ clonotypes persist 8 month post-infection despite reduced clonality.

Abstract Memory T cells play a crucial role in acceleration of immune response during reinfection with the same pathogen. Since the beginning of the COVID-19 pandemic, attention has been focused on determining the epitopes of SARS-CoV-2 that give rise to long-lived memory T cells. We examined the immune response to 12 immunogenic CD8+ epitopes in 26 COVID-19 convalescent patients. Paired blood samples were collected shortly after infection and approximately 8 months after. Using ex vivo T cell expansions with a subsequent MHC-tetramer+ cell-sorting and T cell receptor beta-chain (TCRβ) sequencing we studied the epitope-specific CD8+ response on the clonal level. In total in both time points we identified 756 epitope-specific TCRβ sequences. Nine epitopes were immunodominant while five immunogenic epitopes (YLQ, KCY, LLY, KTF and ALW) yielded response in 100% of tested individuals. This coincided with a high number of clones per individual specific to these epitopes (median = 16.5). Despite the very low frequency of specific cells in the total repertoire most (75.4%) of responses were retained at 8-month post-infection. The median number of clones for the top five most immunogenic epitopes dropped to 6. The TCRβ repertoires of T cells specific to the studied epitopes were characterized by different degrees of CDR3 homology. LLY and YLQ-specific TCRβ had the highest degree of mutual similarity while KCY and KTF-specific TCRβ were the most diverse. Our data demonstrates that despite the overall decrease of T cell reactivity and clonality most epitopes are still recognized due to the ability of memory SARS-CoV-2-specific CD8+ cells to survive over prolonged periods of time. The work was supported by the Russian Science Foundation grant 20-15-00395.

Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination.

Antigen recognition through the T cell receptor (TCR) αβ heterodimer is one of the primary determinants of the adaptive immune response. Vaccines activate naïve T cells with high specificity to expand and differentiate into memory T cells. However, antigen-specific memory CD4 T cells exist in unexposed antigen-naïve hosts. In this study, we use high-throughput sequencing of memory CD4 TCRβ repertoire and machine learning to show that individuals with preexisting vaccine-reactive memory CD4 T cell clonotypes elicited earlier and higher antibody titers and mounted a more robust CD4 T cell response to hepatitis B vaccine. In addition, integration of TCRβ sequence patterns into a hepatitis B epitope-specific annotation model can predict which individuals will have an early and more vigorous vaccine-elicited immunity. Thus, the presence of preexisting memory T cell clonotypes has a significant impact on immunity and can be used to predict immune responses to vaccination.

Longitudinal High-Throughput Sequencing of the T-Cell Receptor Repertoire Reveals Dynamic Change and Prognostic Significance of Peripheral Blood TCR Diversity in Metastatic Colorectal Cancer During Chemotherapy.

Colorectal cancer (CRC) is a major cause of cancer mortality and morbidity. Despite advances in chemotherapy and targeted therapy, unsustainable clinical benefit was noted due to recurrence and therapy resistance. The immune status of the cancer patient may affect the effectiveness of disease treatments. The dynamic change in the T-cell receptor (TCR) repertoire might be a clinical parameter for monitoring treatment responses. In this study, we aimed to determine the characteristics and clinical significance of the TCR repertoire in patients with unresectable metastatic colorectal cancer (mCRC). Herein, we comprehensively profile 103 peripheral blood samples from 20 healthy controls and 16 CRC patients with a follow-up of 98 to 452 days to identify hypervariable rearrangements of the TCRα and TCRβ repertoires using high-throughput sequencing. We found that TCRα repertoires, TCRβ repertoires, and CDR3 clonotypes were altered in mCRC patients compared with healthy controls. The diversity of TCR repertoires and CDR3 clonotypes decreased in most mCRC patients after therapy. Furthermore, compared with baseline TCR diversity, patients whose TCR diversity dropped considerably during therapy had better treatment responses, including lower CEA and CA19-9 levels and smaller tumor sizes. TCR baseline diversity was also significantly associated with partial response (PR) status (odds ratio: 5.29, p = 0.04). In conclusion, the present study demonstrated the association between dynamic changes in TCR diversity during chemotherapy and clinical outcomes as well as the potential utility of the TCR repertoire in predicting the prognosis of cancer treatment.

TIM3+ TRBV11-2 T cells and IFNγ signature in patrolling monocytes and CD16+ NK cells delineate MIS-C.

In rare instances, pediatric SARS-CoV-2 infection results in a novel immunodysregulation syndrome termed multisystem inflammatory syndrome in children (MIS-C). We compared MIS-C immunopathology with severe COVID-19 in adults. MIS-C does not result in pneumocyte damage but is associated with vascular endotheliitis and gastrointestinal epithelial injury. In MIS-C, the cytokine release syndrome is characterized by IFNγ and not type I interferon. Persistence of patrolling monocytes differentiates MIS-C from severe COVID-19, which is dominated by HLA-DRlo classical monocytes. IFNγ levels correlate with granzyme B production in CD16+ NK cells and TIM3 expression on CD38+/HLA-DR+ T cells. Single-cell TCR profiling reveals a skewed TCRβ repertoire enriched for TRBV11-2 and a superantigenic signature in TIM3+/CD38+/HLA-DR+ T cells. Using NicheNet, we confirm IFNγ as a central cytokine in the communication between TIM3+/CD38+/HLA-DR+ T cells, CD16+ NK cells, and patrolling monocytes. Normalization of IFNγ, loss of TIM3, quiescence of CD16+ NK cells, and contraction of patrolling monocytes upon clinical resolution highlight their potential role in MIS-C immunopathogenesis.

Maternal and Infant Immune Repertoire Sequencing Analysis Identifies Distinct Ig and TCR Development in Term and Preterm Infants.

Preterm labor (PTL) is the leading cause of neonatal morbidity and mortality worldwide. Whereas many studies have investigated the maternal immune responses that cause PTL, fetal immune cell activation has recently been raised as an important contributor to the pathogenesis of PTL. In this study, we analyzed lymphocyte receptor repertoires in maternal and cord blood from 14 term and 10 preterm deliveries, hypothesizing that the high prevalence of infection in patients with PTL may result in specific changes in the T cell and B cell repertoires. We analyzed TCR β-chain (TCR-β) and IgH diversity, CDR3 lengths, clonal sharing, and preferential usage of variable and joining gene segments. Both TCR-β and IgH repertoires had shorter CDR3s compared with those in maternal blood. In cord blood samples, we found that CDR3 lengths correlated with gestational age, with shorter CDR3s in preterm neonates suggesting a less developed repertoire. Preterm cord blood displayed preferential usage of a number of genes. In preterm pregnancies, we observed significantly higher prevalence of convergent clones between mother/baby pairs than in term pregnancies. Together, our results suggest the repertoire of preterm infants displays a combination of immature features and convergence with maternal TCR-β clones compared with that of term infants. The higher clonal convergence in PTL could represent mother and fetus both responding to a shared stimulus like an infection. These data provide a detailed analysis of the maternal-fetal immune repertoire in term and preterm patients and contribute to a better understanding of neonate immune repertoire development and potential changes associated with PTL.

Allogeneic NKT Cells Expressing a CD19-Specific CAR in Patients with Relapsed or Refractory B-Cell Malignancies: An Interim Analysis

Autologous T cells engineered to express a CD19-specific chimeric antigen receptor (CAR) mediate high rates of complete response (CR) in patients with B-cell malignancies. However, autologous cell therapy products are time- and resource-intensive to manufacture and vary in potency and toxicity. Unlike polymorphic HLA-restricted T cells, monomorphic CD1d-restricted Vα24-invariant natural killer T cells (NKTs) are not alloreactive, and therefore therapeutic NKTs can be generated from allogeneic donors without the risk of graft-versus-host disease (GvHD). Moreover, pre-clinical models suggest that CAR-NKTs have inherent advantages over CAR-T products including an ability to trans-activate NK cells, cross-prime tumor-specific CD8 T cells, and recognize CD1d-positive B-cell lymphoma cells via their endogenous NKT T cell receptor.We report interim results from five patients treated on a phase 1 dose-escalation trial of allogeneic NKTs engineered to co-express a CD19-specific CAR, IL-15, and shRNA targeting beta-2 microglobulin and CD74 for downregulation of HLA class I and class II molecules, respectively (ANCHOR, NCT00840853). Four patients with relapsed/refractory B-cell non-Hodgkin's lymphoma (NHL, cohort A) were enrolled on dose level (DL) 1 (NHL-1, -2, -3) and DL 2 (NHL-4), and 1 patient with relapsed acute B-lymphoblastic leukemia (ALL, cohort B) was enrolled on DL 1 (ALL-1). Primary and secondary objectives of the trial are to assess safety and anti-tumor responses; immune response evaluation is an additional objective. NKTs were isolated from the leukapheresis product of 1 HLA-unmatched healthy individual, transduced with the CAR, expanded ex vivo for 14 days to a total of 2.7×10 9 CAR-positive cells (99.8% NKT purity, 0.04% T cells), and cryopreserved. Patients received 10 7 (DL 1) or 3×10 7 (DL 2) CAR-NKT cells per square meter of body surface area following lymphodepleting conditioning with cyclophosphamide/fludarabine. Adverse events were evaluated per NCI criteria. When accessible, patients underwent core biopsies of an involved site at 2-5 weeks post-infusion. Response to therapy was assessed at 4 weeks per Lugano Criteria (for NHL) or NCCN guidelines (for ALL).The most common adverse effects observed were nausea and grade 3-4 hematologic toxicities related to the lymphodepletion chemotherapy. There were no early adverse events attributable to the cellular product except grade 1 cytokine release syndrome in 1 patient. Of the 4 NHL patients (all with diffuse large B cell lymphoma), 3 had a partial response (NHL-1, -2, and -4, Figure 1A) that evolved into a CR in 1 case (NHL-2). The ALL patient achieved a CR with incomplete hematologic recovery and showed no evidence of leukemia by morphology, flow cytometry, or next-generation sequencing at 4 weeks. We detected in vivo expansion of donor-derived NKT and CAR-NKT cells in the peripheral blood of NHL-4 and ALL-1 that peaked at 1 week post-infusion in both cases as determined by flow cytometry and qPCR. While CAR-NKTs were not detected in the peripheral blood of the first 3 NHL patients beyond 3 hours post-infusion, they were found in tumor tissues collected from the 2 biopsied NHL patients (Figure 1B). In patient NHL-2, we also observed a 2000-fold expansion of recipient NKTs with a skewed TCRβ repertoire; this population peaked at 6 weeks post-treatment and remained elevated through 12 weeks (Figure 1C). An ELISpot assay performed with peripheral blood from NHL-1 and NHL-2 before treatment and at 4 and 6 weeks post-therapy to detect changes in the frequency of T cells reactive to a set of tumor-associated antigens (TAAs) found no evidence for epitope spreading toward the tested TAAs.In conclusion, our data indicate that allogeneic CAR-NKT cells are well-tolerated and can mediate objective responses in relapsed/refractory NHL and ALL patients even at the low doses tested. Our initial results from this first-in-human clinical evaluation of allogeneic CAR-NKTs suggest that NKTs represent a promising platform for “off-the-shelf” cancer immunotherapy. [Display omitted] DisclosuresRamos: Tessa Therapeutics: Patents & Royalties, Research Funding; Athenex: Research Funding; Genentech: Consultancy; Novartis: Consultancy. Courtney: Athenex: Patents & Royalties, Research Funding. Metelitsa: Athenex: Patents & Royalties, Research Funding. OffLabel Disclosure:Cyclophosphamide and fludarabine are being used as lymphodepleting agents before immune effector infusion.

Impact of HLA type, age and chronic viral infection on peripheral T-cell receptor sharing between unrelated individuals.

The human adaptive immune system must generate extraordinary diversity to be able to respond to all possible pathogens. The T-cell repertoire derives this high diversity through somatic recombination of the T-cell receptor (TCR) locus, a random process that results in repertoires that are largely private to each individual. However, factors such as thymic selection and T-cell proliferation upon antigen exposure can affect TCR sharing among individuals. By immunosequencing the TCRβ variable region of 426 healthy individuals, we find that, on average, fewer than 1% of TCRβ clones are shared between individuals, consistent with largely private TCRβ repertoires. However, we detect a significant correlation between increased HLA allele sharing and increased number of shared TCRβ clones, with each additional shared HLA allele contributing to an increase in ~0.01% of the total shared TCRβ clones, supporting a key role for HLA type in shaping the immune repertoire. Surprisingly, we find that shared antigen exposure to CMV leads to fewer shared TCRβ clones, even after controlling for HLA, indicative of a largely private response to major viral antigenic exposure. Consistent with this hypothesis, we find that increased age is correlated with decreased overall TCRβ clone sharing, indicating that the pattern of private TCRβ clonal expansion is a general feature of the T-cell response to other infectious antigens as well. However, increased age also correlates with increased sharing among the lowest frequency clones, consistent with decreased repertoire diversity in older individuals. Together, all of these factors contribute to shaping the TCRβ repertoire, and understanding their interplay has important implications for the use of T cells for therapeutics and diagnostics.

T-follicular helper cell expansion and chronic T-cell activation are characteristic immune anomalies in Evans syndrome

AbstractPediatric Evans syndrome (pES) is increasingly identified as the presenting manifestation of several inborn errors of immunity. Despite an improved understanding of genetic defects in pES, the underlying immunobiology of pES is poorly defined, and characteristic diagnostic immune parameters are lacking. We describe the immune characteristics of 24 patients with pES and compared them with 22 patients with chronic immune thrombocytopenia (cITP) and 24 healthy controls (HCs). Compared with patients with cITP and HC, patients with pES had increased circulating T-follicular helper cells (cTfh), increased T-cell activation, and decreased naïve CD4+ T cells for age. Despite normal or high immunoglobulin G (IgG) in most pES at presentation, class-switched memory B cells were decreased. Within the cTfh subset, we noted features of postactivation exhaustion with upregulation of several canonical checkpoint inhibitors. T-cell receptor β chain (TCR-β) repertoire analysis of cTfh cells revealed increased oligoclonality in patients with pES compared with HCs. Among patients with pES, those without a known gene defect had a similar characteristic immune abnormality as patients with defined genetic defects. Similarly, patients with pES with normal IgG had similar T-cell abnormalities as patients with low IgG. Because genetic defects have been identified in less than half of patients with pES, our findings of similar immune abnormalities across all patients with pES help establish a common characteristic immunopathology in pES, irrespective of the underlying genetic etiology.

A comprehensive model based on temporal dynamics of peripheral T cell repertoire for predicting post-treatment distant metastasis of nasopharyngeal carcinoma.

Many nasopharyngeal carcinoma (NPC) patients develop distant metastases after treatment, leading to poor outcomes. To date, there are no peripheral biomarkers suitable for all NPC patients to predict distant metastasis. Hence, we purposed to develop a noninvasive comprehensive model for predicting post-treatment distant metastasis of all NPC. Since T-cell receptor β chain (TCRB) repertoire has achieved prognostic prediction in many cancers, the clinical characteristics and parameters of TCRB repertoire of 71 cases of peripheral blood samples (pairwise pre-treatment and post-treatment samples from 40 NPC patients who without (nM, n = 21) or with (M, n = 19) post-treatment distant metastasis) were collected. The least absolute shrinkage and selection operator algorithm was used to construct a distant metastasis prediction model. In terms of TCRB repertoire parameters, the diversity of TCRB repertoire was significantly decreased in M group after treatment but not in nM group. Ascending TCRB diversity and higher similarity between pre- and post-treatment samples showed better distant metastasis-free survival (DMFS). The similarity still had robust DMFS prediction in patients with reduced TCRB diversity. More importantly, the 5-factor comprehensive model consisting of basic clinical characteristics and TCRB repertoire indices showed a higher prognostic accuracy than any one individual factor in DMFS predicting. In conclusion, treatment had different effects on the composition of TCRB repertoire in patients without and with post-treatment distant metastasis. The dynamics of TCRB diversity, the similarity of TCRB repertoires, and combinations of these factors with basic clinical characteristics could serve as noninvasive DMFS predictors for all NPC patients.

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.

The Orally Available Selective Axl Inhibitor BGB324 Induces Diversification of the Immune Repertoire and Specific Changes in Plasma Biomarker Profiles

Introduction: The RTK Axl represents a novel therapeutic target in AML promoting proliferation and therapy resistance. In addition, Axl suppresses immune responses by decreasing antigen presentation and inhibiting NK cells. BGB324 is a first-in-class, orally available, selective Axl kinase inhibitor which is being explored as a therapy for solid tumours and myeloid malignancies. Selective blockade of Axl by BGB324 in AML is currently being explored in the Phase 1/2 trial BGBC003 (NCT02488408). Until now, n=25 patients have been treated in a classical 3+3 dose escalation design and treatment was generally well-tolerated (n=22 with AML and n=3 with MDS). Steady-state levels of BGB324 were achieved between three and six days after initiation of treatment. One AML patient achieved a CRi of five months, a second achieved a PR and another achieved clearance of circulating blasts accompanied by peripheral blood count recovery > three months (ORR 10%). Four additional AML patients (25%) experienced disease stabilisation for more than four months. Two MDS patients experienced a PR.Here, we report the spectrum of plasma proteins modulated by BGB324 in patients with AML/MDS and their relationship to treatment outcome and compound exposure. Furthermore, we analysed the B- and T- cell repertoire in order to assess potential effects of BGB324 on the immune response in AML patients.Methods: We used the DiscoveryMap panel (Myriad RBM) to measure plasma protein biomarker levels in a selection of patients pre-dose and at C2D1 and performed a cross-comparison with samples from an ongoing NSCLC study with BGB324 (BGBC004, NCT02424617) to identify candidates specifically modulated in AML. The TCRß repertoire was quantified by Next Generation Sequencing of DNA isolated from peripheral blood MNCs using an Illumina MiSeq sequencer. TCRß gene containing the entire Vß, Dß and Jß segments were amplified with BIOMED2-TCRß-A and -B primer pools. The IGH repertoire was analysed using BIOMED2-FR1/-FR3 primer pools. Using genomic DNA as template, the amplicons were tagged with Illumina adapters and indices in two consecutive PCR reactions. Demultiplexing and FastQ formated data output was generated by the MiSeq reporter. Analysis of TCRß and IGH data was performed on a Microsoft Cloud using our in-house analysis pipeline Pippa, which relies on MiXCR analysis tools.Results: Treatment with BGB324 significantly modulated Gas6, soluble Axl (sAxl), Angpt2, CXCL1 (GROalpha), SCGB1A1 (uteroglobin, club cell 10kDa protein) and PLAU (uPAR) levels in patient plasma. Of these, Gas6 was modulated after treatment with BGB324 in AML but not in lung cancer patients while CXCL1 and SCGB1A1 were only influenced in lung cancer patients. sAxl levels correlated with BGB324 exposure indicating that the shed Axl receptor represents a novel biomarker of Axl kinase inhibition.Furthermore, we studied the effects of BGB324 on the T- and B- cell receptor repertoire in n=5 available matched PB samples. We could demonstrate diversification of the TCR repertoire in PB by higher abundance of small T-cell clones at C2D1 with BGB324 compared to pre-treatment levels in three out of five analysed patients.The analyses of the IGH repertoire in the PB indicates higher diversity at C2D1 in two out of five available patients.Thus, treatment with BGB324 can increase the diversity of the IGH and TCRb repertoire in the PB of AML patients.Conclusion: BGB324 is well tolerated in AML patients, exhibits anti-leukemic activity and induces exposure-related modulation of biomarker candidates. Furthermore, BGB324 can induce a diversification of the TCR repertoire in AML patients and might hold potential as an immune-activating drug. DisclosuresLoges:BerGenBio ASA: Research Funding. Ben-Batalla:BerGenBio ASA: Research Funding. Micklem:BerGenBio ASA: Employment, Equity Ownership, Patents & Royalties. Brown:BerGenBIo ASA: Employment. Lorens:BerGenBIo ASA: Employment, Equity Ownership, Patents & Royalties, Research Funding. Yule:BerGenBIo ASA: Employment. Fiedler:Amgen: Patents & Royalties; Amgen, ARIAD/Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen, Pfizer: Research Funding; Amgen, Gilead, GSO, Teva, Jazz Pharmaceuticals: Other: Support for meeting attendance. Cortes:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Sun Pharma: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Teva: Research Funding.

Quantitative Assessment of TCR Repertoire and Allo-Reactivity of Human Acute Gvhd Tissue-Infiltrating T Cells

Abstract Introduction: Due to difficulty in isolating T cells from human tissues, the diversity of allo-reactive T cells that are infiltrating human acute GVHD (aGVHD) tissues remains largely uninvestigated. Here, we report the analysis of comprehensive T cell repertoire and allo-reactivity of human aGVHD tissue-infiltrating T cells. Patients and Methods: Skin (n = 7), stomach (n = 3) and colon (n = 2) biopsy samples and blood samples (n = 8) were obtained from eight post-transplant patients when the patients were supposed to develop aGVHD and prior to the fist-line treatment of aGVHD. Total RNA was extracted from tissue and blood samples, and T cell receptor (TCR)-β gene deep sequencing was performed (~100000 reads for each sample). To estimate the TCR-β repertoire diversity, inverse Simpson's diversity index (1/Ds) were calculated. T cells were isolated from tissues and cloned by limiting dilution. This study was approved by the ethics committees of Nagoya University. Results: In the skin of patient #1, the relative abundance of the most and second most frequent clonotypes were 74.9% and 22.8%, respectively, suggesting that only two T cell clones accounted for approximately 98% of GVHD skin-infiltrating T cells. Two T cell clones, termed clones A and B, were isolated from the skin of patient #1. Both clones lysed patient peripheral blood mononuclear cells, but failed to lyse donor cells. Thus, clones A and B were allo-reactive cytotoxic T lymphocytes (CTLs). The IFN-γ production of clone B was significantly increased when stimulated by COS cells transfected with HLA-DRB1*14:54 cDNA, suggesting that the clone B recognized the mismatched patient's HLA-DRB1*14:54 molecule. To confirm the relative abundance of clones A and B in his skin, the TCR-β usage and CDR3 were determined. Surprisingly, clones A and B were the most and second most frequent skin-infiltrating T cell clones. Taken together, these data suggested that a limited allo-reactive CTL clones were involved in GVHD development of patient #1. In all samples, the mean 1/Ds of skin, stomach, colon and all tissues were 9.4 (range, 1.2-21.3), 7.5 (1.9-14.9), 3.3 (1.0-5.5) and 7.9 (1.0-21.3), respectively, while that of all blood samples was 247 (18.8-888.7) (P 10% frequency) clonotypes in a certain tissue were detected in other tissue(s) of the same patient at an extremely low frequency ( 10% frequency) clonotypes in the skin of a certain patient were detected in those of other patients with an extremely low frequency ( Conclusion: In humans, a limited allo-reactive CTL clones, which differ from tissue to tissue and from patient to patient, may induce aGVHD. The degree of skewing of tissue-infiltrating T cell repertoire may be predictive of GVHD outcome. Download : Download high-res image (62KB) Download : Download full-size image Disclosures Kiyoi: Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Eisai Co., Ltd.: Research Funding; Meiji Seika Pharma Co.,Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; JCR Pharmaceuticals Co.,Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; MSD K.K.: Research Funding; ONO Pharmaceutical Co., Ltd.: Research Funding; Phizer Japan Inc.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Celgene Corporation: Consultancy, Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding.

Characterization of Circulating T Cell Receptor Repertoire Provides Information about Clinical Outcome after PD-1 Blockade in Advanced Non-Small Cell Lung Cancer Patients.

Simple SummaryImmune checkpoint blockers (ICBs) have demonstrated durable anti-tumor responses in advanced non-small cell lung cancer (NSCLC). Despite progress in development of new predictive biomarkers, such as PD-L1 expression, TMB, or MSI, there is still an urge for a better selection of patients that will benefit from the blockade of PD-1/PD-L1 axis. In this study, peripheral blood T cell receptor beta chain (TCR-β) repertoire, at baseline (PRE) and first response (FR) assessment, was analyzed with high-throughput sequencing in a cohort of advanced NSCLC patients receiving first-line pembrolizumab. Our results suggest that measuring TCR-β features in peripheral blood may be a potential tool to assess patients’ immune response. Furthermore, the usage of the TRBV20-1 segment highly predicts host response and survival in anti-PD-1 treated NSCLC patients.Despite the success of immunotherapies in lung cancer, development of new biomarkers for patient selection is urgently needed. This study aims to explore minimally invasive approaches to characterize circulating T cell receptor beta chain (TCR-β) repertoire in a cohort of advanced non-small cell lung cancer (NSCLC) patients treated with first-line pembrolizumab. Peripheral blood samples were obtained at two time points: i) pretreatment (PRE) and ii) first response assessment (FR). Next-generation sequencing (NGS) was used to analyze the hypervariable complementary determining region 3 (CDR3) of TCR-β chain. Richness, evenness, convergence, and Jaccard similarity indexes plus variable (V) and joining (J)-gene usage were studied. Our results revealed that increased richness during treatment was associated with durable clinical benefit (DCB; p = 0.046), longer progression-free survival (PFS; p = 0.007) and overall survival (OS; p = 0.05). Patients with Jaccard similarity index ≥0.0605 between PRE and FR samples showed improved PFS (p = 0.021). Higher TRBV20-1 PRE usage was associated with DCB (p = 0.027). TRBV20-1 levels ≥9.14% in PRE and ≥9.02% in FR significantly increased PFS (p = 0.025 and p = 0.016) and OS (p = 0.035 and p = 0.018). Overall, analysis of circulating TCR-β repertoire may provide information about the immune response in anti-PD-1 treated NSCLC patients; in this scenario, it can also offer important information about the clinical outcome.

Comprehensive T-cell receptor repertoire of Chinese lung cancer patients related with age.

e21076 Background: Cancer is most commonly diagnosed disease worldwide and also a leading cause of death. Cancer is more common in older people, but it can also be found in younger people. As the importance of T cell receptor (TCR) repertoire gains appreciation, particularly given their potential utility for cancer immunotherapeutic prognostication, the characteristics of TCR repertoire are needed. We detected the complementarity determining region 3 (CDR3) by TCR sequence to describe the correlation between diversity and clonality in human’s immune system and age. Methods: The TCRβ repertoire of 47 stage Ia lung cancer patients were analyzed. Genomic DNA was extracted from peripheral blood or tissue, and used to amplified and sequenced the CDR3 region of rearranged TCRβ genes. Finally, we got the relative frequencies of patients T cell clones. Shannon index was calculated on the clonal abundance of all productive TCR sequences. Simpson index also was calculated to measure the clonality of TCR repertoire. MOI was a measure of the similarity in the T cell repertoire between tissue and blood taking into account the specific rearrangements and their respective frequencies. Results: In tissue, the Simpson index ranged from 0.00075 to 0.11466 (mean = 0.01343); Shannon index ranged from 2.91 to 8.95 (mean = 6.26). The age ranged from 27 to 78 (median = 63). Patients were divided into two groups based on median age. The older showed higher Shannon index (p = 0.005) but lower Simpson index (p = 0.0072) than younger patients in tissue. Further, the higher Shannon index was found in younger group in blood(p = 0.0411). Interestingly, the MOI of the older group was significantly higher than that of the younger group (p = 0.0181), suggesting that the older group had a relatively better TME immune response. Then, the usage of V and J gene segments were analyzed. Through statistical analysis and the frequencies of V and J gene usage, we identified that more V and J gene usage were different in older and younger patients. The TRBJ1-1 and TRBJ1-2 were more common in younger patients and TRBJ2-1 and TRBJ2-2 were more common in older patients. There was also more V gene usage different in both groups. Conclusions: Given that the use of certain TRBV regions is predominant in tumor tissues, our results suggested that there may be differences in immune responses of TME to tumors in younger and older patients.

Characteristic of genetic and t cell receptor repertoire of Chinese multiple primary lung cancer patients.

e20510 Background: As the incidence of lung cancer soars, the number of patients diagnosed with multiple primary lung cancers (MPLC) was also rising. It was already known that genetic and immune microenvironment were involved in oncogenesis and treatment of tumors. The aim of this study was to investigate genetic and T Cell Receptor (TCR) Repertoire characteristic in patients with multiple primary lung cancers. Methods: We queried 84 lesions analyzed by 1021 gene panel and TCRβ repertoire for 32 patients with MPLC profiled by NGS. Shannon index was calculated on the clonal abundance of all productive TCR sequences. T cell clonality was defined as 1 − (Shannon index)/ln(# of productive unique sequences). MOI was a measure of the similarity in the T cell repertoire between tissue and blood taking into account the specific rearrangements and their respective frequencies. Results: 98.7% (77/78) of lesions from 30 patients were detected somatic mutations and EGFR mutations were detected in 44.2% (34/77) lesions; BRAF were detected in 22.1% (17/77) lesions; and KRAS were detected in 14.3% (11/77) lesions. We found different combinations of three driving genes in 13 patients. Almost all lesions (97.4%) had mutations in the RAS-RTK pathway. Shared mutations were detected between different lesions from 8 patients, and two patients were intrapulmonary metastases; The others were more meet to convergent evolution. The overall immune characteristics of 76 lesions from 30 patients were further studied. The shannon index showed heterogeneity in difference lesions of each patients and the comparing the most and least shannon index lesions variations ranged between 0.066 to 3.344. The lesions with shared mutations showed higher Shannon index than the non-share lesions (7.31 vs 6.96; p = 0.011). The heterogeneity also identified in MOI and 55% MOI of lesions was less than 0.5. The lesions with MOI higher than 0.5 had some pathologically identical and some have shared mutations. Furthermore, the shared TCR between lesions in same patient were analyzed. The more than 78% of T cell clones were restricted to individual lesions, which indicated the presence of heterogeneity between lesions due to T cell clones distribution heterogeneity. Meanwhile, the shared TCR was significantly higher in identical pathology group than difference pathology group (17.18% vs 6.60%; p = 0.0006); Besides, we found that shared TCR was comparable in both shared mutations and non-shared mutations groups (13.1% vs 15.33%, p = 0.537). These results suggested that the pathology between lesions in patients was affected to 100% shared TCR. Conclusions: In conclusion, the multiple lesions of MPLC showed heterogeneity and this heterogeneity is due to the distribution of T cell clones between lesions. Besides, the MOI and shared TCR was higher in MPLC with same pathology, which may increase the understanding of MPLC and influence the treatment choice of MPLC patients.