Lymphocyte Repertoire Research Articles

The Tumor-Infiltrating T-Cell Receptor-b Repertoire Is Linked to Risk of Radiation-Induced Temporal Lobe Necrosis in Locally Advanced Nasopharyngeal Carcinoma

Temporal lobe necrosis (TLN), a late complication of nasopharyngeal carcinoma (NPC) patients after concurrent chemoradiotherapy (CCRT), causes permanent neurological deficits. We aimed to investigate the risk factors for the development of CCRT-induced TLN in locally advanced NPC with intracranial invasion. The impact of CCRT-induced TLN on clinical outcomes was assessed in 73 consecutive patients initially staged T4N0-3M0 NPCs receiving curative intensity-modulated radiotherapy (IMRT) and cisplatin-based chemotherapy with long-term follow-up. The TLN risk was evaluated with the dose-volume histograms (a dosimetric impact of organ injury) and the dynamics of blood-circulating neutrophil-to-lymphocyte ratios (a clinical indicator of systemic inflammation) by linear and logistic regression models. High-throughput unbiased T-cell receptor-beta (TCRbeta) sequencing was performed to correlate the different TCRbeta repertoires of NPC-infiltrating lymphocytes (a biological factor of immune microenvironment) with the TLN incidence. We found that in the era of modern IMRT-based CCRT, radiation dose up to 74 Gy achieved more than 90% of the 5-year local control rate but still induced 30% of TLN incidence in the T4 NPC patients. TLN occurrence did not affect the local recurrence-free survival rate. Univariate and multivariate analyses showed the radiation tolerance dose-volume effect was not an absolutely independent factor for TLN occurrence. However, increased TLN risk was seen in association with higher pre-CCRT baseline and increased post-CCRT levels of neutrophil-to-lymphocyte ratios. There was also a link of intratumoral TCRbeta repertoire subgroups to higher TLN incidence. Combining the inherent TCRbeta genomic susceptibility with clinical variable neutrophil-to-lymphocyte ratios better predicted the risk of TLN for T4 NPC following CCRT. These findings suggest that the immune system should play a role in CCRT-induced TLN. The reciprocal interactions of neutrophils and lymphocytes in the immune microenvironment around NPC, whether modulated or provoked by CCRT, could be a key player or driver in TLN development.

Manufacture of Chimeric Antigen Receptor T Cells from Mobilized Cyropreserved Peripheral Blood Stem Cell Units Depends on Monocyte Depletion

Cytotoxic chemotherapy and radiation can render lymphocyte repertoires qualitatively and quantitatively defective. Thus, heavily treated patients are often poor candidates for the manufacture of autologous chimeric antigen receptor (CAR)-T cell products. In the United States and Europe, children with high-risk neuroblastoma undergo apheresis early in the course of treatment to collect peripheral blood stem cells (PBSCs) for cryopreservation in preparation for high-dose chemotherapy followed by autologous stem cell rescue. Here, we investigate whether these cryopreserved chemotherapy and granulocyte colony-stimulating factor (G-CSF)-mobilized PBSCs can serve as starting material for CAR-T cell manufacturing. We evaluated T cell precursor subsets in cryopreserved PBSC units from 8 patients with neuroblastoma using fluorescent activated cell sorting-based analysis. Every cryopreserved unit collected early in treatment contained both CD4 and CD8 precursors with significant numbers of naïve and central memory precursors. Significant numbers of Ki67+/PD1+ T cells were detected, presumably the result of chemotherapy-induced lymphopenia and subsequent homeostatic proliferation. Cryopreserved PBSC units containing 56 to 112 × 106 T cells were amenable to immunomagnetic selection, CD3 × 28 bead activation, lentiviral transduction, and cytokine-driven expansion, provided that CD14 monocytes were depleted before the initiation of cultures. Second- and third-generation CD171 CAR+ CD4 and CD8 effector cells derived from cryopreserved units displayed antineuroblastoma lytic potency and cytokine secretion comparable to those derived from a healthy donor and mediated in vivo antitumor regression in NSG mice. We conclude that cryopreserved PBSCs procured via standard methods during early treatment can serve as an alternative starting source for CAR-T cell manufacturing, extending the options for heavily treated patients.

The TAB1-p38α complex aggravates myocardial injury and can be targeted by small molecules.

Inhibiting MAPK14 (p38α) diminishes cardiac damage in myocardial ischemia. During myocardial ischemia, p38α interacts with TAB1, a scaffold protein, which promotes p38α autoactivation; active p38α (pp38α) then transphosphorylates TAB1. Previously, we solved the X-ray structure of the p38α-TAB1 (residues 384–412) complex. Here, we further characterize the interaction by solving the structure of the pp38α-TAB1 (residues 1–438) complex in the active state. Based on this information, we created a global knock-in (KI) mouse with substitution of 4 residues on TAB1 that we show are required for docking onto p38α. Whereas ablating p38α or TAB1 resulted in early embryonal lethality, the TAB1-KI mice were viable and had no appreciable alteration in their lymphocyte repertoire or myocardial transcriptional profile; nonetheless, following in vivo regional myocardial ischemia, infarction volume was significantly reduced and the transphosphorylation of TAB1 was disabled. Unexpectedly, the activation of myocardial p38α during ischemia was only mildly attenuated in TAB1-KI hearts. We also identified a group of fragments able to disrupt the interaction between p38α and TAB1. We conclude that the interaction between the 2 proteins can be targeted with small molecules. The data reveal that it is possible to selectively inhibit signaling downstream of p38α to attenuate ischemic injury.

Abstract 136: T-cell clonotype convergence in colorectal cancer driven by tumor-specific neoantigens

Abstract A cornerstone of the biological process which mediates response to immunotherapy in cancer patients is the expression of neoantigens resulting from somatic mutations in cancer cells. Little attention has been paid so far to the neoantigen's immune cell counterpart, the T-cell receptor (TCR). In this study, we aimed at characterizing the tumor infiltrating T-cell repertoire in colorectal cancer (CRC), and to evaluate evidence for neoantigen driven T-cell expansion within the tumor microenvironment. To characterize the T-cell repertoire, we used the Ion AmpliSeq Immune Repertoire Assay Plus with an Ion S5 XL system. This assay works with as little as 10ng of starting total RNA, allows 400bp read length, and offers complete characterization of the CDR1, CDR2 and CDR3 regions. A cohort of 20 CRC cases was used, including microsatellite instability (MSI) positive and MSI-negative cases. Our results show a higher T-cell richness in distal CRC samples compared with proximal samples (p=0.04), and lower T-cell richness in M1 as compared with M0 cases (p=0.0001). No association was observed with MSI status, inflammatory infiltrate, tumor grade or lymph node metastasis. We observed that the T-cell repertoire in CRC samples is highly convergent compared to the peripheral blood lymphocyte repertoire of healthy donors (p<0.0001). In CRC samples we observed evidence of selection of TCR clonotypes coding for the same CDR3 aminoacid sequence, suggesting neoantigen-driven TCR convergence. TCR clone overlap was observed only between pairs of samples (4 pairs of primary tumor and metastasis samples available) from the same patient, and not between samples from different patients, suggesting high tumor specificity of T-cell clonotype selection. A validation cohort of CRC cases, including additional pairs of primary tumor and metastasis samples, is currently being analyzed to validate our findings. In this study we provide evidence of T-cell clonotype selection in CRC driven by tumor-specific neoantigens. The specificity of this process is further reinforced by the T-cell clonotype overlap within patients. It remains to be seen whether it is possible to map convergent T-cell clonotypes to its respective tumor neoantigens. Citation Format: Jose C. Machado, Joana Reis, Margarida Fernandes, Rafaela Silva, Luis Cirnes, Fatima Carneiro, Jose L. Costa. T-cell clonotype convergence in colorectal cancer driven by tumor-specific neoantigens [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 136.

Bohemian T cell receptors: sketching the repertoires of unconventional lymphocytes.

Over the last several decades, novel populations of unconventional T cells have been identified; defined by an invariant (or nearly invariant) T cell receptor (TCR) with a fixed specificity to non-canonical antigens and major histocompatibility (MHC) molecules, they form large, functionally monoclonal populations tasked with surveying for their specific antigens. With residence in both lymphoid and non-lymphoid tissues coupled with their ability to rapidly produce a spectrum of cytokines and effector molecules, the unconventional T cells are poised as some of the first responders to infection/damage and are thought to provide critical coverage before more focused, conventional T cell responses are mobilized. However, new technologies for the measurement and characterization of TCR repertoires have identified an underappreciated amount of TCR diversity in the unconventional T cells. In many cases, the specificities of these diverse TCRs converge on the same or similar antigens as their invariant counterparts, while others have yet to be defined. Here, we will review the current knowledge of the TCR repertoires of unconventional T cells and discuss how repertoires might be used as a framework for their organization, and further our understanding of their role not only during an immune response, but also their contribution in maintaining homeostasis.

Identification of Tumoricidal TCRs from Tumor-Infiltrating Lymphocytes by Single-Cell Analysis.

T-cell receptor (TCR) gene therapy is a promising next-generation antitumor treatment. We previously developed a single-T-cell analysis protocol that allows the rapid capture of paired TCRα and β cDNAs. Here, we applied the protocol to analyze the TCR repertoire of tumor-infiltrating lymphocytes (TIL) of various cancer patients. We found clonally expanded populations of T cells that expressed the same clonotypic TCR in 50% to 70% of CD137+CD8+ TILs, indicating that they responded to certain antigens in the tumor environment. To assess the tumor reactivity of the TCRs derived from those clonally expanded TILs in detail, we then analyzed the CD137+CD8+ TILs from the tumor of B16F10 melanoma cells in six C57BL/6 mice and analyzed their TCR repertoire. We also found clonally expanded T cells in 60% to 90% of CD137+CD8+ TILs. When the tumor reactivity of dominant clonotypic TCRs in each mouse was analyzed, 9 of 13 TCRs induced the secretion of IFNγ in response to, and showed killing of, B16F10 cells in vitro, and 2 of them showed strong antitumor activity in vivo Concerning their antigen specificity, 7 of them reacted to p15E peptide of endogenous murine leukemia virus-derived envelope glycoprotein 70, and the rest reacted to tumor-associated antigens expressed on EL4 lymphoma as well as B16 melanoma cells. These results show that our strategy enables us to simply and rapidly obtain the tumor-specific TCR repertoire with high fidelity in an antigen- and MHC haplotype-independent manner from primary TILs. Cancer Immunol Res; 6(4); 378-88. ©2018 AACR.

Indexing labeled sequences.

BackgroundLabels are a way to add some information on a text, such as functional annotations such as genes on a DNA sequences. V(D)J recombinations are DNA recombinations involving two or three short genes in lymphocytes. Sequencing this short region (500 bp or less) produces labeled sequences and brings insight in the lymphocyte repertoire for onco-hematology or immunology studies.MethodsWe present two indexes for a text with non-overlapping labels. They store the text in a Burrows–Wheeler transform (BWT) and a compressed label sequence in a Wavelet Tree. The label sequence is taken in the order of the text (TL-index) or in the order of the BWT (TLBW-index). Both indexes need a space related to the entropy of the labeled text.ResultsThese indexes allow efficient text–label queries to count and find labeled patterns. The TLBW-index has an overhead on simple label queries but is very efficient on combined pattern–label queries. We implemented the indexes in C++ and compared them against a baseline solution on pseudo-random as well as on V(D)J labeled texts.DiscussionNew indexes such as the ones we proposed improve the way we index and query labeled texts as, for instance, lymphocyte repertoire for hematological and immunological studies.

Dynamic changes of the normal B lymphocyte repertoire in CLL in response to ibrutinib or FCR chemo-immunotherapy

ABSTRACTUsing next-generation immunoglobulin (IGH) sequencing and flow cytometry, we characterized the composition, diversity and dynamics of non-malignant B cells in patients undergoing treatment with the Bruton tyrosine kinase (BTK) inhibitor ibrutinib or chemo-immunotherapy with fludarabine, cyclophosphamide, and rituximab (FCR). During ibrutinib therapy, non-malignant B cell numbers declined, but patients maintained stable IGH diversity and constant fractions of IGH-mutated B cells. This indicates partial preservation of antigen-experienced B cells during ibrutinib therapy, but impaired replenishment of the normal B cell pool with naïve B cells. In contrast, after FCR we noted a recovery of normal B cells with a marked predominance of B cells with unmutated IGH. This pattern is compatible with a deletion of pre-existing antigen-experienced B cells followed by repertoire renewal with antigen-naïve B cells. These opposite patterns in B cell dynamics may result in different responses towards neoantigens versus recall antigens, which need to be further defined.

Apoptosis and Mobilization of Lymphocytes to Cardiac Tissue Is Associated with Myocardial Infarction in a Reperfused Porcine Model and Infarct Size in Post-PCI Patients.

ST-segment elevation myocardial infarction (STEMI) is the most severe outcome of coronary artery disease. Despite rapid reperfusion of the artery, acute irrigation of the cardiac tissue is associated with increased inflammation. While innate immune response in STEMI is well described, an in-depth characterization of adaptive immune cell dynamics and their potential role remains elusive. We performed a translational study using a controlled porcine reperfusion model of STEMI and the analysis of lymphocyte subsets in 116 STEMI patients undergoing percutaneous coronary intervention (PCI). In the animal model, a sharp drop in circulating T lymphocytes occurred within the first hours after reperfusion. Notably, increased apoptosis of circulating lymphocytes and infiltration of proinflammatory Th1 lymphocytes in the heart were observed 48 h after reperfusion. Similarly, in STEMI patients, a sharp drop in circulating T lymphocyte subsets occurred within the first 24 h post-PCI. A cardiac magnetic resonance (CMR) evaluation of these patients revealed an inverse association between 24 h circulating T lymphocyte numbers and infarction size at 1-week and 6-month post-PCI. Our translational approach revealed striking changes in the circulating and tissue-infiltrating T lymphocyte repertoire in response to ischemia-reperfusion. These findings may help in developing new diagnostic and therapeutic approaches for coronary diseases.

IRAGu: A Novel Inducible and Reversible Mouse Model for Ubiquitous Recombinase Activity.

Developing lymphocytes express the recombination activating genes (RAGs) 1 and 2 products that form a site specific recombinase complex (RAG), introducing double strand DNA breaks (DSBs) at recombination signal sequences (RSSs) flanking the V, D, and J gene segments in the antigen receptor loci. The subsequent steps in the reaction consist in the ligation of DSBs by ubiquitous enzymes of the non-homologous end joining DNA repair pathway. This mutagenesis process is responsible for the generation of the very large clonal diversity of T and B lymphocytes, itself allowing the recognition of a virtually open-ended antigenic universe. Sequences resembling RSS are found at high frequency all over the genome, and involved in RAG mediated illegitimate recombination and translocations. Hence, natural and induced ectopic activity of RAG is a threat to the genome only recently underscored. Here, we report and characterize a novel mouse transgenic system for which ubiquitous expression of the recombinase is inducible. In this system, the RAG1 protein is constitutively expressed and functional, while the RAG2 protein, coupled to the estrogen receptor, becomes functionally active upon 4-hydroxytamoxifen (TAM) administration. We describe two transgenic lines. The first one, when introgressed into an endogenous Rag2−/− genetic background is faithfully recapitulating lymphocyte development, repertoire dynamics and cryptic rearrangements, in a TAM-dependent manner. In this model, deprivation of TAM is followed by lymphocyte development arrest, evidencing the reversibility of the system. The second transgenic line is leaky, as the transgenes promote lymphocyte differentiation in absence of TAM treatment. Upon TAM-induction defects in lymphocytes composition and global health reveals the deleterious effect of uncontrolled RAG activity. Overall, this novel transgenic model provides a tool where RAG activity can be specifically manipulated to assess the dynamics of lymphocyte differentiation and the challenges imposed by the recombinase on the vertebrate genome.

Clonal characteristics of paired infiltrating and circulating B lymphocyte repertoire in patients with primary biliary cholangitis.

PBC is a prototypical autoimmune liver disease characterized by portal lymphoplasmacyte infiltration. ALD is a prototypical environment-driven disease, featured by mild lymphocyte infiltration. We hypothesize that B cells are more involved in the pathogenesis of PBC. By analysing the infiltrating B cell repertoire, we aimed to unveil greater oligoclonal expansion and active clonal exchange between liver and periphery in PBC than in ALD patients. Using NGS of Ig H chain genes, we analysed the liver-infiltrating and paired peripheral B lymphocyte repertoire from nine PBC and four ALD patients. In the liver of PBC and ALD patients, (i) roughly 10% of the B lymphocytes were clonally related and highly expressed, and there were also lineages that underwent extensive clonal expansion; (ii) there was different use of IGHV/IGHJ segments between PBC and ALD, suggesting distinct Ag exposure backgrounds, but this did not lead to a significant difference in their clonal expansion level. Analysis of data sets from paired samples further revealed, (iii) direct clonal exchange and evolutionally related B cell clones between the infiltrating and peripheral repertoire; (iv) the seeding of the infiltrating clones to periphery, and peripheral ones to the liver, for further extensive evolution. The oligoclonally expanded nature of the infiltrating B cell repertoire implies B cell immunity is involved in the pathogenesis of both diseases. The observed clonal exchange might provide an approach to identify and monitor the infiltrating B cells through the periphery.

Tracking the interplay between circulating and tumor-infiltrating T cells using AmpliSeq-based Ion Torrent TCRβ immune repertoire sequencing.

e14518 Background: TCRβ immune repertoire analysis by next-generation sequencing is emerging as a valuable tool for research studies of the tumor microenvironment and potential immune responses to future cancer immunotherapy. Here we describe a multiplex PCR-based TCRβ sequencing assay that takes advantage of the exceptionally low base-call error rate and long read capability of the Ion S5 530 chip. The S530 chip provides > 15M reads, allowing for sample sequencing in multiplex. The research assay utilizes AmpliSeq technology and Framework 1 and Constant gene targeting primers to provide comprehensive coverage of TCRβ CDR 1, 2 and 3 from RNA input with a hands-on time of less than 30 minutes. CDR region sequences reveal the antigen specificity of the receptor and may be genetically modified to increase TCR affinity for tumor antigen. Methods: We evaluated assay performance by sequencing TCRβ rearrangements from normal donor peripheral blood leukocyte (PBL) cDNA that had been spiked with 30 reference rearrangements taken from the literature. We then used our assay to evaluate the extent of clonal overlap between matched tumor infiltrating lymphocyte (TIL) and peripheral blood leukocyte repertoires in an individual with squamous cell carcinoma of lung. Results: Results from sequencing of spike-in reference rearrangements indicate that the assay is accurate and sensitive over 5 logs of input template amount while showing minimal amplification bias. Sequencing of matched PBL and TIL repertoires revealed that a subset of the PBL repertoire (8%) consisted of clones also found in TIL. Technical replicates showed high concordance (r > .96) in the frequency of detected clones, indicating that the results were reproducible and samples were sequenced to an appropriate depth. Conclusions: In summary, these data suggest that AmpliSeq-based multiplex PCR and Ion Torrent sequencing provide unbiased, reproducible, scalable, complete, and accurate information for immune repertoire research sequencing applications.

Increased Frequencies of PD-1+ CD8+ Marrow-Infiltrating Lymphocytes Associated with Highly Clonal T-Lymphocyte Expansions in Relapsed and Refractory AML Patients but Not Healthy Adults

Immune checkpoint therapy, particularly inhibition of the PD-1 axis, has shown remarkable efficacy in multiple solid tumor types and some hematological malignancies. Clinical results using anti-PD1 immunotherapy in AML have not yet been reported. Immune checkpoint expression levels and magnitude and/or repertoire of tumor-infiltrating lymphocytes have all been proposed as potential predictive biomarkers of response to such targeted immunotherapy. We therefore examined the marrow infiltrating T-lymphocyte compartment of Relapsed/Refractory Acute Myeloid Leukemia (RR-AML) patients undergoing salvage chemotherapy and that of healthy donors (HD) to determine baseline data for PD-1 expression and T-lymphocyte clonality.Nine adult RR-AML patients recruited to the clinical trial NCT02527447 (Myeloid Malignancies Section, NHLBI, NIH) were analyzed. Cryopreserved bone marrow aspirate mononuclear cells (BMMCs) from these patients prior to salvage chemotherapy (Day 0) and HD (n=9) were assessed using two custom ten-color flow cytometry panels. Frequencies of naive (TN), central memory (TCM), effector memory (TEM), terminal effector (TEMRA), stem cell memory (TSCM), and activated T-lymphocyte subsets, and the expression of PD-1, TIM-3, CTLA-4, 4-1BB on CD8+ T-lymphocyte subpopulations were calculated. DNA extracted from bone marrow of 5 of the RR-AML patients and 5 HD was used for sequencing of the CDR3 region of TCRB gene to evaluate TCR repertoire (ImmunoSEQ, Adaptive Biotechnologies).Immunohistochemistry of pre-treatment bone marrow biopsy sections revealed 10-40% CD3+ cells, typically scattered with focal small aggregations. Flow cytometry of marrow aspirate demonstrated more activated CD8+ CD69+ T-lymphocytes in the marrow of RR-AML patients compared with HD (19.8% vs. 9.4%, p=0.031). Significantly more CD8+ TEMRA T-lymphocytes (60.8% vs. 45.2%, p=0.040) and a trend towards fewer CD8+ TEM T-lymphocytes (19.0% vs. 32.1%, p=0.062) were observed in RR-AML versus HD. An increased proportion of total CD8+ T-lymphocytes in RR-AML had PD-1 expression compared to HD (22.0% vs. 9.2%, p=0.008). This pattern held true for every CD8+ sub-population in AML versus HD: TN (14.0% vs. 4.0%, p=0.014); TCM (24.2% vs. 7.3%, p=0.003); TEMRA (19.6% vs. 10.5%, p=0.060) and TEM (37.3% vs. 16.2%, p=0.004) (Figure 1a). There were no significant differences in frequencies of CD8+ T-lymphocyte subsets or PD-1 expression between Day 0 and Day 28 in AML patients (in 4 patients tested). Furthermore, there was negligible PD-1 co-expression with other immune checkpoint markers and no differences in 4-1BB or CTLA4 expression in AML versus HD. However, we noted significantly higher TIM-3 expression on TN and TCM CD8+ T-lymphocytes in AML (6.5% vs. 2.2%, p=0.001, and 4.5% vs. 0.7%, p=0.021, respectively).Sequencing of productive TCRB gene rearrangements revealed significantly higher average marrow T-cell clonality in AML versus HD (0.232 vs. 0.086, respectively, n=5 AML, n=5 HD). When considering the top 10 most frequent clonotypes for each AML patient and HD, frequencies were found to be much higher in AML than HD (Figure 1b), consistent with large clonal T-lymphocyte expansions in the marrow of RR-AML patients. Furthermore, we identified several TCR clones that were found in AML patients but never seen in HD. Sequencing of an additional cohort of 20 newly diagnosed adult AML patients however discovered that only a subset (25%) of those patients had T cell clonal expansions above the upper limit of that seen in HD.AML is already known to be an immunogenic malignancy, as demonstrated by the measurable efficacy of allogeneic transplantation and donor lymphocyte infusion. Our data demonstrate that the bone marrow tumor microenvironment in RR-AML is enriched for PD-1+ CD8+ marrow-infiltrating lymphocytes, and that large clonal expansions of T-lymphocytes can be found in the bone marrow in these patients. This is suggestive that RR-AML patients, for whom cytotoxic chemotherapy is often suboptimal, may benefit from immune checkpoint blockade therapies, particularly PD-1 axis inhibition, to enable improved immunologic control of AML by autologous T-lymphocytes already resident in the tumor microenvironment. Based in part on these data, a clinical trial of anti-PD-1 immunotherapy in combination with a hypomethylating agent for treatment of relapsed and refractory AML patients will open at our institution. [Display omitted] DisclosuresHourigan:Sellas Life Sciences Group: Research Funding.

Clonal Characteristics of Circulating B Lymphocyte Repertoire in Primary Biliary Cholangitis.

Primary biliary cholangitis (PBC) is an autoimmune liver disease characterized by elevated serum anti-mitochondrial Ab and lymphocyte-mediated bile duct damage. This study was designed to reveal the clonal characteristics of B lymphocyte repertoire in patients with PBC to facilitate better understanding of its pathogenesis and better management of these patients. Using high-throughput sequencing of Ig genes, we analyzed the repertoire of circulating B lymphocytes in 43 patients with PBC, and 34 age- and gender-matched healthy controls. Compared with healthy controls, PBC patients showed 1) a gain of 14 new clones and a loss of 8 clones; 2) a significant clonal expansion and increased relative IgM abundance, which corresponded with the elevated serum IgM level; 3) a significant reduction of clonal diversity and somatic hypermutations in class-switched sequences, which suggested a general immunocompromised status; 4) the reduction of clonal diversity and enhancement of clonal expansion were more obvious at the cirrhotic stage; and 5) treatment with ursodeoxycholic acid could increase the clonal diversity and reduce clonal expansion of the IgM repertoire, with no obvious effect on the somatic hypermutation level. Our data suggest that PBC is a complex autoimmune disease process with evidence of B lymphocyte clonal gains and losses, Ag-dependent ogligoclonal expansion, and a generally compromised immune reserve. This new insight into the pathogenesis of PBC opens up the prospect of studying disease-relevant B cells to better diagnose and treat this devastating disease.

Tissue-resident Eomes(hi) T-bet(lo) CD56(bright) NK cells with reduced proinflammatory potential are enriched in the adult human liver.

The adult human liver is enriched with natural killer (NK) cells, accounting for 30-50% of hepatic lymphocytes, which include tissue-resident hepatic NK-cell subpopulations, distinct from peripheral blood NK cells. In murine liver, a subset of liver-resident hepatic NK cells have altered expression of the two highly related T-box transcription factors, T-bet and eomesodermin (Eomes). Here, we investigate the heterogeneity of T-bet and Eomes expression in NK cells from healthy adult human liver with a view to identifying human liver-resident populations. Hepatic NK cells were isolated from donor liver perfusates and biopsies obtained during orthotopic liver transplantation (N = 28). Hepatic CD56(bright) NK cells were Eomes(hi) T-bet(lo) , a phenotype virtually absent from peripheral blood. These NK cells express the chemokine receptor CXCR6 (chemokine (C-X-C motif) receptor 6), a marker of tissue residency, which is absent from hepatic CD56(dim) and blood NK cells. Compared to blood populations, these hepatic CD56(bright) NK cells have increased expression of activatory receptors (NKp44, NKp46, and NKG2D). They show reduced ability to produce IFN-γ but enhanced degranulation in response to challenge with target cells. This functionally distinct population of hepatic NK cells constitutes 20-30% of the total hepatic lymphocyte repertoire and represents a tissue-resident immune cell population adapted to the tolerogenic liver microenvironment.

Immune function in Amazonian horticulturalists

Background: Amazonian populations are exposed to diverse parasites and pathogens, including protozoal, bacterial, fungal and helminthic infections. Yet much knowledge of the immune system is based on industrialised populations where these infections are relatively rare.Aim: This study examines distributions and age-related differences in 22 measures of immune function for Bolivian forager-horticulturalists and US and European populations.Subjects and methods: Subjects were 6338 Tsimane aged 0–90 years. Blood samples collected between 2004–2014 were analysed for 5-part blood differentials, C-reactive protein, erythrocyte sedimentation rate (ESR) and total immunoglobulins E, G, A and M. Flow cytometry was used to quantify naïve and non-naïve CD4 and CD8 T cells, natural killer cells, and B cells.Results: Compared to reference populations, Tsimane have elevated levels of most immunological parameters, particularly immunoglobulins, eosinophils, ESR, B cells, and natural killer cells. However, monocytes and basophils are reduced and naïve CD4 cells depleted in older age groups.Conclusion: Tsimane ecology leads to lymphocyte repertoires and immunoglobulin profiles that differ from those observed in industrialised populations. These differences have consequences for disease susceptibility and co-vary with patterns of other life history traits, such as growth and reproduction.

A TCRβ Repertoire Signature Can Predict Experimental Cerebral Malaria.

Cerebral Malaria (CM) is associated with a pathogenic T cell response. Mice infected by P. berghei ANKA clone 1.49 (PbA) developing CM (CM+) present an altered PBL TCR repertoire, partly due to recurrently expanded T cell clones, as compared to non-infected and CM- infected mice. To analyse the relationship between repertoire alteration and CM, we performed a kinetic analysis of the TRBV repertoire during the course of the infection until CM-related death in PbA-infected mice. The repertoires of PBL, splenocytes and brain lymphocytes were compared between infected and non-infected mice using a high-throughput CDR3 spectratyping method. We observed a modification of the whole TCR repertoire in the spleen and blood of infected mice, from the fifth and the sixth day post-infection, respectively, while only three TRBV were significantly perturbed in the brain of infected mice. Using multivariate analysis and statistical modelling, we identified a unique TCRβ signature discriminating CM+ from CTR mice, enriched during the course of the infection in the spleen and the blood and predicting CM onset. These results highlight a dynamic modification and compartmentalization of the TCR diversity during the course of PbA infection, and provide a novel method to identify disease-associated TCRβ signature as diagnostic and prognostic biomarkers.

Immune Repertoire Profiling Reveals that Clonally Expanded B and T Cells Infiltrating Diseased Human Kidneys Can Also Be Tracked in Blood.

Recent advances in high-throughput sequencing allow for the competitive analysis of the human B and T cell immune repertoire. In this study we compared Immunoglobulin and T cell receptor repertoires of lymphocytes found in kidney and blood samples of 10 patients with various renal diseases based on next-generation sequencing data. We used Biomed-2 primer panels and ImmunExplorer software to sequence, analyze and compare complementarity determining regions and V-(D)-J elements. While generally an individual’s renal receptor repertoire is different from the repertoire present in blood, 94% (30/32) of the lymphocytes with clonal expansion in kidney can also be traced in blood however, not all of these clonotypes are equally abundant. Summarizing the data of all analyzed patients, 68% of highly expanded T cell clonotypes and 30% of the highly expanded B cell clonotypes that have infiltrated the kidney can be found amongst the five most abundant clonotypes in blood. In addition, complementarity determining region 3 sequences of the immunoglobulin heavy chains are on average more diverse than T cell receptor beta chains. Immune repertoire analysis of tissue infiltrating B and T cells adds new approaches to the assessment of adaptive immune response in kidney diseases. Our data suggest that expanded clonotypes in the tissues might be traceable in blood samples in the course of treatment or the natural history of the disease.

Phenotype, function and T cell receptor repertoire of tumor-infiltrating lymphocytes in patients with pancreatic ductal adenocarcinoma

Meeting abstracts Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a median survival of only about two years even in the 20% of patients that present early enough to be eligible for surgical resection and adjuvant chemotherapy. With this urgent medical need in mind, we are

Characteristics of Tumor Infiltrating Lymphocyte and Circulating Lymphocyte Repertoires in Pancreatic Cancer by the Sequencing of T Cell Receptors.

Pancreatic cancer has a poor prognosis and few effective treatments. The failure of treatment is partially due to the high heterogeneity of cancer cells within the tumor. T cells target and kill cancer cells by the specific recognition of cancer-associated antigens. In this study, T cells from primary tumor and blood of sixteen patients with pancreatic cancer were characterized by deep sequencing. T cells from blood of another eight healthy volunteers were also studied as controls. By analyzing the complementary determining region 3 (CDR3) gene sequence, we found no significant differences in the T cell receptor (TCR) repertoires between patients and healthy controls. Types and length of CDR3 were similar among groups. However, two clusters of patients were identified according to the degree of CDR3 overlap within tumor sample group. In addition, clonotypes with low frequencies were found in significantly higher numbers in primary pancreatic tumors compared to blood samples from patients and healthy controls. This study is the first to characterize the TCR repertoires of pancreatic cancers in both primary tumors and matched blood samples. The results imply that specific types of pancreatic cancer share potentially important immunological characteristics.