Tumor-Infiltrating B- and T-Cell Repertoire in Pancreatic Cancer Associated With Host and Tumor Features

BackgroundInfiltrating B and T cells have been observed in several tumor tissues, including pancreatic ductal adenocarcinoma (PDAC). The majority known PDAC risk factors point to a chronic inflammatory process leading to different forms of immunological infiltration. Understanding pancreatic tumor infiltration may lead to improved knowledge of this devastating disease.MethodsWe extracted the immunoglobulins (IGs) and T cell receptors (TCRs) from RNA-sequencing of 144 PDAC from TCGA and 180 pancreatic normal tissue from GTEx. We used Shannon entropy to find differences in IG/TCR diversity. We performed a clonotype analysis considering the IG clone definition (same V and J segments, same CDR3 length, and 90% nucleotide identity between CDR3s) to study differences among the tumor samples. Finally, we performed an association analysis to find host and tumor factors associated with the IG/TCR.ResultsPDAC presented a richer and more diverse IG and TCR infiltration than normal pancreatic tissue. A higher IG infiltration was present in heavy smokers and females and it was associated with better overall survival. In addition, specific IG clonotypes classified samples with better prognosis explaining 24% of the prognosis phenotypic variance. On the other hand, a larger TCR infiltration was present in patients with previous history of diabetes and was associated with lower nonantigen load.ConclusionsOur findings support PDAC subtyping according to its immune repertoire landscape with a potential impact on the understanding of the inflammatory basis of PDAC risk factors as well as the design of treatment options and prognosis monitoring.

Antibody heavy chain CDR3 length-dependent usage of human IGHJ4 and IGHJ6 germline genes

Background Therapeutic antibody discovery using synthetic diversity has been proved productive, especially for target proteins not suitable for traditional animal immunization-based antibody discovery approaches. In recent years, many lines of evidences suggest that the quality of synthetic diversity design limits the development success of synthetic antibody hits. The aim of our study is to understand the quality limitation and to properly address the challenges with a better design. Methods Using VH3–23 as a model framework, we analyzed the naturally and productively rearranged CDR-H3 diversity in human immune repertoire. With homology modeling, we further built VH3–23-based structural models to understand the spatial paratope and its influencing parameters. Results We observed and quantitatively mapped CDR-H3 loop length-dependent usage of human IGHJ4 and IGHJ6 germline genes in the natural human immune repertoire. Skewed usage of DH2-JH6 and DH3-JH6 rearrangements was quantitatively determined in a CDR-H3 length-dependent manner in natural human antibodies with long CDR-H3 loops. Such CDR-H3 length dependent usage of human germline genes was not impacted by the choices of VH in the V(D)J recombination, ethnic background and health conditions. Structural modeling suggests choices of JH help to stabilize antibody CDR-H3 loop and JH only partially contributes to the paratope. Conclusions We quantitatively determined the CDR-H3 length-dependent usage of human germline genes, which makes it possible to design synthetic diversity fully mimicking that of natural immune repertoire. Our observations shed light on the design of next generation synthetic diversity with improved probability of success.

Response to Hypomethylating Agents in Myelodysplastic Syndrome Is Associated With Emergence of Novel TCR Clonotypes

Aberrant T-cell function is implicated in the pathogenesis of myelodysplastic syndrome (MDS). Monitoring the T-cell receptor (TCR) repertoire can provide insights into T-cell adaptive immunity. Previous studies found skewed TCR repertoires in MDS compared to healthy patients; however these studies that leverage mRNA-based spectratyping have limitations. Furthermore, evaluating the TCR repertoire in context of hypomethylating agents (HMAs) treatment can provide insights into the dynamics of T-cell mediated responses in MDS. We conducted immunosequencing of the CDR3 regions of TCRβ chains in bone marrows of 11 MDS patients prior to treatment (n=11 bone marrows prior to treatment), and in at least 2 timepoints for each patient following treatment (n=26 bone marrow aspirates post-treatment) with (HMA), alongside analyzing bone marrows from 4 healthy donors as controls. TCR repertoires in MDS patients were more clonal and less diverse than healthy donors. However, unlike previous reports, we did not observe significant skewness in CDR3 length or spectratyping. The global metrics of TCR profiling including richness, clonality, overlaps were not significantly changed in responders or non-responders following treatment with HMAs. However, we found an emergence of novel clonotypes in MDS patients who responded to treatment, while non-responders had a higher frequency of contracted clonotypes following treatment. By applying GLIPH2 for antigen prediction, we found rare TCR specificity clusters shared by TCR clonotypes from different patients at pre- or following treatment. Our data show clear differences in TCR repertoires of MDS compared with healthy patients and that novel TCR clonotype emergence in response to HMA therapy was correlated with response. This suggests that response to HMA therapy may be partially driven by T-cell mediated immunity and that the immune-based therapies, which target the adaptive immune system, may play a significant role in select patients with MDS.

The role of chromatin organizer Satb1 in shaping TCR repertoire in adult thymus

T cells recognize the universe of foreign antigens with a diverse repertoire of T cell receptors generated by V (D)J recombination. Special AT-rich binding protein 1 (Satb1) is a chromatin organizer that plays an essential role in T cell development. The previous study showed that Satb1 regulates the re-induction of recombinase Rag1 and Rag2 in CD4+CD8+ thymocytes, affecting the secondary rearrangement of the Tcra gene. Here, we detected the repertoires of four TCR genes, Tcrd, Tcrg, Tcrb, and Tcra in the adult thymus, and explored the role of the Satb1 in shaping the TCR repertoires. We observed a strong bias in the V and J gene usages of the Tcrd and Tcrg repertoires in WT and Satb1-deleted thymocytes. Satb1 deletion had few effects on the V(D)J rearrangement and repertoire of the Tcrg, Tcrd, and Tcrb genes. The Tcra repertoire was severely impaired in Satb1-deleted thymocytes, while the primary rearrangement was relatively normal. We also found the CDR3 length of TCRα chain was significantly longer in Satb1-deleted thymocytes, which can be explained by the strong bias of the proximal Jα usage. Our results showed that Satb1 plays an essential role in shaping TCR repertoires in αβ T cells.

Decrease of Clone Diversity in IgM Repertoires of HBV Chronically Infected Individuals With High Level of Viral Replication

High-throughput antibody sequencing allows in-depth insights into human antibody repertoires. To investigate the characteristics of antibody repertoires in patients with chronic HBV infection, we performed Illumina sequencing and IMGT/HighV-QUEST analysis of B lymphocytes from healthy adults and the HBV carriers with high or low level of viral replication. The comparative study revealed high levels of similarity between the IgM and IgG repertoires of the HBV carriers and the healthy adults, including the somatic mutations in V regions, the average CDR3 length, and the occurrence of junctional modifications. Nevertheless, the diversity of the unique clones decreased and some clusters of unique clones expanded in the IgM repertoire of chronic HBV carriers (CHB) compared with healthy adults (HH) and inactive HBV carriers (IHB). Such difference in clone diversity and expansion was not observed in the IgG repertoires of the three populations. More shared antibody clones were found between the IgM repertoires of IHB and HH than that found between CHB and HH (7079 clones vs. 2304 clones). Besides, the biased used IGHD genes were IGHD2-2 and IGHD3-3 in CHB library but were IGHD3-10 and IGHD3-22 in IHB and HH library. In contrast, for IgG repertories, the preferred used VDJ genes were similar in all the three populations. These results indicated that low level of serum HBV might not induce significant changes in BCR repertoires, and high level of HBV replication could have more impacts on IgM repertories than IgG repertoires. Taken together, our findings provide a better understanding of the antibody repertoires of HBV chronically infected individuals.

An analysis of abnormalities in the B cell receptor repertoire in patients with systemic sclerosis using high-throughput sequencing

Systemic sclerosis is a chronic multisystem autoimmune disease that is associated with polyclonal B cell hyperreactivity. The CDR3 of BCRs is the major site of antigen recognition. Therefore, we analyzed the BCR repertoire of patients with SSc. The BCR repertoires in 12 subjects including eight SSc patients and four healthy controls were characterized by high-throughput sequencing, and bioinformatics analysis were studied. The average CDR3 length in the SSc group was significantly shorter. The SSc patient displayed more diverse BCR. Moreover, SSc patients with mild skin sclerosis, anti-Scl70, interstitial lung disease or female sex were more diversified. B cells from the SSc patients showed a differential V and J gene usage. SSc patients had distinct BCR repertoires.These findings reflected the differences of BCR repertoires between SSc patients and controls. The higher-usage genes for the BCR sequence might be potential biomarkers of B cell-targeted therapies or diagnosis for SSc.

High-throughput sequencing of CD4+ T cell repertoire reveals disease-specific signatures in IgG4-related disease

Background CD4+ T cells play critical roles in the pathogenesis of IgG4-related disease (IgG4-RD). The aim of this study was to investigate the TCR repertoire of peripheral blood CD4+ T cells in IgG4-RD. Methods The peripheral blood was collected from six healthy controls and eight IgG4-RD patients. TCR β-chain libraries of CD4+ T cells were constructed by 5′-rapid amplification of cDNA ends (5′-RACE) and sequenced by Illumina Miseq platform. The relative similarity of TCR repertoires between samples was evaluated according to the total frequencies of shared clonotypes (metric F), correlation of frequencies of shared clonotypes (metric R), and total number of shared clonotypes (metric D). Results The clonal expansion and diversity of CD4+ T cell repertoire were comparable between healthy controls and IgG4-RD patients, while the proportion of expanded and coding degenerated clones, as an indicator of antigen-driven clonal expansion, was significantly higher in IgG4-RD patients. There was no significant difference in TRBV and TRBJ gene usage between healthy controls and IgG4-RD patients. The complementarity determining region 3 (CDR3) length distribution was skewed towards longer fragments in IgG4-RD. Visualization of relative similarity of TCR repertoires by multi-dimensional scaling analysis showed that TCR repertoires of IgG4-RD patients were separated from that of healthy controls in F and D metrics. We identified 11 IgG4-RD-specific CDR3 amino acid sequences that were expanded in at least 2 IgG4-RD patients, while not detected in healthy controls. According to TCR clonotype networks constructed by connecting all the CDR3 sequences with a Levenshtein distance of 1, 3 IgG4-RD-specific clusters were identified. We annotated the TCR sequences with known antigen specificity according to McPAS-TCR database and found that the frequencies of TCR sequences associated with each disease or immune function were comparable between healthy controls and IgG4-RD patients. Conclusion According to our study of CD4+ T cells from eight IgG4-RD patients, TCR repertoires of IgG4-RD patients were different from that of healthy controls in the proportion of expanded and coding degenerated clones and CDR3 length distribution. In addition, IgG4-RD-specific TCR sequences and clusters were identified in our study.

T-cell receptor αβ chain pairing is associated with CD4+ and CD8+ lineage specification

While a highly diverse T-cell receptor (TCR) repertoire is the hallmark of a healthy adaptive immune system, relatively little is understood about how the CD4+ and CD8+ TCR repertoires differ from one another. We here utilize high-throughput single T-cell sequencing to obtain approximately 100,000 TCR αβ chain pairs from human subjects, stratified into CD4+ and CD8+ lineages. We reveal that substantial information about T-cell lineage is encoded by Vαβ gene pairs and, to a lesser extent, by several other TCR features such as CDR3 length and charge. We further find that the strength of association between the β chain and T-cell lineage is surprisingly weak, similar in strength to that of the α chain. Using machine learning classifiers to predict T-cell lineage from TCR features, we demon-strate that αβ chain pairs are significantly more informative than individual chains alone. These findings provide unprecedented insight into the CD4+ and CD8+ TCR repertoires and highlight the importance of αβ chain pairing in TCR function and specificity.

Human TCR-MHC coevolution after divergence from mice includes increased nontemplate-encoded CDR3 diversity

For thymic selection and responses to pathogens, T cells interact through their αβ T cell receptor (TCR) with peptide–major histocompatibility complex (MHC) molecules on antigen-presenting cells. How the diverse TCRs interact with a multitude of MHC molecules is unresolved. It is also unclear how humans generate larger TCR repertoires than mice do. We compared the TCR repertoire of CD4 T cells selected from a single mouse or human MHC class II (MHC II) in mice containing the human TCR gene loci. Human MHC II yielded greater thymic output and a more diverse TCR repertoire. The complementarity determining region 3 (CDR3) length adjusted for different inherent V-segment affinities to MHC II. Humans evolved with greater nontemplate-encoded CDR3 diversity than did mice. Our data, which demonstrate human TCR–MHC coevolution after divergence from rodents, explain the greater T cell diversity in humans and suggest a mechanism for ensuring that any V–J gene combination can be selected by a single MHC II.