Estimating T-cell repertoire diversity: limitations of classical estimators and a new approach

A highly diverse T-cell receptor (TCR) repertoire is a fundamental property of an effective immune system, and is associated with efficient control of viral infections and other pathogens. However, direct measurement of total TCR diversity is impossible. The diversity is high and the frequency distribution of individual TCRs is heavily skewed; the diversity therefore cannot be captured in a blood sample. Consequently, estimators of the total number of TCR clonotypes that are present in the individual, in addition to those observed, are essential. This is analogous to the ‘unseen species problem’ in ecology. We review the diversity (species richness) estimators that have been applied to T-cell repertoires and the methods used to validate these estimators. We show that existing approaches have significant shortcomings, and frequently underestimate true TCR diversity. We highlight our recently developed estimator, DivE, which can accurately estimate diversity across a range of immunological and biological systems.     

T-cell receptor gene-modified cells: past promises,present methodologies and future challenges

Immunotherapy constitutes an exciting and rapidly evolving field, and the demonstration that genetically modified T-cell receptors (TCRs) can be used to produce T-lymphocyte populations of desired specificity offers new opportunities for antigen-specific T-cell therapy.Overall, TCR-modified T cells have the ability to target a wide variety of self and non–self targets through the normal biology of a T cell. Although major histocompatibility complex (MHC)–restricted and dependent on co-receptors, genetically engineered TCRs still present a number of characteristics that ensure they are an important alternative strategy to chimeric antigen receptors (CARs), and high-affinity TCRs can now be successfully engineered with the potential to enhance therapeutic efficacy while minimizing adverse events. This review will focus on the main characteristics of TCR gene-modified cells, their potential clinical application and promise to the field of adoptive cell transfer (ACT), basic manufacturing procedures and characterization protocols and overall challenges that need to be overcome so that redirection of TCR specificity may be successfully translated into clinical practice, beyond early-phase clinical trials.     

Characterization of T-cell repertoire in hairy cell leukemia patients before and after recombinant immunotoxin BL22 therapy

We previously reported that hairy cell leukemia (HCL) patients have high percentages of CD56+/CD57+/CD3+ large granular lymphocytes consistent with cytotoxic T-lymphocytes (CTLs), and other investigators have reported skewing of the T-cell repertoire. In previous studies of up to seven HCL patients, many of the 22 established T-cell receptor (TCR) beta variable region (TRBV) families showed mono- or oligoclonal restriction. To determine whether percentages of CTLs are correlated with TRBV clonal excess, we studied 20 HCL patients with flow cytometry, PCR of TCR gamma and TRBV regions, and fractional gel electrophoresis of PCR-amplified TRBV CDR3 domains (CDR3 spectratyping). Increased percentages of CD3+/CD8+/CD57+ CTLs correlated with more mono/oligoclonal and fewer polyclonal TRBV families (r=0.53; P=0.016). Age correlated with number of mono/oligoclonal TRBV families (r=0.51; P=0.022). Time since last purine analog therapy correlated with number of polyclonal TRBV families (r=0.46; P=0.040), but treatment with the anti-CD22 recombinant immunotoxin BL22 was not related to clonal excess. We conclude that abnormalities in the T-cell repertoire in HCL patients may represent deficient immunity, and may be exacerbated by purine analogs. Increased CD3+/CD57+ T-cells may be a useful marker of abnormal TRBV repertoire in HCL patients, and might prove useful in deciding whether patients should receive biologic antibody-based treatment rather than repeated courses of purine analog for relapsed disease.     

T-cell receptor tetramer binding or the lack there of does not necessitate antigen reactivity in T-cell receptor transduced T cells

Genetic transfer of T-cell receptor (TCR) chains provides a means of transferring tumor antigen specificity onto an alternate T-cell population. To determine which tumor reactive TCRs are best suitable for such adoptive transfer, careful evaluation of the resulting TCR modified populations need to be performed. We have previously cloned, and expressed TCRs from melanoma, EBV, HCV, and HPV reactive T-cell clones and found that several routine indicators of T-cell function do not always predict the relative strength of a TCR. Using a combination of tetramer binding assays and antigen recognition assays, we identified TCRs that fall into three classes. One class of TCR did not bind tetramers yet resulted in cells with high avidity for antigen. A second TCR class bound tetramer but did not secrete cytokines in response to antigen. Finally, the third class of TCRs bound tetramer and reacted to antigen as would be expected. We conclude that tetramer binding is not always a good indicator of the function of a cloned TCR or the avidity of a TCR gene modified T cell. Furthermore, our data indicate that the use of tetramer binding alone to identify antigen reactive TCRs may result in the exclusion of TCRs that may be highly reactive or cross reactive to the relevant tumor antigen.     

Identification of three new bovine T-cell receptor delta variable gene subgroups expressed by peripheral blood T cells

To understand the biology of γδ T cells in ruminants, it is necessary to have a comprehensive picture of γδ T-cell receptor gene diversity and expression. In this study, three new subgroups of bovine T-cell receptor δ (TRD) variable genes were identified by RT-PCR and sequencing and homology with TRDV genes from other mammals determined. Previously unidentified TRDV subgroup genes described in this study include the bovine homologues of ovine TRDV2, TRDV3, and TRDV4 which were named accordingly. TRDV2 subgroup has two genes (TRDV2-1 and TRDV2-2) while we found the previously identified TRDV1 has at least eight genes corresponding to separate genomic sequences. Nucleotide and amino acid sequences for particular gene subgroups between cattle and sheep were more than 87% identical but identities among TRDV subgroups within a species were much less, with bovine TRDV4 having <45% identity to the other three bovine TRDV gene subgroups. Analysis of circulating bovine γδ T cells revealed that genes from all four TRDV subgroups were expressed in combination with TRDJ1, TRDJ3, and TRDC, although TRDV4 was the least represented, and all displayed a variety of CDR3 junctional lengths. Finally, some genes within the TRDV1, TRDV2, and TRDV3 subgroups recombined with TRAV incorporating TRAJs, suggesting dual use.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank database under the accession numbers DQ275147, DQ275148, DQ275149, and DQ280318.     

Cytotoxic activity and T cell receptor repertoire in tumor-infiltrating lymphocytes of adrenal cell carcinomas

The cytotoxic activity and T cell receptor (TCR) V repertoire in tumor-infiltrating lymphocytes (TIL) of three primary adrenal cell carcinomas were analyzed. Fresh, non-cultured TIL from two of the three tumors showed low but significant lysis of the autologous tumor, and for one of the patients this activity was strongly enhanced upon culture in interleukin-2. An allogeneic adrenal cell carcinoma line and the K562 or Daudi targets included as controls were not killed. Phenotypic analysis of freshly isolated TIL demonstrated that the cells from the two patients that demonstrated cytolytic capacity mainly consisted of CD45RO⁺ T cells. In vitro cultured TIL lines from these patients demonstrated a high percentage of CD8⁺ cells expressing either the V6 gene or the V8 gene product, as measured with a panel of mAb specific for TCR V and V gene products. Analysis of the TCR V gene mRNA expression in freshly isolated non-cultured TIL, using a polymerase-chain-reaction-assisted cDNA-amplification assay, confirmed the strong expression of the genes coding for the TCR V6 or the V8. This assay also demonstrated a more restricted TCR V gene usage in the TIL as compared to peripheral blood lymphocytes from the same patient.This study was supported by the Swedish Cancer Society and by the Cancer Society in Stockholm     

Application of T‐cell receptor repertoire as a novel monitor in dynamic tracking and assessment: A cohort‐study based on RA patients

T‐cell receptor repertoire (TCRR) sequencing has been widely applied in many fields as a novel tool. This study explored characteristics of TCRR in detail with a cohort of 598 rheumatoid arthritis (RA) patients before and after anti‐rheumatic treatments. We highlighted the abnormal TCRR distribution in RA characterized by decreased diversity and increased proportion of hyperexpanded clones (HECs), which was potentially attributed to skewed usage of global V/J segments but not a few certain ones. Enriched motifs analysis in RA community demonstrated the huge heterogeneity of CDR3 sequences, so that individual factors are strongly recommended to be taken into consideration when it comes to clinical application of TCRR. Disease‐modifying antirheumatic drugs (DMARDs) can regulate immune system through recovery of TCRR richness to relieve symptoms. Remarkably, sensitive gene profile and advantageous gene profile were identified in this study as new biomarkers for different DMARDs regimens.     

小细胞肺癌患者外周血淋巴细胞亚群分析

目的 探究化疗对小细胞肺癌(small cell lung cancer,SCLC)患者免疫功能的影响。 方法 选择2013年1月到2018年12月我院收治的95例小细胞肺癌患者为研究对象。患者第一周期、第二周期化疗前采用流式细胞术检测患者外周血淋巴细胞亚群水平,分别按照不同疗效及不同化疗方案对患者外周血淋巴细胞亚群进行比较。 结果 (1)化疗后,95例患者CD3+、CD4+、CD8+细胞平均值增加,CD19+、γδT细胞平均值减少,差异均有统计学意义(均P+、CD8+细胞平均值增加,CD19+细胞减少,差异有统计学意义(均P0.05)。(3)依托泊苷联合顺铂(EP)方案组化疗后患者CD3+、CD8+细胞平均值增多,CD19+细胞减少,差异均有统计学意义(均P0.05)。 结论 化疗可以调节小细胞肺癌患者的免疫功能,增强细胞免疫,降低体液免疫,其中EC方案对患者细胞免疫的增强作用较为显著。     

Ferritin-bearing lymphocytes and T-cell levels in peripheral blood of patients with breast cancer

Summary Peripheral blood lymphocytes bearing surface ferritin and thymus-dependent lymphocyte (T cell) levels were determined in 15 breast cancer patients in stage I–II, 5 in stage III, 10 with benign breast disease, 4 with Thalassaemia, and 25 normal controls. The results of this study demonstrate that a subpopulation of lymphocytes (16.6%) bearing surface ferritin was found in patients with breast cancer in stage I–II. None were demonstrated in patients with either benign breast disease, or with Thalassaemia, the latter known to have high serum ferritin levels, and almost none (1.7%) in normal individuals. A significant decrease in the percentage of ERFC as compared with the percentage of T cells, determined with anti-T cell antiserum (P<0.01), was observed in patients with breast cancer in stage I–II. Yet, the mean T-cell percentage in this group of patients was significantly higher than the mean percentage of T cells in normal controls (P<0.01). In patients with benign breast disease, the percentage of T cells corresponded to the percentage of ERFC and did not significantly differ from those in normals. Stage III breast cancer patients seem to constitute a biologically distinct group, since the ferritin-positive lymphocyte subpopulation disappeared and the percentage of ERFC and T cells returned to the values of normal controls.Overnight incubation of lymphocytes from patients exhibiting a ferritin-positive lymphocyte subpopulation in culture media containing 20% FCS resulted in the removal of ferritin from the surface of the cells and in restoration of the percentage of ERFC.     

Cancer of the gastrointestinal tract results in a restricted T-cell repertoire dependent upon tumor differentiation

We investigated the influence of tumor tissue differentiation on the diversity of TCR repertoire. CDR3 spectratypes of CD4⁺ and CD8⁺ T cell subsets were analyzed from 27 patients with gastrointestinal tract tumors exhibiting varying degrees of differentiation. A CDR3 spectratype complexity scoring system was used to quantify the diversity of TCR repertoire. Each patient was matched with an age-matched healthy group to control for age variability. Results show that the complexity scores (TCR repertoire diversity) have a significant correlation with the degree of tumor differentiation, which provides useful information for understanding immune response in cancer patients.     

Analysis of T-cell receptor Vbeta gene from infiltrating T cells in insulitis and myocarditis in encephalomyocarditis virus-infected BALB/C mice

Encephalomyocarditis (EMC) virus induces insulin-dependent diabetes and myocarditis in several strains of mice. The T-cell receptor (TCR) Vbeta genes of infiltrating T cells in the pancreas and myocardium of BALB/C mice infected with EMC virus D-variant (EMC-D virus) were analyzed. Using a nested two-step polymerase chain reaction (PCR), TCR Vbeta cDNAs were cloned and sequenced. Two and four kinds of TCR Vbeta clones were obtained from T cells infiltrating into the pancreas and myocardium of BALB/C mice infected with EMC-D virus, respectively. The infiltrating lymphocytes in the diabetic mice expressed Vbeta 8.1, 8.2, and 8.3 genes predominantly. Previously, the use of Vbeta 8.2 has been reported in autoimmune diseases such as murine experimental allergic encephalomyelitis (EAE) and non-obese diabetic (NOD) mouse. This study suggests that mice infected with EMC virus are a useful animal model for autoimmune diseases such as insulin-dependent diabetes.     

Cellular interaction against autologous tumor cells between IL-2-cultured lymphocytes and fresh peripheral blood lymphocytes in patients with breast cancer given immuno-chemotherapy

In patients with Stage II or III breast cancer and in patients with liver metastases from breast cancer, we examined cellular interaction in the cytotoxicity against autologous tumor cells by interleukin-2(IL-2)-cultured lymphocytes (CL) and fresh peripheral blood lymphocytes (FPBL) treated with immunochemotherapy including OK-432 and cyclophosphamide. In flow cytometric analysis, CD8 + CD11b+ and CD16+ cells significantly decreased after immuno-chemotherapy in both groups of patients. A protocol study in Stage II or III breast cancer patients showed suppressive activity of FPBL on the cytotoxic activity of CL in 3/9 of the non-treatment group but no suppressive activity and enhancing activity in 3/7 in the immuno-chemotherapy group. Moreover, in 19 patients with liver metastases from breast cancer treated with immuno-chemotherapy including adoptive immunotherapy, FPBL in 6/19 showed enhancing activity, and in 8/19 suppressive activity in the lysis of autologous tumor cells. In assaysin vitro using autologous and allogeneic tumor cells, FPBL showed a partial specificity in cellular interaction against autologous tumor cells. CD4-depleted FPBL inhibited cytotoxicity of CL, while CD8-depleted FPBL enhanced cytotoxicity of CL in patients with liver metastases. These results suggest that immuno-chemotherapy eliminates the suppressive population in FPBL and may induce tumor regression if combined with adoptive immunotherapy using CL.Abbreviations IL-2 interleukin-2 - CL IL-2-cultured lymphocytes - FPBL fresh peripheral blood lymphocytes - AIT adoptive immunotherapy     

The analysis of clonal expansions in normal and autoimmune B cell repertoires

Clones are the fundamental building blocks of immune repertoires. The number of different clones relates to the diversity of the repertoire, whereas their size and sequence diversity are linked to selective pressures. Selective pressures act both between clones and within different sequence variants of a clone. Understanding how clonal selection shapes the immune repertoire is one of the most basic questions in all of immunology. But how are individual clones defined? Here we discuss different approaches for defining clones, starting with how antibodies are diversified during different stages of B cell development. Next, we discuss how clones are defined using different experimental methods. We focus on high-throughput sequencing datasets, and the computational challenges and opportunities that these data have for mining the antibody repertoire landscape. We discuss methods that visualize sequence variants within the same clone and allow us to consider collections of shared mutations to determine which sequences share a common ancestry. Finally, we comment on features of frequently encountered expanded B cell clones that may be of particular interest in the setting of autoimmunity and other chronic conditions.     

Production and characterization of monoclonal IgM autoantibodies specific for the T-cell receptor

Natural autoantibodies to the T-cell receptor (Tcr) have been identified in all human sera. However, titer, epitope specificity, and isotype vary with physiological conditions, autoimmune diseases, and retroviral infections. The levels of anti-Tcr autoantibodies in rheumatoid arthritis (RA) patients are significantly higher than in normal individuals, and the autoantibodies are typically IgM. To obtain detailed information on these autoantibodies, we generated B-cell heterohybridomas secreting monoclonal IgM autoantibodies (mAAbs) from the synovial tissue and peripheral blood of RA patients. We selected clones secreting mAAbs that bound a major V epitope defined by a synthetic peptide that contains the CDR1 region of the V 8.1 gene product. From these we isolated a subset of seven mAAbs that bound a recombinant single-chain V/V construct containing the peptide epitope and, also to JURKAT cells which express V 8.1. The mAAbs produced by these clones were distinct from each other in their V-region sequences. However, all the V regions were essentially identical to germline sequences in both the heavy and light chains. Heavy-chain CDR3 segments ranged in length from 17 to 26 residues, did not correspond to any known autoantibodies, and showed extensive N-region diversity in the V(D)J junctions. Five monoclonal autoantibodies use V_H 3 genes, while the remaining two utilized V_H 4 sequences. Light-chain variable regions used were V 3 (two), V 3 (four), and one V 2. These autoantibodies derived their unique features from their CDR3 segments that could not be aligned with any known sequences.     

Characterization of the T-cell receptor gamma locus and analysis of the variable gene segment expression in rabbit

The genomic organization and expression of genes of the T-cell receptor gamma (TRG) locus are described for mice and humans, but not for species such as rabbits (Oryctolagus cuniculus), in which T cells compose a sizeable proportion of T cells in the periphery. We cloned 200 kb of the rabbit TRG locus and determined the TRGV gene usage in adult and newborn rabbits by RT-PCR. We identified two TRGJ genes, one TRGC gene, and 22 TRGV genes, all of which encoded functional variable regions. One TRGV gene is the unique member of the TRGV2 subgroup, whereas the other genes belong to the TRGV1 subgroup. Evolutionary analyses of TRGV1 genes identified three distinct groups that can be explained by separate duplication events in the rabbit genome. Evidence of gene conversion between TRGV1.1 and TRGV1.6 was observed. Both TRGV1 and TRGV2 subgroup genes were expressed in the spleen, intestine, and appendix of adult rabbits, and the repertoire of TRGV genes expressed in these tissues was similar. In these tissues from newborns, and in skin from adults, only the genes from the TRGV1 subgroup were expressed. Greater TRGV-J junctional diversity was found in tissues from adult compared to newborn rabbits. Our analyses indicate rabbits have a larger germ line encoded TRG repertoire compared with that of mice and humans. In addition, we found TRGV gene usage is alike in most tissues of rabbits similar to that found in humans but in contrast to that found in mice.Electronic SupplementaryMaterial Supplementary material is available for this article at The nucleotide sequence data reported in this article have been submitted to GenBank and are assigned the accession numbers AY748325–AY748348     

Cloning,expression, and crystallization of the V delta domain of a human gamma delta T-cell receptor.

T-lymphocytes recognize a wide variety of antigens through highly diverse cell-surface glycoproteins known as T-cell receptors (TCRs). These disulfide-linked heterodimers are composed of alpha and beta or gamma and delta polypeptide chains consisting of variable (V) and constant (C) domains non-covalently associated with at least four invariant chains to form the TCR-CD3 complex. It is well established that alpha beta TCRs recognize antigen in the form of peptides bound to molecules of the major histocompatibility complex (MHC); furthermore, information on the three-dimensional structure of alpha beta TCRs has recently become available through X-ray crystallography. In contrast, the antigen specificity of gamma delta TCRs is much less well understood and their three-dimensional structure is unknown. We have cloned the delta chain of a human TCR specific for the MHC class I HLA-A2 molecule and expressed the V domain as a secreted protein in the periplasmic space of Escherichia coli. Following affinity purification using a nickel chelate adsorbent, the recombinant V delta domain was crystallized in a form suitable for X-ray diffraction analysis. The crystals are orthorhombic, space group P2(1)2(1)2 with unit cell dimensions a = 69.9, b = 49.0, c = 61.6 A. and diffract to beyond 2.3 A resolution. The ability of a V delta domain produced in bacteria to form well-ordered crystals strongly suggests that the periplasmic space can provide a suitable environment for the correct in vivo folding of gamma delta TCRs.