Similar T-cell Receptor Research Articles

Comprehensive TCR repertoire analysis of CD4+ T-cell subsets in rheumatoid arthritis

The pathogenesis of rheumatoid arthritis (RA), a systemic autoimmune disease characterized by autoreactive T-cell accumulation and pro-inflammatory cytokine overproduction, is unclear. Systematically addressing T-cell receptor (TCR) repertoires of different CD4+ T-cell subsets could help understand RA pathogenesis. Here, peripheral CD4+ T cells from treatment-naïve RA patients and healthy controls were sorted into seven subsets including naïve, effector, central memory, effector memory (EMT), Th1, Th17, and regulatory T cells. T-cell receptor β chain repertoires were then analyzed by next-generation sequencing. We identified T-cell clonal expansion in EMT and Th17 cells of RA patients, with highly similar TCR repertoires. Ex vivo experiments demonstrated the preferred differentiation from EMT to Th17 cells in RA. Notably, we showed that TCR diversity and abundance of differentiated T cells of Th17 were significantly correlated with RA disease activity. Based on these observations, we propose that abnormal differentiation from EMT to Th17 and expansion of Th17 play pivotal role in RA pathogenesis.

Analysis of the Whole CDR3 T Cell Receptor Repertoire after Hematopoietic Stem Cell Transplantation in 2 Clinical Cohorts.

A major cause of morbidity and mortality for patients who undergo hematologic stem cell transplantation (HSCT) is acute graft-versus-host disease (aGVHD), a mostly T cell-mediated disease. Examination of the T cell receptor (TCR) repertoire of HSCT recipients and the use of next-generation nucleotide sequencing have raised the question of whether features of TCR repertoire reconstitution might reproducibly associate with aGVHD. We hypothesized that the peripheral blood TCR repertoire of patients with steroid-nonresponsive aGVHD would be less diverse. We also hypothesized that patients with GVHD who shared HLA might also share common clones at the time of GVHD diagnosis, thereby potentially providing potential clinical indicators for treatment stratification. We further hypothesized that HSCT recipients with the same HLA mismatch might share a more similar TCR repertoire based on a potentially shared focus of alloreactive responses. We studied 2 separate patient cohorts and 2 separate platforms for measuring TCR repertoire. The first cohort of patients was from a multicenter Phase III randomized double-blinded clinical trial of patients who developed aGVHD (NCT01002742). The second cohort comprised samples from biobanks from 2 transplantation centers and the Center for International Blood and Marrow Transplant Research of patients who underwent mismatched HSCT. There were no statistically significant differences in the TCR diversity of steroid responders and nonresponders among patients with aGVHD on the day of diagnosis. Most clones in the repertoire were unique to each patient, but a small number of clones were found to be both exclusive to and shared among aGVHD nonresponders. We were also able to show a strong correlation between the presence of Vβ20 and Vβ29 and steroid responsiveness. Using the Bhattacharya coefficient, those patients who shared the same HLA mismatch were shown to be no more similar to one another than to those who had a completely different mismatch. Using 2 separate clinical cohorts and 2 separate platforms for analyzing the TCR repertoire, we have shown that the sampled human TCR repertoire is largely unique to each patient but contains glimmers of common clones of subsets of clones based on responsiveness to steroids in aGVHD on the day of diagnosis. These studies are informative for future strategies to assess for reproducible TCR responses in human alloreactivity and possible markers of GVHD responsiveness to therapy.

Lattice Light-Sheet Microscopy Multi-Dimensional Analyses (LaMDA) of T-Cell Receptor Dynamics Predict T-Cell Signaling States

Cell surface receptors dynamically change in response to cell signaling, but a direct linkage between receptor dynamics and cell state has not been established. Here we report the development of lattice light-sheet microscopy multi-dimensional analyses (LaMDA), a pipeline that combines high spatiotemporal-resolution four-dimensional lattice light-sheet microscopy, machine learning, and diffusion maps to analyze T-cell receptor (TCR) dynamics and predict T-cell signaling states without the need for complex biochemical measurements. LaMDA images thousands of TCR microclusters on the surface of live primary cells to collect high-dimensional dynamic data for machine learning, which extracts key dynamic features to build predictive diffusion maps. LaMDA spatiotemporally reveals global changes of TCRs across the 3D cell surface, accurately differentiates stimulated cells from unstimulated cells, precisely predicts attenuated T-cell signaling after CD4 and CD28 receptor blockades, and reliably discriminates between structurally similar TCR ligands. We anticipate broad usage of this approach for other receptors and cells, as well as for guiding the design and development of future immunotherapies for cancer, infection, and autoimmunity.

TCR Convergence in Individuals Treated With Immune Checkpoint Inhibition for Cancer.

Tumor antigen-driven selection may expand T cells having T cell receptors (TCRs) of shared antigen specificity but different amino acid or nucleotide sequence in a process known as TCR convergence. Substitution sequencing errors introduced by TCRβ (TCRB) repertoire sequencing may create artifacts resembling TCR convergence. Given the anticipated differences in substitution error rates across different next-generation sequencing platforms, the choice of platform could be consequential. To test this, we performed TCRB sequencing on the same peripheral blood mononuclear cells (PBMC) from individuals with cancer receiving anti-CTLA-4 or anti-PD-1 using an Illumina-based approach (Sequenta) and an Ion Torrent-based approach (Oncomine TCRB-LR). While both approaches found similar TCR diversity, clonality, and clonal overlap, we found that Illumina-based sequencing resulted in higher TCR convergence than with the Ion Torrent approach. To build upon this initial observation we conducted a systematic comparison of Illumina-based TCRB sequencing assays, including those employing molecular barcodes, with the Oncomine assay, revealing differences in the frequency of convergent events, purportedly artifactual rearrangements, and sensitivity of detection. Finally, we applied the Ion Torrent-based approach to evaluate clonality and convergence in a cohort of individuals receiving anti-CTLA-4 blockade for cancer. We found that clonality and convergence independently predicted response and could be combined to improve the accuracy of a logistic regression classifier. These results demonstrate the importance of the sequencing platform in assessing TCRB convergence.

TCR sequencing analysis of cancer tissues and tumor draining lymph nodes in colorectal cancer patients

ABSTRACTTumor draining lymph nodes (TDLNs) are located in the routes of lymphatic drainage from a primary tumor and have the highest risk of metastasis in various types of solid tumors. TDLNs are also considered as a tissue to activate the antitumor immunity, where antigen-specific effector T cells are generated. However, T cell receptor (TCR) repertoires in TDLNs have not been well characterized. We collected 23 colorectal cancer tumors with 203 lymph nodes with/without metastatic cancer cells (67 were metastasis-positive and the remaining 136 were metastasis-negative) and performed TCR sequencing. Metastasis-positive TDLNs showed a significantly lower TCR diversity and shared TCR clonotypes more frequently with primary tumor tissues compared to metastasis-negative TDLNs. Principal component analysis indicated that TDLNs with metastasis showed similar TCR repertoires. These findings suggest that cancer-reactive T cell clones could be enriched in the metastasis-positive TDLNs.

MS14.01 Basic Science Rationale of IO in Early Stage NSCLC?

MS14.01 Basic Science Rationale of IO in Early Stage NSCLC?

Tissue-specific functions of invariant natural killer T cells.

Invariant natural killer T cells (iNKT cells) are an innate-like T cell subset that expresses an invariant T cell receptor (TCR) α-chain and recognizes lipids presented on CD1d. They secrete diverse cytokines and can influence many types of immune responses. Despite having highly similar TCR specificities, iNKT cells differentiate in the thymus into distinct subsets that are analogous to T helper 1 (TH1), TH2 and TH17 cell subsets. Additional iNKT cell subsets that may require peripheral activation have also been described, including one that produces IL-10. In general, iNKT cells are non-circulating, tissue-resident lymphocytes, but the prevalence of different iNKT cell subsets differs markedly between tissues. Here, we summarize the functions of iNKT cells in four tissues in which they are prevalent, namely, the liver, the lungs, adipose tissue and the intestine. Importantly, we explain how local iNKT cell responses at each site contribute to tissue homeostasis and protection from infection but can also contribute to tissue inflammation and damage.

Distinct immune signatures in chronic lymphocytic leukemia (CLL) and Richter’s syndrome (RS).

7524 Background: Immune checkpoint blockade with PD-1 inhibitor pembrolizumab was effective in RS but not CLL in a phase 2 study (NCT02332980). Analyzing the immune signatures including PD-L1 expression and T cell diversity is important for understanding the differential responses. Methods: 15 CLL and 14 RS patients in NCT02332980 were included. Expression of PD-L1 in lymph node (LN) was analyzed by immunohistochemistry staining. Peripheral blood (PB) and LN T cell diversity was analyzed using the ImmunoSEQ platform (Adaptive Biotechnology), which quantifies the clonailty of T cells by deep sequencing of the CDR3 region of T cell receptor (TCR). Data analysis (Student’s t-test and Mann-Whiney U test) was done by GraphPad Prism (v7). Results: PD-L1 expression was significantly lower in CLL (n = 6) vs RS (n = 12) patients (mean 5.5% vs 22.9%, P= 0.003). A control CLL cohort (n = 11, Mayo CLL tissue registry) also had lower PD-L1 expression (7.7%, P= 0.002 vs RS). RS Patients progressed on ibrutinib or chemotherapy had similar PD-L1 expression (25.3% vs 19.6%, P= 0.416). PB TCR clonality at trial baseline was significantly lower in RS (n = 13) vs CLL (n = 13) patients (median 0.098 vs 0.342, P= 0.026). Patients progressed on ibrutinib or chemotherapy had similar TCR clonality (CLL: 0.34 vs 0.24, P= 0.524; RS: 0.11 vs 0.09, P= 0.534). 5 RS patients achieved an objective response (1 CR, 2 PR, 1 CMR and 1 PMR), 4 of which were after progression on ibrutinib. For CLL patients, only 1 nodal reduction was seen. 4 RS patients had prior CLL samples available for TCR analysis. 2 patients had a notable decrease of TCR clonality (0.21-0.04 and 0.17-0.03) from CLL (PB) to RS state (LN), and achieved CR and PR, respectively. 2 other patients with stable clonality (0.04-0.05 and 0.03-0.08) had SD and PMR, respectively. PB TCR clonality did not change significantly with pembrolizumab (CLL [n = 11]: P= 0.775; RS [n = 11]: P= 0.830; paired t-test). Conclusions: RS patients had higher expression of PD-L1 and lower TCR clonality (more diverse T cells) compared with CLL patients. The distinct immune signatures may explain their differential responses to PD-1 blockade. The dynamic changes of TCR clonality during Richter’s transformation may predict a response to PD-1 blockade.

Interleukin-18 activates Vγ9Vδ2+ T cells from HIV-positive individuals: recovering the response to phosphoantigen.

The study aimed to identify an immunoregulatory factor that restores the phosphoantigen response of Vγ9Vδ2+ T cells from HIV-positive individuals on antiretroviral therapy. It was designed to characterize the effects of interleukin-18 (IL-18) on proliferation and effector function in Vγ9Vδ2 T cells from HIV-negative individuals and test whether exogenous IL-18 reconstitutes the Vγ9Vδ2 T-cell response to phosphoantigen from HIV-positive donors. Vγ9Vδ2 T cells from HIV-negative individuals responded strongly to phosphoantigen or aminobisphosphonate stimulation of peripheral blood mononuclear cells (PBMC), whereas cells with similar T-cell receptor profiles from HIV-positive individuals only responded to aminobisphosphonate. Interleukin-18 was higher after aminobisphosphonate stimulation due to activation of the inflammasome pathway. Both IL-18 and IL-18 receptor levels were measured and the activity of exogenous IL-18 on HIV-negative and HIV-positive PBMC was evaluated in terms of Vγ9Vδ2 T-cell proliferation, memory subsets, cytokine expression and CD107a expression. Interleukin-18 stimulation increased proliferation, enhanced the accumulation of effector memory cells, and increased expression of cytotoxic markers in HIV-negative controls. When Vγ9Vδ2 T cells from HIV-positive individuals were stimulated with isopentenyl pyrophosphate in the presence of IL-18, there was increased proliferation, accumulation of memory cells, and higher expression of CD56, NKG2D and CD107a (markers of cytotoxic effector phenotype). Interleukin-18 stimulation specifically expanded the Vγ9-JγP+ subset of Vγ9Vδ2 T cells, as was expected for normal responses to phosphoantigen. Interleukin-18 is a potent stimulator of Vγ9Vδ2 T-cell proliferation and effector function. Therapies directed at reconstituting Vγ9Vδ2 T-cell activity in HIV-positive individuals should include stimulators of IL-18 or direct cytokine supplementation.

Plasmodium yoelii infection of BALB/c mice results in expansion rather than induction of CD4(+) Foxp3(+) regulatory T cells.

Recently, we demonstrated elevated numbers of CD4(+) Foxp3(+) regulatory T (Treg) cells in Plasmodium yoelii-infected mice contributing to the regulation of anti-malarial immune response. However, it remains unclear whether this increase in Treg cells is due to thymus-derived Treg cell expansion or induction of Treg cells in the periphery. Here, we show that the frequency of Foxp3(+) Treg cells expressing neuropilin-1 (Nrp-1) decreased at early time-points during P. yoelii infection, whereas percentages of Helios(+) Foxp3(+) Treg cells remained unchanged. Both Foxp3(+) Nrp-1(+) and Foxp3(+) Nrp-1(-) Treg cells from P. yoelii-infected mice exhibited a similar T-cell receptor Vβ chain usage and methylation pattern in the Treg-specific demethylation region within the foxp3 locus. Strikingly, we did not observe induction of Foxp3 expression in Foxp3(-) T cells adoptively transferred to P. yoelii-infected mice. Hence, our results suggest that P. yoelii infection triggered expansion of naturally occurring Treg cells rather than de novo induction of Foxp3(+) Treg cells.

Clonotypically similar hybrid αβ T cell receptors can exhibit markedly different surface expression, antigen specificity and cross-reactivity.

SummaryEmerging data indicate that particular major histocompatibility complex (MHC)‐bound antigenic peptides can be recognized by identical or near‐identical αβ T cell receptors (TCRs) in different individuals. To establish the functional relevance of this phenomenon, we artificially paired α and β chains from closely related TCRs specific for the human leucocyte antigen (HLA)‐B*35:01‐restricted HIV‐1 negative regulatory factor (Nef)‐derived epitope VY8 (VPLRPMTY, residues 74–81). Several hybrid TCRs generated in this manner failed to express at the cell surface, despite near homology with naturally isolated αβ chain combinations. Moreover, a substantial proportion of those αβ TCRs that did express lost specificity for the index VY8 peptide sequence. One such hybrid αβ pair gained neo‐variant specificity in the context of the VY8 backbone. Collectively, these data show that clonotypically similar TCRs can display profound differences in surface expression, antigen specificity and cross‐reactivity with potential relevance for the control of mutable viruses.

Determinants of public T cell responses

Historically, sharing T cell receptors (TCRs) between individuals has been speculated to be impossible, considering the dramatic discrepancy between the potential enormity of the TCR repertoire and the limited number of T cells generated in each individual. However, public T cell response, in which multiple individuals share identical TCRs in responding to a same antigenic epitope, has been extensively observed in a variety of immune responses across many species. Public T cell responses enable individuals within a population to generate similar antigen-specific TCRs against certain ubiquitous pathogens, leading to favorable biological outcomes. However, the relatively concentrated feature of TCR repertoire may limit T cell response in a population to some other pathogens. It could be a great benefit for human health if public T cell responses can be manipulated. Therefore, the mechanistic insight of public TCR generation is important to know. Recently, high-throughput DNA sequencing has revolutionized the study of immune receptor repertoires, which allows a much better understanding of the factors that determine the overlap of TCR repertoire among individuals. Here, we summarize the current knowledge on public T-cell response and discuss future challenges in this field.

Transgenic mice with a diverse human T cell antigen receptor repertoire

Because of tolerance mechanisms, it has been hard to identify the T cell receptors (TCRs) of high-avidity T cells against self (for example, tumor) antigens. TCRs that are specific for foreign human antigens from the nontolerant T cell repertoire can be identified in mice. Moreover, if mice are constructed to express the human TCR repertoire, they can be used to analyze the unskewed repertoire against human self antigens. Here we generated transgenic mice with the entire human TCRalphabeta gene loci (1.1 and 0.7 Mb), whose T cells express a diverse human TCR repertoire that compensates for mouse TCR deficiency. A human major histocompatibility class I transgene increases the generation of CD8+ T cells with human compared to mouse TCRs. Functional CD8+ T cells against several human tumor antigens were induced, and those against the Melan-A melanoma antigen used similar TCRs to those that have been detected in T cell clones from individuals with autoimmune vitiligo or melanoma. These mice will allow researchers to identify pathogenic and therapeutic human TCRs.

Determinants of in vitro expansion of different human virus-specific FoxP3+ regulatory CD8+ T cells in chronic hepatitis C virus infection

It has been shown previously that suppressive virus-specific FoxP3+ regulatory CD8+ T cells can be expanded from human peripheral blood mononuclear cells after in vitro antigen-specific stimulation. This study extended this finding by analysing the mechanisms of virus-specific FoxP3+ regulatory CD8+ T-cell generation during peptide-specific expansion in vitro. It was shown that hepatitis C virus (HCV)-, influenza virus (FLU)-, Epstein-Barr virus (EBV)- and cytomegalovirus (HCMV)-specific FoxP3+ regulatory CD8+ T cells could be expanded differentially from the blood of chronically HCV-infected patients following in vitro peptide-specific stimulation. The different ability of virus-specific CD8+ T-cell populations to express FoxP3 after continuous antigen stimulation in vitro correlated significantly with the ex vivo differentiation status. Indeed, CD27+ CD28+ CD57- HCV-, FLU- and EBV-specific CD8+ T cells displayed a significantly higher ability to give rise to FoxP3+ regulatory CD8+ T cells compared with CD27- CD28- CD57+ HCMV-specific CD8+ T cells. Similar T-cell receptor expression patterns of FoxP3+ versus FoxP3- CD8+ T cells of the same antigen specificity indicated that both cell populations were probably expanded from the same virus-specific CD8+ T-cell precursor. In addition, no specific antigen-presenting cell populations were required for the generation of FoxP3+ CD8+ T cells, as CD8+-selected virus-specific FoxP3+ CD8+ T cells could be expanded by peptide presentation in the absence of antigen-presenting cells. Taken together, these results suggest that the ability to expand FoxP3+ regulatory CD8+ T cells from virus-specific CD8+ T cells differs among distinct virus-specific CD8+ T-cell populations depending on the differentiation status.

Oligoclonal Expansions of CD4 + and CD8 + T-Cells in the Target Organ of Patients With Biliary Atresia

Biliary atresia is an inflammatory, fibrosclerosing neonatal cholangiopathy, characterized by a periductal infiltrate composed of CD4(+) and CD8(+) T cells. The pathogenesis of this disease has been proposed to involve a virus-induced, subsequent autoreactive T cell-mediated bile duct injury. Antigen-specific T-cell immunity involves clonal expansion of T cells expressing similar T-cell receptor (TCR) variable regions of the beta-chain (Vbeta). We hypothesized that the T cells in biliary atresia tissue expressed related TCRs, suggesting that the expansion was in direct response to antigenic stimulation. The TCR Vbeta repertoire of T cells from the liver, extrahepatic bile duct remnants, and peripheral blood of biliary atresia and other cholestatic disease controls were characterized by fluorescent-activated cell sorter analysis, and TCR junctional region nucleotide sequencing was performed on expanded TCR Vbeta regions to confirm oligoclonality. FACS analysis revealed Vbeta subset expansions of CD4(+) and CD8(+) T cells from the liver or bile duct remnant in all patients with biliary atresia and only 1 control. The CD4(+) TCR expansions were limited to Vbeta3, -5, -9, and -12 T-cell subsets and the CD8(+) TCR Vbeta expansions were predominantly Vbeta20. Each Vbeta subset expansion was composed of oligoclonal populations of T cells. Biliary atresia is associated with oligoclonal expansions of CD4(+) and CD8(+) T cells within liver and extrahepatic bile duct remnant tissues, indicating the presence of activated T cells reacting to specific antigenic stimulation. Future studies entail identifying the specific antigen(s) responsible for T-cell activation and bile duct injury.

The Characteristics of T Lymphocytic Clones Correlate with the Pathogenesis of Idiopathic Thrombocytopenic Purpura.

Objective To determine the characteristics of T lymphocytic clones that correlate with the pathogenesis of idiopathic thrombocytopenic purpura (ITP) by investigating complementarity determining region (CDR3) repertoires of T cell receptors (TCRs) β chain variable region (BV).Methods Twenty-five of patients with idiopathic thrombocytopenic purpura (including 15 patients with acute ITP and 10 patients with chronic ITP), twenty of normal peoples and 20 of normal umbilical cord blood samples were enrolled. Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify 24 subfamily genes of TCR BV from peripheral blood lymphocytes of ITP patients and normal controls, the PCR products were run on denatured polyacrylamide sequencing gel, establishing spectratyping of TCR BV CDR3 gene expressing repertoire. The bands that dense or disappeared of TCR BV gene repertoire on the electrophoresis gel were used to analyze the variety of T cell clones in cases of ITP. The dense bands in the gel were cut down and sequencing. Compare with those major sequences of TCRBV gene in normal and abnormal T cell clones to understand the relationship among the TCRBV gene and ITP.Results: In acute ITP (aITP), the spectratyping of TCR BV CDR3 size distribution were similar to normal control (P=0.179), the oligoclonality could be observed with an average value of 2.73±0.88 per person in 15 cases of aITP. TCR BV CDR3 size distribution had little difference compared with aITP and healthy controls. Abnormal spectratyping of TCR BV CDR3 distribution could be observed significantly different between chronic ITP (cITP) patients and healthy controls (P<0.05), with oligoclonality at an average value of 7.2±3.04 per person in 10 cases of cITP. Same T cell clone expansions could be observed in gene subfamilies of TCR BV8,BV13.1,BV14,BV17. In 10 of cITP patients, sequences could be read from 19 out of 20 dense bands on the gene spectratyping of TCR BV CDR3, which suggested some dominant T cell clones expanded in cITPs. In different patients of cITP, the expanded T cell clones shared identical or similar TCR BV gene or CDR3 encoded by the TCR BV gene. Two T cell clones in 2 patients had identical TCR BV8 gene sequences separately. Other 2 T cell clones in another 2 cITP patients had same TCR BV13.1. It showed those expanded T cell clones in the bodies of the cITP patients could recognize common antigen. The similar CDR3 was found in 2/4 expanded TCR BV17 T cell clones, with only 2 amino acids different in framework 4 (FR4). Two T cell clones had almost same sequence of CDR3 in TCR BV17 except for 4 basepairs difference in their full sequences. In analyzing the motifs in CDR3 of different cITPs, we found three common motifs (E/DTQYFGPG;N(K)EQFFGPG;GANVLTFGAG) which were separately used in different TCR BV CDR3 of 19 expanded T cell clones. Among these motifs, TCR BV of 7/19 T cell clones shared common E/DTQYFGPG;TCR BV of 4/19 T cell clones shared common N(K)EQFFGPG; TCR BV17 of 3/4 T cell clones had common GANVLTFGAG. Compared with the various sequence of 10 T cell clones in 6 cases of SLE patients that we had reported (Journal of Chinese Medicine 2003; 83(10):1648–1652), 5/10 T cell clones of the SLEs shared the motif NEQFFGPG in the CDR3.Three of TCR BV13.1 in these T cell clones showed motif DTQYFGPG.Conclusion the cITPs have some abnormal expanded T cell clones that correlate with the pathogenesis, while aITPs have no significant expanded clones. All the cITP patients share 3 common motifs in TCR BV CDR3, which is possibly to recognize similar autoantigens.

Analysis of the T cell receptor Vgamma region gene repertoire in bronchoalveolar lavage (BAL) and peripheral blood of atopic asthmatics and healthy subjects.

We analyzed the T cell receptor (TCR) Vgamma repertoire in BAL and peripheral blood (PBL) of three mild stable atopic asthmatics and two non-asthmatic controls. We used the polymerase chain reaction (PCR) to establish the expression of the four Vgamma families, and to detect oligo or monoclonal expansion of gammadelta+ T cells, we resolved the PCR projects on denaturing and non-denaturing gels to find the extent of junctional diversity arising from differences in the lengths of the V(D)J junctions. We also subcloned and sequenced the PCR products to characterize fully the sequence diversity. BAL T lymphocytes from two asthmatic patients (treated with inhaled steroids) expressed only VgammaII and, in one of them, VgammaIIJgamma usage was restricted to JgammaP and JgammaP1 gene segments, contrasting with the VgammaJgamma repertoire found in his respective PBL. Analyses in denaturing and non-denaturing gels showed that the BAL VgammaIIJgammaP and VgammaIIJgammaP1 PCR products resolved into few bands, suggesting deletions at the juctions due to oligoclonal expansion. BAL T lymphocytes from the third asthmatic (not receiving inhaled steroids) expressed VgammaI, II and III, and the sequences of the in-frame TCR transcripts from this asthmatic and one healthy volunteer who expressed a similar BAL VgammaTCR repertoire showed clonal expansion of T cells expressing all three Vgamma families. Our analyses showed that much of the GammaDeltaT cell population found in BAL fluid of humans derives from clonally expanded T cells.

Stimulation of peripheral blood lymphocytes with Campylobacter jejuni generates a gammadelta T cell response in patients with Guillain-Barrésyndrome.

In three patients whose Guillain-Barré syndrome (GBS) was preceded by gastrointestinal infection due to Campylobacter jejuni, gammadelta T cells were generated from peripheral blood in response to in vitro stimulation with C. jejuni. In one of the patients, where a diagnostic sural nerve biopsy was performed, gammadelta T cells were also isolated following culture of the nerve tissue. Studies with healthy volunteers and C. jejuni gastroenteritis patients also showed preferential enrichment for gammadelta T cells in peripheral blood cells stimulated with C. jejuni, although the response was significantly lower than that seen in GBS patients. In two out of three GBS patients and all of the controls, gammadelta T cell receptor (TCR) gene usage was shown to be Vgamma9/Vdelta2+. In the GBS patient where nerve-infiltrating gammadelta T cells were isolated, these and C. jejuni-specific peripheral blood cells had similar TCR gene usage, predominantly consisting of Vgamma5/Vdelta1+ cells. Sequencing the Vdelta1 products from nerve and peripheral blood showed similarities in CDR3 length, but the single Vdelta1 sequence obtained from nerve was not identified in peripheral blood. These results suggest that the generation of gammadelta T cells is part of a normal immune response to C. jejuni, which, in patients with GBS, may contribute to the pathogenesis of their inflammatory neuropathy.

A Correlation between TCR Vα Docking on MHC and CD8 Dependence: Implications for T Cell Selection

T cell receptors (TCR) adopt a similar orientation when binding with major histocompatibility complex (MHC) molecules, yet the biological mechanism that generates this similar TCR orientation remains obscure. We show here the cocrystallographic structure of a mouse TCR bound to a human MHC molecule not seen by the TCR during thymic development. The orientation of this xenoreactive murine TCR atop human MHC deviates from the typical orientation more than any previously determined TCR/MHC structure. This unique orientation is solely due to the placement of the TCR Vα domain on the MHC. In light of new information provided by this structure, we have reanalyzed the existing TCR/MHC cocrystal structures and discovered unique features of TCR Vα domain position on class I MHC that correlate with CD8 dependence. Finally, we propose that the orientation seen in TCR recognition of MHC is a consequence of selection during T cell development.

Staphylococcal enterotoxin B induces anergy to conventional peptide in memory T cells

Microbial superantigens can alter host immunity through aberrant activation and subsequent anergy of responding naive T cells. We show here that the superantigen, staphylococcal enterotoxin B (SEB), directly induces tolerance in memory CD4 T cells. Murine naive and memory CD4 + T cells were labeled with the fluorescent dye CFSE and the cells were exposed to SEB before they were cultured with specific peptide antigen. Memory, but not naive, T cells became anergic and did not respond to their cognate peptide antigen. The extent and duration of T cell receptor (TCR) clustering was similar to promote naive T cell activation and memory T cell anergy, suggesting similar TCR–SEB interactions led to distinct intracellular signaling processes in the two cell types. Like SEB, soluble anti-CD3 mAb does not stimulate memory cell proliferation. However, unlike SEB, soluble anti-CD3 mAbs did not induce anergy to cognate peptide. Anergy was directly visualized in vivo. CD4 + memory T cells were identified in mice that had been administered SEB. The cells failed to proliferate in response to subsequent immunization with their cognate recall antigen. Hence, one mode of pathogen survival is the modulation of host immunity through selective elimination of memory T cell responses.