T-cell Receptor Vβ Repertoire Research Articles

SARS-CoV-2-Specific T-Cell as a Potent Therapeutic Strategy against Immune Evasion of Emerging COVID-19 Variants.

Despite advances in vaccination and therapies for coronavirus disease, challenges remain due to reduced antibody longevity and the emergence of virulent variants like Omicron (BA.1) and its subvariants (BA.1.1, BA.2, BA.3, and BA.5). This study explored the potential of adoptive immunotherapy and harnessing the protective abilities using virus-specific T cells (VSTs). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) VSTs were generated by stimulating donor-derived peripheral blood mononuclear cells with spike, nucleocapsid, and membrane protein peptide mixtures. Phenotypic characterization, including T-cell receptor (TCR) vβ and pentamer analyses, was performed on the ex vivo-expanded cells. We infected human leukocyte antigen (HLA)-partially matched human Calu-3 cells with various authentic SARS-CoV-2 strains in a Biosafety Level 3 facility and co-cultured them with VSTs. VSTs exhibited a diverse TCR vβ repertoire, confirming their ability to target a broad range of SARS-CoV-2 antigens from both the ancestral and mutant strains, including Omicron BA.1 and BA.5. These ex vivo-expanded cells exhibited robust cytotoxicity and low alloreactivity against HLA-partially matched SARS-CoV-2-infected cells. Their cytotoxic effects were consistent across variants, targeting conserved spike and nucleocapsid epitopes. Our findings suggest that third-party partial HLA-matching VSTs could counter immune-escape mechanisms posed by emerging variants of concern.

Deletion of Vβ3+CD4+ T cells by endogenous mouse mammary tumor virus 3 prevents type 1 diabetes induction by autoreactive CD8+ T cells.

In both humans and NOD mice, type 1 diabetes (T1D) develops from the autoimmune destruction of pancreatic beta cells by T cells. Interactions between both helper CD4+ and cytotoxic CD8+ T cells are essential for T1D development in NOD mice. Previous work has indicated that pathogenic T cells arise from deleterious interactions between relatively common genes which regulate aspects of T cell activation/effector function (Ctla4, Tnfrsf9, Il2/Il21), peptide presentation (H2-A g7, B2m), and T cell receptor (TCR) signaling (Ptpn22). Here, we used a combination of subcongenic mapping and a CRISPR/Cas9 screen to identify the NOD-encoded mammary tumor virus (Mtv)3 provirus as a genetic element affecting CD4+/CD8+ T cell interactions through an additional mechanism, altering the TCR repertoire. Mtv3 encodes a superantigen (SAg) that deletes the majority of Vβ3+ thymocytes in NOD mice. Ablating Mtv3 and restoring Vβ3+ T cells has no effect on spontaneous T1D development in NOD mice. However, transferring Mtv3 to C57BL/6 (B6) mice congenic for the NOD H2 g7 MHC haplotype (B6.H2 g7) completely blocks their normal susceptibility to T1D mediated by transferred CD8+ T cells transgenically expressing AI4 or NY8.3 TCRs. The entire genetic effect is manifested by Vβ3+CD4+ T cells, which unless deleted by Mtv3, accumulate in insulitic lesions triggering in B6 background mice the pathogenic activation of diabetogenic CD8+ T cells. Our findings provide evidence that endogenous Mtv SAgs can influence autoimmune responses. Furthermore, since most common mouse strains have gaps in their TCR Vβ repertoire due to Mtvs, it raises questions about the role of Mtvs in other mouse models designed to reflect human immune disorders.

Deletion of Vβ3 +CD4 +T-cells by endogenous mouse mammary tumor virus 3 prevents type 1 diabetes induction by autoreactive CD8 +T-cells

Abstract In both humans and NOD mice, type 1 diabetes (T1D) develops from the autoimmune destruction of pancreatic beta cells by T-cells. Interactions between both helper CD4 +and cytotoxic CD8 +T-cells are essential for T1D development in NOD mice. Previous work has indicated that pathogenic T-cells arise from deleterious interactions between relatively common genes which regulate aspects of T-cell activation/effector function (Ctla-4, Tnfrsf9, Il2/Il21), peptide presentation (H2-A g7, B2m), and T-cell receptor signaling (Ptpn22). In this study, we used a combination of subcongenic mapping and a CRISPR/Cas9 screen to identify the NOD-encoded Mtv3 provirus as a genetic element affecting CD4 +/CD8 +T-cell interactions through an additional mechanism, altering the T-cell receptor (TCR) repertoire. Mtv3 encodes a superantigen that deletes the majority of Vβ3 +thymocytes in NOD mice. Ablating Mtv3 and restoring Vβ3 +T-cells has no effect on spontaneous T1D development in NOD mice. However, transferring Mtv3 to C57BL/6 (B6) mice congenic for the NOD H2 g7MHC haplotype (B6.H2 g7) completely blocks their normal susceptibility to T1D mediated by transferred CD8 +T-cells transgenically expressing AI4 or NY8.3 TCRs. The entire genetic effect is manifest by Vβ3 +CD4 +T-cells, which unless deleted by Mtv3, accumulate in insulitic lesions and trigger in B6 background mice the pathogenic activation of diabetogenic CD8 +T-cells. Our findings provide evidence that endogenous Mtv superantigens can influence autoimmune responses. Furthermore, since most common mouse strains have gaps in their TCR Vβ repertoire due to Mtvs, it raises questions about the role of Mtvs in other mouse models designed to reflect human disorders. Supported by grants from the American Diabetes Association (1-14-BS-051) and NIH (AI130656)

Naive T cells inhibit the outgrowth of intractable antigen-activated memory T cells: implications for T-cell immunotherapy

BackgroundThe wider application of T cells targeting viral tumor-antigens via their native receptors is hampered by the failure to expand potent tumor-specific T cells from patients. Here, we examine reasons...

Clonal composition and persistence of antigen-specific circulating T follicular helper cells.

T follicular helper (TFH ) cells play an essential role in promoting B cell responses and antibody affinity maturation in germinal centers (GC). A subset of memory CD4+ T cells expressing the chemokine receptor CXCR5 has been described in human blood as phenotypically and clonally related to GC TFH cells. However, the antigen specificity and relationship of these circulating TFH (cTFH ) cells with other memory CD4+ T cells remain poorly defined. Combining antigenic stimulation and T cell receptor (TCR) Vβ sequencing, we found T cells specific to tetanus toxoid (TT), influenza vaccine (Flu), or Candida albicans (C.alb) in both cTFH and non-cTFH subsets, although with different frequencies and effector functions. Interestingly, cTFH and non-cTFH cells specific for C.alb or TT had a largely overlapping TCR Vβ repertoire while the repertoire of Flu-specific cTFH and non-cTFH cells was distinct. Furthermore, Flu-specific but not C.alb-specific PD-1+ cTFH cells had a "GC TFH -like" phenotype, with overexpression of IL21, CXCL13, and BCL6. Longitudinal analysis of serial blood donations showed that Flu-specific cTFH and non-cTFH cells persisted as stable repertoires for years. Collectively, our study provides insights on the relationship of cTFH with non-cTFH cells and on the heterogeneity and persistence of antigen-specific human cTFH cells.

Rapid evaluation of T cell clonality in the diagnostic work-up of mature T cell neoplasms: TRBC1-based flow cytometric assay experience

The identification of mature T cell neoplasms by flow cytometry is often challenging, due to overlapping features with reactive T cells and limitations of currently available T cell clonality assays. The description of an antibody specific for one of two mutually exclusive T cell receptor (TCR) β-chain constant regions (TRBC1) provides an opportunity to facilitate the detection of clonal TCRαβ+ T cells based on TRBC-restriction. Here we prospectively analyzed 14 healthy controls and 63 patients with the flow cytometry protocol currently used for suspected T cell neoplasm implemented with immunostaining targeting TRBC1. Specimens were firstly classified in 3 groups based on clinical records data, laboratory findings and immunophenotypic features. T cell clonality was assessed by TCR Vβ repertoire analysis and the new rapid TRBC1 assay. Results showed that TRBC1 unimodal expression was unequivocally associated with samples presenting with immunophenotypic aberrancies. Moreover, we demonstrated that the use of TRBC1 is useful in solving uncertain cases and confirmed the high sensitivity of the method in identifying small T cell clones of uncertain significance (T-CUS). Finally, we found a high degree of concordance (97%) comparing the currently available clonality assessment methods with the proposed new method. In conclusion, our results provided real-life evidence of the utility of TRBC1 introduction in the flow cytometric clonality evaluation for the routine diagnostic work-up of T cell neoplasms.

T and NK Cells in IL2RG-Deficient Patient 50 Years After Hematopoietic Stem Cell Transplantation

The first successful European hematopoietic stem cell transplantation (HSCT) was performed in 1968 as treatment in a newborn with IL2RG deficiency using an HLA-identical sibling donor. Because of declining naive T and natural killer (NK) cells, and persistent human papilloma virus (HPV)-induced warts, the patient received a peripheral stem cell boost at the age of 37 years. NK and T cells were assessed before and up to 14 years after the boost by flow cytometry. The boost induced renewed reconstitution of functional NK cells that were 14 years later enriched for CD56dimCD27+ NK cells. T-cell phenotype and T-cell receptor (TCR) repertoire were simultaneously analyzed by including TCR Vβ antibodies in the cytometry panel. Naive T-cell numbers with a diverse TCR Vβ repertoire were increased by the boost. Before and after the boost, clonal expansions with a homogeneous TIGIT and PD-1 phenotype were identified in the CD27− and/or CD28− memory population in the patient, but not in the donor. TRB sequencing was applied on sorted T-cell subsets from blood and on T cells from skin biopsies. Abundant circulating CD8 memory clonotypes with a chronic virus-associated CD57+KLRG1+CX3CR1+ phenotype were also present in warts, but not in healthy skin of the patient, suggesting a link with HPV. In conclusion, we demonstrate in this IL2RG-deficient patient functional NK cells, a diverse and lasting naive T-cell compartment, supported by a stem cell boost, and an oligoclonal memory compartment half a century after HSCT.

Recurrent STAT3 Mutations in the Lymphocytes of Pure Red Cell Aplasia without T-Cell Large Granular Lymphocytic Leukemia

▪Background: Activating somatic mutations of STAT3 are one of the most frequently recognized genetic alterations in T-cell lymphoma including T-cell large granular lymphocytic leukemia (T-LGLL). T-LGLL is associated with certain proportions of patients (pts) with acquired aplastic anemia (AA) and/or myelodysplastic syndromes (MDS). Pure red cell aplasia (PRCA), especially acquired PRCA, is a syndrome defined by an anemia with marked reduction or absence of erythroid production associated with immunological mechanisms. PRCA is classified into primary and secondary types including T-LGLL. Previous reports have described that T-LGLL with STAT3 mutations was closely associated with PRCA. However, it is yet to be clear whether STAT3-mutated T cells would be involved in the pathogenesis of other cytopenias, such as AA, MDS, primary PRCA or secondary PRCA without (w/o) T-LGLL. In this study, we analyzed the profiles of STAT3 gene mutations in bone marrow failure syndrome (BMFS)s including AA/MDS and PRCA.Methods: Peripheral blood samples were collected from 94 patients with BMFSs, including AA, MDS, AA/PNH, and PRCA with or w/o T-LGLL. When possible, T-cell receptor (TCR) Vβ repertoire of the patients was determined by flow cytometry. DNA was extracted from mononuclear cells (MNC), and STAT3 Y640F and D661Y, two hot spot mutations, were detected with sensitive allele specific-PCR (AsPCR) methods. Then we performed deep amplicon sequencing analysis of STAT3 in PRCA pts. CD8+ T cells were sorted if possible, and also subjected to the analyses.Results: The recruited pts included 38 AA, 20 MDS, 7 AA/PNH, 11 PRCA with T-LGLL, and 18 PRCA w/o T-LGLL. Among PRCA pts w/o apparent T-LGLL, 10 had primary PRCA and 8 had secondary PRCA including 2 with thymoma. The clinical characteristics of PRCA are shown in Table 1. TCR Vβ repertoire analysis were performed in 8 PRCA w/o T-LGLL and 5 PRCA with T-LGLL. All pts with PRCA associated with T-LGLL showed restricted Vβ repertoire. On the other hand, only 1pt showed monoclonal Vβ pattern in PRCA w/o TLGLL. AsPCR did not detect Y604F or D661Y mutations in any of 65 pts with AA, MDS, or AA/PNH. In contrast, 2 pts with PRCA and 4 pts with PRCA-T-LGLL were positive for either of the mutations, suggesting STAT3 mutations may be unique to PRCA pts. Further analysis with deep amplicon analysis in the 29 PRCA pts revealed STAT3 mutations in 11 pts (61%) of PRCA w/o T-LGLL, which included 6 pts with primary PRCA and 5 pts with secondary PRCA associated with autoimmune diseases (n=2), 2 thymoma (n=2) and thymic cancer (n=1). The median variant allele frequency of STAT3 mutations was 2.3% (0.2-48.9). N538Y and I653V were most frequent mutations in PRCA w/o T-LGLL. Eight pts (73%) with T-LGLL-associated PRCA were also positive for STAT3 mutation. Thirty-nine % and 70% of STAT3 mutation were found in SH2 domain in PRCA w/o T-LGLL and PRCA with T-LGL, respectively. There was no difference in clinical features between pts with and w/o STAT3 mutations.Discussion: The high frequency of STAT3 mutations in PRCA pts regardless of etiology and the absence of the mutation in pts with AA/MDS suggest that STAT3 mutations may serve as a molecular marker of PRCA. The discordant results with regard to AA/MDS pts from a previous report which detected STAT3 mutations in 4.9% of AA/MDS patients by AsPCR methods (Jerez et al. BLOOD 2013) may be due to the difference in ethnic backgrounds of the pts studied. Given very low frequencies of STAT3 mutations in AA and MDS that were revealed by several previous studies, JAK/STAT signaling systems in the axis of STAT3 mutations may have unique roles in the pathogenesis of PRCA.Conclusion: STAT3 mutations are frequently detected in pts with PRCA even in pts w/o T-LGLL and the JAK/STAT pathway may therefore be therapeutic targets of PRCA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Phase II Study of High Dose Cytarabine Followed By Pembrolizumab in Relapsed/Refractory Acute Myeloid Leukemia (AML)

Introduction: Outcomes are dismal for patients (pts) with relapsed/refractory (R/R) AML. Innate and adaptive immune aberrations exist in AML pts leading to immune escape. We have previously shown that there is an increased expression of multiple co-inhibitory molecules including programmed death-1 (PD-1) in the peripheral blood of AML pts at diagnosis, and these levels persist in pts with refractory disease (Knaus H, et al. Blood [Abstract] 2015). We hypothesized that administration of pembrolizumab after high dose cytarabine (HiDAC) salvage chemotherapy would stimulate a T-cell mediated anti-leukemic immune response leading to improved efficacy in R/R AML.Methods: A multicenter phase II study of age-adjusted HiDAC (<60 years: 2 gm/m2 IV Q12hours days 1-5; >=60 years: 1.5 gm/m2 IV Q12hours days 1-5) followed by pembrolizumab 200 mg IV on day 14 is being conducted in R/R AML pts 18-70 years. The primary endpoint of this study is the rate of complete remission (CR)/CR with incomplete recovery (CRi) after HiDAC followed by pembrolizumab. The study design is a Simon's like two-stage design (n=37 pts) with relaxed stopping for futility. Relaxed stopping refers to overall response rate (CR/CRi+partial remission [PR]) when stopping rule for futility is applied in the first stage. The null hypothesis that the CR/CRi rate for HiDAC followed by pembrolizumab is 20% will be tested against a one-sided alternative hypothesis yielding a type 1 error rate = 5% and power = 84% when the CR/CRi rate of HiDAC followed by pembrolizumab = 40%. Continuous monitoring for toxicity will be performed to assess for unacceptable toxicity. Overall responders will receive maintenance phase pembrolizumab 200 mg IV Q3weeks for up to 2 years until relapse/progression. Pts can proceed to allogeneic stem cell transplant (SCT) before or after maintenance phase (SCT must be >=30 days from last dose of pembrolizumab). Correlative studies include serial peripheral blood and bone marrow flow cytometry, mRNA-sequencing, and T cell receptor (TCR) clonality studies to determine predictive biomarkers of response.Results: 13 pts enrolled on this study between August, 2016-July, 2017 (Table 1). There have been 0 unacceptable toxicities to date. Immune-related adverse events have included grade 2 hyperbilirubinemia (n=1), grade 3 hepatic enzymes (n=1), both resolved promptly after systemic corticosteroids, as well as self-resolving grade 3 rash (n=1). Additionally, 1 pt developed grade 3 pulmonary edema without evidence of pneumonitis that resolved spontaneously without steroids. Of the 10 evaluable pts (as of July 1, 2017), overall response rate = 50% (CR/CRi: 4/10= 40%; PR = 1/10=10%), meeting the threshold to proceed to stage 2. Two pts have proceeded to a myeloablative allogeneic SCT in CR (one of whom received 2 cycles of maintenance pembrolizumab) and remain in CR. Post-SCT, both pts developed steroid-responsive acute GVHD of skin, one pt developed a transient increase in hepatic enzymes that was responsive to steroids, and one pt developed moderate chronic GVHD. Four pts have received maintenance pembrolizumab: 2 pts relapsed (median duration of CR = 3.9 months; range: 2-5.7 months), one pt proceeded to SCT after 2 cycles, and one pt initially achieved a PR and remains with stable disease after 11 cycles of maintenance pembrolizumab. No pts have died in remission. 30-day and 60-day mortality = 0% and 10%, respectively. Preliminary findings from the first 6 pts (3 responders vs. 3 non-responders) revealed increased diversity of TCR Vβ repertoire from pre-treatment peripheral blood CD8+ T-cells in responders when compared with non-responders using Shannon entropy analysis (p=0.0058; Figure 1).Conclusions: Early results of a multicenter phase II study demonstrate the feasibility of adding immune checkpoint blockade with pembrolizumab after HiDAC chemotherapy in R/R AML. An overall response rate of 50% is encouraging in a high-risk R/R AML cohort though ongoing findings are imperative to evaluate the efficacy and future role of pembrolizumab in AML. Interestingly, in a small cohort of patients, broadening of the immune repertoire, perhaps by increasing T cell responses beyond endogenous viral responses, was associated with response to PD-1 blockade. Future directions include a comprehensive immune biomarker discovery approach to determine predictors of response to immune checkpoint blockade in AML. [Display omitted] DisclosuresZeidner:Tolero Pharmaceuticals: Honoraria; Agios: Honoraria; Merck: Research Funding; Takeda: Research Funding; Tolero Pharmaceuticals: Research Funding; Celgene: Honoraria. Vincent:Merck: Research Funding; Pharmacyclics: Research Funding; Nanostring: Membership on an entity's Board of Directors or advisory committees. Foster:Shire: Honoraria; Incyte: Honoraria; Macrogenics: Research Funding; Celgene: Research Funding; Amgen: Honoraria; Pfizer: Research Funding; Celator: Research Funding. Coombs:Pharmacyclics: Honoraria; H3 Biomedicine: Honoraria. Gojo:Merck: Research Funding; Amgen: Research Funding; Amgen: Honoraria; Novartis: Research Funding. Serody:Merck: Research Funding.

Issue Highlights — May 2021

Issue Highlights — May 2021

Flow cytometric evaluation of TRBC1 expression in tissue specimens and body fluids is a novel and specific method for assessment of T-cell clonality and diagnosis of T-cell neoplasms.

Flow cytometric detection of T-cell clonality is challenging. The current available methodology for T-cell receptor (TCR) Vβ repertoire evaluation is a complex assay and has limited sensitivity especially for detecting low levels of disease. Therefore, there is an unmet need for a reliable, simple, and rapid assay to identify T-cell clonality. The rearrangement of the TCRB gene involves the random and mutually exclusive expression of one of two constant β chain genes (TRBC1 and TRBC2), analogous to the kappa and lambda gene utilization by B cells. Here, we used a single TRBC1 antibody, in conjunction with other T-cell associated markers, to detect T-cell clonality in tissue biopsies and body fluids. A total of 143 tissue/body fluid specimens from 46 patients with a definitive diagnosis of a T-cell neoplasm and 97 patients with no T-cell malignancy were analyzed with a cocktail of monoclonal antibodies including CD2/CD3/CD4/CD5/CD7/CD8/CD45/TCRγδ/TRBC1. We examined TRBC1 expression on neoplastic T-cell populations identified based on their immunophenotypic aberrancies, and monotypic TRBC1 expression was identified in all 46 known T-cell lymphoma cases. We applied a similar gating strategy to the 97 cases without T-cell neoplasms, and arbitrarily dissected T-cell populations into immunophenotypically distinct subsets; in this group, we found that all cases revealed an expected polytypic TRBC1 expression in all subsets. Single TRBC1 antibody detection of T-cell clonality by flow cytometry is a simple, rapid, and robust assay that could be routinely utilized in flow cytometric laboratories.

088 A phase I/II study on the anti-CD3/CD7 immunotoxin combination for the treatment of steroid-refractory acute GVHD

Effective therapies for treating patients with steroid-refractory acute graft-versus-host-disease (SR-aGVHD) are urgently needed. aGvHD affects mostly the skin and gut, and is a leading cause of morbidity and mortality. We conducted a phase I/II trial in 20 patients with SR-aGVHD, to examine the safety and efficacy of a mixture of anti-CD3 and anti-CD7 antibodies separately conjugated to recombinant ricin A (CD3/CD7-IT). On day 28 after the start of CD3/CD7-IT therapy, the overall response rate was 60%, with 50% complete remissions. The 6-month overall survival rate was 60%. Using flow cytometric analysis and T cell receptor (TCR) sequencing, we show CD3/CD7-IT caused profound but transient depletion of T cells and NK cells, followed by rapid recovery of the immune system with a diverse TCR Vβ repertoire, and preservation of Epstein-Barr virus- and cytomegalovirus-specific T cell clones. Different characteristics of responders (Rs) versus non-responders (NR) were noticed. Rs had a more swift recovery of T cells in the blood after treatment, and CD4 and CD8 subsets reconstitute differently between groups. Before treatment, Rs had significantly higher amounts of regulatory T cells (Tregs), CD4:CD8 ratios and higher clonal T cell diversity. While T cells reconstitution is not associated with disease severity, there seems to be an inverse relationship between disease severity and response rate. We further noticed a correlation between the conditioning regimen and amount Tregs, CD4:CD8 ratio and T cell diversity. These results suggest CD3/CD7-IT is safe and well tolerated, with a relatively low prevalence of reversible adverse events. It can be effective in all aGvHD grades and in patients with different conditioning regimens. Determining the Treg counts, CD4:CD8 ratio and T cell repertoire diversity before CD3/CD7-IT may predict response and select patients that benefit from CD3/CD7-IT therapy.

Frequent STAT3 Mutations in CD8+ T Cells from Patients with Pure Red Cell Aplasia

Background: Dysregulation of T-cell mediated immunity is responsible for acquired pure red cell aplasia (PRCA). Although STAT3 mutations are frequently detected in patients with T-cell large granular lymphocytic leukemia (T-LGLL), which is often complicated by PRCA and is also reported to be associated with acquired aplastic anemia (AA) and myelodysplastic syndrome (MDS), whether STAT3-mutated T cells are involved in the pathophysiology of PRCA and other types of bone marrow failure (BMF) remains unknown.Methods: Patients with AA, AA-paroxysmal nocturnal hemoglobinuria (AA-PNH) syndrome, MDS or PRCA were enrolled in this study. We performed STAT3 mutation analyses of the peripheral blood mononuclear cells using an allele-specific PCR (AsPCR) to detect STAT3 Y640F or D661Y mutations and amplicon sequencing using primers covering entire coding region of STAT3. CD4+ T cells, CD8+ T cells or granulocytes were sorted, if possible, and also subjected to the analyses. The T-cell receptor (TCR) Vβ repertoire was analyzed using whole blood samples by flow cytometry.Results: A total of 124 patients including PRCA (n=42), AA (n=54), AA-PNH (n=7) and MDS (n=21) were enrolled. The subtypes of PRCA were as follows: idiopathic, n= 15, T-LGLL-associated, n=13; thymoma or thymic cancer-associated, n=7; autoimmune disease-associated, n=5; adverse drug reactions, n=1; and human parvovirus B19 infection complicated by T-LGLL, n=1. As an initial step in the STAT3 mutational analysis, we screened all patients with an AsPCR. Among the 42 patients with PRCA, 3 (10%) of 29 patients without T-LGLL and 6 (46%) of 13 patients with T-LGLL were positive for mutations. In contrast, none of the patients with AA, MDS or AA-PNH were positive (P value= 0.000031). Then, we examined MNC-derived DNA from 73 patients (PRCA, n=42 and AA, n=31) using amplicon sequencing. In this sequencing analysis, the median depth of coverage was 6,219x (range; 1,065-14,188). No STAT3 mutations were detected in 31 AA patients. In contrast, 15 (36%) PRCA patients possessed STAT3 mutations. The variant allele frequency of STAT3 mutations ranged from 0.0057 to 0.489. In all of the 7 patients studied, the STAT3 mutations were restricted to sorted CD8+ T cells. Three patients were negative for STAT3 mutations in MNCs but found to be positive when sorted CD8+ T cells were analyzed. The prevalence of STAT3 mutation in idiopathic, thymoma-associated, autoimmune disorder-associated and T-LGLL-associated PRCA was 33% (5/15), 29% (2/7), 20% (1/5), and 77% (10/13), respectively. In total, STAT3 mutations were detected in 8 of 29 (28%) PRCA patients without T-LGLL and 10 of 13 (77%) PRCA patients with T-LGLL. When TCRVβ repertoires of CD8+ T cells sorted from 3 STAT3 mutation(+) PRCA patients without T-LGLL were analyzed, skewed TCRVb repertoires were evident in all patients, and STAT3 mutations were detected in skewed TCRVβ fractions from 2 patients whose samples were available for cell sorting. The STAT3-mutation(+) patients were younger (median age of 63 years vs 73 years, P= 0.026) and less responsive to cyclosporine (CsA) (46% [6/13] vs 100% [8/8], P= 0.0092) in comparison to STAT3-mutation(-) patients. Of note, 4 of 8 STAT3 mutation(+) patients who had been refractory to CsA were treated with CY and all of them responded well, whereas none of 8 STAT3 mutation(-) patients required secondary treatment with CY owing to a sustained response to CsA.Discussion: This study is the first to reveal frequent STAT3 mutations in PRCA patients, even in thouse without T-LGLL, using a large cohort of patients. The presence of STAT3-mutated CD8+ T cells may be unique background of PRCA, irrespective of disease etiology. Poor response to CsA in STAT3 mutation(+) patients suggests that STAT3-mutated CD8+ T cells may be less sensitive to the inhibitory effects of CsA than non-mutated CD8+ T cells. In contrast, our analyses failed to detect STAT3 mutations in any of 52 AA patients, suggesting that STAT3 mutation(+) T cells had little impact on the development of AA in Japanese patients.Conclusion:STAT3 mutations are frequently detected in the CD8+ T cells of PRCA patients, regardless of the presence of T-LGLL. The identification of STAT3 mutations may be useful for appropriately managing patients with PRCA. DisclosuresNakao:Novartis: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

T Cell Phenotype and T Cell Receptor Repertoire in Patients with Major Depressive Disorder.

While a link between inflammation and the development of neuropsychiatric disorders, including major depressive disorder (MDD) is supported by a growing body of evidence, little is known about the contribution of aberrant adaptive immunity in this context. Here, we conducted in-depth characterization of T cell phenotype and T cell receptor (TCR) repertoire in MDD. For this cross-sectional case–control study, we recruited antidepressant-free patients with MDD without any somatic or psychiatric comorbidities (n = 20), who were individually matched for sex, age, body mass index, and smoking status to a non-depressed control subject (n = 20). T cell phenotype and repertoire were interrogated using a combination of flow cytometry, gene expression analysis, and next generation sequencing. T cells from MDD patients showed significantly lower surface expression of the chemokine receptors CXCR3 and CCR6, which are known to be central to T cell differentiation and trafficking. In addition, we observed a shift within the CD4+ T cell compartment characterized by a higher frequency of CD4+CD25highCD127low/− cells and higher FOXP3 mRNA expression in purified CD4+ T cells obtained from patients with MDD. Finally, flow cytometry-based TCR Vβ repertoire analysis indicated a less diverse CD4+ T cell repertoire in MDD, which was corroborated by next generation sequencing of the TCR β chain CDR3 region. Overall, these results suggest that T cell phenotype and TCR utilization are skewed on several levels in patients with MDD. Our study identifies putative cellular and molecular signatures of dysregulated adaptive immunity and reinforces the notion that T cells are a pathophysiologically relevant cell population in this disorder.

Severity of Acute Infectious Mononucleosis Correlates with Cross-Reactive Influenza CD8 T-Cell Receptor Repertoires.

ABSTRACTFifty years after the discovery of Epstein-Barr virus (EBV), it remains unclear how primary infection with this virus leads to massive CD8 T-cell expansion and acute infectious mononucleosis (AIM) in young adults. AIM can vary greatly in severity, from a mild transient influenza-like illness to a prolonged severe syndrome. We questioned whether expansion of a unique HLA-A2.01-restricted, cross-reactive CD8 T-cell response between influenza virus A-M158 (IAV-M1) and EBV BMLF1280 (EBV-BM) could modulate the immune response to EBV and play a role in determining the severity of AIM in 32 college students. Only ex vivo total IAV-M1 and IAV-M1+EBV-BM cross-reactive tetramer+ frequencies directly correlated with AIM severity and were predictive of severe disease. Expansion of specific cross-reactive memory IAV-M1 T-cell receptor (TCR) Vβ repertoires correlated with levels of disease severity. There were unique profiles of qualitatively different functional responses in the cross-reactive and EBV-specific CD8 T-cell responses in each of the three groups studied, severe-AIM patients, mild-AIM patients, and seropositive persistently EBV-infected healthy donors, that may result from differences in TCR repertoire use. IAV-M1 tetramer+ cells were functionally cross-reactive in short-term cultures, were associated with the highest disease severity in AIM, and displayed enhanced production of gamma interferon, a cytokine that greatly amplifies immune responses, thus frequently contributing to induction of immunopathology. Altogether, these data link heterologous immunity via CD8 T-cell cross-reactivity to CD8 T-cell repertoire selection, function, and resultant disease severity in a common and important human infection. In particular, it highlights for the first time a direct link between the TCR repertoire with pathogenesis and the diversity of outcomes upon pathogen encounter.

Circulating and Tissue-Resident CD4+ T Cells With Reactivity to Intestinal Microbiota Are Abundant in Healthy Individuals and Function Is Altered During Inflammation

Background & AimsInteractions between commensal microbes and the immune system are tightly regulated and maintain intestinal homeostasis, but little is known about these interactions in humans. We investigated responses of human CD4+ T cells to the intestinal microbiota. We measured the abundance of T cells in circulation and intestinal tissues that respond to intestinal microbes and determined their clonal diversity. We also assessed their functional phenotypes and effects on intestinal resident cell populations, and studied alterations in microbe-reactive T cells in patients with chronic intestinal inflammation.MethodsWe collected samples of peripheral blood mononuclear cells and intestinal tissues from healthy individuals (controls, n = 13−30) and patients with inflammatory bowel diseases (n = 119; 59 with ulcerative colitis and 60 with Crohn’s disease). We used 2 independent assays (CD154 detection and carboxy-fluorescein succinimidyl ester dilution assays) and 9 intestinal bacterial species (Escherichia coli, Lactobacillus acidophilus, Bifidobacterium animalis subsp lactis, Faecalibacterium prausnitzii, Bacteroides vulgatus, Roseburia intestinalis, Ruminococcus obeum, Salmonella typhimurium, and Clostridium difficile) to quantify, expand, and characterize microbe-reactive CD4+ T cells. We sequenced T-cell receptor Vβ genes in expanded microbe-reactive T-cell lines to determine their clonal diversity. We examined the effects of microbe-reactive CD4+ T cells on intestinal stromal and epithelial cell lines. Cytokines, chemokines, and gene expression patterns were measured by flow cytometry and quantitative polymerase chain reaction.ResultsCirculating and gut-resident CD4+ T cells from controls responded to bacteria at frequencies of 40−4000 per million for each bacterial species tested. Microbiota-reactive CD4+ T cells were mainly of a memory phenotype, present in peripheral blood mononuclear cells and intestinal tissue, and had a diverse T-cell receptor Vβ repertoire. These cells were functionally heterogeneous, produced barrier-protective cytokines, and stimulated intestinal stromal and epithelial cells via interleukin 17A, interferon gamma, and tumor necrosis factor. In patients with inflammatory bowel diseases, microbiota-reactive CD4+ T cells were reduced in the blood compared with intestine; T-cell responses that we detected had an increased frequency of interleukin 17A production compared with responses of T cells from blood or intestinal tissues of controls.ConclusionsIn an analysis of peripheral blood mononuclear cells and intestinal tissues from patients with inflammatory bowel diseases vs controls, we found that reactivity to intestinal bacteria is a normal property of the human CD4+ T-cell repertoire, and does not necessarily indicate disrupted interactions between immune cells and the commensal microbiota. T-cell responses to commensals might support intestinal homeostasis, by producing barrier-protective cytokines and providing a large pool of T cells that react to pathogens.

Overexpression of Vα14Jα18 TCR promotes development of iNKT cells in the absence of miR-181a/b-1.

Expression of microRNA miR-181a/b-1 is critical for intrathymic development of invariant natural killer T (iNKT) cells. However, the underlying mechanism has remained a matter of debate. On the one hand, growing evidence suggested that miR-181a/b-1 is instrumental in setting T-cell receptor (TCR) signaling threshold and thus permits agonist selection of iNKT cells through high-affinity TCR ligands. On the other hand, alterations in metabolic fitness mediated by miR-181a/b-1-dependent dysregulation of phosphatase and tensin homolog (Pten) have been proposed to cause the iNKT-cell defect in miR-181-a/b-1-deficient mice. To re-assess the hypothesis that modulation of TCR signal strength is the key mechanism by which miR-181a/b-1 controls the development of iNKT cells, we generated miR-181a/b-1-deficient mice expressing elevated levels of a Vα14Jα18 TCRα chain. In these mice, development of iNKT cells was fully restored. Furthermore, both subset distribution of iNKT cells as well as TCR Vβ repertoire were independent of the presence of miR-181a/b-1 once a Vα14Jα18 TCRα chain was overexpressed. Finally, levels of Pten protein were similar in Vα14Jα18 transgenic mice irrespective of their miR-181a/b-1 status. Collectively, our data support a model in which miR-181 promotes development of iNKT cells primarily by generating a permissive state for agonist selection with alterations in metabolic fitness possibly constituting a secondary effect.

End stage renal disease patients have a skewed T cell receptor Vβ repertoire.

BackgroundEnd stage renal disease (ESRD) is associated with defective T-cell mediated immunity. A diverse T-cell receptor (TCR) Vβ repertoire is central to effective T-cell mediated immune responses to foreign antigens. In this study, the effect of ESRD on TCR Vβ repertoire was assessed.ResultsA higher proportion of ESRD patients (68.9 %) had a skewed TCR Vβ repertoire compared to age and cytomegalovirus (CMV) – IgG serostatus matched healthy individuals (31.4 %, P < 0.001). Age, CMV serostatus and ESRD were independently associated with an increase in shifting of the TCR Vβ repertoire. More differentiated CD8+ T cells were observed in young ESRD patients with a shifted TCR Vβ repertoire. CD31-expressing naive T cells and relative telomere length of T cells were not significantly related to TCR Vβ skewing.ConclusionsESRD significantly skewed the TCR Vβ repertoire particularly in the elderly population, which may contribute to the uremia-associated defect in T-cell mediated immunity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12979-015-0055-7) contains supplementary material, which is available to authorized users.

Donor lymphocytes expressing the herpes simplex virus thymidine kinase suicide gene: detailed immunological function following add-back after haplo-identical transplantation

Background aimsHaplo-identical hematopoietic stem cell transplantation (HSCT) with add-back of donor lymphocytes expressing the herpes simplex virus thymidine kinase suicide gene (TK cells) is one of the most widely applied promising new gene therapy approaches. However, the immunological status of added-back TK cells after HSCT has yet to be well characterized. MethodsWe investigated TK cells through the use of flow cytometry, T-cell receptor (TCR) Vβ repertoire spectratyping and linear amplification-mediated polymerase chain reaction followed by insertion site analysis in a patient enrolled in our clinical trial. ResultsA comparison of onset with remission of acute graft-versus-host disease confirmed that TK cells were predominantly eliminated and that proliferative CD8+ non-TK cells were also depleted in response to ganciclovir administration. The TCR Vβ-chain repertoire of both TK cells and non–TK cells markedly changed after administration of ganciclovir, and, whereas the TCR repertoire of non–TK cells returned to a normal spectratype long after transplantation, that of TK cells remained skewed. With the long-term prophylactic administration of acyclovir, TK cells oligoclonally expanded and the frequency of spliced variants of TK cells increased. Known cancer-associated genes were not evident near the oligoclonally expanded herpes simplex virus (HSV)-TK insertion sites. ConclusionsWe demonstrate obvious differences in immunological status between TK cells and non-TK cells. In addition, we speculate that long-term prophylactic administration of acyclovir increases the risk of oligoclonal expansion of spliced forms of TK cells.

Characteristics of the TCR Vβ repertoire in imatinib-resistant chronic myeloid leukemia patients with ABL mutations.

Diversity in the T cell receptor (TCR) repertoire provides a miniature defense ability for the T cell immune system that may be related to tumor initiation and progression. Understanding the T cell immune status of leukemia patients is critical for establishing specific immunotherapies. Previous studies have reported abnormal TCR repertoires and clonally expanded TCR Vβ T cells in chronic myeloid leukemia in chronic phase (CP-CML). In this study, we investigated the distribution and clonality of the TCR Vβ repertoire in 4 cases with imatinib-resistant CML in blast crisis (BC-CML) with abelson murine leukemia viral oncogene homolog 1 (ABL1) kinase domain mutations (KDMs). Examination of TCR V expression and clonality was performed by reverse transcription-polymerase chain reaction (RT-PCR) and GeneScan analysis. Significantly skewed TCR Vβ repertoires were observed in BC-CML patients with different KDMs, and 4 to 8 oligoclonally expanded TCR Vβ subfamilies could be identified in each sample. Intriguingly, a relatively highly expanded Vβ9 clone with the same length as complementarity- determining region 3 (CDR3) (139 bp) was found in all three CML patients in lymphoid blast crisis (LBC-CML) who had different KDMs, but the clone was not detected in the only CML patient in myeloid blast crisis (MBC-CML). In conclusion, restricted TCR Vβ repertoire expression and decreased clone complexity was a general phenomenon observed in the BC-CML patients with different KDMs, indicating the T-cell immunodeficiency of these patients. In addition, clonally expanded Vβ9 T cell clones may indicate a specific immune response to leukemia-associated antigens in LBC-CML patients.