T-cell Repertoire In Patients Research Articles

Risk HLA Variants Affect the T-Cell Repertoire in Multiple Sclerosis.

The major histocompatibility complex (MHC) locus has a predominant role in the genetic predisposition to multiple sclerosis (MS), with 32 associations found to be involved. We aimed to investigate the impact of MHC MS-risk alleles on T-cell repertoire in patients with MS. We studied 161 untreated patients with relapsing-remitting MS for whom Class I and II human leukocyte antigen (HLA) alleles were inferred from whole-genome genotyping data, and T-cell receptor (TCR) CDR3 sequences were obtained through next-generation sequencing. T-cell repertoire features including diversity, public clones, and architecture were evaluated. We identified 5 MS-risk loci associated with TCR diversity: HLA-DRB1*15:01 (7.65 × 10-3), rs9271366 (1.96 × 10-3), rs766848979 A (1.89 × 10-2), rs9277626 (2.95 × 10-2), and rs11751659 (1.92 × 10-2), with evidence of expanded clonotypes in carriers of risk alleles. Moreover, HLA-DRB1*15:01 (4.99 × 10-3), rs9271366 (6.54 × 10-3), rs1049079 C (4.37 × 10-2), AA DQΒ1 position -5 L (1.05 × 10-3), and AA DQΒ1 position 221 Q (9.39 × 10-4) showed an association with the CDR3 aminoacidic sequence architecture, suggesting an impact on the antigen recognition breadth as well. Evaluating the sharing of clones across MS-risk allele carrier individuals revealed the presence of highly shared clonotypes predicted to target viral antigens, including Epstein-Barr virus. Our study supports the association between MHC-risk alleles and macrofeatures of the T-cell repertoire in the context of MS. Further studies are needed to understand the underlying molecular mechanisms.

Patients with CD3G mutations reveal a role for human CD3γ in Treg diversity and suppressive function

AbstractIntegrity of the T-cell receptor/CD3 complex is crucial for positive and negative selection of T cells in the thymus and for effector and regulatory functions of peripheral T lymphocytes. In humans, CD3D, CD3E, and CD3Z gene defects are a cause of severe immune deficiency and present early in life with increased susceptibility to infections. By contrast, CD3G mutations lead to milder phenotypes, mainly characterized by autoimmunity. However, the role of CD3γ in establishing and maintaining immune tolerance has not been elucidated. In this manuscript, we aimed to investigate abnormalities of T-cell repertoire and function in patients with genetic defects in CD3G associated with autoimmunity. High throughput sequencing was used to study composition and diversity of the T-cell receptor β (TRB) repertoire in regulatory T cells (Tregs), conventional CD4+ (Tconv), and CD8+ T cells from 6 patients with CD3G mutations and healthy controls. Treg function was assessed by studying its ability to suppress proliferation of Tconv cells. Treg cells of patients with CD3G defects had reduced diversity, increased clonality, and reduced suppressive function. The TRB repertoire of Tconv cells from patients with CD3G deficiency was enriched for hydrophobic amino acids at positions 6 and 7 of the CDR3, a biomarker of self-reactivity. These data demonstrate that the T-cell repertoire of patients with CD3G mutations is characterized by a molecular signature that may contribute to the increased rate of autoimmunity associated with this condition.

Azacitidine improves the T-cell repertoire in patients with myelodysplastic syndromes and acute myeloid leukemia with multilineage dysplasia

Patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with multilineage dysplasia show several immunological abnormalities. In this clinical setting, by combining flow cytometry and CDR3 spectratyping we monitored the kinetic of the T-cell repertoire during Azacitidine treatment, in order to explore its potential ability to reverse the immune derangement typical of these disorders. We firstly demonstrated by flow cytometry an increase in both CD4+ and CD8+ T-cell frequencies after starting treatment. Moreover, when monitored by spectratyping our patients showed significant changes in their T-cell receptor (TCR) CDR3 profiles, which were much more evident in helper T-cells. In fact, the frequency of BV (beta variable) subfamilies showing a skewed CDR3 profile significantly decreased from baseline to the following evaluations in CD4+ T-cells (81% vs. 70%). This pattern was even more pronounced in patients responding to Azacitidine (90% vs. 61%). Our data show that the overall derangement of the T-cell repertoire detectable in patients with MDS and AML with multilineage dysplasia gradually improves during Azacitidine treatment. These findings therefore suggest that Azacitidine could be potentially able, not only to restore the hematopoietic function, but also to reverse the immune derangement typical of these hematologic disorders.

Vascular effects of circulating CD4-T cells in patients with unstable angina

T cells are involved in the process of endothelial dysfunction contributing to plaque destabilization [ [1] George J. Mechanisms of disease: the evolving role of regulatory T cells in atherosclerosis. Nat Clin Pract Cardiovasc Med. 2008; 5: 531-540 Crossref PubMed Scopus (69) Google Scholar ] and several studies have shown that the predominant population of T cells that infiltrates atherosclerotic plaques is CD4+ T cells [ [2] Chan W.L. Pejnovic N. Hamilton H. et al. Atherosclerotic abdominal aortic aneurysm and the interaction between autologous human plaque-derived vascular smooth muscle cells, type 1 NKT, and helper T cells. Circ Res. 2005; 96: 675-683 Crossref PubMed Scopus (75) Google Scholar ]. An increased number of CD4+ T cells have already been found in the peripheral blood of patients presenting with acute coronary syndromes [ [3] Liuzzo G. Goronzy J.J. Yang H. et al. Monoclonal T-cell proliferation and plaque instability in acute coronary syndromes. Circulation. 2000; 101: 2883-2888 Crossref PubMed Scopus (454) Google Scholar ]. Particularly, a subset of CD4+ T cells characterized by a loss of the CD31 molecule seems to have a crucial role in plaque destabilization and thrombotic complications. Furthermore, an increased number of CD4+ T lymphocytes characterized by a defective cell surface expression of CD28 have been detected in patients with unstable angina (UA). CD4+CD28null T cells were found to expand in the peripheral blood of patients with UA and infiltrate unstable coronary plaques, where they undergo clonal expansion, probably due to their activation by specific antigens [ [4] Liuzzo G. Kopecky S.L. Frye R.L. et al. Perturbation of the T-cell repertoire in patients with unstable angina. Circulation. 1999; 100: 2135-2139 Crossref PubMed Scopus (342) Google Scholar ]. Therefore, we aimed to investigate whether CD4+CD31null and CD4+CD28null T cell subpopulations contribute to vascular function in patients with UA.

Clinical Use of Next-Generation Sequencing of TRG Gene Rearrangements Has Arrived

In this issue of the Journal , Schumacher and colleagues1 describe important principles to consider when applying next-generation sequencing (NGS) for the identification of clonal sequences to support the diagnosis of T-cell neoplasms. NGS has been shown to be clinically useful in identifying germline mutations in hereditary conditions and somatic mutations in numerous cancers. In 2009, one of the first reports of the use of NGS methods to describe the repertoire of TRB gene rearrangements was published.2 Since then, it has been used to characterize the T-cell repertoire in patients with various diseases. The next application described was the identification of unique sequences of minimal residual disease in acute lymphoblastic leukemia.3,4 It was a logical step to go from defining a T-cell repertoire to the application of NGS in clonality detection, whether at diagnosis1 or in follow-up.3,4 One must aim for certain goals in preparing unbiased libraries based on polymerase chain reaction (PCR) amplified products of any gene targets. The first desirable goal is to use primers that give proportional representation of the variable (V) and joining (J) region genes that are used in the TRG gene rearrangements. Second, the primers selected and the products produced must have equal amplification efficiencies. Proportional representation for each of the V and J gene groups is largely achieved by the primers selected, but it will be less than 100%, since specific primers for each of the individual group I variable genes were not designed in the set of primers that the …

Ex vivo characterization of γδ T-cell repertoire in patients after adoptive transfer of Vγ9Vδ2 T cells expressing the interleukin-2 receptor β-chain and the common γ-chain

Adoptive immunotherapy is emerging as a potent anti-tumor treatment modality; Vγ9Vδ2 T cells may represent appropriate agents for such cancer immunotherapy. To improve the currently limited success of Vγ9Vδ2 T-cell-based immunotherapy, we examined the in vivo dynamics of these adoptively-transferred cells and hypothesized that interleukin (IL)-15 is the potential factor for Vγ9δ2 T cell in vivo survival. We conducted a clinical trial of adoptive Vγ9Vδ2 T-cell transfer therapy in six colorectal cancer patients who received pulmonary metastasectomy. Patients' peripheral blood mononuclear cells were stimulated with zoledronate (5 μmol/L) and IL-2 (1000 IU/mL) for 14 d. Harvested cells, mostly Vγ9Vδ2 T cells, were given intravenously weekly without additional IL-2 eight times in total. The frequency, phenotype and common γ-chain cytokine receptor expression of Vγ9Vδ2 T cells in peripheral blood was monitored by flow cytometry at each time point during treatment and 4 and 12 weeks after the last administration. Adoptively transferred Vγ9Vδ2 T cells expanded well without exogenous IL-2 administration or lymphodepleting preconditioning. They maintained effector functions in terms of interferon-γ secretion and prompt release of cytotoxic granules in response to PMA/ionomycin or isopentenyl pyrophosphate-positive cells. Because they are IL-2Rα(-)IL-7Rα(-)IL-15Rα(-)IL-2Rβ(+)γc(+), it is likely that IL-2 or IL-15 is required for their maintenance. The persistence of large numbers of functionally active adoptively transferred Vγ9Vδ2 T cells in the absence of exogenous IL-2 implies that an endogenous factor, such as IL-15 transpresentation, is adequate to support these cells in vivo.

Recovery of the T-cell repertoire in CIDP by IV immunoglobulins

To investigate changes in the T-cell repertoire in patients with chronic inflammatory demyelinating polyneuropathy (CIDP) without and with treatment of IV immunoglobulins (IVIg). The T-cell receptor (TCR) repertoire of CD4+ and CD8+ T cells in the peripheral blood was analyzed using CDR3 spectratyping. Patients with CIDP were included without (n = 14) and with IVIg treatment (n = 11) cross-sectionally and longitudinally (n = 2). While the TCR length distribution of patients with CIDP was only moderately altered for most of the Vβ elements of CD4+ T cells, the CD8+ population displayed extensive oligoclonal expansions in all analyzed 24 Vβ elements. A public expansion of a distinct TCR length in one Vβ element within a majority of affected patients was not detectable. Treatment with IVIg reduced the oligoclonal expansions within both the CD4+ and CD8+ population. Our data demonstrate that cytotoxic CD8+ T cells exhibit a much broader activation than CD4+ T cells, indicating a potentially crucial role of CD8+ T cells in the immunopathogenesis of CIDP. The profound oligoclonal response in T-cell activation suggests that multiple peptides may induce and propagate this autoimmune-driven disease. The observed reduction of highly activated T cells may contribute to the therapeutic effects of IVIg.

Profound loss of T-cell receptor repertoire complexity in cutaneous T-cell lymphoma

AbstractCutaneous T-cell lymphoma (CTCL) is a malignancy of skin-homing T cells. A major feature of CTCL is profound immunosuppression, such that patients with advanced mycosis fungoides or Sézary syndrome have been compared with patients with advanced HIV disease and are susceptible to opportunistic infection. The etiology of this immunosuppression is unclear. We analyzed peripheral blood T cells of patients with CTCL with stage I to IV disease, using a sensitive beta-variable complementarity-determining region 3 spectratyping approach. Our data revealed a profound disruption of the complexity of the T-cell repertoire, which was universally observed in patients with advanced disease (stages III and IV), and present in up to 50% of patients with early-stage disease (stages I and II). In most patients, multiple monoclonal and oligoclonal complementarity-determining region 3 (CDR3) spectratype patterns in many different beta-variable families were seen. Equally striking was a reduction of normal T cells (as judged by absolute CD4 counts) across multiple beta-variable families. In general, CTCL spectratypes were reminiscent of advanced HIV spectratypes published elsewhere. Taken together, these data are most consistent with a global assault on the T-cell repertoire in patients with CTCL, a process that can be observed even in early-stage disease. (Blood. 2003;102:4059-4066)

A prospective study on the evolution of the T-cell repertoire in patients with Sézary syndrome treated by extracorporeal photopheresis

Sézary syndrome is a leukemic form of epidermotropic cutaneous T-cell lymphoma related to the malignant proliferation of clonal CD4+ T cells. Extracorporeal photochemotherapy may induce a transient improvement of the clinical signs, but its efficiency is discussed. To investigate the frequency of the T-cell clone in the peripheral blood of patients with Sézary syndrome and to monitor its evolution in patients treated using extracorporeal photopheresis or chemotherapy, we used the immunoscope technique. In one patient, we observed a decrease of the relative frequency of the clone from 15.6% to 0%, paralleling a complete remission of the clinical disease and a disappearance of the circulating Sézary cells. In the other cases, the evolution of the relative frequency paralleled the initial improvement of the clinical status and the absence of long-term efficiency in patients treated with extracorporeal photopheresis or chemotherapy. We observed a quick-acting direct cytotoxicity of the association 8MOP + UVA on the T-cell clone. The immunoscope technique appears to be an efficient tool to appreciate the amount of tumoral cells and to monitor the evolution of the clonal component in the Sézary syndrome.

A prospective study on the evolution of the T-cell repertoire in patients with Sézary syndrome treated by extracorporeal photopheresis

AbstractSézary syndrome is a leukemic form of epidermotropic cutaneous T-cell lymphoma related to the malignant proliferation of clonal CD4+ T cells. Extracorporeal photochemotherapy may induce a transient improvement of the clinical signs, but its efficiency is discussed. To investigate the frequency of the T-cell clone in the peripheral blood of patients with Sézary syndrome and to monitor its evolution in patients treated using extracorporeal photopheresis or chemotherapy, we used the immunoscope technique. In one patient, we observed a decrease of the relative frequency of the clone from 15.6% to 0%, paralleling a complete remission of the clinical disease and a disappearance of the circulating Sézary cells. In the other cases, the evolution of the relative frequency paralleled the initial improvement of the clinical status and the absence of long-term efficiency in patients treated with extracorporeal photopheresis or chemotherapy. We observed a quick-acting direct cytotoxicity of the association 8MOP + UVA on the T-cell clone. The immunoscope technique appears to be an efficient tool to appreciate the amount of tumoral cells and to monitor the evolution of the clonal component in the Sézary syndrome.

Rapid reconstitution of Epstein-Barr virus–specific T lymphocytes following allogeneic stem cell transplantation

Epstein-Barr virus (EBV)-specific CD8 T lymphocytes are present at remarkably high frequencies in healthy EBV(+) individuals and provide protection from EBV-associated lymphoproliferative diseases. Allogeneic peripheral blood stem cell transplantation (allo-PBSCT) is a commonly used therapy in which T-cell surveillance for EBV is temporarily disrupted. Herein, human leukocyte antigen (HLA) class I tetramers were used to investigate the reestablishment of the EBV-specific CD8 T-cell repertoire in patients following allo-PBSCT. CD8(+) T cells specific for lytic and latent cycle-derived EBV peptides rapidly repopulate the periphery of matched sibling allo-PBSCT patients. The relative frequencies of T cells specific for different EBV peptides in transplantation recipients closely reflect those of their respective donors. Investigation of patients at monthly intervals following unmanipulated allo-PBSCT demonstrated that the frequency of EBV-specific T cells correlates with the number of EBV genome copies in the peripheral blood and that expansion of EBV-specific T-cell populations occurs even in the setting of immunosuppressive therapy. In contrast, patients undergoing T-cell-depleted or unrelated cord blood transplantation have undetectable EBV-specific T cells, even in the presence of Epstein-Barr viremia. The protective shield provided by EBV-specific CD8 T cells is rapidly established following unmanipulated matched sibling allo-PBSCT and demonstrates that HLA class I tetramers complexed with viral peptides can provide direct and rapid assessment of pathogen-specific immunity in this and other vulnerable patient populations. (Blood. 2000;96:2814-2821)

Rapid reconstitution of Epstein-Barr virus–specific T lymphocytes following allogeneic stem cell transplantation

AbstractEpstein-Barr virus (EBV)–specific CD8 T lymphocytes are present at remarkably high frequencies in healthy EBV+individuals and provide protection from EBV-associated lymphoproliferative diseases. Allogeneic peripheral blood stem cell transplantation (allo-PBSCT) is a commonly used therapy in which T-cell surveillance for EBV is temporarily disrupted. Herein, human leukocyte antigen (HLA) class I tetramers were used to investigate the reestablishment of the EBV-specific CD8 T-cell repertoire in patients following allo-PBSCT. CD8+ T cells specific for lytic and latent cycle–derived EBV peptides rapidly repopulate the periphery of matched sibling allo-PBSCT patients. The relative frequencies of T cells specific for different EBV peptides in transplantation recipients closely reflect those of their respective donors. Investigation of patients at monthly intervals following unmanipulated allo-PBSCT demonstrated that the frequency of EBV-specific T cells correlates with the number of EBV genome copies in the peripheral blood and that expansion of EBV-specific T-cell populations occurs even in the setting of immunosuppressive therapy. In contrast, patients undergoing T-cell–depleted or unrelated cord blood transplantation have undetectable EBV-specific T cells, even in the presence of Epstein-Barr viremia. The protective shield provided by EBV-specific CD8 T cells is rapidly established following unmanipulated matched sibling allo-PBSCT and demonstrates that HLA class I tetramers complexed with viral peptides can provide direct and rapid assessment of pathogen-specific immunity in this and other vulnerable patient populations.

Perturbation of the T-cell repertoire in patients with unstable angina.

Monocytes are constitutively activated in unstable angina (UA), resulting in the production of IL-6 and the upregulation of acute phase proteins. Underlying mechanisms are not understood. To explore whether the production of the potent monocyte activator IFN-gamma is altered in UA, we compared cytokine production by T lymphocytes in patients with UA (Braunwald's class IIIB) and with stable angina (SA). Peripheral blood lymphocytes were collected at the time of hospitalization and after 2 and 12 weeks. Cytokine-producing CD4(+) and CD8(+) T cells were quantified by 3-color flow cytometry after stimulation with phorbol myristate acetate and ionomycin. UA was associated with an increased number of CD4(+) and CD8(+) T cells producing IFN-gamma, whereas patients with SA had higher frequencies of IL-2(+) and IL-4(+) CD4(+) T cells. Expansion of the IFN-gamma( +) T-cell population in UA persisted for at least 3 months. Increased production of IFN-gamma in UA could be attributed to the expansion of an unusual subset of T cells, CD4(+)CD28(null) T cells. Patients with UA are characterized by a perturbation of the functional T-cell repertoire with a bias toward IFN-gamma production, suggesting that monocyte activation and acute phase responses are consequences of T-cell activation. IFN-gamma is produced by CD4(+)CD28(null) T cells, which are expanded in UA and distinctly low in SA and controls. The emergence of CD4(+)CD28(null) T cells may result from persistent antigenic stimulation.

Association of clonally expanded T cells with the syndrome of primary biliary cirrhosis and limited scleroderma.

Clinical features of the CREST (calcinosis cutis, Raynaud's syndrome, esophageal dysmotility, sclerodactyly, and telangiectasias) syndrome are sometimes exhibited in patients with primary biliary cirrhosis (PBC), but the postulated autoimmune mechanisms behind these conditions are poorly understood. Clonally expanded T cells may play an important role in disease pathogenesis. In this study, overrepresentation of one T-cell receptor beta chain variable region, TCRBV3, was documented in patients with PBC and/or CREST. Overrepresentation of the TCRBV3 gene mRNA was demonstrated by semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR). T cells expressing TCRBV3 were analyzed by flow cytometry, were primarily CD8(+), and contained activated cells as assessed by expression of CD69. Clonally expanded T cells within this population were documented by both complementarity determining region 3 (CDR3) length polymorphism analysis and sequencing of T-cell receptor CDR3 cDNA. TCRBV3(+) clonal expansions were stable when followed for up to 5 years. The results of this study demonstrate that the T-cell repertoire of patients with PBC and CREST is characterized by expanded clonal populations of CD8(+) TCRBV3(+) T cells. These clonal expansions provide evidence that stimulation of clonal populations of CD8(+) T cells is associated with the clinical syndrome of PBC with CREST.

T-cell receptor Vbeta gene utilization in primary biliary cirrhosis

Semiquantitative reverse-transcriptase polymerase chain reaction (RT- PCR) was used to study the T-cell receptor (TCR) beta-chain variable (Vbeta) region gene families expressed by T cells in 28 patients with primary biliary cirrhosis (PBC), 20 normal controls, and 9 patients with other chronic inflammatory hepatic diseases. We hypothesized that activation and clonal proliferation of T cells would lead to biases in the T-cell repertoire of patients with PBC. Freshly harvested T cells from both peripheral blood and liver tissue were examined for evidence of biased Vbeta utilization. Individuals varied considerably in their pattern of Vbeta expression, but several significant differences were noted in the PBC group. In peripheral blood, the mean level of Vbeta6.1,3 expression was greater in PBC patients than in normal controls. In the liver of PBC patients, the mean level of Vbeta6.1,3 expression was even higher than in the peripheral blood, indicating intrahepatic accumulation of these T cells. The mean level of Vbeta6.1,3 expression was not significantly different in the blood compared with liver in patients with other liver diseases. Expression of Vbeta7 and Vbeta13.1 was also significantly greater in the liver than in the blood of PBC patients, but similar trends were also seen in the control liver group. These data show that specific alterations in the TCR repertoire are present in the blood and liver of patients with PBC. (Hepatology 1996 Nov;24(5):1148-55)

T-cell receptor Vbeta gene utilization in primary biliary cirrhosis.

Semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) was used to study the T-cell receptor (TCR) beta-chain variable (Vbeta) region gene families expressed by T cells in 28 patients with primary biliary cirrhosis (PBC), 20 normal controls, and 9 patients with other chronic inflammatory hepatic diseases. We hypothesized that activation and clonal proliferation of T cells would lead to biases in the T-cell repertoire of patients with PBC. Freshly harvested T cells from both peripheral blood and liver tissue were examined for evidence of biased Vbeta utilization. Individuals varied considerably in their pattern of Vbeta expression, but several significant differences were noted in the PBC group. In peripheral blood, the mean level of Vbeta6.1,3 expression was greater in PBC patients than in normal controls. In the liver of PBC patients, the mean level of Vbeta6.1,3 expression was even higher than in the peripheral blood, indicating intrahepatic accumulation of these T cells. The mean level of Vbeta6.1,3 expression was not significantly different in the blood compared with liver in patients with other liver diseases. Expression of Vbeta7 and Vbeta13.1 was also significantly greater in the liver than in the blood of PBC patients, but similar trends were also seen in the control liver group. These data show that specific alterations in the TCR repertoire are present in the blood and liver of patients with PBC.

T-cell repertoire complexity after allogeneic bone marrow transplantation

We analyzed the T-cell repertoire in patients transplanted with bone marrow from an HLA identical sibling by determining the TCR diversity through Vβ-CDR3-size spectratyping with Vβ/Cβ- and Vβ/Jβspecific primers. Using the Vβ/Cβ primers, we observed limited TCR diversity only in recipients of a T-celldepleted graft, whereas the TCR diversity of patients transplanted with an unmanipulated graft seemed to be indistinguishable from the one of a normal individual. However, with Vβ/Jβ-specific primers, increase of the resolution by approximately 10-fold also allowed the detection of imbalances in the TCR repertoire of recipients of an unmanipulated graft. This demonstrates that when high numbers of T cells are cotransfused with marrow, the TCR repertoire is more complete but still not as complete as in normal individuals, thereby emphasizing the important role of coinfused mature T cells in the restoration of the T-cell compartment after bone marrow transplantation.