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Somatic mosaicism, the presence of genetically distinct cells within an organism, has been increasingly associated with human morbidity, ranging from being a cause of rare syndromes to a risk factor for common disorders such as malignancy and cardiovascular disease. Previous studies interrogating the normal prevalence of somatic mosaicism have focused on adults. We here present an estimate of the baseline frequency of somatic mosaic copy number variation (CNV) at the time around birth, by sampling eight different organs from a total of five fetuses and newborns. Overall we find a significantly lower frequency of organ specific (i.e. mosaic) CNVs as compared to adults (p = 0.003; Mann-Whitney U-test). The rate of somatic CNV in adults has been estimated to around 2.2 CNV per organ assayed. In contrast, after stringent filtering, we found no organ-private CNVs in fetuses or newborns with exception of the thymus. This organ exhibited a specific genome profile in the form of deletions resulting from polyclonal T-cell receptor rearrangements. This implies that somatic non-immune related CNVs, if present at birth, are typically confined to very small cell populations within organs.

Cytomegalovirus (CMV) is the most common congenital infection in the United States. The major target of congenital CMV is the brain, with clinical manifestations including mental retardation, vision impairment and sensorineural hearing loss. Previous reports have shown that CD8+ T cells are required to control viral replication and significant numbers of CMV-specific CD8+ T cells persist in the brain even after the initial infection has been cleared. However, the dynamics of CD8+ T cells in the brain during latency remains largely undefined. In this report, we used T cell receptor (TCR) sequencing to track the development and maintenance of neonatal clonotypes in the brain and spleen of mice during chronic infection. Given the discontinuous nature of tissue resident memory CD8+ T cells, we hypothesized that neonatal TCR clonotypes would be ‘locked-in’ the brain and persist into adulthood. Surprisingly, we found that the antigen-specific T cell repertoire in neonatal-infected mice diversified during persistent infection in both the brain and spleen, while maintaining substantial similarity between the CD8+ T cell populations in the brain and spleen in both early and late infection. However, despite the diversification of, and potential interchange between, the spleen and brain antigen-specific T cell repertoires, we observed that germline-encoded TCR clonotypes, characteristic of neonatal infection, persisted in the brain, albeit sometimes in low abundance. These results provide valuable insights into the evolution of CD8+ T cell repertoires following neonatal CMV infection and thus have important implications for the development of therapeutic strategies to control CMV in early life.

Multiple sclerosis is a neuroimmunological disease in which etiologic agents have not been identified yet. The etiology of MS is complex in its nature and may involve many different agents acting simultaneously or in a cascade manner leading to the development of the disease. The causes of MS development were sought among the factors associated with HLA and TCR genes and human endogenous retroviruses (HERV). Environmental factors such as bacterial, fungal and viral infections as well as potential participation of vitamin D in the pathogenesis of the disease have also been examined. The current state of knowledge concerning potential factors participating in the etiopathogenesis of multiple sclerosis has been reviewed in this paper.

Epstein–Barr virus (EBV) infects various types of lymphocytes and is associated with not only B cell‐origin lymphoma, but also T or natural killer cell lymphoproliferative diseases (T/NK LPD). Recently, we established a novel assay to identify EBV‐infected cells using FISH. Using this assay, dual staining with antibodies to both surface antigens and an EBV‐encoded small RNA (EBER) probe can be performed. In the present study, we applied this recently developed FISH assay to EBV‐associated T/NK LPD to confirm its diagnostic utility. Using FISH, we prospectively analyzed peripheral blood from patients with suspected EBV‐associated T/NK LPD. The results were compared with those obtained using immunobead sorting followed by quantitative PCR. In all, 26 patients were included study. Using FISH, 0.15–67.0% of peripheral blood lymphocytes were found to be positive for EBER. Dual staining was used to determine EBER‐positive cell phenotypes in 23 of 26 subjects (88.5%). In five of seven patients with hydroa vacciniforme‐like lymphoma (an EBV‐positive cutaneous T cell lymphoma), EBER‐positive cells were identified as CD3(+) CD4(−) CD8(− ) TCRγδ(+) T cells. Furthermore, in a 25‐year‐old male patient with systemic EBV‐positive T cell LPD, two lymphocyte lineages were positive for EBER: CD4(+) CD8(−) and CD4(−) CD8(+) T cells. Thus, we confirmed that our newly developed assay is useful for quantifying and characterizing EBV‐infected lymphocytes in EBV‐associated T/NK LPD and that it can be used not only to complement the pathological diagnosis, but also to clarify the pathogenesis and to expand the spectrum of EBV‐associated diseases. (Cancer Sci, doi: 10.1111/j.1349‐7006.2012.02305.x, 2012)

Polymerase chain reaction (PCR) based clonality assays are an important tool to differentiate neoplastic from reactive lymphocyte populations. A recent description of the canine T cell receptor γ locus identified a large number of formerly unknown genes, and determined the locus topology consisting of 8 cassettes with up to 3 variable (V) genes, 2 joining (J) genes and one constant (C) gene each. Given that these data were not available when existing canine T cell clonality assays were developed, it is likely that they will fail to detect a subset of clonal lymphocyte populations. The objective of this study was to gauge the potential of canine T cell clonality assays to detect all rearranged T cell receptor γ genes and to develop an improved clonality assay. The primer sequences of existing clonality assays were aligned to the reference sequences of all rearranged genes and genes were scored as to the likelihood of being recognized by a primer. All four assays likely recognized subgroup Vγ2 and Vγ6 genes but 3 out of 4 assays were unlikely to detect subgroup Vγ3 and Vγ7 genes. All assays likely recognized Jγx-2 genes, but only two assays were likely to detect most Jγx-1 genes. Two assays had forward primers located as close as four nucleotides to the junctional region. A new multiplex PCR was designed with all primers combined in a single tube. An alternative primer set allowed identification of variable gene usage through gene specific forward primers. The coverage of all rearranged genes facilitated the detection of multiple clonal rearrangements per neoplastic sample. The new assay detected clonal DNA at a concentration of 5% within polyclonal background but detection thresholds were dependent on the gene usage of clonal rearrangements as well as the position of the clonal peak in respect to the polyclonal background. The new multiplex assay recognized 12/12 (100%) of confirmed neoplastic samples as compared to 2/12 (17%) by an existing assay. On a series of 60 diagnostic samples the concordance rate of both assays was 41/60 (68.3%). In 14/60 (23.3%) of the cases, the new multiplex assay yielded a clonal result while the existing assay gave a non-clonal result. In 5/60 (8.3%) of cases, the new assay yielded a non-clonal result while the existing primer set gave a clonal result. These findings suggest that the new multiplex assay has an improved sensitivity over traditional assays and is suited to reduce the rate of false-negative results.

The T-cell receptor (TCR) is the key player within the so called immunological synapse and the analysis of its repertoire offers a picture of both versatility and wideness of the whole immune T-cell compartment. Among the different approaches applied to its study the so-called spectratyping identifies the pattern of the third complementarity determining region (CDR3) length distribution in each one of the beta variable (TRBV) subfamilies encoded by the corresponding genes. This technique consists in a CDR3 fragment analysis through capillary electrophoresis, performed after cell separation, RNA extraction and reverse transcriptase PCR. This review will run through the most relevant studies which have tried to dissect the TCR repertoire usage in patients with different immune-mediated and infective diseases as well as solid or haematologic malignancies.

Mice with a homozygous p53 gene deletion develop thymic lymphomas by 9 wk of age. Using the sequence of the rearranged T-cell receptor gene from each clone of cells in the thymus, one can determine the number of independent transformation events. These tumors are oligoclonal, occurring at a frequency of 0.13-0.8 new cancer clones per day. By 20 wk only a few clones are detected, indicating competition among transformed cell clones. DNA sequencing of these tumors demonstrates a point mutation frequency of one per megabase and many genes that are consistently amplified or deleted in independent tumors. The tumors begin with an inherited p53 gene deletion. Next is a PTEN mutation in a stem cell or progenitor cell, before the rearrangement of the T-cell receptor. After that, the T-cell clone selects gene amplifications in cyclin D and cdk-6, and in Ikaros in the Notch pathway. Humans heterozygous for the p53 mutant gene in the germline (Li-Fraumeni syndrome) develop cancers at an early age. The penetrance of heterozygous p53 mutations is ∼93% of individuals developing tumors over their lives. At older ages the remaining 7% of this Li-Fraumeni population actually have a lower risk of developing tumors than the population at large with wild-type p53 genes.

This work considers biological sequences that exhibit combinatorial structures in their composition: groups of positions of the aligned sequences are "linked" and covary as one unit across sequences. If multiple such groups exist, complex interactions can emerge between them. Sequences of this kind arise frequently in biology but methodologies for analyzing them are still being developed. This article presents a nonparametric prior on sequences which allows combinatorial structures to emerge and which induces a posterior distribution over factorized sequence representations. We carry out experiments on three biological sequence families which indicate that combinatorial structures are indeed present and that combinatorial sequence models can more succinctly describe them than simpler mixture models. We conclude with an application to MHC binding prediction which highlights the utility of the posterior distribution over sequence representations induced by the prior. By integrating out the posterior, our method compares favorably to leading binding predictors.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a distinct Hodgkin lymphoma subtype composed of few neoplastic lymphocyte-predominant (LP) cells in a back-ground of reactive small B and T cells. We have seen occasional NLPHL cases that contain background T cells with prominent cytologic atypia, raising the differential diagnosis of peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) or a composite lymphoma. We sought to characterize the clinicopathologic features of such cases. Eleven NLPHL cases with atypical T cells diagnosed from 1977 to 2010 were identified at 2 institutions and compared with 24 control NLPHL cases lacking atypical T cells. All 9 male patients and 2 female patients presented with localized peripheral lymphadenopathy. In comparison with control patients, they were younger (median age, 13.8 vs. 36.1 y; P = 0.015), with more frequent cervical lymph node involvement (54.5% vs. 8.3%, P = 0.015). In all 11 cases, areas of NLPHL with typical B-cell-rich nodules containing LP cells were present. Nine cases contained sheets of atypical T cells surrounding primary and secondary follicles in a pattern mimicking the T-zone pattern of PTCL-NOS; the remaining 2 cases contained atypical T cells presented as large clusters at the periphery of B-cell-rich nodules. In all cases, the atypical T-cell-rich areas contained rare scattered LP cells, which were IgD+ in 5 of 7 cases (71.4%). The atypical T cells showed no pan-T-cell antigen loss or aberrant T-cell antigen expression in any case, and polymerase chain reaction or Southern blot analysis showed no evidence of T-cell clonality in 6 cases tested. The atypical T cells exhibited a variable immunophenotype with respect to germinal center, follicular T-helper, T-regulatory, and cytotoxic T-cell markers. Among 8 patients with clinical follow-up (median follow-up: 6.4 y), 5 patients had recurrent NLPHL at 6 months to 12 years after diagnosis and 6 patients are alive without disease at 9 months to 18 years after diagnosis. In comparison with control patients, NLPHL patients with atypical T cells were more likely to develop recurrent NLPHL (71.4% vs. 13.6%, P = 0.008) and to have a shorter time to relapse (P = 0.04). Our findings suggest that some cases of NLPHL, occurring predominantly in younger patients, contain prominent populations of morphologically atypical T cells that may raise the possibility of concurrent nodal involvement by PTCL-NOS, a rare diagnosis in children. The clinical behavior of these cases appears similar to that of NLPHL with T-cell-rich diffuse areas, with a higher risk of disease recurrence and no difference in overall survival; however, this finding warrants confirmation in studies of larger numbers of patients.

It has been reported that brain-infiltrating T lymphocytes play critical roles in the clearance of West Nile virus (WNV) from the brains of mice. We characterized brain-infiltrating T lymphocytes by analyzing the TCR α- and β-chain repertoires, T cell clonality, and CDR3 sequences. CD3+CD8+ T cells were localized in the WNV-infected brains. The expression of CD3, CD8, CD25, CD69, perforin, and granzymes positively correlated with viral RNA levels, and high levels of expression of IFN-γ, TNF-α, and IL-2 were detected in the brains, suggesting that Th1-like cytotoxic CD8+ T cells are expanded in the brains in response to WNV infection. The brain-infiltrating T lymphocytes dominantly used TCR genes, VA1-1, VA2-1, VB5-2, and VB8-2, and exhibited a highly oligoclonal TCR repertoire. Interestingly, the brain-infiltrating T lymphocytes had different patterns of TCR repertoire usages among WNV-, Japanese encephalitis virus-, and tick-borne encephalitis virus-infected mice. Moreover, CD8+ T cells isolated from the brains of WNV-infected mice produced IFN-γ and TNF-α after in vitro stimulation with peritoneal cells infected with WNV, but not with Japanese encephalitis virus. The results suggest that the infiltrating CD8+ T cells were WNV-specific, but not cross-reactive among flaviviruses. T cells from the WNV-infected brains exhibited identical or similar CDR3 sequences in TCRα among tested mice, but somewhat diverse sequences in TCRβ. The results indicate that WNV-specific CD3+CD8+ T cells expanding in the infected brains are highly oligoclonal, and they suggest that TCR α-chains play a dominant and critical role in Ag specificity of WNV-specific T cells.

During the first 2 years of newborn screening (NBS) for severe combined immunodeficiency (SCID), 39 infants with an abnormal or inconclusive newborn screening test for SCID died prior to assessment of immune function. We investigated if SCID or primary T-cell lymphopenia likely contributed to the death of these neonates.This study is a detailed retrospective chart review.Medical records were available in all 39 infants. Three neonates were full-term infants whose deaths were due to congenital anomalies. Thirty-three infants were born33 weeks estimated gestational age, and the majority of these infants died from complications of prematurity. Six infants died from sepsis: two due to maternal chorioamnionitis, two due to necrotizing enterocolitis, one due to early onset group B strep sepsis, and one from a likely nosocomial infection.There was no evidence that SCID contributed to the cause of death in neonates with an abnormal of inconclusive NBS test for SCID.

The function of T-cell receptor (TCR) gene modified T cells is dependent on efficient surface expression of the introduced TCR α/β heterodimer. We tested whether endogenous CD3 chains are rate-limiting for TCR expression and antigen-specific T-cell function. We show that co-transfer of CD3 and TCR genes into primary murine T cells enhanced TCR expression and antigen-specific T-cell function in vitro. Peptide titration experiments showed that T cells expressing introduced CD3 and TCR genes recognized lower concentration of antigen than T cells expressing TCR only. In vivo imaging revealed that TCR+CD3 gene modified T cells infiltrated tumors faster and in larger numbers, which resulted in more rapid tumor elimination compared with T cells modified by TCR only. After tumor clearance, TCR+CD3 engineered T cells persisted in larger numbers than TCR-only T cells and mounted a more effective memory response when rechallenged with antigen. The data demonstrate that provision of additional CD3 molecules is an effective strategy to enhance the avidity, anti-tumor activity and functional memory formation of TCR gene modified T cells in vivo.

Juvenile idiopathic arthritis (JIA) is an autoimmune disease where T cells are key players. It can be classified into two main clinical diseases: polyarticular and pauciarticular, based on the number of joints involved. Oligoarthritis, which is considered a pauciarticular subtype since it involves up to four joints upon presentation, is further divided into persistent or extended forms based on disease progression.Here we assessed the T-cell compartment in synovial fluid obtained from 33 JIA patients with active disease and correlated the analyzed parameters with the patients' clinical characteristics. The T-cell compartment was determined by the representation of T-cell receptor (TCR) repertoires and the amount of TCR excision circles (TRECs).Patients with polyarticular disease have more a clonal pattern of their TCR repertoire. These findings were consistent in all tested TCR-Vγ consensus primers. Similarly, patients with polyarticular disease had lower TREC levels than patients with pauciarticular disease. A predictive value of TRECs may be suggested, as lower TREC levels were observed in patients in whom disease modifying anti rheumatic drugs were initiated subsequently during the follow-up.In pediatric JIA patients, we showed an alteration in the T cells from synovial fluid, which correlated with disease phenotype, assumedly secondary to enhanced proliferation, clonal TCR restriction, and reduced T-cell production, possibly reflecting a different disease or a different course of disease progression.

Purpose: In human leukocyte antigen (HLA)–matched stem cell transplantation (SCT), it has been shown that beneficial immune response mediating graft-versus-tumor (GVT) responses can be separated from graft-versus-host disease (GVHD) immune responses. In this study, we investigated whether it would be possible to dissect the beneficial immune response of allo-HLA–reactive T cells with potent antitumor reactivity from GVHD-inducing T cells present in the detrimental immune response after HLA-mismatched SCT. Experimental Design: The presence of specific tumor-reactive T cells in the allo-HLA repertoire was analyzed at the time of severe GVHD after HLA-mismatched SCT, using tetramers composed of different tumor-associated antigens (TAA). Results: High-avidity allo-HLA-restricted T cells specific for the TAA preferentially expressed antigen on melanomas (PRAME) were identified that exerted highly single-peptide–specific reactivity. The T cells recognized multiple different tumor cell lines and leukemic cells, whereas no reactivity against a large panel of nonmalignant cells was observed. These T cells, however, also exerted low reactivity against mature dendritic cells (DC) and kidney epithelial cells, which was shown to be because of low PRAME expression. Conclusions: On the basis of potential beneficial specificity and high reactivity, the T-cell receptors of these PRAME-specific T cells may be effective tools for adoptive T-cell therapy. Clinical studies have to determine the significance of the reactivity observed against mature DCs and kidney epithelial cells. Clin Cancer Res; 17(17); 5615–25. ©2011 AACR.

The large (LI) and small intestine (SI) differ in patterns of susceptibility to chronic mucosal inflammation. In this study, we evaluated whether this might, in part, reflect differences in resident mucosal CD11c+ T cells. These cells comprised 39–48% (SI) and 12–17% (LI) of the intraepithelial compartment, most of which were T-cell receptor-αβ+. In the SI, the majority of these cells were CD103+ CD8+ NK1.1−, whereas the opposite phenotype prevailed in the LI. In transfer models of CD4+ T cell-induced colitis, small numbers (2.5 × 105) of SI CD11c+ CD8+ T cells suppressed proinflammatory cytokine-producing CD4+ T cells in mesenteric lymph nodes and mucosa-associated lymphoid compartments (SI and LI) and protected mice from chronic inflammation. On a per-cell basis, the regulatory function of SI CD11c+ T cells in CD4+ T cell colitis was potent compared with other reported regulatory CD4+ or CD8+ T cells. In contrast, neither LI CD11c+ T cells nor SI CD11c− T cells were effective in such immunoregulation. SI CD11c+ CD8+ T cells were similarly effective in suppressing CD4+CD45RBhi T cell colitis, as evidenced by inhibition of intracellular proinflammatory cytokine expression and histological inflammation. These findings indicate that SI CD11c+ CD8+ T cells are a distinct intestinal T cell population that plays an immunoregulatory role in control of proinflammatory CD4+ T cells and maintenance of intestinal mucosal homeostasis.

Wilms' tumor gene (WT1), which is expressed in human pancreatic cancer (PC), is a unique tumor antigen recognized by T-cell-mediated antitumor immune response. Gemcitabine (GEM), a standard therapeutic drug for PC, was examined for the regulation of WT1 expression and the sensitizing effect on PC cells with WT1-specific antitumor immune response. Expression of WT1 was examined by quantitative PCR, immunoblot analysis, and confocal microscopy. Antigenic peptide of WT1 presented on HLA class I molecules was detected by mass spectrometry. WT1-specific T-cell receptor gene-transduced human T cells were used as effecter T cells for the analysis of cytotoxic activity. GEM treatment of human MIAPaCa2 PC cells enhanced WT1 mRNA levels, and this increase is associated with nuclear factor kappa B activation. Tumor tissue from GEM-treated MIAPaCa2-bearing SCID mice also showed an increase in WT1 mRNA. Some human PC cell lines other than MIAPaCa2 showed up-regulation of WT1 mRNA levels following GEM treatment. GEM treatment shifted WT1 protein from the nucleus to the cytoplasm, which may promote proteasomal processing of WT1 protein and generation of antigenic peptide. In fact, presentation of HLA-A*2402-restricted antigenic peptide of WT1 (CMTWNQMNL) increased in GEM-treated MIAPaCa2 cells relative to untreated cells. WT1-specific cytotoxic T cells killed MIAPaCa2 cells treated with an optimal dose of GEM more efficiently than untreated MIAPaCa2 cells. GEM enhanced WT1 expression in human PC cells and sensitized PC cells with WT1-specific T-cell-mediated antitumor immune response.

Objective— Programmed cell death-1 (PD-1) is a member of the CD28 superfamily that delivers negative signals on interaction with its 2 ligands, PD-L1 and PD-L2. We studied the contribution of the PD-1 pathway to regulation of T cells that promote atherosclerotic lesion formation and inflammation. Methods and Results— We show that compared with Ldlr −/− control mice, Pd1 −/− Ldlr −/− mice developed larger lesions with more abundant CD4 + and CD8 + T cells and macrophages, accompanied by higher levels of serum tumor necrosis factor-α. Iliac lymph node T cells from Pd1 −/− Ldlr −/− mice proliferated more to αCD3 or oxidized low-density lipoprotein stimulation compared with controls. CD8 + T cells from Pd1 −/− Ldlr −/− mice displayed more cytotoxic activity compared with controls in vivo and in vitro. Administration of a blocking anti-PD-1 antibody increased lesional inflammation in hypercholesterolemic Ldlr −/− mice with more lesional T cells and more activated T cells in paraaortic lymph nodes. The changes in lesional T-cell content when PD-1 was absent or blocked were also observed in bone marrow chimeric Ldlr −/− mice lacking PD-L1 and PD-L2 on hematopoietic cells. Conclusion— PD-1 has an important role in downregulating proatherogenic T-cell responses, and blockade of this molecule for treatment of viral infections or cancer may increase risk of cardiovascular complications.

A 70-year-old man complained of fever and sore throat accompanied by hoarseness of voice. On physical examination, there was no systemic abnormality but a mild lymphadenopathy of cervical lymph nodes. With laryngoscopy, there was a marked outgrowth of the bilateral palatine tonsils proximal to the vocal cord. The histology of the resected tumor was compatible with angioimmunoblastic T cell lymphoma (AITL), revealing the effacement of normal tonsillar architecture and small to medium-sized neoplastic cell proliferation around marked vascular proliferation and atrophic lymphoid follicles. Tumor cells were positive for conventional T-cell antigens as well as for the follicular helper T-cell marker, PD-1, and CXCL13. Large hodgkinoid cells, but no tumor cells, were positive for latent membrane protein-1 and Epstein-Barr virus-encoded small RNA (EBER)-1 (in situ hybridization). Non-neoplastic, double positive cells for EBER-1 and CD20 were also scattered. Southern blot analysis revealed dual TCR-Cβ1 and IGH-JH gene rearrangements. Although the swelling of bilateral inguinal and perigastric lymph nodes developed later, the radical resection of tumor and chemotherapy appeared to be effective for the treatment of AITL with clinical stage IIIa. We here report a rare case of AITL involving palatine tonsil as primary site and give a review of the literature.

of CTLs generated ex vivo would be expected to exert a clinical effect superior to that of cancer vaccines. Based on this concept, various types of T-cell adoptive transfer have been performed and various degrees of clinical responses have been obtained. Donor lymphocyte infusion (DLI) has been widely performed after allogeneic hematopoietic stem cell transplantation for patients with therapy-resistant malignancies, especially chronic myelogenous leukemia (CML). The major concern regarding DLI is that its antitumor effect is strongly dependent on graft-versus-host disease (GVHD) [3]. To control severe GVHD induced by DLI, clinical trials of infusion of donor lympho cytes transduced with a suicide gene, such as herpes simplex thymidine kinase, have been performed [4]. Furthermore, a more defined type of adoptive T-cell transfer, involving infusion of virus-specific T cells, has been investigated. Infusion of Epstein–Barr virus (EBV)-reactive T cells, generated by stimulating peripheral blood lymphocytes with autologous EBV-transformed B cells, has been applied for the treatment of EBV-associated post-transplantation lymphoproliferative disease (PTLD) and EBVassociated malignancies, including Hodgkin’s lymphoma and naso pharyngeal carcinoma [5,6]. Previous studies have revealed that adoptive transfer of EBV-specific T cells as monotherapy is effective in inducing remission of EBV-associated PTLD with low-risk features. For high-risk EBVassociated diseases, a combination of adoptive transfer of EBV-specific T cells with conventional chemotherapies has yielded superior outcomes [7]. In addition, infusion of CTLs specific for antigens, uniquely or preferentially expressed by cancer cells, has received marked attention for its antitumor effectiveness with minimal adverse events including GVHD. More than 10 years ago, Falkenburg et al. reported that infusion Adoptive T-cell immunotherapy It is well known that the immune surveillance system plays an important role in resistance to tumor cell growth. Therefore, if the antitumor immune response can be augmented in vivo, improved survival of cancer patients would be expected. The immune surveillance system is harmonized with interactions between various kinds of immune cells, and among them, cytotoxic T lymphocytes (CTLs) play the most important role in antitumor effect. On the basis of this concept, various types of cell-mediated immunotherapy aimed at induction of tumorspecific CTLs have been developed. These include vaccination with peptides derived from tumor-associated antigens, or with dendritic cells pulsed with peptide or tumor cell lysate, adoptive transfer of tumor-specific CTLs, and gene-engineered T-cell immunotherapy. Among these approaches, various trials of cancer peptide vaccines have been ongoing in many countries [1]. The results obtained from these clinical trials have revealed no severe adverse events and peptide-specific immune responses have been detected in the majority of the vaccinated patients. However, the clinical responses have failed to meet expectations. A variety of immunosuppressive mechanisms, particularly those mediated by regulatory T cells and myeloid-derived suppressor cells, operate in cancer patients. This suppressive influence may play a role in limiting the antitumor effectiveness of cancer vaccines [2].

Carp kidney leukocytes co-cultured with a supporting cell layer resulted in the rapid proliferation of various types of leukocytes including immature leukocytes. Expressions of marker genes for multiple blood cell lineages were observed in the primary culture. However, after several passages, the proliferating cells expressed only T cell and macrophage marker genes. Further RT-PCR analysis revealed that the proliferating cells expressed TCR constant regions (Cα, Cβ, Cγ, Cδ), CD3γ/δ and CD4 (CD4L-1), but did not express CD8α and CD8β. Additionally, in situ hybridization analysis showed that the majority of proliferating cells expressed Cα, Cβ, Cγ, Cδ and CD4. Moreover, 5'-RACE sequences of TCR variable regions (Vα, Vβ, Vγ, Vδ) revealed that the proliferating cells contained a polyclonal T cell repertoire, and most of the Vα and Vβ sequences were functional, but the Vγ and Vδ sequences were non-functional with frame shifts and stop codons. Taken together, these results indicate that the proliferating cells after serial passages predominantly contained CD4+ CD8- αβT cells that simultaneously co-expressed non-functional γδTCR. To obtain CD4+ αβT cell (helper T cell) clones, single cells were picked up from the bulk culture, seeded into each well of 96-well plates and cultured in the presence of supporting cells and conditioned media. T cell colonies formed from single cells after 2-3 weeks. These colony cells expressed Cα, Cβ, Cδ and CD4, and weakly expressed Cγ, but did not express CD8α, CD8β and CD4L-2. Taken together, these results indicate that these clonal T cells resemble a subpopulation of mammalian CD4+ helper T cells.

T cells undergo a series of complex phenotypic changes before achieving maturation. Discrete stages of T-cell differentiation are simplified to four stages (pro-, pre-, cortical and mature-T cell) and used in the classification of T-cell leukaemia. HLA-DR has been reported to be expressed in immature T-cell acute lymphoblastic leukemia (ALL) and also confer a poorer treatment outcome. Simultaneously, the genotype goes through distinct pattern changes due to rearrangement of T-cell receptor (TCR) genes. TCR gene rearrangement is important in the diagnosis of clonality and used as markers to detect minimal residual disease in lymphoproliferative disorders. We identified a subset within Pro-T and Pre-T cell cases distinguished by the expression of HLA-DR. These subgroups appeared to be more immature as rearrangement of the TCR-gamma gene was either at germline or involved only the first constant region (C1) unlike a more rearranged pattern in the HLA-DR-subgroups. We also observed a higher incidence of mediastinal mass (67%) in the HLA-DR-subgroup in the Pre-T stage. These characteristics may be useful as markers to further refine staging of T-cell ALL and determine prognosis.

BACKGROUND Real-time quantitative PCR (qPCR) targeting a specific marker of functional T cells, the T-cell–receptor excision circle (TREC), detects the absence of functional T cells and has a demonstrated clinical validity for detecting severe combined immunodeficiency (SCID) in infants. There is need for a qPCR TREC assay with an internal control to monitor DNA quality and the relative cellular content of the particular dried blood spot punch sampled in each reaction. The utility of the qPCR TREC assay would also be far improved if more tests could be performed on the same newborn screening sample. METHODS We approached the multiplexing of qPCR for TREC by attenuating the reaction for the reference gene, with focus on maintaining tight quality assurance for reproducible slopes and for prevention of sample-to-sample cross contamination. Statewide newborn screening for SCID using the multiplexed assay was implemented, and quality-assurance data were recorded. RESULTS The multiplex qPCR TREC assay showed nearly 100% amplification efficiency for each of the TREC and reference sequences, clinical validity for multiple forms of SCID, and an analytic limit of detection consistent with prevention of contamination. The eluate and residual ghost from a 3.2-mm dried blood spot could be used as source material for multiplexed immunoassays and multiplexed DNA tests (Multiplex Plus), with no disruption to the multiplex TREC qPCR. CONCLUSIONS Population-based SCID newborn screening programs should consider multiplexing for quality assurance purposes. Potential benefits of using Multiplex Plus include the ability to perform multianalyte profiling.

Type 1 diabetes (T1D) is caused by autoimmune destruction of insulin-producing pancreatic β cells in the islets of Langerhans. Although defects in various T cell subsets have been linked to the disease pathogenesis, mechanisms initiating or enhancing the autoimmunity in prediabetes remain poorly understood. To unravel genes and molecular pathways affected by the diabetes-associated autoimmunity, we investigated transcriptomic profiles of prospective whole-blood samples from children who have developed T1D-associated autoantibodies and eventually clinical T1D. Gene-level investigation of the data showed systematic differential expression of 520 probesets. A network-based analysis revealed then a highly significant down-regulated network of genes involved in antigen presentation as well as T-cell receptor and insulin signaling. Finally, detection of dynamic changes in the affected pathways at the early or late phases of autoimmunity showed down-regulation of several novel T1D-associated pathways as well as known key components of immune response. The longitudinal genome-wide data generated in the present study allows the detection of dynamic changes relevant to the disease that may be completely missed in conventional cross-sectional studies or in genome-wide association studies. Taken together, our analysis showed systemic high-level repression of immune response pathways associated with T1D autoimmunity.

International audience; In the context of the cross-talk between the neuroendocrine and immune systems, it is well known that growth hormone (GH) exerts physiological effects in central as well as peripheral compartments of the immune system. GH modulates a variety of thymic functions, including proliferation of thymocytes and thymic epithelial cells (TEC). Accordingly, GH-transgenic mice, as well as animals and humans treated with exogenous GH, exhibit an enhanced cellularity in the organ. GH also stimulates the secretion of cytokines and chemokines by the thymic microenvironment, as well as the production of extracellular matrix proteins. These effects lead to an increase in thymocyte migratory responses and intrathymic traffic of developing T cells, including the export of thymocytes from the organ, as ascertained by experimental studies with intrathymic injection of GH in normal mice and with GH-transgenic animals. Most likely, GH effects in the thymus are mediated by an IGF-1/IGF-1 receptor circuitry, which physiologically operates in nonstimulated conditions in both thymocytes and TECs. Since GH enhances thymus replenishment and increases intrathymic T-cell traffic, ultimately modulating thymocyte exit, it should be placed as a potential adjuvant therapeutic agent in the treatment of immunodeficiencies associated with thymic atrophy, and examples recently appeared in the literature are promising and strongly indicate that GH can be beneficial for individuals suffering severe immunodeficiency.

Accessibility of chromosomal recombination signal sequences to the RAG protein complex is known to be essential for V(D)J recombination at Ag receptor loci in vivo. Previous studies have addressed the roles of cis-acting regulatory elements and germline transcription in the covalent modification of nucleosomes at Ag receptor loci. However, a detailed picture of nucleosome organization at accessible and inaccessible recombination signal sequences has been lacking. In this study, we have analyzed the nucleosome organization of accessible and inaccessible Tcrb and Tcra alleles in primary murine thymocytes in vivo. We identified highly positioned arrays of nucleosomes at Dβ, Jβ, and Jα segments and obtained evidence indicating that positioning is established at least in part by the regional DNA sequence. However, we found no consistent positioning of nucleosomes with respect to recombination signal sequences, which could be nucleosomal or internucleosomal even in their inaccessible configurations. Enhancer- and promoter-dependent accessibility was characterized by diminished abundance of certain nucleosomes and repositioning of others. Moreover, some changes in nucleosome positioning and abundance at Jα61 were shown to be a direct consequence of germline transcription. We suggest that enhancer- and promoter-dependent transcription generates optimal recombinase substrates in which some nucleosomes are missing and others are covalently modified.

Primary effusion lymphoma is a human herpes virus 8 (HHV-8)-associated large cell lymphoma of body cavities. Detailed large-scale clinicopathological studies are rarely reported, and the underlying mechanism of lymphomagenesis remains elusive. In the present report, we studied the clinicodemographic, immunophenotypic, and cytomorphological features on a cohort of 12 cases of primary effusion lymphoma. In contrast to HHV-8, which was positive in all nine cases tested (100%), HIV was found in 75% (9/12) of cases, whereas the three HIV-negative cases were either in elderly patients (one with hepatitis C virus infection and one with asbestoses exposure) or in a heart transplantation recipient. By flow cytometry, the antigens expressed in descending order were CD38, CD71, HLA-DR, CD30, and CD45RO. B-cell markers were largely negative. Cytomorphologically, all cases showed atypical to anaplastic morphology. Notch1, a member of transmembrane signal transduction family, was found in six of seven HHV-8-positive cases (86%). In agreement with in vitro studies using human primary effusion lymphoma cell lines, we have found that Notch1 was expressed in the majority of HHV-8-positive primary effusion lymphoma cases, corroborating the notion that Notch1 may have an important role in HHV-8-mediated lymphomagenesis of primary effusion lymphoma.

The distinction between mycosis fungoides (MF) and inflammatory dermatoses (ID) by clinicopathologic criteria can be challenging. There is limited information regarding the performance characteristics and utility of TCRG and TCRB clonality assays in diagnosis of MF and ID from paraffin-embedded tissue sections. In this study, PCR tests were performed with both TCRG and TCRB BIOMED-2 clonality methods followed by capillary electrophoresis and Genescan analysis using DNA samples from 35 MF and 96 ID patients with 69 and 133 paraffin-embedded specimens, respectively. Performance characteristics were determined for each test individually and in combination. TCRG and TCRB tests demonstrated identical sensitivity (64%) and specificity (84%) when analyzed as individual assays. The positive predictive value, negative predictive value, and change of posttest MF probability over a range of MF pretest probabilities were obtained. These data were used to construct an algorithm for sequential use of TCRG and TCRB. As single tests, commercially available BIOMED-2 PCR-based TCRG and TCRB clonality tests on paraffin-embedded tissue have no significant difference in terms of sensitivity and specificity. Combined use of the two tests in patients with intermediate pretest probabilities as proposed in the algorithm could improve test utility.

Monoclonal expansions of immunoglobulin (Ig) and T-cell receptor (TCR) genes are, respectively, important markers for B- and T-cell malignancies. We used BIOMED-2 protocols to detect clonality of these genes in B- (BCL) and T-cell lymphoma (TCL) in southern Taiwan. Heteroduplex analysis was used after PCR reactions. Ig/TCR clonality rates were 95% (22 in 23 cases) for BCL and 76% (19 in 25 cases) for TCL. These results reveal overall satisfactory detection rates for both BCL and TCL. None of the four natural killer (NK)/T-cell lymphomas detected showed TCR gene clonality, which suggested that BIOMED-2 protocols distinguished the NK/T-cell lymphoma from TCL. In addition, three of the five tested precursor T-cell lymphoblastic lymphomas showed no TCR gene clonality, probably because the immature T-cells in this type of TCL had not undergone TCR gene rearrangements. We conclude that BIOMED-2 protocols are suitable for detecting Ig and TCR clonalities in B- and T-cell malignancies in southern Taiwan.

The recent recognition of the importance of transforming growth factor beta (TGF-beta) in mediating many cellular immune functions has popularized the use of mouse models where TGF-beta signaling is blocked in specific cell types. However, there are some caveats affiliated with each model. Here we describe and show evidence for some of the limitations of these models and provide insight into ways to improve the utility of these animals for in vivo studies.

International audience; Translocations of proto-oncogenes to the B-cell or T-cell antigen receptor loci in acute T- or B-cell leukemia and lymphoma have been, in most cases, accredited to V(D)J or switch recombination depending on the location of the breakpoint at the receptor locus. Only in rare instances, the reports take into account mechanistic characteristics of the translocation mechanism. To assess the functional ability of several sites implicated in supposedly V(D)J-mediated translocations, we tested five sites at four proto-oncogene loci in an ex vivo recombination substrate assay for their potential to act as direct target for V(D)J recombination. Our results show that the LMO2/RBTN2/TTG2 site and one LCK/P56 site readily engage in recombination with a genuine TCR element with the majority of breakpoint junctions showing the characteristics of V(D)J recombination, which strongly supports the involvement of this mechanism in the pathogenesis of the corresponding translocations in vivo. The site at the TLX1/HOX11 locus yielded 0.8% V(D)J-specific junctions. Sites at the LCK/P56 and TCF3/E2A proto-oncogenes resulted in exclusively unspecific breakpoints scattered over part of or the entire proto-oncogene region tested, marking them as unlikely V(D)J recombination targets. Our data suggest that, while being a potentially dangerous mechanism due to the introduction of DNA breaks, V(D)J recombination is a tightly controlled mechanism allowing for only few direct mistakes.

Focal myositis is an uncommon inflammatory pseudotumor of skeletal muscle that can be confused with a variety of neoplastic and inflammatory diseases. It is often misunderstood because it presents as a tumor-like mass, but histologically resembles a skeletal muscle myopathy or dystrophy. We wanted to discuss the detailed morphologic and immunophenotypic features of the largest reported group of focal myositis patients.Two hundred and six cases coded as "focal myositis" were culled from our files. Only 115 cases with adequate material, a solitary lesion, and correct diagnosis were included. A variety of immunohistochemical studies were performed, as were polymerase chain reaction for T cell receptor gene rearrangement and immunoglobulin heavy chain rearrangement.Age ranged from 7 to 94 years (mean 41, median 36 y). Most patients were otherwise healthy, and with the exception of 10 cases, lacked antecedent trauma. Masses that ranged in size from 1.0 to 20.0 cm (median 3.0 cm, mean 3.9 cm) were reported in specific muscles of the lower extremities (including vastus lateralis, adductor muscle, and groin muscles, n=39; gastrocnemius, n=22), followed by the trunk, neck (mentalis, n=8; sternocleidomastoid muscle, n=8), and upper extremity. Histologically, these were solitary intramuscular processes composed of variable myopathic (93%) and focal neurogenic (89%) changes, fibrosis, and inflammation (97%), occasionally accompanied by prominent eosinophils (n=20). By immunohistochemistry, most cases had CD163-positive macrophages that were negative for S100 protein and CD1a. Lymphocytes were mostly CD3, CD4-positive lymphocytes that were negative for cytotoxic markers, TIA-1 and granzyme-B. Polymerase chain reaction did not show B cell or T cell rearrangement. In situ studies for Epstein-Barr-encoded receptor were negative, as was ALK-1 immunohistochemistry. Major histocompatibility complex-1 and weak IgG4 were focally positive in skeletal muscle. Cases with severe inflammation had increased numbers of CD20-positive B cells and CD123-positive plasmacytic dendritic cells. S100 was strongest in skeletal muscle fibers with vacuolar change. Clinical diagnostic considerations ranged from benign entities such as rhabdomyoma, intramuscular lipoma, fibromatosis, myositis ossificans, proliferative myositis, inflammatory myofibroblastic tumor, and inflammatory myopathy to malignant entities such as rhabdomyosarcoma, leiomyosarcoma, liposarcoma, and lymphoma. Available follow-up revealed spontaneous regression.Focal myositis occurs in specific muscle groups of young adults of both sexes without significant trauma. It is a largely unrecognized entity with specific histology including myopathic, focal neurogenic, fibrosis, and inflammatory features. It can be easily mistaken for an inflammatory myopathy, dystrophy, alternate reactive, or even neoplastic process. Focal myositis seems to be a macrophage and T-cell-rich lesion that changes to B cell and dendritic plasmacytoid cells when markedly inflamed, but does not seem to have a known viral or molecular etiology. IgG4 presence may be linked to the fibrosis in these lesions; a possible transient autoimmune etiology cannot be excluded. Careful attention to reproducible clinicopathologic features can aid diagnosis and spare patients from excessive surgery or adverse therapy.

A considerable number of studies on hematological malignancies have recently demonstrated that the identification of rearrangements in immunoglobulin (Ig) and T-cell receptor (TCR) genes are important tools for diagnosis and follow-up of B- and T-cell disorders. The heterogeneity of these malignancies makes it difficult to carry out a precise assessment in all patients despite well-established morphological and immunophenotyping criteria. Clonal analysis of hematological malignancies is supported by the fact that all malignant cells have a common clonal origin with identically rearranged Ig and/or TCR genes. Identification of B- or T-cell clonality in polyclonal tissue such as the blood is indicative of a lymphoproliferative process. Germline gene segments of Ig heavy chain (IGH), Ig kappa (IGK), Ig lambda (IGL) and TCR are rearranged in each lymphocyte during B- and T-cell differentiation. A specific combination of gene segments and somatic mutations occurring during this process is responsible for the wide diversity of antigen-specific receptors and antibodies. Ig and TCR rearrangements are considered the "fingerprint" of each lymphocyte and therefore can be used as tumor-specific PCR targets for detection of residual malignant cells present after treatment. Determination of minimal residual disease (MRD) has a proven prognostic value and enables effective early interventional treatment. This is becoming routinely implemented in several treatment protocols and is increasingly used in guidelines for drug therapy and stem cell transplantation. In this review we focus on: (1) the process of gene rearrangements in B- and T-cells, (2) principles of polymerase chain reaction (PCR)-based assays and real-time PCR methods commonly used to detect and follow clonal Ig and TCR rearrangements, (3) multiplex primer sets recently designed by the BIOMED-2 concerted action group, and (4) application of these techniques in MRD detection.

The elusive etiology of germline bias of the T cell receptor (TCR) for major histocompatibility complex (MHC) has been clarified by recent 'proof-of-concept' structural results demonstrating the conservation of specific TCR-MHC interfacial contacts in complexes bearing common variable segments and MHC allotypes. We suggest that each TCR variable-region gene product engages each type of MHC through a 'menu' of structurally coded recognition motifs that have arisen through coevolution. The requirement for MHC-restricted T cell recognition during thymic selection and peripheral surveillance has necessitated the existence of such a coded recognition system. Given these findings, a reconsideration of the TCR-peptide-MHC structural database shows that not only have the answers been there all along but also they were predictable by the first principles of physical chemistry.

The international ImMunoGeneTics information system® (IMGT) (http://imgt.cines.fr), created in 1989, by the Laboratoire d'ImmunoGenetique Moleculaire LIGM (Universite Montpellier II and CNRS) at Montpellier, France, is a high-quality integrated knowledge resource specializing in the immunoglobulins (IGs), T cell receptors (TRs), major histocompatibility complex (MHC) of human and other vertebrates, and related proteins of the immune systems (RPI) that belong to the immunoglobulin superfamily (IgSF) and to the MHC superfamily (MhcSF). IMGT includes several sequence databases (IMGT/LIGM-DB, IMGT/PRIMER-DB, IMGT/PROTEIN-DB and IMGT/MHC-DB), one genome database (IMGT/GENE-DB) and one three-dimensional (3D) structure database (IMGT/3Dstructure-DB), Web resources comprising 8000 HTML pages (IMGT Marie-Paule page), and interactive tools. IMGT data are expertly annotated according to the rules of the IMGT Scientific chart, based on the IMGT-ONTOLOGY concepts. IMGT tools are particularly useful for the analysis of the IG and TR repertoires in normal physiological and pathological situations. IMGT is used in medical research (autoimmune diseases, infectious diseases, AIDS, leukemias, lymphomas, myelomas), veterinary research, biotechnology related to antibody engineering (phage displays, combinatorial libraries, chimeric, humanized and human antibodies), diagnostics (clonalities, detection and follow up of residual diseases) and therapeutical approaches (graft, immunotherapy and vaccinology). IMGT is freely available at http://imgt.cines.fr.

T cell responses to MHC-mismatched transplants can be mediated via direct recognition of allogeneic MHC molecules on the cells of the transplant or via recognition of allogeneic peptides presented on the surface of recipient APCs in recipient MHC molecules - a process known as indirect recognition. As CD4(+)CD25(+) Tregs play an important role in regulating alloresponses, we investigated whether mouse Tregs specific for allogeneic MHC molecules could be generated in vitro and could promote transplantation tolerance in immunocompetent recipient mice. Tregs able to directly recognize allogeneic MHC class II molecules (dTregs) were obtained by stimulating CD4(+)CD25(+) cells from C57BL/6 mice (H-2(b)) with allogeneic DCs from BALB/c mice (H-2(d)). To generate Tregs that indirectly recognized allogeneic MHC class II molecules, dTregs were retrovirally transduced with TCR genes conferring specificity for H-2K(d) presented by H-2A(b) MHC class II molecules. The dual direct and indirect allospecificity of the TCR-transduced Tregs was confirmed in vitro. In mice, TCR-transduced Tregs, but not dTregs, induced long-term survival of partially MHC-mismatched heart grafts when combined with short-term adjunctive immunosuppression. Further, although dTregs were only slightly less effective than TCR-transduced Tregs at inducing long-term survival of fully MHC-mismatched heart grafts, histologic analysis of long-surviving hearts demonstrated marked superiority of the TCR-transduced Tregs. Thus, Tregs specific for allogeneic MHC class II molecules are effective in promoting transplantation tolerance in mice, which suggests that such cells have clinical potential.

A Vbeta TCR repertoire is analyzed for understanding the T-cell population in the immune response. However, the TCR repertoire of the Valpha-Vbeta pair is difficult to analyze because no suitable analytical method is available. Here, we have applied the single-cell 5'-RACE method for amplifying TCR cDNAs from single T-cells and analyzed the repertoire of Valpha-Vbeta pairs in human T-cells that responded to a superantigen, SEB. We found that the TCR Vbeta profile of the SEB-stimulated CD4(+) T-cells was in accordance with the previous reports, that the TCR Valpha profile also exhibited a prominent difference, and that the TCR Valpha-Vbeta pairs of the SEB-responding T-cells were promiscuous. We have also found a significant dual TCRalpha expression in single T-cells. This is the first report of a comprehensive analysis of the functional repertoire of Valpha-Vbeta pairs at the single T-cell level. This novel method may contribute to TCR-based immunotherapeutics.