TCR Alpha Research Articles

Stage and Tissue Specific Expression of Four TCR Subunits in Olive Flounder (Paralichthys olivaceus)

TCR subunits are members of membrane-bound receptors which allow the fast and efficient elimination of the specific fish pathogens have regulated function in adaptive immunity. Sequence structure of TCR subunits have been reported for various teleosts, but the information of each TCR subunit functional characterization through expression analysis in fish was unknown. In this study, we examined the gene expression of TCR subunits in the early developmental stages and observed transcript levels in various tissues from healthy adult olive flounder by RT-PCR. The mRNA expression of alpha subunit was already detected in the previous hatching step. But the transcripts of another TCR subunit were not observed during embryo development and increased after hatching and maintained until metamorphosis at the same level. It was found that all TCR subunits mRNAs are commonly expressed in the immune-related organ such as spleen, kidney and gill, also weak expressed in fin and eye. TCR alpha and beta subunit were expressed in brain, whereas gamma and delta were not expressed same tissue. The sequence alignment analysis shows that there are more than 80% sequence homology between TCR subunits. Because it has a high similarity of amino acid sequence to expect similar in function, but expression analysis show that will have may functional diversity due to different time and place of expression.

Autoantibodies against distinct neuronal populations in narcoleptic patients – A possible link to vaccine induced autoimmunity?

Introduction Narcolepsy is a neurological sleep disorder that is characterized by excessive daytime sleepiness and cataplexy. The aetiology is unknown but genetic analyses have revealed a strong link to the HLA DQB1 ∗ 06:02 and T-cell receptor alpha loci. Thus, narcolepsy is proposed to be an autoimmune disease. During 2009–2010, a link between vaccination with the adjuvanted H1N1 influenza vaccine Pandemrix and onset of narcolepsy was observed in Finland and Sweden. We hypothesized that vaccination triggered generation of autoantibodies against orexinergic neurons in the lateral hypothalamus (LH) of affected patients. We searched for autoantibodies using immunohistochemistry using a rat brain ‘neuroanatomical array’. Materials and methods Sera from 45 narcoleptic patients, 20 patients with other sleep related disorders (OSRD) and 20 healthy controls were included in the study, all of Finnish origin. All patients were HLA DQB1 ∗ 06:02 positive. Clinical information was collected. Sections of formalin- fixed rat brains were processed for immunohistochemistry using patient sera as primary antibody, followed by FITC-conjugated antibodies. Double-labelling with antisera to various relevant markers was carried out: Orexin, melanocyte-concentrating hormone (MCH), s-endorphin (marker for pro-opiomelanocortin (POMC) neurons), glutamic acid decarboxylase and more. Results Three distinct groups of immunoreactivity were recorded: group A (4/45) encompassed the MCH, but not the orexin neurons in the LH and the POMC neurons in the hypothalamic arcuate nucleus; group B (5/45) GABAergic interneurons in the cortex, many of them being calretinin or calbindin immunoreactive; and group C (4/45) neurons in the globus pallidus. In total 13/45 (29%) of narcoleptic sera stained some of the groups A, B or C. In the OSRD-group one positive serum was found (group B), whereas none of the sera in the healthy controls showed any staining. There was no clear link between the staining patterns and a previous history of Pandemrix-vaccination. Conclusion Almost one third of narcoleptic sera of Finnish patients contains autoantibodies against neuronal subpopulations, at least one of which has been associated with sleep-regulation. Acknowledgements The study was funded by the Swedish Medical Product Agency, the Swedish Research Council (VR) and Academy of Finland (26063) is a part of the NARPA- NORD consortium between Finland and Sweden.

Copy Number Abnormalities Of The Interferon Regulatory Factor-4 (IRF4) Gene Are Associated With IRF4/MUM1 Expression In Peripheral T-Cell Lymphomas

BackgroundPeripheral T-cell lymphomas (PTCLs) are aggressive non-Hodgkin lymphomas with poor overall survival rates. The genetics of PTCLs are poorly understood. We have described t(6;14)(p25.3;q11.2) translocations in PTCLs involving the T-cell receptor-alpha (TRA@) and interferon regulatory factor-4 (IRF4) genes and overexpressing IRF4 protein (also called MUM1). IRF4/MUM1 is a lymphoid transcription factor involved in differentiation and growth, the expression of which is tightly regulated in normal T cells. However, nearly half of PTCLs constitutively express IRF4/MUM1 and this expression is associated with adverse overall survival in several PTCL subtypes. Because IRF4/TRA@ translocations are rare among PTCLs (<1%), we investigated the presence of other genetic abnormalities of IRF4 in PTCLs and examined their association with IRF4/MUM1 protein expression. MethodsStudies were performed on paraffin tissue sections of PTCL specimens diagnosed by standard WHO criteria. IRF4/MUM1 immunohistochemical staining (IHC) was performed using the MUM1p antibody and considered positive if nuclear staining was present in >30% of tumor cell nuclei. Fluorescence in situ hybridization (FISH) was performed using breakapart probes constructed from bacterial artificial chromosome DNA that hybridized to loci flanking the IRF4 locus on 6p25.3. Cases with 3 or more fusion signals were considered to have extra copies of the IRF4 gene. In a subset of cases, PCR and Sanger sequencing was performed on DNA extracted from frozen PTCL tumor tissue with primer sets that amplified exons 1 to 10 of the IRF4 gene. Data were analyzed using the chi-square test or t test. ResultsPTCLs from 277 patients (175 M:102 F; median age, 61 y) were examined by both IHC and FISH. The subtype distribution was: PTCL, not otherwise specified (NOS), 93; anaplastic large cell lymphoma (ALCL), 89; angioimmunoblastic T-cell lymphoma (AITL), 27; cutaneous T-cell lymphoma (CTCL), 43; and cytotoxic PTCL, 25. IRF4/MUM1 was positive in 116 cases (42%). Extra copies of IRF4 were identified in 18 PTCLs (7%), 83% of which were IRF4/MUM1 protein-positive. In contrast, 39% of cases without extra copies of IRF4 were IRF4/MUM1 protein-positive (p=0.0002). The mean percentage of tumor cells with IRF4/MUM1 staining was 69% in cases with extra copies of IRF4, compared to 30% in cases without extra copies (p=0.0001). Extra copies of IRF4 were seen in 13% of ALCLs, 4% each of PTCLs, NOS and cytotoxic PTCLs, 2% of CTCLs, and 0% of AITLs (p=0.02). Two IRF4/MUM1-positive cases had an IRF4/TRA@ translocation (1%). We selected 30 IRF4/MUM1-positive PTCLs for Sanger sequencing. In this subset, we identified a previously undescribed non-synonymous variant in exon 5 (S309R) in 1 case (3%) without an extra copy of IRF4. ConclusionsExtra copies of IRF4 are seen in ∼7% of PTCLs, are significantly associated with IRF4/MUM1 expression, and are preferentially observed in ALCLs. Both IRF4/TRA@ translocations and IRF4 mutations are rare in PTCLs. These data suggest that genetic abnormalities of the IRF4 gene – specifically extra copies of IRF4 – may contribute to IRF4/MUM1 expression in some PTCLs. Since IRF4/MUM1 expression is seen in ∼42% of PTCLs, these data also imply the existence of other regulatory mechanisms. Characterizing these mechanisms may be important clinically, since IRF4/MUM1 has been proposed as a therapeutic target in human cancers but drugs that directly inhibit its function are currently not available. Disclosures:No relevant conflicts of interest to declare.

Measuring T cell receptor and T cell gene expression diversity in antigen-responsive human CD4+ T cells

T cells have diversity in TCR, epitope recognition, and cytokine production, and can be used for immune monitoring. Furthermore, clonal expansion of TCR families in disease may provide opportunities for TCR-directed therapies. We developed methodology for sequencing expressed genes of TCR alpha and beta chains from single cells and applied this to vaccine (tetanus-toxoid)-responsive CD4+ T cells. TCR alpha and beta chains were both successfully sequenced in 1309 (43%) of 3038 CD4+ T cells yielding 677 different receptors. TRAV and TRBV gene usage differed between tetanus-toxoid-responsive and non-responsive cells (p=0.004 and 0.0002), and there was extensive TCR diversity in tetanus-toxoid-responsive cells within individuals. Identical TCRs could be recovered in different samples from the same subject: TCRs identified after booster vaccination were frequent in pre-booster memory T cells (31% of pre-booster TCR), and also identified in pre-booster vaccination naïve cells (6.5%). No TCR was shared between subjects, but tetanus toxoid-responsive cells sharing one of their TCR chains were observed within and between subjects. Coupling single-cell gene expression profiling to TCR sequencing revealed examples of distinct cytokine profiles in cells bearing identical TCR. Novel molecular methodology demonstrates extensive diversity of Ag-responsive CD4+ T cells within and between individuals.

Pre-T cell receptor for improved expansion of TCR alpha disrupted T cells

Meeting abstracts Recent data have emerged from adoptive T-cell therapies where exogenous expression of a chimeric antigen receptor (CAR) has been shown to confer cancer recognition on autologous T cells. However, the ability to apply this technology in an allogeneic setting would permit the

Past Visits Present: TCF/LEFs Partner with ATFs for β-Catenin–Independent Activity

Past Visits Present: TCF/LEFs Partner with ATFs for β-Catenin–Independent Activity

Pairing of T‐cell receptor chains via emulsion PCR

Our ability to analyze adaptive immunity and engineer its activity has long been constrained by our limited ability to identify native pairs of heavy-light antibody chains and alpha-beta T-cell receptor (TCR) chains--both of which comprise coupled "halves of a key", collectively capable of recognizing specific antigens. Here, we report a cell-based emulsion RT-PCR approach that allows the selective fusion of the native pairs of amplified TCR alpha and beta chain genes for complex samples. A new type of PCR suppression technique was developed that makes it possible to amplify the fused library with minimal noise for subsequent analysis by high-throughput paired-end Illumina sequencing. With this technique, single analysis of a complex blood sample allows identification of multiple native TCR chain pairs. This approach may be extended to identify native antibody chain pairs and, more generally, pairs of mRNA molecules that are coexpressed in the same living cells.

TCR repertoire analysis of La/SS-B autoantigen-responsive peripheral blood CD4+ T cells using single-cell RT-PCR: a proof of principle case study (P3249)

Abstract TCR repertoire analysis in a variety of biological settings has largely consisted of TCR spectratyping of V-beta expression or V-specific antibody staining. Here, we combine in vitro stimulation of CFSE-labeled antigen specific T cells with a single cell nested multiplex RT-PCR to efficiently capture the unique T cell repertoire of autoantigen La/SS-B specific CD4+ T cells in a systemic lupus erythematosus and Sjögren’s syndrome patient with precipitating levels of anti-La and Ro antibodies. CFSE-labeled peripheral blood cells were cultured with either purified La protein or a mixture of human La peptides, and CD4+CFSElow cells were single sorted into PCR plates. TCR alpha and beta chain cDNAs were amplified and sequenced. The efficiency of paired TCR chain recovery was 60-65%, and was similar between the populations analyzed. While the magnitude of proliferative responses to the two types of La antigen-specific stimuli were similar, the responding T-cells displayed little overlap in TCR sequence. The sequences derived from the peptide pool-stimulated CD4+ cells were pauci-clonal, with 2 clones dominating more than half the response (58/93 cells). Conversely, the purified protein-responsive CD4+ TCRs were distributed among multiple clonotypes. Our approach maps the T cell repertoire of human antigen responsive cells and may have great utility for elucidating the properties of a variety of antigen-specific T cells in diverse clinical settings.

Narrowing of influenza A virus specific T-cell receptor alpha and beta repertoire with increasing age (P6176)

Abstract Alterations in memory CD8 T cell responses may contribute to the high morbidity and mortality caused by seasonal influenza A virus (IAV) infections in older individuals. We questioned whether memory CD8 responses changed overtime with increasing age to this non-persistent virus, to which recurrent exposure with new strains is common. Here, for the first time we show that with age the HLA-A2-restricted IAV M158-66-specific Vα T cell repertoire was significantly narrowed leading to oligoclonal expansions including the usage of a single identical VA12 clonotype in all 8 older donors. The VA repertoire of older individuals also had longer CDR3 regions with increased usage of alanine/glycine runs which may enhance TcR promiscuity. Collectively these results suggest that CD8 memory responses in humans to non-persistent viruses like IAV are dynamic, and with aging become narrower with preferential retention of T cell repertoires with features of enhanced promiscuity.

A genome‐wide association study of recipient genotype and medium‐term kidney allograft function

We examined, through genome-wide association studies (GWAS), the correlation between recipient genetic variation and renal function at fiveyr. Our cohort contained 326 Irish, first time, kidney-only, deceased donor, transplant recipients on calcineurin inhibitors (263 had a functioning graft at fiveyr) between 1993 and 2002. Outcomes were creatinine at fiveyr and long-term graft function. Two variants were identified showing borderline genome-wide significance - one on chromosome 18 (p=4.048e-08, rs6565887) and another on chromosome 14 (p=7.631e-08, rs3811321). Individually, the two SNPs explained up to 8.8% and 11.29% of five-yr creatinine variance, respectively, while together they explained up to 17.4% of trait variance. Both variants were predictors of long-term allograft function (p=0.004, 70% vs 30% survival at 10yr). The chromosome 14 variant is located in the intergenic region of the T-Cell Receptor Alpha locus. Using a genome-wide approach, we have identified two associations with five-yr creatinine levels in renal transplant recipients treated with calcineurin inhibitors. Independent replication is now warranted to clarify the clinical significance of these results.

Down-regulation of Macrophage Immune Activity by Natural CD8+ Regulatory T Cells*

To evaluate the influence of natural regulatory CD8+ T cells on macrophages we investigated in vitro production of cytokines, reactive oxygen intermediates (ROIs) and expression of CD80 surface costimulatory molecules by macrophages (MF) of wild type (WT) B10PL and syngeneic knock-out (KO) strains, TCRalpha-/-, beta2m-/- and CD1d-/- mice. MF of TCR alpha-/- (CD4- and CD8-) and beta2m-/- (CD8-) animals produced higher levels ofTNF-a, IL-6, IL-12 and ROIs and showed increased expression of CD80 costimulatory molecules in comparison to MF of WT or CD1d-/- (NKT-) mice. When MF of these strains were conjugated with TNP hapten and injected i.v. into WT mice to test either induction of contact sensitivity (CS) or tolerance, only TNP-MF of TCRalpha-/- and beta2m-/- animals induced a significant CS reaction, while cells of WT and CD1d-/- strains were tolerogenic. MF of the tested strains can be classified functionally as resembling either proinflammatory (TCRalpha-/- and beta2m-/-mice) or immunosuppressive (WT and CD1d-/-) phenotypes. We suggest the presence of an in vivo regulatory loop in which innate CD8+ Treg cells control the transition between MF phenotypes and thus adjust the magnitude of the inflammatory response to strictly local requirements.

Molecular and Functional Analysis of Peripheral Lymphocytes in Churg Strauss Syndrome Reveals Several Monoclonal Expansions of CD8+ Cells with a Th1/Proinflammatory Profile

AbstractAbstract 1051Churg Strauss Syndrome (CSS) is a systemic small-vessel necrotizing vasculitis characterized by asthma, chronic rhinosinusitis, pulmonary infiltrations, eosinophil-rich inflammation, vascular and extra-vascular granulomas. We recently documented in the peripheral blood of CSS patients the presence of one or two numerically expanded V-Beta (V-B) families of CD8+ lymphocytes with an effector memory phenotype and with a monoclonal or oligoclonal T-cell receptor (TCR), as shown by TCR-γ rearrangement analysis.To define the characteristics of numerically expanded families at a molecular level, we purified the expanded V-B family of two CSS patients and cloned purified T-cells by limiting dilution. Ten clones from each patient were randomly selected for molecular analysis and the Complementarity-Determining Region 3 (CDR3) of their TCR alpha e beta chain was sequenced. In each patient, 9/10 clones had an identical CDR3 sequence. We also amplified by PCR the cDNA obtained from whole PBMC with specific primers for the expanded V-B family of the same patients (i.e. V-B1 and V-B8) and sequenced the PCR product. The sequence obtained from the entire V-B family (as detected by PCR amplification from whole PBMC) was identical to that obtained from each T-cell clone. These data indicate the presence of a dominant clonotype within a numerically expanded V-B family, which is detectable by molecular analysis not only of single cells but also of whole PBMC.To compare the TCR V-B diversity of CSS patients with healthy donors (HD), we performed TCR-CDR3 length analysis by spectratyping of purified PBMC, CD4 and CD8 cells in CSS patients and in age-matched HD. Analysis was performed by determining the complexity score of 22 V-B families in 8 patients and 8 HD. The TCR repertoire of CSS circulating T-cells was skewed (i.e. with a significantly lower complexity score than HD) and this skewing was found exclusively within the CD8+ subset. Therefore, the CD8+ repertoire of CSS patients is composed by one or two TCR V-B families that are numerically expanded and by several other V-B families that have a skewed spectrum, but are not numerically expanded.In one patient, we also analyzed the cDNA of 11 different V-B families with a low complexity score on a polyacrylamide gel. Five discrete bands were detected, that were purified and sequenced. Sequences from 2/5 PCR-amplified V-B families were readable and representative of a productive and clonal VDJ rearrangement of the CDR3 region. This confirmed that a skewed V-B family at spectratyping is very often the result of an antigen-driven proliferation of a single cell with a distinct TCR.These molecular data revealed that alterations of the T-cell repertoire in CSS patients are more profound than initially expected, involving many CD8+ V-B families. We hypothesized that functional analysis of the whole CD8+ fraction could reveal the main functional characteristics of the several clonal expansions observed within this subset. We therefore analyzed cytokines and chemokine receptor expression of CD8+ and CD4+ cells from the same CSS patients and HD. A significantly higher percentage of both CD8+ and CD4+ cells producing INFγ and TNFα was observed in CCS compared to HS. IL1β, IL2, IL4, IL6, IL13, IL17, GM-CSF, and TGF-β in CD4+ and CD8+ cells were not different between CSS patients and controls, even if a trend toward a higher expression of IL-13 was observed. CD4+ cells, but not CD8+ cells, from CSS patients had a significantly higher production of IL5 and IL10 than HS. These data suggested that patients’ CD8+ cells have a Th1/proinflammatory profile, while the functional profile of CD4+ T-cells is less clearly polarized. Expression of CCR5 and, to a lesser extent, of CXCR3 by total and Vβ-expanded CD8+ cells was significantly higher in CSS than in HS. Expression of CRTH2, but not of CCR4, was significantly higher in CD4+ cells of CSS patients than in controls.In conclusion, the molecular profiling of T-cells revealed that monoclonal expansion of CD8+ lymphocytes in CSS patients are frequent and involve several V-B families. CD8+ cells of patients have a Th1/proinflammatory profile, potentially involved in vasculitic damage with the cooperation of CD4+/Th1 cells. CD4+/Th2 cells (but not CD8+ cells) are functionally apt to mediate eosinophil-rich inflammation and asthma. Disclosures:No relevant conflicts of interest to declare.

Autoreactive T Cells From Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) Specifically Recognize Glycosyl- Phosphatidyl-Inositol (GPI)

AbstractAbstract 647PNH is caused by somatic mutations in the PIG-A gene in hematopoietic stem cells leading to disruption of the synthesis of GPI, a glycolipid used for post-translational modification of proteins. PNH is classically characterized by the triad of intravascular hemolysis, severe acquired thrombophilia and multiple cytopenias. Whereas the first two components are the consequence of increased susceptibility to activated complement, due to the deficiency on red cells of GPI-linked CD55 and CD59, cytopenias are an expression of bone marrow failure. This fact, together with strong clinical evidence associating PNH with aplastic anemia, has led to the notion that a T cell-mediated attack underlies both conditions whereby in PNH the PIG-A mutant clone is able to escape the attack. Consistent with this, we previously found skewing of the spectratyping pattern of T cells, and recurrent TCR beta chain sequences. Here, using complementary assays, we tested the hypothesis that PIG-A mutant cells escape an auto-immune T cell attack targeted to the GPI molecule itself, which would be presented by CD1d.First, CD8+ T cells from PNH patients and from normal donors were co-cultured with C1R-CD1d B cells pulsed with either trypanosomal GPI (t-GPI) or with solvent. Reactive T cells producing interferon gamma (IFNG) were then enumerated by ELISPOT technology. We found that in the presence of t-GPI-pulsed C1R-CD1d cells, the frequency of reactive T cells was significantly increased (up to 5-fold) in patients on each of days 1, 7, 14 but not in controls. To confirm that T cell reactivity to GPI depended on the presence of CD1d, when similar experiments were carried out with the untransfected, CD1d-negative C1R cells, no such increase was seen. Similar results were obtained using as antigen presenting cells (APC) K562 cells and appropriate derivatives.Next, we used for antigen presentation, in addition to the C1R-CD1d cell line, a derivative that lacks the ability to synthesize GPI (GPI- C1R-CD1d). In these experiments, reactive (IFNG-producing) T cells were counted by intracellular staining and flow-cytometry:in PNH patients, but not in controls, we found a 3-fold increase in the frequency of reactive CD8+ T cells when the APC were GPI+ C1R-CD1d compared to when they were GPI- C1R-CD1d. This increase was not seen when APC were the C1R cells. By contrast, with GPI- C1R-CD1d the increase was restored by the addition of exogenous human GPI (h-GPI).Next, we tested the reactivity of T cells from 11 PNH patients using as APC mature dendritic cells (DC) obtained from autologous peripheral blood monocytes. By selecting CD14-neg cells – that are the majority in PNH patients - we took advantage of the fact that in this way we had APC that were naturally GPI negative. In the absence of added GPI we could not demonstrate reactive T cells; however, in 7 out of 11 patients there was a significant increment when the DC were loaded with h-GPI (p =0,0149; Mann-Whitney test).Since CD1d is required for development and function of iNKT cells, immunoregulatory, glycolipid-specific T cells characterized by an invariant TCRValpha24Jalpha18 chain, we tested the hypothesis that CD1d-restricted, GPI-specific T cells might also possess an invariant TCR alpha chain. Global TCR alpha chain repertoire analysis of flow-sorted dimer CD1d/GPI+ T cells identified a novel TCRValpha21Jalpha31 invariant chain (9/9 clones) in 1 out of 3 patients tested. Further analysis of the TCRValpha21 repertoire of patients and controls by next generation sequencing (GS Junior, Roche) revealed that the invariant TCRValpha21Jalpha31 chain was present but not expanded in 5/8 controls (frequency<0.5% of TCRValpha21-Calpha T cell repertoire); by contrast, it was expanded in 6/11 patients (median frequency: 1.86%, range: 1.05–75% of TCRValpha21-Calpha T cell repertoire).In conclusion, we provide the first direct evidence that GPI-specific, CD1d-restricted T cells, enriched in a novel invariant TCR alpha chain sequence, are increased in patients with PNH but not normal controls. Disclosures:No relevant conflicts of interest to declare.

Gene expression profile suggests that pigs (Sus scrofa) are susceptible to Anaplasma phagocytophilum but control infection

BackgroundAnaplasma phagocytophilum infects a wide variety of hosts and causes granulocytic anaplasmosis in humans, horses and dogs and tick-borne fever in ruminants. Infection with A. phagocytophilum results in the modification of host gene expression and immune response. The objective of this research was to characterize gene expression in pigs (Sus scrofa) naturally and experimentally infected with A. phagocytophilum trying to identify mechanisms that help to explain low infection prevalence in this species.ResultsFor gene expression analysis in naturally infected pigs, microarray hybridization was used. The expression of differentially expressed immune response genes was analyzed by real-time RT-PCR in naturally and experimentally infected pigs. Results suggested that A. phagocytophilum infection affected cytoskeleton rearrangement and increased both innate and adaptive immune responses by up regulation of interleukin 1 receptor accessory protein-like 1 (IL1RAPL1), T-cell receptor alpha chain (TCR-alpha), thrombospondin 4 (TSP-4) and Gap junction protein alpha 1 (GJA1) genes. Higher serum levels of IL-1 beta, IL-8 and TNF-alpha in infected pigs when compared to controls supported data obtained at the mRNA level.ConclusionsThese results suggested that pigs are susceptible to A. phagocytophilum but control infection, particularly through activation of innate immune responses, phagocytosis and autophagy. This fact may account for the low infection prevalence detected in pigs in some regions and thus their low or no impact as a reservoir host for this pathogen. These results advanced our understanding of the molecular mechanisms at the host-pathogen interface and suggested a role for newly reported genes in the protection of pigs against A. phagocytophilum.

Narrowing of influenza A virus specific T-cell receptor alpha and beta repertoire with increasing age (105.46)

Abstract Alterations in memory CD8 T cell responses may contribute to the high morbidity and mortality caused by seasonal influenza A virus (IAV) infections in older individuals. We questioned whether memory CD8 responses changed overtime with increasing age. Here, for the first time we show that the HLA-A2-restricted IAV M158-66-specific Vα T cell repertoire was significantly narrowed leading to oligoclonal expansions including the usage of a single identical VA12 clonotype. This was the case for all 8 older donors. The VA repertoire of older individuals also had longer CDR3 regions with increased usage of alanine/glycine runs which may enhance TCR promiscuity. Collectively these results suggest that CD8 memory responses in humans to non-persistent viruses like IAV are dynamic, and become narrower with preferential retention of T cell repertoires with features of enhanced promiscuity with age.

Simultaneous Translocation of Both TCR Loci (14q11) with Rare Partner Loci (Xq22 and 12p13) in a Case of T-lymphoblastic Leukemia

The most common recurrent cytogenetic abnormalities in T-lymphoblastic leukemia (T-acute lymphoblastic leukemia [T-ALL]) involve T-cell receptor (TCR) loci and a variety of partner genes, including HOX11, HOX11L2, MYC, and TAL1. In this report, we present a rare case involving simultaneous translocation of the TCR α/δ loci with different partner loci (Xq22 and 12p13); this resulted in a poor prognosis. Chromosomal analysis showed 46,Y,t(X;14)(q22;q11.2),t(12;14)(p13;q11.2) and FISH analysis by using a T-cell receptor alpha delta DNA probe, Split Signal (DakoCytomation, Denmark), showed translocations at the same TCR α/δ locus on both chromosomes. FISH with 2 bacterial artificial chromosome clones showed break apart signal, which suggests involvement of the IRS4 gene. To our knowledge, this is the first report of T-ALL in which both TCR α/δ loci were translocated with different partner loci, and 1 of the partner loci, Xq22, was a rare translocation partner locus that included IRS4 gene.

Chitin microparticles for the control of intestinal inflammation

Chitin is a polymer of N-acetylglucosamine with the ability to regulate innate and adaptive immune responses. However, the detailed mechanisms of chitin-mediated regulation of intestinal inflammation are only partially known. In this study chitin microparticles (CMPs) or phosphate-buffered saline (PBS) were orally administered to acute and chronic colitis models every 3 days for 6 consecutive weeks beginning at weaning age. The effects of this treatment were evaluated by histology, cytokine production, coculture study, and enteric bacterial analysis in dextran sodium sulfate (DSS)-induced colitis or T-cell receptor alpha (TCRα) knockout chronic colitis models. Histologically, chitin-treated mice showed significantly suppressed colitis as compared with PBS-treated mice in both animal models. The production of interferon-gamma (IFN-γ) was upregulated in the mucosa of chitin-treated mice compared with control mice. The major source of IFN-γ-producing cells was CD4+ T cells. In mouse dendritic cells (DCs) we found that CMPs were efficiently internalized and processed within 48 hours. Mesenteric lymph nodes (MLNs) CD4+ T cells isolated from chitin-treated mice produced a 7-fold higher amount of IFN-γ in the culture supernatant after being cocultured with DCs and chitin as compared with the control. Proliferation of carboxyfluorescein succinimidyl ester (CFSE)(low) CD4+ T cells in MLNs and enteric bacterial translocation rates were significantly reduced in chitin-treated mice when compared with the control. In addition, CMPs improved the imbalance of enteric bacterial compositions and significantly increased interleukin (IL)-10-producing cells in noninflamed colon, indicating the immunoregulatory effects of CMPs in intestinal mucosa. CMPs significantly suppress the development of inflammation by modulating cytokine balance and microbial environment in colon.

Thymus and immune reconstitution after allogeneic hematopoietic stem cell transplantation in humans: never say never again

Assessment of the host immune status is becoming a key issue in allogeneic hematopoietic stem cell transplantation (allo-HSCT). In the long-term follow-up of these patients, severe post-transplant infections, relapse or secondary malignancies may be directly related to persistent immune defects. In allo-HSCT, T-cell differentiation of donor progenitors within the recipient thymus is required to generate naive recent T-cell emigrants (RTE). These cells account for a durable T-cell reconstitution, generating a diverse T-cell receptor (TCR) repertoire and robust response to infections. It is now possible to quantify the production of RTE by measuring thymic T-cell receptor excision circles or 'TREC' which are small circular DNA produced during the recombination of the genomic segments encoding the TCR alpha chain. Here we discuss the role of thymic function in allo-HSCT. The pre-transplant recipient thymic function correlates with clinical outcome in terms of survival and occurrence of severe infections. Post-transplant, TREC analysis showed that the thymus is a sensitive target to the allogeneic acute graft-versus-host disease (GvHD) reaction but is also prone to recovery in young adult patients. In all, thymus is a key player for the quality of immune reconstitution and clinical outcome after allo-HSCT. Thymic tissue is plastic and it is a future challenge to halt or reverse thymic GVHD therapeutically by acting at the level of T-cell progenitors generation, thymic homing and/or epithelial thymic tissue preservation.

TRAV gene expression in PBMCs and TILs in patients with breast cancer analyzed by a DNA melting curve (FQ-PCR) technique for TCR alpha chain CDR3 spectratyping

To explore the expression of the TRAV gene in peripheral blood mononuclear cells (PBMCs) and in tumor-infiltrating lymphocytes (TILs) in the patients with breast cancer using a DNA melting curve (FQ-PCR) technique for T cell receptor (TCR) alpha chain CDR3 spectratyping. Peripheral blood samples and tissue samples were obtained from thirty breast cancer patients. Total RNA was extracted from PBMCs and tumor tissues and then reverse transcribed into cDNA. FQ-PCR was used to amplify the human TCR alpha chain CDR3 region with the primers to the TRAV and TRAC genes. TCR alpha chain CDR3 spectratyping and partial CDR3 sequencing were used to determine use of TRAV gene product in T cell responses. TCR alpha CDR3 spectratyping showed preferential usage of certain TRAV genes in the PBMCs and TILs of all patients with breast cancer. The frequencies of TRAV1.1, TRAV9, and TRAV29 exceeded 30% in PBMCs and the frequencies of TRAV1.1 and TRAV22 exceeded 30% in TILs. More than three quarters of the patients (23/30) overexpressed the same gene in both PBMCs and TILs; for example, patient-1 highly expressed TRAV9 in the PBMCs and TILs. Patients with positive or negative tumor markers of estrogen receptor (ER), progesterone receptor (PR), pS2, C-erbB-2, nm23, P53, and Ki-67 showed no significant common TRAV gene expression, but some TRAV gene preferential usage frequencies exceeded 20%. For example, five of seven patients positive for ER had high levels of expression of TRAV1.1 and TRAV3. Finally, the amino acid sequence of TCR CDR3 region showed some common motifs in some of the patients. TRAV gene expression was complex and diverse in the patients with breast cancer. The TRAV gene usage may be closely related to the diversity of breast tumor antigens and the differential immune responses observed in individual patients. Research into the immunological mechanism of T cells may provide guidance for individual T cell-directed therapy for breast cancer.

A remarkable effect of alemtuzumab in a patient suffering from narcolepsy with cataplexy

We made a remarkable clinical observation in a patient suffering from narcolepsy with cataplexy that may add to the understanding of the pathophysiology of this disorder. Our patient, born in 1929, developed all classical symptoms of narcolepsy in 1946, and came to our attention in 1997. Cataplectic attacks were present on a daily basis, mostly partial, involving the face, neck and knees. Typical triggers were making witty remarks and laughter. He was overweight and used no medication because various treatments in the past had been ineffective or not tolerated. Nocturnal polysomnography showed fragmentation but no co-morbid disorders, and the multiple sleep latency test gave a mean sleep latency of 2.1 min and two SOREMs. He was HLA DQB1-0602*positive, and hypocretin-1 was undetectable in the cerebrospinal fluid. He refused to try any newly available treatment. We contacted him irregularly for participation in research protocols. In 2008, he developed a low grade T-cell lymphoma. A year later, he informed us that he had started treatment with alemtuzumab several months before, after which he had observed a complete absence of his cataplectic attacks, whereas prior to this treatment they had been present on a daily basis. The diagnosed lymphoma and initial therapy with methotrexate had not had any influence on the frequency, duration or pattern of his cataplectic attacks. He stated that as far as joking and witty remarks were concerned, his life had not changed. His wife who had observed his attacks for decades confirmed this. All other narcoleptic symptoms remained unchanged with alemtuzumab. His cataplexy did not return during the 1.5 years he was treated with alemtuzumab, until his death. Narcolepsy with cataplexy is considered to be an autoimmune disorder with T-cell involvement, although there is no final proof for this hypothesis. The main arguments are: the tight association with HLA (Mignot et al., 2001); the presence of HLA DQB1*0602 that is almost conditional; and a trans DRB1*1301-DQB1*0603 haplotype that is highly protective (Hor et al., 2010); the identification of Trib2 reactive autoantibodies; the association with polymorphisms in the T-cell receptor alpha locus in a genome-wide association study