Vβ Gene Research Articles

T Cell Receptor Clonotype Influences Epitope Hierarchy in the CD8+ T Cell Response to Respiratory Syncytial Virus Infection

CD8+ T cell responses are important for recognizing and resolving viral infections. To better understand the selection and hierarchy of virus-specific T cell responses, we compared the T cell receptor (TCR) clonotype in parent and hybrid strains of respiratory syncytial virus-infected mice. K(d)M2(82-90) (SYIGSINNI) in BALB/c and D(b)M(187-195) (NAITNAKII) in C57Bl/6 are both dominant epitopes in parent strains but assume a distinct hierarchy, with K(d)M2(82-90) dominant to D(b)M(187-195) in hybrid CB6F1/J mice. The dominant K(d)M2(82-90) response is relatively public and is restricted primarily to the highly prevalent Vβ13.2 in BALB/c and hybrid mice, whereas D(b)M(187-195) responses in C57BL/6 mice are relatively private and involve multiple Vβ subtypes, some of which are lost in hybrids. A significant frequency of TCR CDR3 sequences in the D(b)M(187-195) response have a distinct "(D/E)WG" motif formed by a limited number of recombination strategies. Modeling of the dominant epitope suggested a flat, featureless structure, but D(b)M(187-195) showed a distinctive structure formed by Lys(7). The data suggest that common recombination events in prevalent Vβ genes may provide a numerical advantage in the T cell response and that distinct epitope structures may impose more limited options for successful TCR selection. Defining how epitope structure is interpreted to inform T cell function will improve the design of future gene-based vaccines.

Complementarity-Determining Region 3 Size Spectratypes of T Cell Receptor β Chains in CD8 + T Cells following Antiviral Treatment of Chronic Hepatitis B

An increased CD8(+) T cell response to hepatitis B virus (HBV) peptides occurs between 12 and 24 weeks after starting antiviral therapy for chronic hepatitis B. It is not known whether these cells have antiviral function. The aim of this study was to determine whether clonal expansions of CD8(+) T cells at these time points predict the virological response to therapy. Peripheral blood CD8(+) T cells were obtained from 20 patients treated with lamivudine or telbivudine for chronic hepatitis B at baseline, 12 weeks, and 24 weeks. The CDR3 spectratype of each T cell receptor (TCR) β chain variable region (Vβ) gene family was analyzed, and the changes in the numbers of Vβ families with clonal expansions were compared in subjects with (n = 12) and without (n = 8) a virological response (52 week HBV DNA < 300 copies/ml). The number of CD8(+) TCR Vβ families with clonal expansions at 12 weeks relative to baseline (median [10th to 90th percentile], +2.5 [0 to +7] versus +1 [0 to +2], P = 0.03) and at 24 weeks relative to 12 weeks (+1 [0 to +2] versus -1 [-3 to +4], P = 0.006) was higher in subjects with a virological response versus subjects without a virological response, as were interleukin-2 (IL-2) but not IL-21 mRNA levels in peripheral blood mononuclear cells. The duration of new expansions at 12 weeks was higher (P < 0.0001) in responders. Increased numbers of CD8(+) T cell expansions after antiviral therapy are associated with a virological response to treatment. These CD8(+) T cells are a potential target for a therapeutic vaccine for chronic hepatitis B.

Non-equilibrium and differential function between intraepithelial and lamina propria virus-specific TCRαβ+ CD8αβ+ T cells in the small intestinal mucosa

The gastrointestinal mucosa regularly encounters commensal and pathogenic microbiota. Gut mucosal lymphocytes consist of two phenotypically different populations residing in the intestinal intraepithelial (IEL) compartment and lamina propria (LP). Little is known about compositional and functional differences of antigen-specific T cells from these mucosal compartments after mucosal infection, or the degree of trafficking between them. We here studied the B8R20−27-specific CD8 T-cell response in LP and IEL compartments after intrarectal immunization with modified vaccinia virus Ankara (MVA). CD8+ T cells in the IEL compartment had much lower avidity than in the LP or spleen during acute and memory phases. Surprisingly, the TCR Vβ-chain distribution of antigen-specific T cells and the length of the CDR3 region of the dominant Vβ genes showed substantial dissimilarities between IEL and LP antigen-specific CD8αβ T cells in individual mice, increasing with time. We show functional and compositional differences between these mucosal compartments during the effector and memory phases of the immune response, indicating limited crosstalk and microenvironmental differences between the IEL, LP, and spleen. The restricted migration of cells from each of these mucosal compartments could partly account for a founder effect we observed in the IEL TCRαβ CD8αβ epitope-specific repertoire that might impact protective efficacy.

Diverse Cross-Reactive Potential and Vβ Gene Usage of an Epitope-Specific Cytotoxic T-Lymphocyte Population in Monkeys Immunized with Diverse Human Immunodeficiency Virus Type 1 Env Immunogens

An ideal human immunodeficiency virus type 1 (HIV-1) vaccine would elicit potent cellular and humoral immune responses that recognize diverse strains of the virus. In the present study, combined methodologies (flow cytometry, Vbeta repertoire analysis, and complementarity-determining region 3 sequencing) were used to determine the clonality of CD8(+) T lymphocytes taking part in the recognition of variant epitope peptides elicited in Mamu-A*01-positive rhesus monkeys immunized with vaccines encoding diverse HIV-1 envelopes (Envs). Monkeys immunized with clade B Envs generated CD8(+) T lymphocytes that cross-recognized both clade B- and clade C-p41A epitope peptides using a large degree of diversity in Vbeta gene usage. However, with two monkeys immunized with clade C Env, one monkey exhibited p41A-specific cytotoxic T-lymphocytes (CTL) with the capacity for cross-recognition of variant epitopes, while the other monkey did not. These studies demonstrate that the cross-reactive potential of variant p41A epitope peptide-specific CTL populations can differ between monkeys that share the same restricting major histocompatibility complex class I molecule and receive the same vaccine immunogens.

Restricted Autoantigen Recognition Associated with Deletional and Adaptive Regulatory Mechanisms

Autoimmune diabetes (T1D) is characterized by CD4(+) T cell reactivity to a variety of islet-associated Ags. At-risk individuals, genetically predisposed to T1D, often have similar T cell reactivity, but nevertheless fail to progress to clinically overt disease. To study the immune tolerance and regulatory environment permissive for such autoreactive T cells, we expressed TCR transgenes derived from two autoreactive human T cells, 4.13 and 164, in HLA-DR4 transgenic mice on a C57BL/6-derived "diabetes-resistant" background. Both TCR are responsive to an immunodominant epitope of glutamic acid decarboxylase 65(555-567), which is identical in sequence between humans and mice, is restricted by HLA-DR4, and is a naturally processed self Ag associated with T1D. Although both TCR use the identical Valpha and Vbeta genes, differing only in CDR3, we found stark differences in the mechanisms utilized in vivo in the maintenance of immune tolerance. A combination of thymic deletion (negative selection), TCR down-regulation, and peripheral activation-induced cell death dominated the phenotype of 164 T cells, which nevertheless still maintain their Ag responsiveness in the periphery. In contrast, 4.13 T cells are much less influenced by central and deletional tolerance mechanisms, and instead display a peripheral immune deviation including differentiation into IL-10-secreting Tr1 cells. These findings indicate a distinct set of regulatory alternatives for autoreactive T cells, even within a single highly restricted HLA-peptide-TCR recognition profile.

T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system

Multipotent hematopoietic cell line EML can differentiate into myeloid, erythroid, megakaryocytic, and B-lymphoid lineages, but it remained unknown whether EML cells have T-cell developmental potential as well. The goal of this study was to determine whether the coculture with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) could induce differentiation of EML cells into T-cell lineage. EML cells were cocultured with control OP9 or OP9-DL1 stromal cells in the presence of cytokines (stem cell factor, interleukin-7, and Fms-like tyrosine kinase 3 ligand). Their T-cell lineage differentiation was assessed through flow cytometry and reverse transcription polymerase chain reaction expression analysis of cell surface markers and genes characterizing and associated with specific stages of T-cell development. The phenotypic, molecular, and functional analysis has revealed that in EML/OP9-DL1 cocultures with cytokines, but not in control EML/OP9 cocultures, EML cell line undergoes T-cell lineage commitment and differentiation. In OP9-DL1 cocultures, EML cell line has differentiated into cells that 1) resembled double-negative, double-positive, and single-positive stages of T-cell development; 2) initiated expression of GATA-3, Pre-Talpha, RAG-1, and T-cell receptor-Vbeta genes; and 3) produced interferon-gamma in response to T-cell receptor stimulation. These results support the notion that EML cell line has the capacity for T-cell differentiation. Remarkably, induction of T-lineage gene expression and differentiation of EML cells into distinct stages of T-cell development were very similar to previously described T-cell differentiation of adult hematopoietic stem cells and progenitors in OP9-DL1 cocultures. Thus, EML/OP9-DL1 coculture could be a useful experimental system to study the role of particular genes in T-cell lineage specification, commitment, and differentiation.

The T-cell receptor Vβ gene repertoire and clonal expansion from peripheral blood T cells in benzene-exposed workers in China

The toxicity of benzene to lymphocytes is well known; however, little is known about the variety of T-cells that are rearranged after benzene exposure. In order to further understand the relative toxicity of benzene on T-cells, we investigated the features of the complementarity determining region 3 (CDR3) of T cells from benzene exposed workers. CDR3 of the T-cell receptor variable β-chain (TCRVβ) subfamily genes were amplified using reverse transcriptase-polymerase chain reaction. The polymerase chain reaction products were further subjected to genescan analysis to evaluate the clonality of T cells. Results revealed 2–12 Vβ subfamilies identified in 16 benzene-exposed workers. The number of detectable Vβ subfamilies present in the benzene-exposed group was significantly lower than in the control group (p<0·05). The most frequently used Vβ genes were Vβ3, Vβ21, Vβ6, and Vβ16. We observed a skewed distribution and clonal expansion of TCR Vβ subfamilies in benzene-exposed workers.

Selective expansion of autoreactive T cell clonotypes drives autoimmune diabetes (137.9)

Abstract It is well established that T cells are the primary mediators of β cell destruction in Type 1 diabetes (T1D). However, the relative role for different β cell-specific T cell clonotypes in the progression of the diabetogenic response, and the molecular basis for their expansion remain poorly understood. CD4+ T cells expressing the BDC2.5 clonotypic T cell receptor (TCR) and/or which recognize the mimetic BDC (mBDC) peptide are thought to have a key albeit undefined role in β cell autoimmunity of the nonobese diabetic (NOD) mouse. Accordingly, using sIAg7-mBDC multimers, BDC2.5 clonotypic CD4+ T cells were tracked and/or isolated in a temporal manner and analyzed at the single cell level. sIAg7-mBDC-binding (g7-mBDC+) CD4+ T cells were detected in peripheral blood lymphocytes (PBL) and the islets at the onset of β cell autoimmunity in NOD female mice, with the frequency and/or number of g7-mBDC+ T cells increasing with age. A comparison of TCR gene usage by single, g7-mBDC+ T cells isolated from 16 week-old NOD female mice demonstrated dominate usage of TRBV15 and monoclonality based on variable β (Vβ) gene complementary determinant region 3 (CDR3) sequences of BDC2.5 clonotypic CD4+ T cells in the islets but not PBL. Furthermore, the analysis of TRBV15+ g7-mBDC+ BDC T cells revealed the biased TRAV13 Vα gene usage. Furthermore, diabetes could be transferred into NOD.scid recipients by g7-mBDC+ T cells purified from the islets but not from PBL. These data demonstrate that g7-mBDC+ T cells are an early indicator and one of the main mediator of the development of destructive insulitis, and that both clonotypic expansion and preferential usage of TCR characterize islet infiltrating g7-mBDC+ T cells.

Identification of Idiotype TCR Vα6, Vα10 and Vβ23 Relate to APL-Associated Antigen

Differentiation therapy with all-trans retinoic acid in combination with chemotherapy has significantly improved survival in patient with acute promyelocytic leukaemia (APL). However, relapse remains a major problem. In recent years, specific adoptive immunotherapy with tumor-specific T-cells was considered as a new approach to malignancy therapy. Recently, researches are focus on searching the generation of effective tumor antigen specific T-cells. The tumor antigen specific T cells could be determined by analysis of the T-cell receptor (TCR) Vα or Vβ repertoire, which display monoclonal or oligoclonal pattern. In order to develop the specific cellular immunotherapy in APL, in this present study, we analysis the clonality and the pattern of CDR3 sequence of TCR Vα and Vβ repertoire in peripheral blood T cells by RT-PCR and genescan technique, which was expected to define the APL associated TCR Vα or Vβ subfamily T cells. Oligoclonal T cells expressed TCR Vα6, Vα10 or Vβ23 genes were identified from one APL patient. The molecular characteristics of the CDR3 of Vα and Vβ genes rearrangement were Vα6-N-Jα17, Vα10-N-Jα35 and Vβ23-N-Dβ1-NJβ2.7. And the amino acid sequence of CDR3 region in TCR Vα6, Vα10 and Vβ23 subfamilies were CAMRENSAGNK, YLCAGDELLWECA or CASSSKWAGGTYEQY, respectively. These sequences were accepted by GenBank (Accession Number EU544946, EU544947 and EU647219). In conclusions, three idiotype Vα and Vβ sequences were identified in a case with APL, which was thought that may mediate the host specific immunity response for leukemia cells in APL. Farther investigation will be performed on construction the vector containing the antigen specific TCRs for gene transfer to establish TCR modified-T cells for specific immunotherapy of APL.

Monoclonality and oligoclonality of T cell receptor β gene in angioimmunoblastic T cell lymphoma

Angioimmunoblastic T cell lymphoma (AILT) is an aggressive T cell lymphoma with an incidence of approximately 1–2% of all non-Hodgkin lymphoma. The detection of clonal T cell receptor (TCR) gene...

E proteins are required to activate germline transcription of the TCR Vβ8.2 gene

Each TCR Vbeta gene is regulated by an individual Vbeta promoter, which becomes active prior to V(D) J recombination and drives germline transcription. It has been shown that Vbeta gene locus activation and recombination are dependent on the Vbeta promoter. However, transcription factors that regulate Vbeta germline transcription remain largely undefined. A major challenge in studying Vbeta gene germline transcription is the quantitative assessment of relatively low-level transcripts in T-cell progenitors. Here we used the established Vbeta8.2(CD2) knock-in mouse model to assess functions of E-protein transcription factors in Vbeta8.2 germline transcription. We show that E proteins are required for the activation but not the maintenance of the Vbeta8.2 germline transcription during thymocyte development. The activation of Vbeta8.2 germline transcription depends more on the E proteins encoded by the E2A gene than by the HEB gene. We further show that IL-7 receptor (IL-7R)-mediated signals are essential for Vbeta8.2 germline transcription. We provide evidence that IL-7R expression is only partially controlled by E2A, suggesting a role for E2A in driving Vbeta8.2 germline transcription independent of IL-7R activation.

Analysis of the interindividual conservation of T cell receptor α- and β-chain variable regions gene in the peripheral blood of patients with systemic lupus erythematosus

The aim of this study was to find conserved motifs in specific T cell receptor (TCR) alpha- and beta-chains, and to analyse the association between complementarity determining region 3 (CDR3) spectratype and systemic lupus erythematosus (SLE) activity. TCR alpha-and beta-chain CDR3 spectratypes were analysed in 20 SLE patients. The CDR3 spectratypes of three patients were monitored over time, and the CDR3 regions of clonally expanded T cells were sequenced. CDR3 spectratype analysis showed prominent usage of TCR AV8, AV14, AV23, AV30, AV31, BV2, BV8, BV11, BV14, BV16, BV19 and BV24 families in SLE patients. The CDR3 spectratype showed dynamic change correlating with SLE activity. The sequence of the CDR3 region in clonally expanded T cells suggested a conserved GGX amino acid motif in both alpha- and beta-chains. The Ja34 and Jb2s1 region genes were found in high frequency. Both TCR Valpha and Vbeta gene usage is highly restricted in SLE, suggesting that the TCRs recognize a limited number of antigenic epitopes. The conserved motifs and limited use of joining region genes may indicate the recognition of similar antigenic epitopes in multiple individuals.

Glomerular T cells in Heymann nephritis.

Active Heymann nephritis (HN) is a rat model of human membranous nephropathy. The appearance of T cells within the glomeruli of HN rats suggests a role for these cells in the pathogenesis of the disease. The aims of this study were to investigate T cells infiltrating the glomerulus in HN in Lewis rats by polymerase chain reaction (PCR) of their Vbeta chains, CDR3 spectratyping and sequencing. HN was induced in Lewis rats by immunization with renal tubular antigen (Fx1A) in CFA. Kidneys were collected between 4 and 10 weeks. The glomeruli were separated, homogenized and RNA extracted. RT-PCR, CDR3 spectratyping and sequencing were used to further characterize the infiltrating T cells. Multiple Vbeta families showed restriction of their CDR3 spectratypes in each animal. Several TCR Vbeta families had identical-sized restricted spectratypes across several different animals. Four Vbeta families were sequenced. In three of those four families, the dominant clones showed identical sized CDR3 regions and a striking over-expression of Jbeta2.6. Further analysis of the CDR3 regions of the Jbeta2.6 clones showed a significant restriction of the amino acids at four of the six CDR3 positions. Glomerular T cells bearing similar CDR3 sequences, using Jbeta2.6 and expressing at least two, and possibly more, Vbeta genes are involved in the pathogenesis of HN.

T cell receptor V beta variable gene family expression in human peripheral blood lymphocytes at the mRNA and membrane protein level.

A polymerase chain reaction (PCR) was developed, using combinations of an oligonucleotide primer for a T cell receptor V beta gene family and one for the constant C beta gene segments, to assess the expression of each of 20 V beta gene families in RNA after reverse transcription into cDNA. The detection was done after agarose gel electrophoresis of PCR products and ethidium bromide staining. The positive identification of the PCR products was done by hybridization with a J beta oligonucleotide probe. For T cell lines, a signal was observed in the V beta 8 combination for Jurkat cells, V beta 5a in HSB cells, V beta 2 and V beta 12a in Molt-3 cells and V beta 2, V beta 5a and V beta 12a in Molt-4 cells. Using mixtures of RNA from different cell lines, the sensitivity of the method was in the range of 0.1-0.5%. In peripheral blood mononuclear cells from four donors, taken at three different occasions, all V beta families were detectable. The intensity of the PCR product varied between various V beta gene families. Flow cytometric analysis of blood mononuclear cells from the same donors with a restricted series of V beta gene family-specific antibodies also revealed the presence of all families. The approach to assess V beta gene family expression in heterogeneous populations opens the possibility to study T cell receptor variable gene expression in relation to physiology and pathologic processes.

Diverse T cell receptor beta gene usage by infiltrating T cells in the lacrimal glands of Sjögren's syndrome.

Sjögren's syndrome (SS) is an autoimmune disease characterized by T cell infiltration into the salivary and lacrimal glands (LG). Previous studies on T cell receptor (TCR) usage in the minor salivary glands (SG) have yielded controversial results. We studied TCR beta gene usage of the T cells infiltrating to LG, which is the other major target organ of SS. Total RNA was extracted from fresh LG and SG biopsy samples, and peripheral blood mononuclear cells from five SS patients, and converted to cDNA. TCR V beta gene repertoire was then assessed with quantitative polymerase chain reaction (PCR) assay. Oligoclonality was studied by sequencing V-D-J junctional regions of the PCR products. The TCR V beta gene usage in LG was diverse in every patient irrespective of disease duration, and similar to that of peripheral lymphocytes from a corresponding patient. The junctional region sequences of over-expressed V beta families in LG T cells were heterogeneous. We did not find any identical clones shared by LG, SG and peripheral blood. These results showed that the infiltrating T cells in LG of SS patients are polyclonal, and LG and SG do not share the same dominant T cell clonotypes. These suggest that TCR-targeted disease manipulation may have a limited effect on SS.

The human T cell antigen receptor repertoire: skewed use of V beta gene families by CD8+ T cells.

The TCR repertoire of human CD8+ peripheral blood lymphocytes has been determined using MoAbs to the V beta 2, 3, 5.1, 5.2/5.3, 6.7, 8, 12 and 19(17)V beta gene families. The CD8T cell repertoire for V beta 2 and V beta 3 is shown to be skewed, with an excess of individuals having higher values than are consistent with a normal distribution. A significant majority of these individuals are over the age of 40. High values of V beta CD8+ cells were found for each V beta family studied except for 6.7a. Individual high values are stable for at least 12 months. In addition, the total percentage of CD4 and CD8 cells reacting with this panel of reagents was determined. There is a significant excess of V beta + CD4+ cells (33%) over CD8+V beta + cells (21.9%). Thus the human CD8 V beta repertoire differs from the human CD4 repertoire in a number of important ways.

T cell receptor repertoire and mitotic responses of lamina propria T lymphocytes in inflammatory bowel disease.

Human intestinal lamina propria T lymphocytes (LPL-T) physiologically exhibit minimal proliferation in response to antigen receptor stimulation in vitro. This is thought to occur as a consequence of regulatory influences which are exerted by the mucosal microenvironment. The present study is aimed at investigating whether proliferative responses of intestinal LPL-T to antigen receptor stimulation are altered in patients with inflammatory bowel disease. Accordingly, proliferative responses of LPL-T in patients with Crohn's disease and ulcerative colitis to stimulation with CD3 MoAb plus IL-2 were examined and compared with controls. In addition, T cell receptor (TCR) repertoires of LPL-T and peripheral blood T lymphocytes were determined by indirect immunofluorescence using a panel of 11 TCR V beta specific antibodies. In most patients with inflammatory bowel disease, LPL-T showed enhanced proliferation to antigen receptor stimulation compared with controls. Moreover, perhaps as a consequence, an enhanced frequency of in vivo preactivated T cells was seen as judged from an increased spontaneous proliferative response to low concentrations of exogenous IL-2. LPL-T and peripheral blood T lymphocytes exhibited similar percentages of TCR V beta gene usage both in controls and in patients. In summary, polyclonal activation of LPL-T due to impairment of local adjustment, i.e. insufficient down-regulation of TCR/CD3-dependent signalling processes, may contribute to the pathogenesis of inflammatory bowel disease.

Vβ5 andVβ8 memory T cells in adults and infancy: co-ordinated increase in response to early antigen stimulus

Blood mononuclear cells (MNC) expressing the T cell receptor V beta gene families were identified with the monoclonal antibodies 1C1 (V beta 5) or Mx6 (V beta 8). These cells were typed for expression of the T200 common leucocyte antigen with the CD45RA antibody defining the 220-kD variant (which characterizes naive cells) and the UCHL1 antibody which defines the 180-kD variant (characterizing memory cells). Of adult MNC, 3 +/- 0.44% stained for V beta 5 and 4.12 +/- 0.26% stained for V beta 8; similar results were obtained in a group of infants aged less than 3 months, who were exposed to antigen stimulus in the form of infection and/or blood transfusion. While the total percentages of V beta 5 and V beta 8 cells in the blood of adults were not correlated, the proportions of these cells expressing CD45RO was positively correlated. A similar trend which did not reach statistical significance was present in a group of infants. A minority of the infants studied showed transient deviations from the mean established for adults. The results suggest that naive and memory T cell populations have similar frequencies of V beta 5 and V beta 8 expressing cells, and that age does not affect the frequency of usage of either of these V beta families.

An altered repertoire of T cell receptor V gene expression by rheumatoid synovial fluid T lymphocytes.

The pattern of T cell receptor V gene expression by lymphocytes from rheumatoid synovial fluid and paired peripheral blood samples was compared using a polymerase chain reaction (PCR)-based assay. Eight rheumatoid arthritis (RA) patients who had varying durations of disease (from 2 to 20 years) were studied. In all patients there was evidence of a different pattern of V gene expression between the two compartments. Significantly increased expression of at least one V alpha or V beta gene family by synovial fluid T cells was observed in all the patients studied. Three different V alpha (V alpha 10, 15 and 18) and three V beta (V beta 4, 5 and 13) families were commonly elevated. Sequencing of synovial V beta transcripts demonstrated that the basis of increased expression of selected V gene families in the synovial fluid was due to the presence of dominant clonotypes within those families, which constituted up to 53% of the sequences isolated from one particular synovial V gene family. There were considerable differences in the NDJ sequences found in synovial and peripheral blood T cell receptor (TCR) transcripts of the same V beta gene family. These data suggest that the TCR repertoire in the two compartments differs, and that antigen-driven expansion of particular synovial T cell populations is a component of rheumatoid synovitis, and is present in all stages of the disease.

Human TCR α/β+ CD4−CD8− Double-Negative T Cells in Patients with Autoimmune Lymphoproliferative Syndrome Express Restricted Vβ TCR Diversity and Are Clonally Related to CD8+ T Cells

The peripheral expansion of alpha/beta+-CD4-CD8- double negative (DN) T cells in patients with autoimmune lymphoproliferative syndrome (ALPS) is a consistent feature of this disease, and part of the diagnostic criteria of ALPS. The origin of these cells remains undetermined. They could derive from mature T cells that have lost coreceptor expression, or represent a special minor cell lineage. To investigate relationship of DN and single positive (SP) T cells in ALPS, we used Immunoscope technology to analyze the TCRVbeta repertoire diversity of sorted DN and SP T cells, and we performed CDR3 sequence analyses of matching clonotypes. We show that DN T cells express all the Vbeta gene families that are used by their SP counterparts, though they dominantly use some Vbeta genes. Analysis of CDR3 length distribution revealed a diverse polyclonal TCR repertoire for sorted CD4+ T cells, whereas both DN and CD8+ T cells showed a skewed TCR repertoire with oligoclonal expansions throughout most of the Vbeta families. CDR3 sequencing of matching clonotypes revealed a significant sharing of CDR3 sequences from selected Vbeta-Jbeta transcripts between DN and CD8+ T cells. Altogether, these data strongly argue for a CD8 origin of DN T cells in ALPS.