The TCR Repertoire Reconstitution in Multiple Sclerosis: Comparing One-Shot and Continuous Immunosuppressive Therapies.

Natalizumab (NTZ) and autologous hematopoietic stem cell transplantation (AHSCT) are two successful treatments for relapsing-remitting multiple sclerosis (RRMS), an autoimmune T-cell-driven disorder affecting the central nervous system that is characterized by relapses interspersed with periods of complete or partial recovery. Both RRMS treatments have been documented to impact T-cell subpopulations and the T-cell receptor (TCR) repertoire in terms of clone frequency, but, so far, the link between T-cell naive and memory populations, autoimmunity, and treatment outcome has not yet been established hindering insight into the post-treatment TCR landscape of MS patients. To address this important knowledge gap, we tracked peripheral T-cell subpopulations (naïve and memory CD4+ and CD8+) across 15 RRMS patients before and after two years of continuous treatment (NTZ) and a single treatment course (AHSCT) by high-throughput TCRß sequencing. We found that the two MS treatments left treatment-specific multidimensional traces in patient TCRß repertoire dynamics with respect to clonal expansion, clonal diversity and repertoire architecture. Comparing MS TCR sequences with published datasets suggested that the majority of public TCRs belonged to virus-associated sequences. In summary, applying multi-dimensional computational immunology to a TCRß dataset of treated MS patients, we show that qualitative changes of TCRß repertoires encode treatment-specific information that may be relevant for future clinical trials monitoring and personalized MS follow-up, diagnosis and treatment regimes. Natalizumab (NTZ) and autologous hematopoietic stem cell transplantation (AHSCT) are two successful treatments for relapsing-remitting multiple sclerosis (RRMS), an autoimmune T-cell-driven disorder affecting the central nervous system that is characterized by relapses interspersed with periods of complete or partial recovery. Both RRMS treatments have been documented to impact T-cell subpopulations and the T-cell receptor (TCR) repertoire in terms of clone frequency, but, so far, the link between T-cell naive and memory populations, autoimmunity, and treatment outcome has not yet been established hindering insight into the posttreatment TCR landscape of MS patients. To address this important knowledge gap, we tracked peripheral T-cell subpopulations (naive and memory CD4+ and CD8+) across 15 RRMS patients before and after 2 years of continuous treatment (NTZ) and a single treatment course (AHSCT) by high-throughput TCRß sequencing. We found that the two MS treatments left treatment-specific multidimensional traces in patient TCRß repertoire dynamics with respect to clonal expansion, clonal diversity, and repertoire architecture. Comparing MS TCR sequences with published datasets suggested that the majority of public TCRs belonged to virus-associated sequences. In summary, applying multidimensional computational immunology to a TCRß dataset of treated MS patients, we show that qualitative changes of TCRß repertoires encode treatment-specific information that may be relevant for future clinical trials monitoring and personalized MS follow-up, diagnosis, and treatment regimens.

Case report for recurrent and new-onset SLE patients treated by high-dose glucocorticoid therapy: Characteristics of peripheral TCR beta chain CDR3 repertoires.

RATIONALE: High-dose glucocorticoid therapy has been widely applied in clinical practice in systemic lupus erythematosus (SLE)patients, but less is known about the changes of T cells, especially the T cell receptor (TCR) repertoires, during the treatment. The aim of this paper is to describe the changes of TCR that recurrent and new-onset SLE patients treated by high-dose glucocorticoid therapy. PATIENT CONCERNS: Drugs of clinical treatment of SLE mainly include glucocorticoid, immunosuppressive agents, nonsteroidal anti-inflammatory drugs and B cell targeted drugs, etc, but the clinical symptoms were in remission and recurrent of onset patients with SLE. DIAGNOSES: Refer to the diagnostic criteria for SLE in 2011 by the American society of rheumatology. INTERVENTIONS: All patients were treated with High-dose glucocorticoid therapy and surveyed the TCR repertoires at 3 monitoring moments (before treatment, one month after treatment, and 3 months after treatment) to analyze the relationship between the characteristics of TCR repertoire and the highdose glucocorticoid therapy. OUTCOMES: We found that high-dose glucocorticoid therapy resulted in clinical symptom remission, as well as change of diversity, highly expanded clones (HEC), usage of TCR beta chain variable gene (TRBV)/TCR beta chain joining gene (TRBJ), and overlapped sequences of TCR beta chain complementarity determining region 3 (CDR3) repertoires. This suggests that the effect of high-dose glucocorticoids on TCR repertoires is closely related to individual autoimmune T cells. LESSONS: In this study, we have shown that we could evaluate the effect of therapy, the pathogenesis, and the prognosis for the patients with SLE by monitoring the TCR CDR3 repertoires. It could afford a new method to find the therapeutic target of SLE.

Assessing a theoretical risk of dolutegravir-induced developmental immunotoxicity in juvenile rats.

HIV-1 integrase inhibitors (INIs) are a promising class of antiretrovirals for the treatment of HIV in adults; there is interest in expanding their use into pediatric populations. A theoretical concern for developmental immunotoxicity was raised after a publication suggested that two HIV INI tool compounds inhibited in vitro cleavage activity of recombination activating genes 1 and 2 (RAG1/2) through the inhibition of their binding to recombination signal sequences. RAG1/2 are required for the development of mature B and T lymphocyte populations. The potential effects of the investigational INI dolutegravir on RAG1/2 were addressed by developing assays in juvenile rats to measure T cell receptor (TCR) Vß usage by flow cytometry as an indicator of TCR repertoire diversity and a T cell dependent antibody response (TDAR) as an indicator of immunosuppression. These endpoints were incorporated into a juvenile rat toxicity study, along with immunophenotyping, hematology, and histopathology of immunologic organs. Dose levels of 0, 0.5, 2, or 75mg/kg/day dolutegravir were given via oral gavage from postnatal day 4 through 66. At the highest dose, there was decreased body weight gain and two preweanling deaths; however, there were no treatment-related effects on developmental parameters. There were no effects on immunologic competence, as measured by TDAR, and no effects on lymphocyte subsets or CD4 and CD8 TCR Vß usage in peripheral blood. Histopathology of immunologic organs (spleen, thymus, lymph nodes) and hematology evaluation revealed no effects. The no observed adverse effect level for immunotoxicity endpoints was 75mg/kg/day.

T cell receptor repertoire in BALB/c mice varies according to tissue type, sex, age, and hydrocortisone treatment.

Diversity in T cell recognition of antigens is determined by diverse usage of T cell receptor (TCR) repertoire. TCR repertoire analysis provides fundamental information for understanding T cell immune responses in the pathogenesis of various diseases. In the present study, we examined the TCR repertoire in various tissues in normal BALB/c mice. The TCR alpha chain variable region repertoires were consistent among the spleen, lymph nodes, and the thymus. The TCR beta chain variable region (TCRBV) repertoires were consistent between the spleen and lymph nodes, but different in the thymus. The TCR repertoires also differed in the lungs and the intestinal tract. The TCR repertoires were consistent between male and female mice, except for TCRBV15-1. TCR repertoire was almost similar in 3- and 7-week-old mice, except for TCRBV1-1, 8-3, and 14-1. The present findings suggest that the TCR repertoire of mice varies according to tissue type, sex and age. Additional analysis of the TCR repertoire, i.e., the effect of hydrocortisone (HC), was carried out. After the HC treatment, although the thymic T cells decreased to one-tenth, only a small fraction of CD4(+)CD8(+) T cells survived the treatment. Furthermore, the percentages of thymic T cells bearing TCRBV3-1, 5-1, 5-2, and 16-1 substantially decreased, but the percentage of cells bearing TCRBV12-1 did not decrease. The present findings suggest that the HC susceptibility of immature thymic T cells is different between TCR families.

In vivo effects of zoledronic acid on peripheral gammadelta T lymphocytes in early breast cancer patients.

INTRODUCTION: Amino-bisphosphonates are potent activators of human gammadelta T cells. The aim of our study was to evaluate the immunomodulating properties of a single-dose of zoledronic acid (ZA) on gammadelta T cells in a select group of disease-free breast cancer patients with osteopenia. MATERIALS AND METHODS: Blood samples were obtained, from 23 patients, before and 7, 28, 56, 90 and 180 days after a single-dose (4 mg) of ZA and analyzed by flow cyometry. RESULTS: A significant decrease of the different gammadelta T cell subsets was observed: Naïve (CD3+/Vdelta2+/CD45RA+/CD27+) after 180 days (P < 0.01); Central Memory (CD3+/Vdelta2+/CD45RA-CD27+) after 28 (P < 0.05), 90 (P < 0.01) and 180 days (P < 0.01); and Effector Memory (CD3+/Vdelta2+/CD45RA-/CD27-) after 56 (P < 0.01) and 90 (P < 0.05) days. Based on the observed gammadelta T cells kinetics patients could be divided in two groups: "responders" that showed a significant decrease in total numbers of gammadelta T cells and "non-responders" that showed no significant change. However, in vitro phosphoantigen stimulation of patients cells did not show significant differences in terms of IFN-gamma response by Vdelta2 T cells. CONCLUSION: We describe for the first time a long-lasting activation of effector subsets of gammadelta T cells in disease-free breast cancer patients after a single-dose of ZA. Our results highlight the need to further investigate the clinical significance of the immunomodulating properties of N-BPs.

Redox modulation inhibits CD8 T cell effector function.

The evolutionary preservation of reactive oxygen species in innate immunity underscores the important roles these constituents play in immune cell activity and as signaling intermediates. In an effort to exploit these pathways to achieve control of aberrant immune activation we demonstrate that modulation of redox status suppresses cell proliferation and production of IL-2, IFN-gamma, TNF-alpha, and IL-17 in two robust CD8 T-cell-dependent in vitro mouse models: (1) response to alloantigen in an mixed leukocyte reaction and (2) CD8 T cell receptor transgenic OT-1 response to cognate peptide (SIINFEKL). To correlate these findings with cytotoxic lymphocyte (CTL) function we performed cytotoxicity assays and found that redox modulation diminishes the ability of alloantigen-specific and antigen-specific OT-1 CTLs to kill their corresponding antigen-expressing target cells. To further examine the mechanisms of redox-mediated repression of CTL target cell lysis, we analyzed the expression of the effector molecules IFN-gamma, perforin, and granzyme B and the degranulation marker CD107a (LAMP-1). In both models, redox modulation reduced the expression of these effector components by at least fivefold. These results demonstrate that redox modulation quells the CD8 T cell response to alloantigen and the T cell receptor transgenic CD8 T cell response to its cognate antigen by inhibiting proliferation, proinflammatory cytokine synthesis, and CTL effector mechanisms.

Comparison of proportional and differential odds models for mixed-effects analysis of categorical data.

In this work a model for analyzing categorical data is presented; the differential odds model. Unlike the commonly used proportional odds model, this model does not assume that a covariate affects all categories equally on the log odds scale. The differential odds model was compared to the proportional odds model, by assessing statistical significance and improvement of predictive performance when applying the differential odds model to data previously analyzed using the proportional odds model. Three clinical studies; 3-category T-cell receptor density data, 5-category diarrhea data and 6-category sedation data, were re-analyzed with the differential odds model. As expected, no improvements were seen with T-cell receptor density and diarrhea data. However, for the more complex measurement sedation, the differential odds model provided both statistical improvements and improvements in simulation properties. The estimated actual critical value was for all data lower than the nominal value, using the number of added parameters as the degree of freedom, i.e. the differential odds model is statistically indicated to a less extent than expected. The differential odds model had the desired property of not being indicated when not necessary, but it may provide improvements when the data does not represent a categorization of continuous data.

Effects of long-term cyclosporine A on rat intestinal intraepithelial lymphocytes.

The aim of this study was to assess the influence of long-term administration of cyclosporine (CsA) on rat intestinal intraepithelial lymphocytes (IEL). Ten male Lewis rats were randomly assigned to either a treatment or a control group. For 12 weeks the treatment group received CsA (5 mg/kg per day) while the control group received the same volume of vehicle. Peripheral blood lymphocytes were counted and IEL were isolated for flow cytometric analysis. Peripheral blood lymphocytes numbers were significantly decreased in the CsA compared with the control group (4.80 +/- 1.02 x 10(6)/mL and 3.34 +/- 0.70 x 10(6)/mL, respectively, P = .029). IEL numbers were increased significantly in CsA compared with the control group. (23.2 +/- 8.0 x 10(6) and 7.8 +/- 1.8 x 10(6), respectively, P = .012). There were no significant differences between the two groups considering IEL phenotypes. In conclusion, CsA did not influence the subpopulation but significantly increased the number of IEL.

Glatiramer acetate (GA) therapy induces a focused, oligoclonal CD8+ T-cell repertoire in multiple sclerosis.

We have demonstrated that GA therapy induces a differential upregulation of GA-specific, cytotoxic/suppressor CD8+ T-cell responses in MS patients. We utilized a novel combination of flow sorting and anchored PCR to analyze the evolving clonal composition of GA-specific CD4+ and CD8+ T-cells. TCRbeta chain analysis revealed the development of an oligoclonal GA-specific CD8+ repertoire with persistence of dominant clones over long periods. Interestingly, some sequences resembled published oligoclonal CD8+ TCR sequences from MS lesions. In contrast, GA-specific CD4+ responses were polyclonal and showed continual evolution of their repertoire. This clonotypic and functional analysis provides mechanistic insights into GA therapy.

Regulated costimulation in the thymus is critical for T cell development: dysregulated CD28 costimulation can bypass the pre-TCR checkpoint.

Expression of CD28 is highly regulated during thymic development, with CD28 levels extremely low on immature thymocytes but increasing dramatically as CD4- CD8- cells initiate expression of TCRbeta. B7-1 and B7-2, the ligands for CD28, have a restricted distribution in the thymic cortex where immature thymocytes reside and are more highly expressed in the medulla where the most mature thymocytes are located. To determine the importance of this regulated CD28/B7 expression for T cell development, we examined the effect of induced CD28 signaling of immature thymocytes in CD28/B7-2 double-transgenic mice. Strikingly, we found that differentiation to the CD4+ CD8+ stage in CD28/B7-2 transgenics proceeds independent of the requirement for TCRbeta expression manifest in wild-type thymocytes, occurring even in Rag- or CD3epsilon- knockouts. These findings indicate that signaling of immature thymocytes through CD28 in the absence of TCR- or pre-TCR-derived signals can promote an aberrant pathway of T cell differentiation and highlight the importance of finely regulated physiologic expression of CD28 and B7 in maintaining integrity of the "beta" checkpoint for pre-TCR/TCR-dependent thymic differentiation.

Using a small molecule inhibitor of peptide: N-glycanase to probe its role in glycoprotein turnover.

Peptide:N-glycanase (PNGase) is ostensibly the sole enzyme responsible for deglycosylation of unfolded N-linked glycoproteins dislocated from the ER to the cytosol. Here we show the pan-caspase inhibitor, Z-VAD-fmk, to be an active site-directed irreversible inhibitor of yeast and mammalian PNGase at concentrations below those used to inhibit caspases in vivo. Through chemical synthesis we determined that the P1 residue, electrophile position, and leaving group are important structural parameters for PNGase inhibition. We show that Z-VAD-fmk inhibits PNGase in living cells and that degradation of class I MHC heavy chains and TCRalpha, in an identical cellular setting, is markedly different. Remarkably, proteasome-mediated turnover of class I MHC heavy chains proceeds even when PNGase is completely inhibited, suggesting that the function of PNGase may be to facilitate more efficient proteasomal proteolysis of N-linked glycoproteins through glycan removal.

Early activation events differentiate the reactivity of two T-cell families to Staphylococcus enterotoxin A.

Analysis of early activation events in two SEA responsive T-cell families demonstrated that low doses of SEA induced CD4+Vbeta22 T-cells to down-regulate their TCR and express CD69, considerably earlier than CD4+Vbeta5 T-cells. The rapid down-regulation of Vbeta22 TCR led to its proliferation, whereas even a 10-fold higher dose of toxin induced only a partial down-regulation of Vbeta5 TCR. Stimulation with SEA induced a significantly higher percentage of Vbeta22 T-cells to produce IFN-gamma compared to Vbeta5 T-cells. SEAF47A, a mutant of SEA, known to have a lower binding affinity for the MHC class II molecule, failed to activate Vbeta5 T-cells whereas Vbeta22 T-cell activation was slightly decreased. Hence, early activation events highlighted the differential requirements of T-cell families to respond to SEA.

Streptococcus dysgalactiae-derived mitogen (SDM), a novel bacterial superantigen: characterization of its biological activity and predicted tertiary structure.

A mitogenic substance, designated Streptococcus dysgalactiae-derived mitogen (SDM), was purified from S. dysgalactiae culture supernatant, and the gene encoding the mitogen was cloned. Both native and recombinant SDM expressed in Escherichia coli significantly activated human V beta 1+ and V beta 23+ T cells in association with major histocompatibility complex (MHC) class II molecules on accessory cells, indicating that SDM possesses superantigenic properties. The sdm gene consists of two segments encoding a signal peptide and a mature 25 kDa protein composed of 212 amino acids. Three of 34 S. dysgalactiae strains but none of 28 Streptococcus pyogenes strains examined carried sdm. Phylogenetic analysis indicated that SDM belongs to a family distinct from established bacterial superantigens. SDM showed around 30% homology with other superantigens at the amino acid sequence level. The tertiary structure of SDM was predicted by modelling onto streptococcal pyrogenic exotoxin C and streptococcal mitogenic exotoxin Z-2, both of which share highly homologous structure-determining regions. SDM showed overall structural similarity to both these superantigens. This is the first study to characterize fully a bacterial superantigen from S. dysgalactiae.

Effect of immunomodulatory treatment of multiple sclerosis on lymphocyte surface immunomarkers.

The aim of this study was to analyze the effect of immunomodulatory treatment of multiple sclerosis (MS) on lymphocyte surface immunomarkers. The special attention was given to TCR alpha/beta, gamma/delta and alpha/beta HLA-DR markers. Peripheral blood was obtained from 39 patients with clinically definite R-R MS, fulfilling the criteria of McDonald et al.[5]. The group of 15 patients was treated with interferon beta-1a (Avonex) intramuscularly once a week. The blood was obtained before and after two years of treatment. The other group of 10 patients was treated every day with 20 mg of glatiramer acetate (Copaxone) intracutaneously. Subsets of lymphocytes were analyzed by the method of flow cytometry, using monoclonal antibodies produced by Ortho Diagnostic System. The relative results were evaluated using Immuno Count II program. The frequency of the studied subsets in MS was markedly different from that in healthy persons. The higher number of CD4, TCR alpha/beta positive cells and higher CD4/CD8 ratio was observed. In comparison to healthy individuals, in MS patients a decreased number of TCR gamma/delta, and alpha/beta HLA-DR was found. After therapy with glatiramer acetate, CD3 and CD8 positive lymphocytes were more frequently observed than before the drug administration. The CD4/CD8 ratio was markedly decreased. The effect of interferon beta-1a treatment was similar as in the previous group, i.e. a slight increase in CD3 and CD8 was noticed after therapy. Despite the differences in action of both immunomodulatory drugs, which was established in several studies, we like to stress some similarity in their effect on CD3, CD8, alpha/beta HLA-DR and gamma/delta HLA-DR immunomarkers frequency in lymphocyte, and on the CD4/CD8 ratio. This may mean that there are some common immunological steps of special importance for the clinical effect in MS.

Preferential survival of an MBP-specific T cell clone in an HLA-DR2 multiple sclerosis patient.

Anti-myelin basic protein (MBP) autoreactive T cells play a key role in the pathogenesis of multiple sclerosis. Thus, we applied the Immunoscope strategy to cerebrospinal fluid (CSF) and peripheral blood lymphocytes (PBLs) of an HLA-DR2 patient. Both compartments showed major expansion for the V(beta)13S5 chain, which was associated in peripheral blood with significant proliferation of PBLs in response to MBP and the 84-102 HLA-DR2-restricted peptide. Sequencing revealed a unique nucleotide sequence in the CSF that gives rise to the amino acid sequence V(beta)13S5-RPGQGDQETQ-J(beta)2.5 if translated. This CDR3 sequence had already been reported to be reactive against the 84-102 peptide. This specific sequence was not detected in PBLs on day 0, whereas it was readily detectable on day 6 culture samples. Thus, cell culture may lead to enrichment in a T cell clone identified as autoreactive.

Delayed-type hypersensitivity reaction to paraphenylenediamine is mediated by 2 different pathways of antigen recognition by specific alphabeta human T-cell clones.

BACKGROUND: Allergic contact dermatitis to paraphenylenediamine (PPD) is a frequent cause of morbidity and occupational disability. OBJECTIVE: The aim of the study was to characterize T-cell responses to PPD and Bandrowski's base (BB), an autoxidation product of PPD, by using polyclonal and monoclonal T-lymphocyte cultures. METHODS: PPD- and BB-driven proliferation of PBMCs and T-cell clones (TCCs) was assessed by means of tritiated thymidine incorporation. Surface markers were studied by means of flow cytometry, and cytokine generation was assessed with an ELISA. RESULTS: TCCs, with one exception, were CD4+/CD45RO+, and T-cell receptors were alphabeta+. Three of 6 TCCs expressed Vbeta 16. TCC stimulation was HLA-DP restricted, and TCCs secreted IL-4, IL-5, and marginal levels of IFN-gamma. TCCs reacted to both PPD and BB. Presentation of BB to TCCs was dependent on viable antigen-presenting cells (APCs) pulsed for 4 hours, and fixed APCs failed to stimulate TCCs. Moreover, polyclonal responses to BB were enhanced by metabolically active enzymes, such as cytochrome P450 enzymes. BB has to be metabolized and processed. In contrast, fixation of APCs did not impair their ability to present PPD to TCC, whereas pulsing of APCs with PPD failed to stimulate TCCs. Thus PPD had to be present during the process, and polyclonal stimulation was not enhanced by cytochromes. CONCLUSION: These results suggest that PPD itself can be recognized by T cells through a processing-independent pathway, whereas its autoxidation product, BB, required processing and possibly metabolism to stimulate the same TCC. Our data demonstrate that 2 distinct pathways of antigen presentation to activate specific TCCs are involved in the immune response to PPD.

Suicide gene therapy of graft-versus-host disease: immune reconstitution with transplanted mature T cells.

After allogeneic hematopoietic stem cell transplantation (HSCT), mature transplanted T cells play a major role in restoration of the immune system. However, they can also induce a life-threatening complication: graft-versus-host disease (GVHD). Suicide gene therapy of GVHD aims to selectively eliminate alloreactive T cells mediating GVHD while sparing nonalloreactive T cells that should contribute to immune reconstitution. It was demonstrated previously that treatment with ganciclovir (GCV) can control GVHD in mice by killing donor T cells engineered to express the thymidine kinase (TK) suicide gene. TK allows phosphorylation of nontoxic GCV into triphosphate GCV, which is selectively toxic for dividing cells. Thus, in the TK-GCV system, the specificity of cell killing depends on the cycling status of TK T cells rather than allogeneic recognition. This is a potential drawback because in recipients of lymphopenic allogeneic HSCT, alloreactive and homeostatic signals drive the proliferation of donor T cells. It is shown here that the onset of alloreactive T-cell division occurs earlier than that of nonalloreactive T cells, thus establishing a time frame for GCV administration. A 7-day GCV treatment initiated at the time of HSCT allowed efficient prevention of GVHD, while sparing a pool of nondividing donor TK T cells. These cells later expanded and contributed to the replenishment of the recipient immune system with a diversified T-cell receptor repertoire. These results provide a rationale for designing the therapeutic scheme when using TK-GCV suicide gene therapy in allogeneic HSCT.

Longitudinal analysis of T-cell receptor gene use by CD8(+) T cells in early human immunodeficiency virus infection in patients receiving highly active antiretroviral therapy.

The effects of early antiretroviral therapy on the peripheral CD8(+) T-cell population were assessed by sequentially determining the T-cell receptor (TCR) repertoire complexity in a cohort of 15 individuals recently diagnosed with human immunodeficiency virus infection. Analysis was based on quantitative TCR variable B gene (TCRBV) usage and complementary-determining region 3 length assessment. Repertories were assessed at baseline and at weeks 2, 4, 12, 24, and 72 after initiation of therapy. Early administration of highly active antiretroviral therapy has a positive effect on the preservation and homeostasis of the CD8(+) cell repertoire. Nevertheless, differences from average baseline and control TCR profiles and initial development of repertoire perturbations were observed. The findings suggest that additional therapeutic protocols will be required during primary infection to significantly prevent long-term erosion of the T-cell-mediated immune response.

Antigen contacts by Ni-reactive TCR: typical alphass chain cooperation versus alpha chain-dominated specificity.

VB17(+) TCR dominate in Ni-driven T cell cultures from highly Ni-sensitized patients. Using transfection of TCR from three CD4(+), VB17(+), Ni-specific human T cell clones, we studied their Ni-MHC contacts by site-directed TCR mutation and combination of alpha and ss chains between different TCR. All three TCR exhibited N-nucleotide-determined Arg-Asp motifs in their CDR3-ss sequences. Two of them were specifically restricted to HLA-DR13, while the third one accepted a variety of HLA-DR alleles. The highly similar alpha or ss chains of the DR13-restricted TCR were interchangable without loss of specificity, but alpha or ss chains of other TCR were not tolerated. Mutations of their Arg-Asp motif revealed loss of reactivity upon exchanging Asp for Glu or Ala and of Arg for Ala but not of Arg for Lys or the Ni binding His. Reactivity was also destroyed by mutation of alpha chain position 51, proposed as a general contact site for MHC. Hence, in these two TCR the Arg-Asp motif is clearly involved in contacting Ni-MHC complexes, and close cooperation between alpha and ss chain is required. In contrast, the third TCR retained Ni reactivity upon mutation of alpha chain position 51 or of its ss chain Arg-Asp motif, which rather affected the pattern of DR cross-restriction. Moreover, its alpha chain paired with various ss chains from other, even mouse TCR, irrespective of their specificity, retaining Ni reactivity as well as promiscuous HLA-DR restriction. This preponderance of an alpha chain in defining specificity indicates fundamental differences in Ni interactions of individual TCR and implies that ss chain similarities may not necessarily result from antigen selection.

Induction of NK1.1(+) alpha beta TCR(+) T cells by bypassing TCR signals in ZAP-70 deficient mice.

The mechanism of development of a unique subset of T cells, thymic NK1.1(+) alpha beta T cells, has been poorly understood. We found that the development of thymic NK1.1(+) alpha beta T cells was defective in mice deficient in ZAP-70. Instead, an accumulation of NK1.1(+) TCR beta(-) NK-like population was detected in the thymus and spleen of the ZAP-70 deficient (ZAP -/-) mouse. In the present report, we examined whether biochemical treatments that replace TCR-mediated positive selection signals could restore the generation of thymic NK1.1(+) alpha beta T cells in ZAP -/- mice using the thymus organ culture. We found that a higher concentration of phorbol ester (PMA) than that required for CD4(+) T cell generation and ionomycin induced the generation of NK1.1(+) alpha beta T cells. Phenotypic analysis of the induced NK1.1(+) alpha beta T cell population suggested that these cells expressed CD8 but not CD4 molecules, which is a different characteristic from ordinary thymic NK1.1(+) alpha beta T cells. These results suggest that differential signaling is required for the generation of mainstream T cells and thymic NK1.1(+) alpha beta T cells.