T-cell Receptor Alpha Beta Research Articles

Development and selection of γδ T cells

Two main lineages of T cells develop in the thymus: those that express the alphabeta T-cell receptor (TCR) and those that express the gammadelta TCR. Whereas the development, selection, and peripheral localization of newly differentiated alphabeta T cells are understood in some detail, these processes are less well characterized in gammadelta T cells. This review describes research carried out in this laboratory and others, which addresses several key aspects of gammadelta T-cell development, including the decision of precursor cells to differentiate into the gammadelta versus alphabeta lineage, the ordered differentiation over the course of ontogeny of functional gammadelta T-cell subsets expressing distinct TCR structures, programming of ordered Vgamma gene rearrangement in the thymus, including a molecular switch that ensures appropriate Vgamma rearrangements at the appropriate stage of development, positive selection in the thymus of gammadelta T cells destined for the epidermis, and the acquisition by developing gammadelta T cells of cues that determine their correct localization in the periphery. This research suggests a coordination of molecularly programmed events and cellular selection, which enables specialization of the thymus for production of distinct T-cell subsets at different stages of development.

Monoclonal Antibody against T‐Cell Receptor αβ Induces Self‐Tolerance in Chronic Experimental Autoimmune Encephalomyelitis

The therapeutic effect of monoclonal antibody (H57-597 MoAb) against T-cell receptor (TCR) alphabeta has been investigated on MOG(35-55)-induced experimental autoimmune encephalomyelitis (EAE), as a model system for T-cell-mediated chronic inflammation in the central nervous system (CNS). Short-term administration of the anti-TCR alphabeta immediately after immunization protected the mice from EAE. Furthermore, anti-TCR alphabeta treatment on an established disease restored the self-tolerance which led to a complete remission of EAE and a dramatic reduction of inflammatory cells in the CNS, while treatment with control antibody (hamster IgG) was ineffective. The remission was durable and not associated with disappearance of autoreactive T cells as measured by persistence of MOG-reactive T-cell proliferation in vitro. However, MOG-reactive T cells from anti-TCR-treated animals produced significantly lower amounts of inflammatory TNF-alpha and IFN-gamma. In addition, while a transient deletion of CD4(+) and CD8(+) T cells was observed, a population of T cells expressing CD3, NK1.1 and CD69 (NKT cells) were expanding. By transfer of spleen cells from anti-TCR MoAb-treated animals, we could show that the tolerogenic capacity can be transferred to untreated recipients with EAE. The data indicate therapeutic effect of anti-TCR alphabeta MoAb (H57-597), which represents a promising approach in treatment of T-cell-mediated autoimmune diseases.

Abdominal Aortic Aneurysm Is a Specific Antigen‐Driven T Cell Disease

To determine whether monoclonal/oligoclonal T cells are present in abdominal aortic aneurysm (AAA) lesions, we amplified beta-chain T cell receptor (TCR) transcripts from these lesions by the nonpalindromic adaptor (NPA)-polymerase chain reaction (PCR)/V-beta-specific PCR followed by cloning and sequencing. Sequence analysis revealed the presence of substantial proportions of identical beta-chain TCR transcripts in AAA lesions in 9 of 10 patients examined, strongly suggesting the presence of oligoclonal populations of alphabeta TCR+ T cells. We have also shown the presence of oligoclonal populations of gammadelta TCR+ T cells in AAA lesions. Sequence analysis after appropriate PCR amplification and cloning revealed the presence of substantial proportions of identical VgammaI and VgammaII TCR transcripts in 15 of 15 patients examined, and of Vdelta1 and Vdelta2 TCR transcripts in 12 of 12 patients. These clonal expansions were very strong. All these clonal expansions were statistically significant by the binomial distribution. In other studies, we determined that mononuclear cells infiltrating AAA lesions express early- (CD69), intermediate- (CD25, CD38), and late- (CD45RO, HLA class II) activation antigens. These findings suggest that active ongoing inflammation is present in the aortic wall of patients with AAA. These results demonstrate that oligoclonal alphabeta TCR+ and gammadelta TCR+T cells are present in AAA lesions. These oligoclonal T cells have been clonally expanded in vivo in response to yet unidentified antigens. Although the antigenic specificity of these T cells remains to be determined, these T cells may play a significant role in the initiation and/or the propagation of the AAA. It appears that AAA is a specific antigen-driven T cell disease.

Hepatosplenic T-Cell Lymphoma: Clinical Characteristics and Treatment Outcome.

Hepatosplenic T-cell lymphoma is a rare peripheral T-cell lymphoma, and less than 100 cases have been described in the literature since the entity was first proposed in 1990. This condition is characterized by malignant T-cell proliferation in the sinusoids of the liver, sinuses and red pulp of the spleen, and sinuses of the bone marrow. The commonly reported T-cell phenotype is CD2+, CD3+, CD4−, CD5−, CD7±, CD8−, with either gamma-delta or alpha-beta T-cell receptor expression. Associated cytogenetic abnormalities include isochromosome 7q with or without trisomy 8. Approximately 10–20% of patients have a history of immunocompromise. Treatment to date with standard anthracycline containing chemotherapy regimens has been disappointing with variable response rates, high relapse rates, and a short median survival. An optimal treatment strategy for this small patient population has not yet been determined.Methods: Eleven cases of pathologically confirmed hepatosplenic T-cell lymphoma seen at M.D. Anderson Cancer Center between January 1990 and June 2006 were identified in the institution's database. Clinical characteristics and treatment results were retrospectively reviewed.Results: The mean age at the time of diagnosis was 35.3 years (range 8 – 58 years). Six patients were male and five were female. Seven patients had the gamma-delta T-cell receptor phenotype, and three patients had the alpha-beta T-cell receptor phenotype. Cytogenetic abnormalities observed included t(7;14)(q34q13), 46,idem,del(2)(q32q37), 45XY,der(13;14)(q10;q10), 45XY,i(7)(q10),der(13;14)(q10;q10), del(22q).ish22(WCP22x2). One patient had lymphomatous involvement of the skin, a finding not previously reported in hepatosplenic T cell lymphoma. Two patients had a history of significant immunocompromise, including one patient with Sjogren's disease treated intermittently with prednisolone and methotrexate, and one patient with Crohn's disease on chronic treatment with mercaptopurine. Complete responses (CR) were achieved in four out of eight patients who received chemotherapy and whose complete records were available. CRs were achieved with the following regimens: (1) Sequential IDSHAP/AMDBIDCOS/MINE, (2) CHOP/methotrexate and autologous stem cell transplant, (3) CHOP, (4) pentostatin/campath and allogeneic stem cell transplant. PET scans in 3 patients and Gallium scans in 5 patients did not correlate with disease activity or clinical response. Mean duration of CR was 8 months (range 2 – 14+ months). Mean overall survival was 11.4 months (range 3 – 25 months), with one patient still alive and in remission at 20 months.Conclusion: In one of the largest series of hepatosplenic T-cell lymphoma patients, we confirm the aggressive clinical course of this subtype of peripheral T-cell lymphoma occurring in a young patient population. We discovered a near-equal incidence of this disease in males and females, contrary to results in earlier series which suggested this disease is usually diagnosed in males. Standard chemotherapy treatment strategies resulted in poor outcomes. However, survival may be improved upon with newer strategies such as the use of alemtuzumab, pentostatin, and high dose chemotherapy with stem cell rescue. New therapeutic strategies are needed to improve the outcome of patients with hepatosplenic T-cell lymphoma.

The Structure of the ζζ Transmembrane Dimer Reveals Features Essential for Its Assembly with the T Cell Receptor

The T cell receptor (TCR) alphabeta heterodimer communicates ligand binding to the cell interior via noncovalently associated CD3gammaepsilon, CD3deltaepsilon, and zetazeta dimers. While structures of extracellular components of the TCR-CD3 complex are known, the transmembrane (TM) domains that mediate assembly have eluded structural characterization. Incorporation of the zetazeta signaling module is known to require one basic TCRalpha and two zetazeta aspartic acid TM residues. We report the NMR structure of the zetazeta(TM) dimer, a left-handed coiled coil with substantial polar contacts. Mutagenesis experiments demonstrate that three polar positions are critical for zetazeta dimerization and assembly with TCR. The two aspartic acids create a single structural unit at the zetazeta interface stabilized by extensive hydrogen bonding, and there is evidence for a structural water molecule (or molecules) within close proximity. This structural unit, representing only the second transmembrane dimer interface solved to date, serves as a paradigm for the assembly of all modules involved in TCR signaling.

Applications of Bilateral Vascularized Femoral Bone Marrow Transplantation for Chimerism Induction Across the Major Histocompatibility (MHC) Barrier

Bilateral vascularized bone marrow transplant (VBMT) model was designed to induce chimerism across the major histocompatibility (MHC) barrier under combined alphabeta T-cell receptor monoclonal antibody and cyclosporine A (alphabeta-TCRmAb/CsA) protocol. Seventeen transplants were performed between BN(RT1) donors and Lewis(RTI) recipients. Group I, isograft controls; Group II, allografts rejection controls; Group III, allografts under 7-day protocol of alphabeta-TCRmAb/CsA. Donor bilateral femoral bones were bilaterally anastomosed to the abdominal aorta and inferior vena cava of recipient. At day 7 posttransplantation, all bone flaps were viable. Groups I and III survived without signs of rejection. In Group III, peak level of chimerism in peripheral blood was evaluated at day 21 (24.2%), at day 63 declined to 1.5%, and was maintained at this level thereafter. Donor-derived cells were present in the bone marrow of recipients at 28.2% at day 21 posttransplant. Histology confirmed viability of bone marrow cells in isograft during the entire follow-up and up to 35 days in treatment Group III. Bilateral VBMT induced donor-specific chimerism across the MHC barrier under the immunomodulatory protocol of alphabeta-TCRmAb/CsA.

Interactions between CD8αβ and the TCRαβ/CD3‐receptor Complex

CD8+ T cells recognize antigenic peptides bound to major histocompatibility complex (MHC) class I molecules on normal antigen-presenting cells (APC), as well as on virus-infected cells or tumour cells (pMHC). At least two receptor types participate in recognition of these complexes: T-cell receptor (TCR) alphabeta heterodimers and CD8alphabeta molecules. The former molecules react with antigenic peptide and variable regions of MHC class I molecules, whereas the latter molecules react with constant alpha3 regions of MHC class I molecules. As the avidity of both receptor-MHC interactions is low, it is believed that TCRalphabeta and CD8alphabeta heterodimers collaborate in T-cell recognition. We have established a TCR/CD3-CD8 capture ELISA, which can measure the interaction of pMHC with CD8alphabeta molecules and with TCR/CD3 complexes. The major findings are: (1) TCR/CD3 complexes derived from in vitro activated T cells and captured by anti-CD3 MoAb, do bind specific pMHC and (2) CD8+ T cells express at least three forms of CD8alphabeta molecules: single CD8alphabeta, CD3-CD8 and TCR/CD3-CD8 complexes. Only the latter complexes are associated with CD3zeta homodimers, and the quantity of TCR/CD3-CD8 complexes relative to total CD8alphabeta molecules appears to increase and to be selected into sucrose-gradient microdomains as a function of TCRalphabeta-mediated T-cell activation.

Characterization of chicken epidermal dendritic cells

It has been known for 15 years that the chicken epidermis contains ATPase+ and major histocompatibility complex class II-positive (MHCII+) dendritic cells. These cells were designated as Langerhans cells but neither their detailed phenotype nor their function was further investigated. In the present paper we demonstrate a complete overlapping of ATPase, CD45 and vimentin staining in all dendritic cells of the chicken epidermis. The CD45+ ATPase+ vimentin+ dendritic cells could be divided into three subpopulations: an MHCII+ CD3- KUL01+ and 68.1+ (monocyte-macrophage subpopulation markers) subpopulation, an MHCII- CD3- KUL01- and 68.1- subpopulation and an MHCII- CD3+ KUL01- and 68.1- subpopulation. The first population could be designated as chicken Langerhans cells. The last population represents CD4- CD8- T-cell receptor-alphabeta- and -gammadelta- natural killer cells with cytoplasmic CD3 positivity. The epidermal dendritic cells have a low proliferation rate as assessed by bromodeoxyuridine incorporation. Both in vivo and in vitro experiments showed that dendritic cells could be mobilized from the epidermis. Hapten treatment of epidermis resulted in the decrease of the frequency of epidermal dendritic cells and hapten-loaded dendritic cells appeared in the dermis or in in vitro culture of isolated epidermis. Hapten-positive cells were also found in the so-called dermal lymphoid nodules. We suggest that these dermal nodules are responsible for some regional immunological functions similar to the mammalian lymph nodes.

Graft‐versus‐host disease in recipients of grafts from natural killer T cell‐deficient (Jα281–/–) donors

Valpha14i natural killer T cells (NKT cells) are a CD1-restricted subset of NKT cells that express an invariable Valpha14+ Jalpha281+ alphabeta T-cell receptor. To determine whether the absence of Valpha14i NKT cells from the graft affects the development of acute GVHD, we induced GVH reactions using Jalpha281(-/-) mice as donors in the C57BL/6-->(C57BL/6 x DBA/2)F1-hybrid strain combination. Recipients of grafts from Jalpha281(-/-) donors were not protected from either the morbidity or the severe wasting syndrome associated with the development of acute GVHD, but the concentrations of some T helper 1 (Th1) and Th2 cytokines were different from those seen in recipients of grafts from wild-type donors. Interferon-gamma was seen earlier (day 4) in recipients of grafts from Jalpha281(-/-) donors but did not reach the concentrations seen in recipients of grafts from wild-type donors on day 8 (P < 0.02). On day 8, the amount of tumour necrosis factor-alpha released into the serum following the injection of a small amount of lipopolysaccharide was lower in recipients of grafts from Jalpha281(-/-) donors (P < 0.02). The amount of interleukin (IL)-5 was also lower in recipients of grafts from Jalpha281(-/-) donors, when compared to the concentration seen in recipients of grafts from wild-type donors (P < 0.002). IL-13 was seen in recipients of grafts from Jalpha281(-/-) donors but not in recipients of grafts from wild-type donors. Our findings show that the absence of donor Valpha14i NKT cells is associated with lower concentrations of some Th1 cytokines. We also observed higher IL-13 concentrations and lower IL-5 concentrations in recipients of grafts from Jalpha281(-/-) donors indicating a variable effect on Th2 cytokine production.

Selection of the cutaneous intraepithelial γδ+ T cell repertoire by a thymic stromal determinant

Intraepithelial lymphocytes constitute a group of T cells that express mainly monospecific or oligoclonal T cell receptors (TCRs). Like adaptive TCR alphabeta+ T cells, intraepithelial lymphocytes, a subset enriched in TCR gammadelta+ T cells, are proposed to be positively selected by thymically expressed self agonists, yet no direct evidence for this exists at present. Mouse dendritic epidermal T cells are prototypic intraepithelial lymphocytes, displaying an almost monoclonal TCR gammadelta+ repertoire. Here we describe an FVB substrain of mice in which this repertoire was uniquely depleted, resulting in cutaneous pathology. This phenotype was due to failure of dendritic epidermal T cell progenitors to mature because of a heritable defect in a dominant gene used by the thymic stroma to 'educate' the natural, skin-associated intraepithelial lymphocyte repertoire to be of physiological use.

A unique lymphotoxin {alpha}beta-dependent pathway regulates thymic emigration of V{alpha}14 invariant natural killer T cells.

Natural killer (NK) T cells using an invariant Valpha14 (Valpha14i) T cell receptor rearrangement form a distinct immunoregulatory T cell lineage. Several studies indicated that a NK1.1(-) Valpha14i NKT precursor cell differentiates and expands within the thymus before export to the peripheral tissues occurs. However, little is known about the signals that cause the emigration of Valpha14i NKT cells from the thymus to the periphery. Here we show that signaling of lymphotoxin (LT) alphabeta through the LTbeta receptor (LTbetaR) is indispensable for regulating peripheral but not thymic Valpha14i NKT cell numbers. Homing to and homeostatic proliferation of thymic Valpha14i NKT cells in peripheral organs, however, was not dependent on LTbetaR. Instead, our data indicate that a LTbetaR-expressing thymic stromal cell regulates the thymic emigration of Valpha14i NKT cells but not conventional T cell receptor alphabeta cells.

Gene transfer of RANTES and MCP-1 chemokine antagonists prolongs cardiac allograft survival

Vascularized organ allografts are rapidly destroyed by host immune cells that are recruited along chemokine gradients. Among chemokines, Regulated on Activation, Normal T-cell Expressed and Secreted (RANTES) CC chemokine ligand (CCL5) and monocyte chemoattractant protein (MCP)-1 (CCL2) are upregulated in rejecting cardiac allografts. To antagonize these chemokines, we constructed adenoviral vectors expressing NH(2)-terminal deletion (8ND) mutants of the respective genes. Using the F344-to-LEW rat model, intragraft gene transfer of chemokine analogs prolonged cardiac allograft survival from 10.1+/-0.7 and 10.4+/-0.7 days using non-coding adenovirus and vehicle alone, respectively, to 17.0+/-0.7 days for 8ND-RANTES (P<0.001) and 14.2+/-0.8 days for 8ND-MCP-1 (P<0.01). 8ND-RANTES reduced graft infiltration by monocytes/macrophages, cluster of differentiation (CD) 8alpha(+) and T-cell receptor alphabeta(+) cells, while 8ND-MCP-1 reduced monocytes/macrophages. In mixed leukocyte reactions in vitro, proliferation of host lymphocytes from regional lymph nodes in response to donor splenocytes was unaffected by 8ND-RANTES gene transfer. Using a two-gene approach, the contribution of 8ND-MCP-1 was negligible, consistent with available evidence that 8ND-RANTES inhibits both RANTES and MCP-1 activities. 8ND-RANTES gene transfer and a short course of low-dose cyclosporine A synergistically prolonged graft survival to 37.8+/-5.5 vs 15.4+/-0.5 days with cyclosporine alone (P<0.001). These results suggest a role for anti-chemokine gene therapy as an adjuvant therapy in heart transplantation.

Rat Thymectomy Effects on Leptin Receptor and T-bet: Erythroid Hyperplasia with Maturation Arrest and Suppressed T-Cell-Mediated Hepatotoxicity

Thymectomy is an inevitable therapy for thymoma. Therefore, determining hemato-immune changes post-thymectomy is important. Twenty-six normal LEW/Sea rats thymectomized (Tx) at the ages of 38 +/- 5 days were followed without any treatment for 4 months (experiments [Exp] A and B). In addition, 16 LEW/Sea rats Tx at the age of 42 days (Exp C and D) and 10 non-Tx control LEW/Sea rats (Exp E) were immunized with syngeneic male liver cells 2 to 3 times and followed 2.7 months after the first immunization. Flow cytometric (FCM) analysis of mesenteric lymph nodes (MLN) and peripheral blood (PB) showed as follows: among the 26 Tx rats (Exp A and B), MLN lymphocyte population at 4 months postthymectomy was characterized by decreased numbers of CD4+ cells (22%-36%) or alphabeta T-cell receptor (TCR)-positive cells (16%-54%) and increased numbers of interleukin 2 receptors (IL-2R) (>90%). In the 16 Tx-immunized rats (Exp C and D), both alphabeta TCR (MLN) and CD45R (PB) expression on lymphocytes was suppressed with rather high numbers of CD4. Bone marrow (BM) and PB hematological studies of the partially and totally Tx rats indicated the following: 8 BM of the 10 totally Tx males (Exp A) and the 7/10 male spleen showed erythroid hyperplasia with maturation arrest at the stage of basophilic erythroblasts and reticulocytopenia in the PB. One of the 10 males, which had both BM myeloid to erythroid (M/E) ratio of 4.3 and spleen myeopoiesis, was in a more advanced stage, a prestage of pure red cell aplasia (PRCA) with 22% of CD4+ MLN cells. Syngeneic liver cell immunization resulted in the Tx rats as follows: hepatotoxicity based on the immunization was weaker in the 16 Tx rats (Exp C and D) than in the 10 non-Tx rats (Exp E). Polymerase chain reaction (PCR) of PB and MLN showed compensatory activated leptin receptor (LR) and T-bet DNA in 19 Tx rats (Exp A and B) with abnormal FCM findings. The ineffective erythropoiesis at 4 months after thymectomy was explained by the erythroblast LR/erythropoietin receptor (EPOR) dysfunction. The low grade hepatotoxicity in the Tx rats (Exp C and D) was explained by the disturbed Th1 reactions (or the disturbed T-bet gene transcription) at postthymectomy.

Genetic alterations in caspase-10 may be causative or protective in autoimmune lymphoproliferative syndrome

Autoimmune lymphoproliferative syndrome (ALPS) is characterized by lymphadenopathy, elevated numbers of T cells with alphabeta-T cell receptors but neither CD4 nor CD8 co-receptors, and impaired lymphocyte apoptosis in vitro. Defects in the Fas receptor are the most common cause of ALPS (ALPS Ia), but in rare cases other apoptosis proteins have been implicated, including caspase-10 (ALPS II). We investigated the role of variants of caspase-10 in ALPS. Of 32 unrelated probands with ALPS who did not have Fas defects, two were heterozygous for the caspase-10 missense mutation I406L. Like the previously reported ALPS II-associated mutation L285F, I406L impaired apoptosis when transfected alone and dominantly inhibited apoptosis mediated by wild type caspase-10 in a co-transfection assay. Other variants in caspase-10, V410I and Y446C, were found in 3.4 and 1.6% of chromosomes in Caucasians, and in 0.5 and <0.5% of African Americans, respectively. In contrast to L285F and I406L, these variants had no dominant negative effect in co-transfection assays into the H9 lymphocytic cell line. We found healthy individuals homozygous for V410I, challenging the earlier suggestion that homozygosity for V410I alone causes ALPS. Moreover, an association analysis suggested protection from severe disease by caspase-10 V410I in 63 families with ALPS Ia due to dominant Fas mutations (P<0.05). Thus, different genetic variations in caspase-10 can produce contrasting phenotypic effects.

Role of the T-cell receptor in kidney ischemia–reperfusion injury

T cells have been demonstrated to modulate ischemia-reperfusion injury (IRI) in kidney, lung, liver and intestine. The underlying mechanisms for T-cell engagement in IRI are unknown. We hypothesized that the T-cell receptor (TCR) plays a role in renal IRI, and examined the effects of TCR alpha/beta (alphabeta) and gamma/delta (gammadelta) deficiency on ischemic acute renal failure (ARF). TCR-specific deficiency in specific mice was confirmed by fluorescence-activated cell sorting analysis using monoclonal antibodies (Abs). IRI was induced by bilateral clamping of kidney pedicles for 30 min, followed by reperfusion. Serum creatinine and kidney histopathology were used to assess the severity of experimental ARF. TCR alphabeta-deficient mice were significantly protected from kidney dysfunction compared to wild-type (WT) littermates after IRI (P<0.05). Histologic analysis demonstrated a significant reduction in renal tubular injury in both TCR alphabeta- and gammadelta-deficient mice compared to WT mice postischemia. TCR alphabeta-deficient mice had reduced tumor necrosis factor-alpha and interleukin-6 protein expression in kidney tissue compared to WT mice at 24 h postischemia using a microbead-based protein detection platform. Relative protection from kidney IRI did not correlate with neutrophil and macrophage infiltration of kidney tissue. Thus, the TCR plays a direct but modest pathophysiological role in kidney IRI. These data suggest that alloantigen-independent activation in IRI can lead to engagement of antigen-specific molecules on T cells. Furthermore, given that the TCR is already a target for diagnostics and therapeutic strategies in immune diseases, these approaches can now be harnessed for IRI.

免疫システムの新たな実体:基本免疫と獲得免疫

Recently, it has been turned out that our internal defense system is composed of two distinct components; innate/natural immune system and acquired/adaptive immune system. The former innate immunity is principally located at the surface area such as skin and mucosal compartment, while the latter acquired immunity is observed mainly in the circulating blood and lymphoid organs. The critical difference between those two systems exists in the receptors as well as their ligands. Rearranged gene-derived receptors like immunoglobulin (Ig) and MHC molecule-restricted alphabeta-type of T-cell receptors (TCR) with high specificities and memories are used to recognize peptide antigens in the acquired immunity, whereas non-rearranged invaliant receptors such as toll-like receptors (TLR), gammasigmaTCR and CD1 molecule-restricted alphabeta TCR are employed to detect lipid/glycolipid or nucleic acid-related antigens in the innate immunity. Based on such new findings, the actual roles of immunity are discussed.

T cell‐mediated hypersensitivity to quinolones: mechanisms and cross‐reactivity

Quinolones are widely used, broad spectrum antibiotics that can induce immediate- and delayed-type hypersensitivity reactions, presumably either IgE or T cell mediated, in about 2-3% of treated patients. To better understand how T cells interact with quinolones, we analysed six patients with delayed hypersensitivity reactions to ciprofloxacin (CPFX), norfloxacin (NRFX) or moxifloxacin (MXFX). We confirmed the involvement of T cells in vivo by patch test and in vitro by means of the lymphocyte proliferation test (LTT). The nature of the drug-T cell interaction as well as the cross-reactivity with other quinolones were investigated through the generation and analysis (flow cytometry and proliferation assays) of quinolone-specific T cell clones (TCC). The LTT confirmed the involvement of T cells because peripheral blood mononuclear cells (PBMC) mounted an enhanced in vitro proliferative response to CPFX and/or NRFX or MXFX in all patients. Patch tests were positive after 24 and 48 h in three out of the six patients. From two patients, CPFX- and MXFX-specific CD4(+)/CD8(+) T cell receptor (TCR) alphabeta(+) TCC were generated to investigate the nature of the drug-T cell interaction as well as the cross-reactivity with other quinolones. The use of eight different quinolones as antigens (Ag) revealed three patterns of cross-reactivity: clones exclusively reacting with the eliciting drug, clones with a limited cross-reactivity and clones showing a broad cross-reactivity. The TCC recognized quinolones directly without need of processing and without covalent association with the major histocompatability complex (MHC)-peptide complex, as glutaraldehyde-fixed Ag-presenting cells (APC) could present the drug and washing quinolone-pulsed APC removed the drug, abrogating the reactivity of quinolone-specific TCC. Our data show that T cells are involved in delayed immune reactions to quinolones and that cross-reactivity among the different quinolones is frequent.

Class II-restricted T cell receptor engineered in vitro for higher affinity retains peptide specificity and function.

The T cell receptor (TCR) alphabeta heterodimer determines the peptide and MHC specificity of a T cell. It has been proposed that in vivo selection processes maintain low TCR affinities because T cells with higher-affinity TCRs would (i) have reduced functional capacity or (ii) cross-react with self-peptides resulting in clonal deletion. We used the class II-restricted T cell clone 3.L2, specific for murine hemoglobin (Hb/I-E(k)), to explore these possibilities by engineering higher-affinity TCR mutants. A 3.L2 single-chain TCR (Vbeta-linker-Valpha) was mutagenized and selected for thermal stability and surface expression in a yeast display system. Stabilized mutants were used to generate a library with CDR3 mutations that were selected with Hb/I-E(k) to isolate a panel of affinity mutants with K(D) values as low as 25 nM. Kinetic analysis of soluble single-chain TCRs showed that increased affinities were the result of both faster on-rates and slower off-rates. T cells transfected with the mutant TCRs and wild-type TCR responded to similar concentrations of peptide, indicating that the increased affinity was not detrimental to T cell activation. T cell transfectants maintained exquisite hemoglobin peptide specificity, but an altered peptide ligand that acted as an antagonist for the wild-type TCR was converted to a strong agonist with higher-affinity TCRs. These results show that T cells with high-affinity class II reactive TCRs are functional, but there is an affinity threshold above which an increase in affinity does not result in significant enhancement of T cell activation.

An early intestinal mucosal source of gamma interferon is associated with resistance to and control of Cryptosporidium parvum infection in mice.

Resistance to and control of Cryptosporidium parvum infection in mice in the absence of adaptive immunity appears to be gamma interferon (IFN-gamma) dependent. Using an IFN-gamma-neutralizing antibody in a murine model, we demonstrated increased susceptibility to infection within 24 h. We correlated this early resistance and control with increased mucosal expression of IFN-gamma and demonstrate that CD8+ T-cell receptor alphabeta intestinal intraepithelial lymphocytes express and secrete this cytokine shortly after infection. The rapid kinetics of IFN-gamma expression and secretion by naive CD8+ T cells in response to a protozoan pathogen have not previously been demonstrated.

The frequency of double‐positive thymocytes expressing an αβ TCR clonotype regulates peripheral CD4 T cell compartment homeostasis

The present study aimed to determine whether the frequency of double positive (DP) thymocytes expressing alphabeta T-cell receptor (TCR) clonotypes at the time of selection regulates peripheral CD4 T-cell compartment size. Scid recipients were inoculated with various ratios of TCR Calpha(0/0) and wild-type bone marrow (BM) stem cells. Increasing the frequency of TCR Calpha(0/0) thymocytes at steady-state introduced a graded decrease in the maturation probability of the total DP thymocyte pool. At 12-14 weeks following BM inoculation, the frequency of TCR Calpha(0/0) DP thymocytes was inversely correlated with that of CD4 single positive (SP) thymocytes. Notwithstanding, a decreased frequency of wild-type DP thymocytes led to a marked increase in their transit efficiency from the DP to SP compartments. The frequency-dependent increase in thymocyte transit efficiency was associated with a CD4 SP cell surface phenotype indicative of increased antigenic experience. Importantly, the frequency of DP thymocytes capable of expressing TCR clonotypes dictated the steady-state size of the peripheral CD4 T cell compartment and its potential for homeostatic proliferation. Collectively, these results indicate that the efficiency of DP to CD4 SP transit is a frequency dependent process, which determines (1) the steady-state size of the peripheral T cell compartment and (2) the threshold for homeostatic expansion of peripheral CD4 T cells.