Immunogenicity of Peptide-25 of Ag85B in Th1 development: role of IFN-gamma

Ag85B (also known as alpha antigen or MPT59) is immunogenic, and induces expansion and differentiation of TCRVbeta11(+)CD4(+) T cells to IFN-gamma-producing cells in C57BL/6 (I-A(b)) mice. We reported that Peptide-25 (amino acids 240-254) of Ag85B is a major T cell epitope, and its amino acid residues at position 244, 247, 249 and 252 are I-A(b) contact residues. Here we examined roles of IFN-gamma in the generation of Peptide-25-reactive CD4(+) TCRVbeta11(+) T cells and the efficacy of mutant peptides of Peptide-25 for T(h)1 development in mice other than C57BL/6 mice. Immunization of C57BL/6 mice with Peptide-25 included in incomplete Freund's adjuvant led to preferential induction of CD4(+) TCRVbeta11(+) IFN-gamma- and tumor necrosis factor-alpha-producing T cells. Compared with other I-A(b)-binding peptides such as Peptide-9 of Ag85B, 50V of pigeon cytochrome c and ovalbumin (OVA)(265-280) peptide, only Peptide-25 was capable of inducing enormous expansion of TCRVbeta11(+) IFN-gamma-producing T cells. Treatment of C57BL/6 mice with anti-Vbeta11 antibody before Peptide-25 immunization reduced the development of CD4(+) IFN-gamma-producing T cells. Furthermore, B10.A(3R) mice, I-A(b)-positive and TCRVbeta11(-) strain, showed remarkably lower response to Peptide-25 immunization than C57BL/6 mice. Peptide-25-primed IFN-gamma(-/-) cells showed significantly decreased expansion of CD4(+) TCRVbeta11(+) T cells as compared with wild-type cells. Interestingly, Peptide-25-primed cells from MyD88-deficient mice responded to Peptide-25 and differentiated into IFN-gamma-producing cells to a similar extent as wild-type mice, indicating Toll-like receptor-independent IFN-gamma production. These results imply that IFN-gamma plays important roles for the generation and expansion of CD4(+) TCRVbeta11(+) T cells in response to Peptide-25. Although Peptide-25 was non-immunogenic in C3H/HeN mice, a substituted mutant of Peptide-25, 244D247V, capable of binding to I-A(k), induced T(h)1 development. These results clearly demonstrate important roles of IFN-gamma in the expansion of CD4(+) TCRVbeta11(+) T cells, and will provide useful information for delineating the regulatory mechanisms of T(h)1-cell development and for analyzing mechanisms on T(h)1-dominant immune responses.     

Mapping of V beta 11+ helper T cell epitopes on mycobacterial antigen in mouse primed with Mycobacterium tuberculosis

Antigenic epitopes for Mycobacterium tuberculosis-reactive T cell immune responses have been mapped using the purified Mycobacterium protein antigen. Lymph node cells from C57BL/6 mice that had been immunized with heat-killed M. tuberculosis were cultured with various Mycobacterium protein antigens and their reactivity was monitored by proliferative response. Usage of the TCR beta chain repertoire was analyzed by flow cytometry. Stimulation of M. tuberculosis-primed lymph node cells with MPT59 (antigen 85B, alpha antigen) induced proliferative response, production of IL-2 and IFN-gamma, and the expansion of V beta 11+ CD4+ T cells in conjunction with antigen- presenting cells in an I-Ab-restricted manner. Lymph node cells from non-primed mice failed to proliferate in response to MPT59. Using peptides covering the complete mature 285 amino acids long MPT59 protein as 15-mer molecules overlapping by five amino acids, we identified the antigenic epitope for MPT59-specific V beta 11+ T cells. The 15-mer peptide, covering amino acid residues 240-254 of MPT59 [peptide-25 (amino acids 240-254)], contains the motif that is conserved for I-Ab and requires processing by antigen-presenting cells to trigger peptide-25-specific V beta 11+ CD4+ T cells. We conclude from these results that MPT59 and peptide-25 (amino acids 240-254) are not superantigens and require antigen processing in order to stimulate V beta 11+ Th1 cells. This experimental system will provide us with a useful tool for delineating the regulation of T cell development in a particular subset of M. tuberculosis infection and for developing antigenic peptides for Th1-dominant immune responses.      

In Vitro Expansion of T-Cell-Receptor Vα2.3+ CD4+ T Lymphocytes in HLA-DR17(3), DQ2+ Individuals upon Stimulation with Mycobacterium tuberculosis

The T-cell receptor (TCR) Valpha/beta gene product expression upon in vitro stimulation with mycobacteria was investigated to assess whether T-cell proliferation was associated with any specific TCR V gene usage. T-cell-enriched populations from peripheral blood of Mycobacterium bovis BCG-vaccinated healthy blood donors were stimulated in vitro with live or killed M. tuberculosis or with a soluble extract thereof. TCR Valpha/beta repertoire analysis of reactive CD4(+) and CD8(+) T cells revealed a selective HLA-DR17(3), DQ2-restricted expansion of Valpha2.3(+) CD4(+) T cells upon stimulation with live M. tuberculosis or its soluble extract. Third-complementarity-determining-region (CDR3) length analysis of the expanded Valpha2.3(+) T cells indicated an oligoclonal pattern with short CDR3 lengths in six of seven HLA-DR17(3), DQ2(+) individuals tested. In addition, Valpha/Vbeta repertoire analysis of T lymphocytes from a DR17(3), DQ2(+) donor before and after BCG vaccination revealed that positivity of skin test reactivity was associated with expansion of Valpha2.3(+) CD4(+) T lymphocytes with preferential use of a short CDR3 peak length after in vitro stimulation. Separation of M. tuberculosis soluble extract by fast protein liquid chromatography (FPLC) purification indicated that fractions corresponding to molecular masses of 60 to 70 and 15 to 25 kDa were particularly effective in eliciting Valpha2.3(+) CD4(+) T-cell expansion.     

Schistosoma mansoni phosphoenolpyruvate carboxykinase, a novel egg antigen: immunological properties of the recombinant protein and identification of a T-cell epitope

In schistosomiasis mansoni, hepatic granulomatous inflammation surrounding parasite eggs is mediated by CD4(+) T helper (Th) cells sensitized to schistosomal egg antigens (SEA). We previously showed that a prominent lymphoproliferative response of CD4(+) Th cells from schistosome-infected C57BL/6 (BL/6) mice was directed against a 62-kDa component of SEA. A partial amino acid sequence of the 62-kDa component was found to be identical with one present in the enzyme phosphoenolpyruvate carboxykinase (PEPCK). Based on this sequence, a cDNA clone containing the entire coding region of PEPCK was identified, and the full recombinant Schistosoma mansoni PEPCK (rSm-PEPCK) of 626 amino acids was purified from a prokaryotic expression system. rSm-PEPCK strongly stimulated a specific T-cell hybridoma, 4E6, as well as CD4(+) Th cells from SEA-immunized BL/6 mice and from infected BL/6, CBA, and BALB/c mice. In the infected mice, rSm-PEPCK elicited significant gamma interferon production as well as, to a lesser extent, production of interleukin-2 and -5. In BL/6 and BALB/c mice, the CD4(+) Th cell response to rSm-PEPCK was greater than that directed against the egg antigen Sm-p40; conversely, CBA mice responded better to Sm-p40 than to Sm-PEPCK. A 12-amino-acid region (residues 398 to 409: DKSKDPKAHPNS) was demonstrated to contain a T-cell epitope; synthetic peptides containing this epitope significantly stimulated specific hybridoma 4E6 and polyclonal CD4(+) Th cells. The identification and characterization of immunogenic egg components will contribute to the understanding and possible control of T-cell-mediated schistosomal disease.     

Specific donor Vbeta-associated CD4 T-cell responses correlate with severe acute graft-versus-host disease directed to multiple minor histocompatibility antigens.

CXB-2/By (CXB-2) recombinant inbred mice express a subset of the minor histocompatibility antigen (miHA) repertoire expressed by C.B10-H2(b)/LiMcdJ (BALB.B) mice. On lethal irradiation and the transplantation of H2(b)-matched C57BL/6 (B6) T cell-depleted bone marrow cells, along with naive unfractionated T cells, both strains succumb to acute graft-versus-host disease (GVHD). Although alloreactive B6 CD4(+) T cells are a necessary source of T-cell help for the B6 CD8(+) component of the GVHD response in both recipient strains, they are capable of mediating severe GVHD by themselves only in BALB.B mice. Previous CD4(+) T-cell receptor repertoire analysis demonstrated overlapping oligoclonal Vbeta use between the CD4(+) B6 anti-BALB.B and B6 anti-CXB-2 responses, with indications of additional BALB.B unique T-cell responses (Vbeta2 and Vbeta11). We report here that the more severe B6 anti-BALB.B response is not due to a quantitative difference in the responding cells, because the frequency of alloreactive donor CD4(+) T cells over time was equivalent in the spleens of BALB.B versus CXB-2 recipients. The responses were also similar in the number of infiltrating B6 CD4(+) T cells in the lingual epithelium of the 2 recipients. In contrast, a significantly greater degree of infiltration and injury of BALB.B intestinal epithelium correlated with the increased level of clinical GVHD severity. Of most significance, despite the involvement of at least 11 Vbeta-associated CD4(+) T-cell families in the overall B6 anti-BALB.B response, the development of severe GVHD correlated with the presence of Vbeta2- and Vbeta11-positive donor T cells. Transplantation of donor CD4(+) T cells from Vbeta-associated families that were shared between the B6 anti-BALB.B and anti-CXB-2 responses resulted in minimal GVHD potential. These data suggest that severe GVHD across miHA barriers depends on the involvement of a restricted number of potent T-cell specificities and implies that there are only a limited number of corresponding responsible miHAs.     

I-A alpha k transgene pairs with I-A beta b gene and protects C57BL10 mice from developing autoimmune myasthenia gravis.

The I-A beta gene has been implicated in the pathogenesis of experimental autoimmune myasthenia gravis (EAMG), with amino acids at positions 67, 70, and 71 of the I-A beta b chain that play a crucial role. In addition, the cell surface expression of the I-E molecule in C57BL10.E alpha k transgenic mice was associated with the partially suppressed serum anti-acetylcholine receptor antibody and clinical expression of EAMG. In this study, the contribution of the I-A alpha gene and the A beta b:A alpha k transpair in EAMG pathogenesis is assessed. The I-A alpha k transgene was introduced into the B10 (A beta b:A alpha b) strain, and the I-A alpha k chain was paired with I-A beta b; therefore, the A beta b:A alpha k complex was expressed on the cell surface. Expression of the A beta b:A alpha k transpair in C57BL10 transgenic mice suppressed the cellular and humoral autoimmune responses to acetylcholine receptors (AChR), reduced the amount of muscle AChR bound with antibody, and significantly reduced the incidence of muscle weakness and its associated abnormal electrophysiological response.     

Instruction of naive CD4+ T-cell fate to T-bet expression and T helper 1 development: roles of T-cell receptor-mediated signals

Using T-cell receptor (TCR) transgenic mice, we demonstrate that TCR stimulation of naive CD4(+) T cells induces transient T-bet expression, interleukin (IL)-12 receptor beta2 up-regulation, and GATA-3 down-regulation, which leads to T helper (Th)1 differentiation even when the cells are stimulated with peptide-loaded I-A(b)-transfected Chinese hamster ovary cells in the absence of interferon-gamma (IFN-gamma) and IL-12. Sustained IFN-gamma and IL-12 stimulation augments naive T-cell differentiation into Th1 cells. Intriguingly, a significant Th1 response is observed even when T-bet(-/-) naive CD4(+) T cells are stimulated through TCR in the absence of IFN-gamma or IL-12. Stimulation of naive CD4(+) T cells in the absence of IFN-gamma or IL-12 with altered peptide ligand, whose avidity to the TCR is lower than that of original peptide, fails to up-regulate transient T-bet expression, sustains GATA-3 expression, and induces differentiation into Th2 cells. These results support the notion that direct interaction between TCR and peptide-loaded antigen-presenting cells, even in the absence of T-bet expression and costimulatory signals, primarily determine the fate of naive CD4(+) T cells to Th1 cells.     

Identification of murine H2-Dd- and H2-Ab-restricted T-cell epitopes on a novel protective antigen, MPT51, of Mycobacterium tuberculosis

Both CD4(+) type 1 helper T (Th1) cells and CD8(+) cytotoxic T lymphocytes (CTL) play pivotal roles in protection against Mycobacterium tuberculosis infection. Here, we identified Th1 and CTL epitopes on a novel protective antigen, MPT51, in BALB/c and C57BL/6 mice. Mice were immunized with plasmid DNA encoding MPT51 by using a gene gun, and gamma interferon (IFN-gamma) production from the immune spleen cells was analyzed in response to a synthetic overlapping peptide library covering the mature MPT51 sequence. In BALB/c mice, only one peptide, p21-40, appeared to stimulate the immune splenocytes to produce IFN-gamma. Flow cytometric analysis with intracellular IFN-gamma and the T-cell phenotype revealed that the p21-40 peptide contains an immunodominant CD8(+) T-cell epitope. Further analysis with a computer-assisted algorithm permitted identification of a T-cell epitope, p24-32. In addition, a major histocompatibility complex class I stabilization assay with TAP2-deficient RMA-S cells transfected with K(d), D(d), or L(d) indicated that the epitope is presented by D(d). Finally, we proved that the p24-32/D(d) complex is recognized by IFN-gamma-producing CTL. In C57BL/6 mice, we observed H2-A(b)-restricted dominant and subdominant Th1 epitopes by using T-cell subset depletion analysis and three-color flow cytometry. The data obtained are useful for analyzing the role of MPT51-specific T cells in protective immunity and for designing a vaccine against M. tuberculosis infection.     

Identification of an I-Ed-restricted T-cell epitope of Escherichia coli outer membrane protein F.

A predominant T-cell epitope of Escherichia coli outer membrane protein F (OmpF) that encompasses amino acids 295 to 314 was identified in H-2(d) mice. BALB/c-derived T-cell hybridomas generated against this region were CD3(+), CD4(+), CD8(-), and T-cell receptor alphabeta(+) and secreted TH-1-associated cytokines (interleukin-2 [IL-2] and gamma interferon), but not a TH-2-associated cytokine (IL-4), when restimulated with peptide 295-314. Class II(+) mouse lymphoma (A20) cells, but not class II(-) mouse mastocytoma (P815) cells, supported IL-2 secretion of hybridomas when substituted for syngeneic splenocytes as antigen-presenting cells (APCs). Antibodies specific for I-E(d) blocked IL-2 secretion by hybridomas, but I-A(d)-specific antiserum did not. When transfected L cells expressing I-A(d) (AalphaAbeta(d)), I-E(d) (EalphaEbeta(d)), or the hybrid molecule I-EalphaAbeta(d) were used as APCs, hybridomas recognized peptide only when presented by the I-E(d)-transfected cells. When peptide 295-314 truncated at either the C or the N terminus of the sequence was used, the minimal epitope was determined. Critical residues were determined by using alanine-substituted peptide analogues. T-cell hybridomas were only stimulated by peptides that encompassed amino acids 295 to 303 (9-mer), and the core sequence required a minimum of three additional amino acids at either the amino or the carboxy terminus to induce IL-2 secretion. Critical residues were determined to be phenylalanine at position 295, threonine at position 300, and tyrosines at positions 301 and 302. This study is the first to identify a minimal T-cell epitope and major histocompatibility complex restriction element of the OmpF protein and confirms previous observations that there is considerable degeneracy in the length of peptides that can bind I-E(d) and variability in the amino acid composition of the C and N termini of these peptides.     

Induction of Thymus-Derived γδ T Cells by Escherichia coli Enterotoxin B Subunit in Peritoneal Cavities of Mice

We examined the activation of intraperitoneal T cells in BALB/c mice by the Escherichia coli enterotoxin B subunit, which induced a specific Th2 type of T-cell response to intraperitoneally coadministered bovine immunoglobulin G. The numbers of both γδ and αβ T cells increased significantly after intraperitoneal administration of the B subunit in a time-dependent manner; these numbers were not affected by the B-subunit G33D mutant, which is defective in GM₁ ganglioside-binding ability. Early after administration a small number of γδ T cells produced either interleukin-4 (IL-4) or gamma interferon, while late after administration primarily IL-10-producing γδ T cells were detected. γδ T cells induced by the B subunit did not express a characteristic V gene over the time course of the study. The induction of γδ T cells did not occur in athymic nu/nu mice but could be induced upon transplantation of fetal AKR thymus-like αβ T cells. γδ T cells in athymic nu/nu mice with a fetal thymic graft predominantly expressed the donor Thy-1.1 antigen but not the host Thy-1.2 antigen. The induction of these T cells, however, could not be restored by coadministration of the B subunit with peritoneal cells from normal mice. These results suggest that the B subunit activates intraperitoneal γδ and αβ T cells in a manner dependent upon its ability to bind to GM₁ ganglioside. γδ T cells induced by the B subunit are Th2-type cells derived from the thymus. These γδ T cells may be functionally involved in specific Th2 responses to the B subunit, which possibly acts as an adjuvant through the influence of αβ T cells.     

Identification and HLA restriction of naturally derived Th1-cell epitopes from the secreted Mycobacterium tuberculosis antigen 85B recognized by antigen-specific human CD4(+) T-cell lines

Antigen 85B (Ag85B/MPT59) is a major secreted protein from Mycobacterium tuberculosis which is a promising candidate antigen for inclusion in novel subunit vaccines against tuberculosis (TB). The present study was undertaken to map naturally derived T-cell epitopes from M. tuberculosis Ag85B in relation to major histocompatibility complex (MHC) class II restriction. Antigen-specific CD4(+) T-cell lines were established from HLA-typed TB patients and Mycobacterium bovis BCG vaccinees by stimulation of peripheral blood mononuclear cells with purified Ag85B in vitro. The established T-cell lines were then tested for proliferation and gamma interferon (IFN-gamma) secretion in response to 31 overlapping synthetic peptides (18-mers) covering the entire sequence of the mature protein. The results showed that the epitopes recognized by T-cell lines from TB patients were scattered throughout the Ag85B sequence whereas the epitopes recognized by T-cell lines from BCG vaccinees were located toward the N-terminal part of the antigen. The T-cell epitopes represented by peptides p2 (amino acids [aa] 10 to 27), p3 (aa 19 to 36), and p11 (aa 91 to 108) were frequently recognized by antigen-specific T-cell lines from BCG vaccinees in both proliferation and IFN-gamma assays. MHC restriction analysis demonstrated that individual T-cell lines specifically recognized the complete Ag85B either in association with one of the self HLA-DRB1, DRB3, or DRB4 gene products or nonspecifically in a promiscuous manner. At the epitope level, panel studies showed that peptides p2, p3, and p11 were presented to T cells by HLA-DR-matched as well as mismatched allogeneic antigen-presenting cells, thus representing promiscuous epitopes. The identification of naturally derived peptide epitopes from the M. tuberculosis Ag85B presented to Th1 cells in the context of multiple HLA-DR molecules strongly supports the relevance of this antigen to subunit vaccine design.     

Increase of γδ T Lymphocytes in Murine Lungs Occurs during Recovery from Pulmonary Infection by Nocardia asteroides

Previous studies have demonstrated that gammadelta T lymphocytes are important for host resistance to pulmonary infection of the murine lung by log-phase cells of Nocardia asteroides. To study the role of gammadelta T cells in nocardial interactions in the murine lung, C57BL/6J wild type and C57BL/6J-Tcrd (gammadelta T-cell knockout mice) were infected intranasally with log-phase cells of N. asteroides GUH-2. At 3, 5, and 7 days after infection, the gammadelta T cells were quantified by multiparameter flow cytometry. At the same time, Gram and hematoxylin-eosin stains of paraffin sections were performed to monitor the host responses. The data showed that gammadelta T lymphocytes increased significantly within the lungs after intranasal infection, and the peak of this cellular increase occurred at 5 days. Furthermore, at this time, greater than 50% of the CD3 T-cell receptor (TCR)-positive (CD3+) cells were gammadelta TCR positive. Histological examination clearly showed divergent inflammatory responses in the lungs of wild-type mice compared to gammadelta T-cell knockout mice. The C57BL/6J-Tcrd mice were less capable of clearing the organism, and the polymorphonuclear leukocyte response lasted longer than in wild-type C57BL/6J mice. These results showed that gammadelta T cells were actively involved in modulating the innate host responses to murine pulmonary infection by N. asteroides.     

A novel 62-kilodalton egg antigen from Schistosoma mansoni induces a potent CD4(+) T helper cell response in the C57BL/6 mouse

In infection with Schistosoma mansoni, hepatic granuloma formation is mediated by CD4(+) T helper (Th) cells sensitized to schistosomal egg antigens. There is considerable variation among infected individuals with respect to both severity of disease and the T-cell response to egg antigens. In the BL/6 mouse, the egg granulomas are relatively small and the relevant sensitizing egg antigens are largely unknown. We investigated the CD4(+) Th cell response of infected BL/6 mice to egg antigens fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and found a prominent lymphoproliferative response to be directed against a 62-kDa component. With the aid of a specific T-cell hybridoma, 4E6, the 62-kDa antigen was isolated; following partial digestion with endoproteinase Glu-C, an internal amino acid sequence was found to be identical with one present in the enzyme phosphoenolpyruvate carboxykinase (PEPCK) of the organisms Caenorhabditis elegans and Treponema pallidum and to differ by one residue from PEPCK of various other species. In CD4(+) Th cells from 7.5- 8.5-week-infected BL/6 mice, the purified 62-kDa molecule elicited a potent proliferative response which, based on cytokine analysis, was of a mixed Th-1 and Th-2 type. Our results reveal a novel egg antigen of particular prominence in the BL/6 mouse and suggest that the immune response in schistosomiasis is a product of sensitization to egg antigens that may vary considerably in immunogenicity from strain to strain.     

Promiscuous T cell recognition of an H-2 IA-presented mycobacterial epitope

Genetically permissive T cell epitopes are an important prerequisite for the development of peptide-based vaccines or immunodiagnostic reagents. We have investigated the structural requirements of permissive T cell recognition of peptide p350—369 from the 38-kDa antigen of Mycobacterium tuberculosis. This peptide was found to be immunogenic in mice of the H-2^{b, bm12, d. s and k}, but not of the H-2^f genotype. T cell responses were restricted by I-A class II molecules. The same epitope core was recognized in the H-2^{b, d and k} genotypes. T cell hybrids from BALB/c and C57BL/10 mice were used to determine: (i) the critical residues using substituted peptide derivatives and (ii) the degree of T cell promiscuity. Two out of five BALB (H-2^d)-derived hybridomas tested displayed promiscuous peptide recognition in the context of H-2^b and H-2^bm12 antigen-presenting cells. The recognition of critical residues was found to be uniform for all five hybridomas when tested with syngeneic antigen-presenting cells; additional critical residues were identified when the peptide was recognized in the context of allogeneic antigen-presenting cells. Only one of the four tested C57BL/10 (H-2^b) hybridomas showed promiscuity in the context of H-2^bm12. Each of these C57BL/10-derived clones had a distinct response profile toward the critical residues. We propose that the demonstrated T cell promiscuity involves peptide interaction with polymorphic H-2 I-A residues.     

T cells recognize a peptide derived from α-gliadin presented by the celiac disease-associated HLA-DQ (α10501, β10201) heterodimer

CD is unique among the HLA-associated diseases since (a) the disease-promoting agent (gliadin) is known and (b) the disease is precipitated mainly in individuals carrying a particular cis- or trans-encoded HLA-DQ heterodimer; i.e., DQ(α1^*0501, β1^*0201). Further, a preponderance of gliadin-specific T cells derived from the small intestinal mucosa of CD patients are restricted by this DQ heterodimer. T-cell recognition of gliadin peptides presented by the DQ(α1^*, β1^*0201) heterdimer may thus be of importance in CD. Here we report that a T-cell clone from a patient with CD recognizes a synthetic α-gliadin peptide, when presented by the cis- or trans-encoded CD-associated DQ(α1^*0501, β1^*0201) heterdimer. The minimal peptide recognized by the T-cell clone corresponds to residues 31–47 of α-gliadin, which is included in the part of α-gliadin previously shown to have disease-promoting activity. When testing analogue peptides derived from other α-gliadin sequences, one peptide differing by one amino acid was recognized by the T-cell clone, whereas the other peptide differing by two amino acids was not recognized. Our findings demonstrate that the CD-associated DQ(α1^*0501, β1^*0201) heterodimer may serve as an antigen-presenting molecule to T cells for certain gliadin peptides.     

Both B and gammadelta TCR(+) lymphocytes regulate alphabeta TCR(+) lymphocytes involved in superantigen specific responses

Endogenous superantigens (SAg) presented by MHC class II IA molecules induce slow-evolving negative selection of alpha beta T cells. The role of both B and gamma delta T cells on the regulation of these SAg-specific alpha beta T cell responses was addressed in IA(b+)IE(b-) C57BL/6 mice bearing genetically induced B cell and gamma delta T cell deficiencies. B lymphocytes were required in the negative selection of Vbeta5(+)/Vbeta12(+) CD4(+) T cells. In contrast, gamma delta T cells positively stimulated the utilization of the same SAg-responsive alpha beta T cell subsets. These differences started in mature CD4(+) thymocytes and extended to naive T cell pools for B cell negative selection, and up to memory T cells for gamma deltaT cell influences. The levels of SAg-responsive T cells did not vary between C57BL/6 and double deficient (B cell and gamma delta T cell-deficient) congenic mice, implying that both B and gamma delta T cells acted through independent mechanisms.     

Selection of Distinct Vα/β T-Cell Receptor Families During In Vivo and In Vitro T-Cell Maturation

The experimental conditions influencing the use of Valphabeta TCR families were examined in lymph node (LN) cells from peptide-immunized C57BL/6 and Vbeta8.2 transgenic mice. Expanded proportions of Vbeta5, Vbeta8.2, Vbeta9, Vbeta12 and Vbeta14 positive cells and an association of Vbeta8.2 with Valpha11 was found in freshly harvested 8-day or 34-day immune LN cells. In contrast, peptide-specific T-cell lines generated in vitro from 8-day immune lymph node cells were found to be almost exclusively of the Valpha2/Vbeta12 family. However, T-cell lines originating from Vbeta8.2 transgenic mice did not show preferential Valpha usage. Anti-Vbeta8.2 antibody produced different effects: when added to cultures of LN cells from C57BL/6 or Vbeta8.2 transgenic strains, the peptide-induced proliferation was suppressed; however, following the injection of mice, subsequent in vitro proliferation and cytokine production induced by both peptide and Concanavalin A was suppressed in Vbeta8.2 transgenic, but much less in C57BL/6 mice. Hence, compensatory expansion of different Vbeta gene products occurred in vivo, but not under the employed in vitro conditions. In conclusion, these results suggest that the TCR family usage is influenced by the experimental conditions in which the T cells are selected and expanded and by the genetic potentials of the precursor pool.     

Role of interleukin-1beta in activating the CD11c(high) CD45RB- dendritic cell subset and priming Leishmania amazonensis-specific CD4+ T cells in vitro and in vivo

Cutaneous leishmaniasis associated with Leishmania amazonensis infection is characterized by uncontrolled parasite replication and profound immunosuppression; however, the underlying mechanisms remain largely unclear. One possibility is that the L. amazonensis parasite modulates antigen-presenting cells, favoring the generation of pathogenic Th cells that are capable of recruiting leukocytes but insufficient to fully activate their microbicidal activities. To test this possibility, we infected bone marrow-derived dendritic cells (DCs) of C57BL/6 mice with L. amazonensis or Leishmania major promastigotes and assessed the activation of DC subsets and their capacity in priming CD4(+) T cells in vitro. In comparison to L. major controls, L. amazonensis-infected DCs secreted lower levels of interleukin-1alpha (IL-1alpha) and IL-1beta, were less potent in activating the IL-12p40-producing CD11c(high) CD45RB(-) CD83(+) CD40(+) DC subset, and preferentially activated CD4(+) T cells with a IFN-gamma(low) IL-10(high) IL-17(high) phenotype. Although the addition of IL-1beta at the time of infection markedly enhanced DC activation and T-cell priming, it did not skew the cytokine profile of DCs and pathogenic Th cells, as local injection of IL-1beta following L. amazonensis infection accelerated Th cell activation and disease progression. This study suggests that intrinsic defects at the level of DC activation are responsible for the susceptible phenotype in L. amazonensis-infected hosts and that this parasite may have evolved unique mechanisms to interfere with innate and adaptive immunity.     

A T-cell clone recognizing an MLC stimulating epitope located on the DRw11β1 chain

An alloreactive proliferative T-cell clone, 6065 WS, was obtained in an intrafamilial cell combination where the stimulator was a homozygous DRw11, DRw52, DQw3 son and the responder his haploidentical mother. Proliferation assays on eight local DRw11 families, 75 homozygous B-cell lines (Tenth Histocompatibility Workshop panel), and blocking assays with monoclonal antibodies showed that clone 6065 WS recognizes an epitope on the DRw11β1 chain. Comparison of the reactivity of clone 6065 WS with cells expressing the three known DRw11β1 amino acid sequences identified two unique amino acids at positions 71 and 86 which contribute to determining the specific recognition by the T-cell clone 6065 WS. Our data suggest that one or both of these amino acids can either be directly involved in the recognition by the T-cell receptor or responsible for critical conformation of the determinants on the DR molecule. Alternatively, they could affect recognition of a self peptide bound to the major histocompatibility complex class II molecule.