Experimental Trypanosoma cruzi infection alters the shaping of the central and peripheral T-cell repertoire

We investigated the thymic and peripheral T-lymphocyte subsets in BALB/c mice undergoing acute or chronic Trypanosoma cruzi infection, in terms of expression of particular Vbeta rearrangements of the T-cell receptor. We first confirmed the severe depletion of CD4(+)CD8(+) thymocytes following acute T. cruzi infection. By contrast, the numbers of CD4(+)CD8(+) cells in subcutaneous lymph nodes increased up to 16 times. In subcutaneous lymph nodes, we found CD4(+)CD8(+) cells that expressed prohibited segments TCRVbeta5 and TCRVbeta12 (which are physiologically deleted in the thymus of BALB/c mice), as did some mature single-positive cells (CD4(+)CD8(-) and CD4(-)CD8(+)). In the thymus of infected animals, although higher numbers of immature cells bearing such Vbeta segments were seen, they were no longer detected in the mature single-positive stage, suggesting that negative selection occurs normally. We also found increased numbers of cells bearing the potentially autoreactive phenotype TCRVbeta5(+) and TCRVbeta12(+) in T-lymphocyte subsets from subcutaneous lymph nodes of T. cruzi chronically infected mice. In conclusion, our data indicate that immature T lymphocytes bearing prohibited TCRVbeta segments leave the thymus and gain the lymph nodes, where they further differentiate into mature CD4(+) or CD8(+) cells. Conjointly, these findings show changes in the shaping of the central and peripheral T-cell repertoire in both acute and chronic phases of murine T. cruzi infection. The release of potentially autoreactive T cells in the periphery of the immune system may contribute to the autoimmune process found in both murine and human Chagas' disease.     

A large number of T lymphocytes recognize Moloney-murine leukemia virus-induced antigens, but a few mediate long-lasting tumor immunosurveillance

The CD8(+) T cell response to Moloney-murine leukemia virus (M-MuLV)-induced Ags is almost entirely dominated by the exclusive expansion of lymphocytes that use preferential TCRVbeta chain rearrangements. In mice lacking T cells expressing these TCRVbeta, we demonstrate that alternative TCRVbeta can substitute for the lack of the dominant TCRVbeta in the H-2-restricted M-MuLV Ag recognition. We show that, at least for the H-2(b)-restricted response, the shift of TCR usage is not related to a variation of the immunodominant M-MuLV epitope recognition. After virus immunization, all the potentially M-MuLV-reactive lymphocytes are primed, but only the deletion of dominant Vbeta rescues the alternative Vbeta response. The mechanism of clonal T cell "immunodomination" that guides the preferential Vbeta expansion is likely the result of a proliferative advantage of T cells expressing dominant Vbeta, due to differences in TCR affinity and/or cosignal requirements. In this regard, a CD8 involvement is strictly required for the virus-specific cytotoxic activity of CTL expressing alternative, but not dominant, Vbeta gene rearrangements. The ability of T cells expressing alternative TCRVbeta rearrangements to mediate tumor protection was evaluated by a challenge with M-MuLV tumor cells. Although T cells expressing alternative Vbeta chains were activated and expanded, they were not able to control tumor growth in a long-lasting manner due to their incapacity of conversion and accumulation in the T central memory pool.     

Identification of CD7 as a cognate of the human K12 (SECTM1) protein

CD7 is a 40-kDa protein found primarily on T, NK, and pre-B cells; the function of the CD7 protein in the immune system is largely unknown. The K12 (SECTM1) protein was originally identified by its location just upstream of the CD7 locus. The K12 gene encodes a transmembrane protein of unknown function. In order to clone a K12-binding protein, we generated a soluble version of the human K12 protein by fusing its extracellular domain to the Fc portion of human IgG(1). Flow cytometry experiments showed that the K12-Fc fusion protein bound at high levels to both human T and NK cells. Precipitation experiments using K12-Fc on (35)S-radiolabeled NK cells lysates indicated that the K12 cognate was an approximately 40-kDa protein. A human peripheral blood T cell cDNA expression library was screened with the K12-Fc protein, and two independent, positive cDNA clones were identified and sequenced. Both cDNAs encoded the same protein, which was CD7. Thus, K12 and CD7 are cognate proteins that are located next to each other on human chromosome 17q25. Additionally, we have cloned the gene encoding the mouse homologue of K12, shown that it maps near the mouse CD7 gene on chromosome 11, and established that the mouse K12 protein binds to mouse, but not human, CD7. Mouse K12-Fc inhibited in a dose-dependent manner concanavalin A-induced proliferation, but not anti-TcRalpha/beta induced proliferation, of mouse lymph node T cells. Human K12-Fc stimulated the up-regulation of CD25, CD54, and CD69 on human NK cells in vitro.     

Phenotype and effector function of CC chemokine receptor 9-expressing lymphocytes in small intestinal Crohn's disease

CCL25/CCR9 chemokine ligand/receptor pair has been reported to play an important role in small bowel (SB) immunity and inflammation. We have previously reported an aberrant SB expression of CCL25 in Crohn's disease (CD) and an increased frequency of CCR9(+) T cells in the peripheral blood of patients with SB inflammatory diseases such as CD and celiac disease. In this study, we have characterized the phenotype and effector function of CCR9(+) T cells in mucosal lymphoid tissues in CD. We show that CCR9(+) T cells isolated from mesenteric lymph nodes (MLN) draining CD SB express a more activated phenotype compared with MLN draining normal SB. Stimulation of CCR9(+) T cells isolated from CD SB lamina propria produced more IFN-gamma and IL-17 in response to anti-CD3 or IL-12/IL-18 stimulation compared with those isolated from normal SB. The addition of TL1A to the cytokine combination markedly augmented the secretion of IFN-gamma, but not IL-17, by CD lamina propria CCR9(+) T cells. CCL25 incubation of CD SB lamina propria lymphocytes and MLN lymphocytes increased their adhesion to VCAM-1/Fc in vitro. Finally, the TCRVbeta analysis of CCR9(+) T cells revealed a diverse TCRVbeta repertoire among MLN CCR9(+) T cells in patients with SB CD. Our data indicate that CCR9(+) T cells in SB CD are proinflammatory and support the rationale for the use of CCR9 antagonists for the treatment of human SB CD.     

Characterization and distribution of a new enterotoxin-related superantigen produced by Staphylococcus aureus

Staphylococcal enterotoxins (SEs) are a family of structurally related pyrogenic exotoxins consisting of the five prototypic SEs (types A to E) and three newly characterized SEs (types G to I) produced by Staphylococcus aureus (S. aureus). They also work as superantigens and cause food poisoning and shock symptoms in humans. In this study, we cloned a new variant gene of the seg and characterized its superantigenic properties and distribution among the clinical isolates of S. aureus. The gene encodes a 233 amino acid protein which is highly homologous to SEG (97.7%). The variant SEG (SEGv) expressed by the cloned gene exerted mitogenic activity on human peripheral blood mononuclear cells at the concentration of 100 pg/ml. T cells bearing Vbeta3, 12, 13.1, 13.2, 14 and 15 were preferentially expanded after stimulation with the recombinant protein. The mRNA of the variant seg gene was detected in the total RNA of the organisms bearing this gene. By PCR, 27 out of 48 clinical isolates of S. aureus (56%) possessed either the seg or variant seg gene. These findings suggest that SEG, or SEGv, is one of the most frequently produced superantigen exotoxins by S. aureus and may participate in the inflammatory process of the host by activating a distinct set of Vbeta families of T cells.     

Identification of stimulating and inhibitory epitopes within the heat shock protein 70 molecule that modulate cytokine production and maturation of dendritic cells

The 70-kDa microbial heat shock protein (mHSP70) has a profound effect on the immune system, interacting with the CD40 receptor on DC and monocytes to produce cytokines and chemokines. The mHSP70 also induces maturation of dendritic cells (DC) and thus acts as an alternative ligand to CD40L on T cells. In this investigation, we have identified a cytokine-stimulating epitope (peptide 407-426), by activating DC with overlapping synthetic peptides (20-mers) derived from the sequence of mHSP70. This peptide also significantly enhances maturation of DC stimulated by mHSP70 or CD40L. The epitope is located at the base of the peptide-binding groove of HSP70 and has five critical residues. Furthermore, an inhibitory epitope (p457-496) was identified downstream from the peptide-binding groove that inhibits cytokine production and maturation of DC stimulated by HSP70 or CD40L. The p38 MAP kinase phosphorylation is critical in the alternative CD40-HSP70 pathway and is inhibited by p457-496 but enhanced by p407-426.     

A novel functional T cell hybridoma recognizes macrophage cell death induced by bacteria: a possible role for innate lymphocytes in bacterial infection

We have established a novel TCRalphabeta (TCRVbeta6)(+)CD4(-)CD8(-) T cell hybridoma designated B6HO3. When the B6HO3 cells were cocultured with bacterial-infected J774 macrophage-like cells, IFN-gamma production by B6HO3 cells was triggered through direct cell-cell contact with dying J774 cells infected with Listeria monocytogenes (LM), Shigella flexneri, or Salmonella typhimurium that expressed the type III secretion system, but not with intact J774 cells infected with heat-killed LM, nonhemolytic lysteriolysin O-deficient (Hly(-)) LM, plasmid-cured Shigella, or stationary-phase Salmonella. However, the triggering of B6HO3 cells for IFN-gamma production involved neither dying hepatoma cells infected with LM nor dying J774 cells caused by gliotoxin treatment or freeze thawing. Cycloheximide and Abs to H-2K(d), H-2D(d), Ia(d), CD1d, TCRVbeta6, and IL-12 did not inhibit the contact-dependent IFN-gamma response, indicating that this IFN-gamma response did not require de novo protein synthesis in bacterial-infected J774 cells and was TCR and IL-12 independent. Thus, in an as yet undefined way, B6HO3 hybridoma recognizes a specialized form of macrophage cell death resulting from bacterial infection and consequently produces IFN-gamma. Moreover, contact-dependent interaction of minor subsets of splenic alphabeta T cells, including NKT cells with dying LM-infected J774 and bone marrow-derived macrophage (BMM) cells, proved to provide an IFN-gamma-productive stimulus for these minor T cell populations, to which the parental T cell of the B6HO3 hybridoma appeared to belong. Unexpectedly, subsets of gammadelta T and NK cells similarly responded to dying LM-infected macrophage cells. These results propose that innate lymphocytes may possess a recognition system sensing macrophage cell "danger" resulting from bacterial infection.     

Induction of a regulatory phenotype in human CD4+ T cells by streptococcal M protein

Regulatory T cells (Tregs) participate in the control of the immune response. In the human system, an IL-10-secreting, T regulatory type 1 cell (Tr1)-like subset of Tregs can be induced by concurrent cross-linking of the TCR and CD46 on naive CD4(+) T cells. Because many viral and bacterial pathogens, including the major human pathogen Streptococcus pyogenes, bind to CD46, we asked whether this bacterium can directly induce Tr1-like cells through the streptococcal ligand for CD46, the M protein. The M5 and M22 proteins were found to induce T cells to develop into the IL-10-producing Tr1-like phenotype. Moreover, whole M5-expressing bacteria, but not isogenic M-negative bacteria, led to proliferation and IL-10 secretion by T cells. The interaction between the M5 protein and T cells was dependent on CD46 and the conserved C repeat region of M5. Supernatants derived from T cells stimulated with M proteins or M protein-expressing bacteria suppressed bystander T cell proliferation through IL-10 secretion. In addition, activation of CD46 through streptococcal M protein induced the expression of granzyme B, providing a second means for these cells to regulate an immune response. These findings suggest that binding to CD46 and exploiting its signaling pathway may represent a strategy employed by a number of important human pathogens to induce directly an immunosuppressive/regulatory phenotype in T cells.     

Isolation of the receptor for the Amaranthus leucocarpus lectin from human T lymphocytes

Amaranthus leucocarpus lectin (ALL) is specific for GalNAc, and recognizes human T cells. The receptor for ALL was purified from T cells using biotin-labeled lectin and avidin-agarose as affinity matrix. It is a 70-kDa glycoprotein, constituted mainly by serine, glycine, and glutamic acid; its glycosidic portion contains mainly GalNAc; galactose, sialic acid, mannose, and GlcNAc were identified at a lower proportion. By ionic strength chromatography, as well as double dimension electrophoresis, we identified four isoforms of the ALL-receptor. N-terminal amino acid was blocked both in the ALL-receptor and its isoforms, therefore, tryptic peptides of ALL-receptor, analyzed through MALDI-TOF, were compared with the relative values obtained from the NCBInr (ProFound 2004/06/01) database. Our results indicated that the tryptic peptides obtained showed 54% homology with a DnaK-core molecular chaperone, 47% with human KIAA protein, and 44% with heat shock protein 8. The most frequent phenotype of the CD4 or CD8 ALL+ T cells was CD45RA+ CD27+; 26% of ALL+ T cells were CD25+ and 13% were CD69+, indicating that the glycoprotein recognized by ALL is present mainly on naive or quiescent T cells.     

STEAP, a prostate tumor antigen, is a target of human CD8+ T cells

STEAP is a recently identified protein shown to be particularly overexpressed in prostate cancer and also present in numerous human cancer cell lines from prostate, pancreas, colon, breast, testicular, cervical, bladder and ovarian carcinoma, acute lymphocytic leukemia and Ewing sarcoma. This expression profile renders STEAP an appealing candidate for broad cancer immunotherapy. In order to investigate if STEAP is a tumor antigen that can be targeted by specific CD8⁺ T cells, we identified two high affinity HLA-A*0201 restricted peptides (STEAP_86–94 and STEAP_262–270). These peptides were immunogenic in vivo in HLA-A*0201 transgenic HHD mice. Peptide specific murine CD8 T cells recognized COS-7 cells co-transfected with HHD (HLA-A*0201) and STEAP cDNA constructs and also HLA-A*0201⁺ STEAP⁺ human tumor cells. Furthermore, STEAP_86–94 and STEAP_262–270 stimulated specific CD8⁺ T cells from HLA-A*0201⁺ healthy donors, and these peptide specific CD8⁺ T cells recognized STEAP positive human tumor cells in an HLA-A*0201-restricted manner. Importantly, STEAP_86–94-specific T cells were detected and reactive in the peripheral blood mononuclear cells in NSCLC and prostate cancer patients ex vivo. These results show that STEAP can be a target of anti-tumor CD8⁺ T cells and that STEAP peptides can be used for a broad-spectrum-tumor immunotherapy.     

Spontaneous development of a pancreatic exocrine disease in CD28-deficient NOD mice

Autoimmune pancreatitis (AIP) is a heterogeneous autoimmune disease in humans characterized by a progressive lymphocytic and plasmacytic infiltrate in the exocrine pancreas. In this study, we report that regulatory T cell-deficient NOD.CD28KO mice spontaneously develop AIP that closely resembles the human disease. NOD mouse AIP was associated with severe periductal and parenchymal inflammation of the exocrine pancreas by CD4(+) T cells, CD8(+) T cells, and B cells. Spleen CD4(+) T cells were found to be both necessary and sufficient for the development of AIP. Autoantibodies and autoreactive T cells from affected mice recognized a approximately 50-kDa protein identified as pancreatic amylase. Importantly, administration of tolerogenic amylase-coupled fixed spleen cells significantly ameliorated disease severity, suggesting that this protein functions as a key autoantigen. The establishment and characterization of this spontaneous pancreatic amylase-specific AIP in regulatory T cell-deficient NOD.CD28KO mice provides an excellent model for the study of disease pathogenesis and development of new therapies for human autoimmune pancreatitis.     

The role of a heat shock protein from V. cholerae 0139 in the gut immune response

An immunodominant heat shock protein (Hsp 24) was purified from Vibrio cholerae O139 at 42 degrees C and used as an immunomodulator for studying the gut immune response. T cell clone and T cell line specific for the Hsp 24 were generated from the lymphocytes of lamina propria and intra-epithelial lymphocytes of mice orally infected with V. cholerae O139, respectively. The T cell clone was TCR alphabeta(+), CD4(+) and appeared to play an important role in the functioning of gut B-lymphocytes. The T cell line had heterogenous population of CD8+ and CD4+ cells, most of which were found to be TCR alphabeta(+) and a minor population was TCR gammadelta(+). The lymphokine profile of T cell line showed IFN-gamma to be the most abundant lymphokine followed by IL-2 and IL-4. The possible involvement of alternative pathway of activation for T cell clone was also addressed in this study. The splenocytes showed an up-regulation of their CD2 receptor expression on stimulation with the Hsp-24. The pattern of lymphokines released by splenocytes stimulated with the Hsp-24 showed no particular cell type to be responsible for mounting immune response. Thus, there is involvement of both, mucosal and peripheral arm of the immune system.     

The surface protein superfamily of Trypanosoma cruzi stimulates a polarized Th1 response that becomes anergic

Trypanosoma cruzi is an obligate intracellular parasite that chronically infects mammals. Extracellular mammalian stage trypomastigotes simultaneously express and release multiple members of the parasite's surface protein superfamily; these extracellular proteins should stimulate MHC class II-restricted CD4 T cells. The surface protein superfamily, however, encodes variant epitopes that may inhibit this CD4 response. In this report the surface protein-specific CD4 response was investigated. CD4 cells isolated from acutely and chronically infected mice did not proliferate when stimulated with surface proteins. Adoptive transfer of surface protein-specific CD4 clones or immunization with a peptide encoding a surface protein T cell epitope protected mice during T. cruzi infection. These data strongly suggested that surface proteins were expressed and presented to CD4 cells during infection. Limiting dilution analysis identified an expanded population of surface protein-specific CD4 cells during the acute and chronic infection. These surface protein-specific CD4 cells did not produce IL-2 or IL-4, but did produce IFN-gamma. Enzyme-linked immunospot analyses confirmed that many of the surface protein-specific CD4 cells produce IFN-gamma. Together these results suggest that during T. cruzi infection a potentially protective CD4 response becomes anergic. It is possible that this anergy is induced by variant T cell epitopes encoded by the surface protein superfamily.     

CD154 as a potential early molecular biomarker for rapid quantification analysis of active Staphylococcus enterotoxin A

Staphylococcus aureus is a major bacterial pathogen producing a group of 21 enterotoxins (SEs). These enterotoxins have two separate but related biological activities. They cause gastroenteritis, and they function as superantigens that activate large numbers of T cells. In the current study, we demonstrate that short-term ex vivo exposure of primary na?ve CD4(+) T-cells to SEA induces differential expression of the T cell surface receptor CD154 in a time- and dose-dependent manner. In addition, we show that SEA induces higher CD154 protein expression and higher splenocyte cell proliferation compared with SEB. We also demonstrate that expression of CD154 can be used for rapid detection of active SEA in milk.     

c-FLIPR, a new regulator of death receptor-induced apoptosis

c-FLIPs (c-FLICE inhibitory proteins) play an essential role in regulation of death receptor-induced apoptosis. Multiple splice variants of c-FLIP have been described on the mRNA level; so far only two of them, c-FLIP(L) and c-FLIP(S,) had been found to be expressed at the protein level. In this report, we reveal the endogenous expression of a third isoform of c-FLIP. We demonstrate its presence in a number of T and B cell lines as well as in primary human T cells. We identified this isoform as c-FLIP(R), a death effector domain-only splice variant previously identified on the mRNA level. Impor-/tantly, c-FLIP(R) is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex upon CD95 stimulation. Several properties of c-FLIP(R) are similar to c-FLIP(S): both isoforms have a short half-life, a similar pattern of expression during activation of primary human T cells, and are strongly induced in T cells upon CD3/CD28 costimulation. Taken together, our data demonstrate endogenous expression of c-FLIP(R) and similar roles of c-FLIP(R) and c-FLIP(S) isoforms in death receptor-mediated apoptosis.     

The expression,regulation and adhesion function of a novel CD molecule,CD226, on human endothelial cells

CD226 is a 67 kDa type I transmembrane glycoprotein mainly expressed on activated T cells, NK cells and platelets, and involved in the differentiation of cytotoxic T lymphocytes (CTL) and NK, as well as platelet activation and aggregation. Here we found that the expression of CD226 protein and CD226mRNA were very weak in resting HUVEC and ECV304 cells, whereas high level expression could be observed when these cells were stimulated. The binding activities between activated endothelial cells and activated Jurkat cells could be partly blocked by CD226/Ig fusion protein. Similarly, CD226/Ig could also partly block the adhesion between activated endothelial cells and some leukocytes or colo205 cells. These data provided the evidence that activated endothelial cells could express high level of CD226, and CD226 was involved in the endothelial cells' adhesion. The above findings suggested that CD226 is a novel inducible adhesion molecule on human endothelial cells.     

Dendritic cell-associated lectin-1: a novel dendritic cell-associated,C-type lectin-like molecule enhances T cell secretion of IL-4

We have characterized dendritic cell (DC)-associated lectin-1 (DCAL-1), a novel, type II, transmembrane, C-type lectin-like protein. DCAL-1 has restricted expression in hemopoietic cells, in particular, DCs and B cells, but T cells and monocytes do not express it. The DCAL-1 locus is within a cluster of C-type lectin-like loci on human chromosome 12p12-13 just 3' to the CD69 locus. The consensus sequence of the DCAL-1 gene was confirmed by RACE-PCR; however, based on sequence alignment with genomic DNA and with various human expressed sequence tags, we predict that DCAL-1 has two splice variants. C-type lectins share a common sequence motif of 14 invariable and 18 highly conserved aa residues known as the carbohydrate recognition domain. DCAL-1, however, is missing three of the cysteine residues required to form the standard carbohydrate recognition domain. DCAL-1 mRNA and protein expression are increased upon the differentiation of monocytes to CD1a(+) DCs. B cells also express high levels of DCAL-1 on their cell surface. Using a DCAL-1 fusion protein we identified a population of CD4(+) CD45RA(+) T cells that express DCAL-1 ligand. Coincubation with soluble DCAL-1 enhanced the proliferation of CD4(+) T cells in response to CD3 ligation and significantly increased IL-4 secretion. In contrast, coincubation with soluble DC-specific ICAM-3-grabbing nonintegrin (CD209) fusion protein as a control had no effect on CD4(+) T cell proliferation or IL-4 and IFN-gamma secretion. Therefore, the function of DCAL-1 on DCs and B cells may act as a T cell costimulatory molecule, which skews CD4(+) T cells toward a Th2 response by enhancing their secretion of IL-4.     

Immune mechanism of the antitumor effects generated by bortezomib

Bortezomib, a proteasome inhibitor, is a chemotherapeutic drug that is commonly used to treat a variety of human cancers. The antitumor effects of bortezomib-induced tumor cell immunogenicity have not been fully delineated. In this study, we examined the generation of immune-mediated antitumor effects in response to treatment by bortezomib in a murine ovarian tumor model. We observed that tumor-bearing mice that were treated with bortezomib had CD8(+) T cell-mediated inhibition of tumor growth. Furthermore, the comparison of tumor cell-based vaccines that were produced from tumor cells treated or untreated with bortezomib showed vaccination with drug-treated tumor cell-based vaccines elicited potent tumor-specific CD8(+) T cell immune response with improved therapeutic antitumor effect in tumor-bearing mice. Conversely, the untreated tumor cell-based vaccines led to no appreciable antitumor response. Treatment of tumor cells with bortezomib led to the upregulation of Hsp60 and Hsp90 on the cell surface and promoted their phagocytosis by dendritic cells (DCs). However, cell surface expression of Hsp60, instead of Hsp90, is the more important determinant of whether bortezomib-treated tumor cells can generate tumor-specific CD8(+) T cells. CD11c(+) DCs that were treated with bortezomib in vitro had enhanced phagocytic activities. In addition, CD11c(+) DCs from bortezomib-treated tumor-bearing mice had increased maturation. At lower concentrations, bortezomib had no inhibitory effects on T cell proliferation. Taken together, our data indicate that bortezomib can render tumor cells immunogenic by upregulating the cell surface expression of heat shock protein 60 and heat shock protein 90, as well as improve DC function, which results in potent immune-mediated antitumor effects.