The myeloid immune signature of enterotoxigenic Bacteroides fragilis-induced murine colon tumorigenesis.

Enterotoxigenic Bacteroides fragilis (ETBF), a human commensal and candidate pathogen in colorectal cancer (CRC), is a potent initiator of interleukin-17 (IL-17)-dependent colon tumorigenesis in MinApc+/- mice. We examined the role of IL-17 and ETBF on the differentiation of myeloid cells into myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages, which are known to promote tumorigenesis. The myeloid compartment associated with ETBF-induced colon tumorigenesis in Min mice was defined using flow cytometry and gene expression profiling. Cell-sorted immature myeloid cells were functionally assayed for inhibition of T-cell proliferation and inducible nitric oxide synthase expression to delineate MDSC populations. A comparison of ETBF infection with that of other oncogenic bacteria (Fusobacterium nucleatum or pks+Escherichia coli) revealed a specific, ETBF-associated colonic immune infiltrate. ETBF-triggered colon tumorigenesis is associated with an IL-17-driven myeloid signature characterized by subversion of steady-state myelopoiesis in favor of the generation of protumoral monocytic-MDSCs (MO-MDSCs). Combined action of the B. fragilis enterotoxin BFT and IL-17 on colonic epithelial cells promoted the differentiation of MO-MDSCs, which selectively upregulated Arg1 and Nos2, produced NO, and suppressed T-cell proliferation. Evidence of a pathogenic inflammatory signature in humans colonized with ETBF may allow for the identification of populations at risk for developing colon cancer.

The 'kiss of death' by dendritic cells to cancer cells.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) specialized in the stimulation of naïve T lymphocytes, which are key components of antiviral and antitumor immunity. DCs are 'sentinels' of the immune system endowed with the mission to (1) sense invading pathogens as well as any form of tissue distress and (2) alert the effectors of the immune response. They represent a very heterogeneous population including subsets characterized by their anatomical locations and specific missions. Beyond their unique APC features, DCs exhibit a large array of effector functions that play critical roles in the induction and regulation of the cell-mediated as well as humoral immune responses. In the course of the antitumor immune response, DCs are unique in engulfing tumor cells killed by natural killer (NK) cells and cross-presenting tumor-associated antigens to cytotoxic T lymphocytes (CTLs). However, while DCs mediate antitumor immune responses by stimulating tumor-specific CTLs and NK cells, direct tumoricidal mechanisms have been recently evoked. This review addresses the other face of DCs to directly deliver apoptotic signals to stressed cells, their role in tumor cell death, and its implication in the design of DC-based cancer immunotherapies.

N-linked carbohydrates in tyrosinase are required for its recognition by human MHC class II-restricted CD4(+) T cells.

Glycosylation of mammalian proteins is known to influence their intracellular trafficking, half life, and susceptibility to enzymatic degradation. Rare instances of natural T cell epitopes dependent upon glycosylation for recognition have been described. We report here on human CD4(+) T lymphocyte cultures and clones from two melanoma patients that recognize the melanoma-associated Ag tyrosinase in the context of HLA-DR4 and -DR8. These T cells recognize tyrosinase, normally a heavily glycosylated molecule, when expressed constitutively in melanoma cells or in COS-7 transfectants pulsed as lysates onto autologous APC. However, these T cells fail to recognize tyrosinase expressed in bacteria, nor do they react with overlapping peptides covering full-length tyrosinase, suggesting a critical role for glycosylation in the processing and / or composition of the stimulatory epitopes. The requirement for glycosylation was demonstrated by the failure of tyrosinase-specific CD4(+) T cells to recognize tyrosinase synthesized in the presence of glycosylation inhibitors, or deglycosylated enzymatically. Site-directed mutagenesis of each of seven potential N-glycosylation sites showed that four sites were required to generate forms of tyrosinase that could be recognized by individual T cell clones. These data indicate that certain carbohydrate moieties are required for processing the tyrosinase peptides recognized by CD4(+) T cells. Post-translational modifications of human tumor-associated proteins such as tyrosinase could be a critical factor for the development of antitumor immune responses.

Prostate cancer chemoprevention agents exhibit selective activity against early stage prostate cancer cells.

Preclinical models for the identification of prostate cancer chemoprevention agents are lacking. Based upon the notion that clinically useful chemoprevention agents should exhibit selective activity against early stage disease, studies were undertaken to assess whether chemoprevention agents selectively inhibited the growth of early stage prostate cancer, as compared to late stage cancer. First, a series of cell and molecular studies were performed, which, when taken together, validated the use of a panel of prostate cell lines as a model of the different stages of carcinogenesis. Next, therapeutic responsiveness to ten different cytotoxic or chemoprevention agents was evaluated. Chemoprevention agents exhibited selective activity against normal and early transformed prostate tissue, whereas cytotoxic agents were non-specific. Selective activity against early versus advanced prostate cancer cells is identified as a potential screening method for chemoprevention agents.Prostate Cancer and Prostatic Diseases (2001) 4, 81-91

Recognition of a shared human prostate cancer-associated antigen by nonclassical MHC-restricted CD8+ T cells.

To identify prostate cancer-associated Ags, tumor-reactive T lymphocytes were generated using iterative stimulations of PBMC from a prostate cancer patient with an autologous IFN-gamma-treated carcinoma cell line in the presence of IL-2. A CD8+ T cell line and TCR alphabeta+ T cell clone were isolated that secreted IFN-gamma and TNF-alpha in response to autologous prostate cancer cells but not to autologous fibroblasts or lymphoblastoid cells. However, these T cells recognized several normal and malignant prostate epithelial cell lines without evidence of shared classical HLA molecules. The T cell line and clone also recognized colon cancers, but not melanomas, sarcomas, or lymphomas, suggesting recognition of a shared epithelium-associated Ag presented by nonclassical MHC or MHC-like molecules. Although Ag recognition by T cells was inhibited by mAb against CD8 and the TCR complex (anti-TCR alphabeta, CD3, Vbeta12), it was not inhibited by mAb directed against MHC class Ia or MHC class II molecules. Neither target expression of CD1 molecules nor HLA-G correlated with T cell recognition, but beta2-microglobulin expression was essential. Ag expression was diminished by brefeldin A, lactacystin, and cycloheximide, but not by chloroquine, consistent with an endogenous/cytosolic Ag processed through the classical class I pathway. These results suggest that prostate cancer and colon cancer cells can process and present a shared peptidic Ag to TCR alphabeta+ T cells via a nonclassical MHC I-like molecule yet to be defined.

Contrasting patterns of regulation of the antioxidant selenoproteins, thioredoxin reductase, and glutathione peroxidase, in cancer cells.

There is strong evidence that selenium protects against certain human cancers, but the underlying mechanism is unknown. Glutathione peroxidase (GPX1) and thioredoxin reductase (TR), the most abundant antioxidant selenium-containing proteins in mammals, have been implicated in this protection. We analyzed the expression of TR and GPX1 in the following model cancer systems: (1) liver tumors in TGFalpha/c-myc transgenic mice; (2) human prostate cell lines from normal and cancer tissues; and (3) p53-induced apoptosis in a human colon cancer cell line. TR was induced while GPX1 was repressed in malignancies relative to controls in transgenic mice and prostate cell lines. In the colon cell line, p53 expression resulted in elevated GPX1, but repressed TR. The data indicate that TR and GPX1 are regulated in a contrasting manner in the cancer systems tested and reveal the p53-dependent regulation of selenoprotein expression. The data suggest that additional studies on selenoprotein regulation in different cancers are required to evaluate future implementation of selenium as a dietary supplement in individuals at risk for developing certain cancers.

MHC-dependent cytolysis of autologous tumor cells by lymphocytes infiltrating urothelial carcinomas.

Tumor-infiltrating lymphocytes (TIL) were grown from 23 urothelial carcinomas. Phenotyping analysis showed that the TIL cultures were mainly CD3+. Although CD4+ and CD8+ T-cell sub-sets were grown in culture, CD4+ T-cell sub-sets predominated over CD8+ T cells. Immunohistochemical studies performed on 5 tumor specimens confirmed this observation, and indicated that CD4+ T cells surrounded the tumor islets, whereas CD8+ T lymphocytes were localized among the tumor cells. Five short-term carcinoma cell lines established from these urothelial tumors were used as target cells in cytolysis assays in order to investigate the functional anti-tumor activity of autologous TIL. TIL from 4/5 tumors were lytic and 3 TIL lines displayed MHC-class-I-dependent cytotoxicity directed against autologous tumor cells. CD4+ T-cell-depletion experiments performed on TIL line 07 confirmed that CD8+ MHC-class-I-dependent CTL were the predominant effectors. Finally, experiments performed on 6 allogeneic urothelial-cancer cell lines matched for HLA-class-I molecules showed that TIL07 exhibited selective lytic activity toward tumor 07. These data indicate that CD8+ MHC-class-I-dependent CTL present in urothelial carcinomas are functional and may participate in the anti-tumor immune response.

Antigen-presenting function of murine gonadal epithelial cell lines.

Murine Leydig (TM3) and Sertoli (TM4) cell lines were studied as nonprofessional antigen-presenting cells using the antigen model of human choriogonadotropin (hCG alpha/beta) and specific T-cell hybridomas. Both cell lines were treated with IFN-gamma to induce I-A(d) and I-E(d) molecules expression. Only the TM3 cell line, which expressed MHC-class II molecules upon IFN-gamma stimulation, was able to uptake, process, and present the human choriogonadotropin beta subunit to related T-cell hybridomas. Interestingly, the TM3 cell line was incapable of presenting the human choriogonadotropin alpha subunit, the presentation of which, by classical APC, is highly efficient. Using T-cell hybridomas directed against the immunogenic regions of hCG alpha/beta previously described in BALB/c mice, we showed that the TM3 cell line generated a narrower peptide repertoire than classical APC (i.e., B cells, macrophages, and dendritic cells). This experimental system suggests that Leydig cells could initiate, in vivo, an autoimmune process directed against gonadal tissues. In particular, such a mechanism has been evoked in experimental autoimmune orchitis.

Reaction of peripheral-blood lymphocytes to the human chorionic gonadotropin beta sub-unit in patients with productive tumors.

Human chorionic gonadotropin (hCG) and its beta sub-unit (hCG beta) are secreted by trophoblast cells during pregnancy, and by tumoral cells of trophoblastic and non-trophoblastic origin. In contrast to hCG, the free hCG beta sub-unit is consistently undetectable in healthy non-pregnant subjects. With this in mind, we sought to determine whether an immune response to hCG beta can be detected in patients with bladder or germ-cell testis cancers. Peripheral-blood mononuclear cells (PBMC) from 31% of patients with hCG beta-productive bladder cancers and 33% of testis-tumor-bearing patients displayed an hCG beta-specific proliferative response, whereas no patients with non-hCG beta-productive cancers had a proliferative response. PBMC from pregnant women and healthy controls did not elicit significant reactivity. By the use of overlapping synthetic peptides, the immunogenic regions of hCG beta were delineated within the central 20-65 portion. Moreover, in 2 bladder-cancer patients with the HLA DR7, DQ2 haplotype, the T-cell response to hCG beta was focused on the hCG beta (20-47) peptide. Taken together, these results indicate that hCG beta is a tumor-associated antigen capable of inducing a cell-mediated immune response in patients with productive tumors.

Deficient antigen processing of a protein quaternary structure can be overcome by receptor-mediated uptake.

Human chorionic gonadotropin (hCG) is a dimer of non-covalently associated alpha (hCG-alpha) and beta (hCG-beta) subunits. This molecule was used to study whether receptor-mediated uptake influences the presentation of a protein quaternary structure. Unprimed splenocytes and a B cell lymphoma were capable of presenting only the free (hCG-alpha) but not the combined (hCG) alpha subunit to hCG-alpha T cell hybridomas, while hCG-alpha-primed lymph node cells (LNC) responded to both hCG-alpha and hCG. As antigen (Ag)-specific antigen-presenting cells (APC) present in the hCG-alpha-primed LNC population may be potentially effective for presenting hCG, we investigated the role of specific Ag capture, through mIg and Fc gamma R, in the processing and presentation of hCG and hCG-alpha to HAG5, a T cell hybridoma directed against the immunodominant region (amino acids 61-81) of hCG-alpha. Results showed that only B cells bearing membrane immunoglobulin capable of recognizing hCG-alpha and hCG, and present in hCG-alpha-primed mice, were extremely effective in presenting the free as well as the combined alpha subunit. The effect of FcR-mediated uptake was analyzed using a B cell line transfected with the Fc gamma RII-B2 gene to present immune complexes of either hCG-alpha or hCG. We found that hCG-alpha and hCG were presented equally well, whatever the Ag-binding site of each antibody to hCG or its alpha subunit. Using HBG 6, an hCG-beta T cell hybridoma, we performed similar experiments with the Fc gamma RII-B2 cell line and determined that the potentiation of hCG presentation to HBG 6 was similar to that observed with HAG 5. Then kinetic experiments were performed to examine the effect of Ag uptake through FcR on processing. Results demonstrated that the uptake pathway drastically influenced the expression of alpha T cell determinants in the alpha/beta dimer. In addition, treatment with cycloheximide, a protein synthesis inhibitor, only impaired the ability of APC to present specifically captured Ag. Thus, the processing pathway for specifically captured Ag might be different from the pathway used to process nonspecifically captured Ag. This observation might explain why receptor-enhanced uptake bypasses the inefficient processing of the hCG quaternary structure and enables similar efficiency in the presentation of alpha and beta T cell specificities. These findings provide new insight into the antigenicity of oligomeric molecules, which is modified whether antigen capture is specific or not.

Influence of protein-quaternary structure on antigen processing.

T cell recognition of the quaternary structure of human chorionic gonadotropin (hCG), resulting from the association between its alpha (hCG-alpha) and beta (hCG-beta) subunits, was analyzed using hCG-alpha and hCG-beta T cell hybridomas produced in BALB/c mice. First, the fine specificity of these T cell hybridomas was determined, enabling us to divide hCG-alpha-specific T cell hybridomas into two groups. Group I recognized the hCG-alpha(61-81) region, and group II responded to the hCG-alpha(50-70) part of the molecule. Two groups of hCG-beta-specific T cell hybridomas, designated groups III and IV, were analyzed and found to respond to the C- and the N-terminal parts of the hCG-beta(1-22) peptide, respectively. Moreover, we observed that the nature of APC influenced Ag recognition by hCG-beta T cell hybridomas from group IV, but not by other selected T cell hybridomas. We then showed that recognition of the hCG alpha/beta dimer by alpha-specific T cell hybridomas was dramatically reduced compared to both free hCG-alpha and heat-dissociated hCG alpha/beta molecules. In contrast, hCG-beta hybridomas exhibited comparable responses to the free beta subunit and the hCG dimer. Experiments using a dimeric molecule assembled from the alpha-subunit of human follicle-stimulating hormone, which is identical to hCG-alpha, and the beta-subunit of human follicle-stimulating hormone, which is homologous to hCG-beta, confirmed that the three-dimensional structure of the complex rather than the primary structure of the beta-subunit plays a critical role in the processing pathway. Finally, kinetic experiments showed that the presentation of hCG-alpha T cell epitopes differed depending upon whether the alpha-subunit was in its free or combined form. In contrast, the kinetic expression of hCG-beta T cell epitopes appeared to be independent of the quaternary structure of hCG. Thus, conformational alterations resulting from the alpha/beta subunit association mainly influenced processing of the alpha-subunit in its complexed form, rather than processing of the combined beta-subunit. The effect of protein-quaternary structure on T cell recognition may represent a new element in our understanding of the processing and presentation of oligomeric molecules.

[Evans' syndrome caused by D-penicillamine in rheumatoid arthritis. Value of the corticoids-danazol combination]. / Syndrome d'Evans sous D-pénicillamine au cours d'une polyarthrite rhumatoïde. Intérêt de l'association corticoïdes-danazol.

The authors report the development of thrombocytopenia purpura in one patient with seropositive and erosive rheumatoid arthritis treated successfully for 11 months with D-penicillamine. Anti-platelet-bound antibodies were present, but also: anti-erythrocyte antibodies with hemolytic anemia (then defining Evans's syndrome): higher level of antinuclear antibodies; intermittent neutropenia. The responsibility of D-penicillamine is discussed, but thrombocytopenia purpura evolved for itself. Glucocorticoids alone, intravenous immunoglobulin, vincristine did not induced remission, which at least occurred under the association danazol-glucocorticoids, without toxicity, especially on the liver function.