Publications

A subset of CD161 (NK1) T cells express an invariant Valpha14Jalpha281 TCR-alpha chain (Valpha(invt) T cells) and produce Th2 and Th1 cytokines rapidly in response to CD1d, but their physiological function(s) remain unclear. We have found that CD1d-reactive T cells mediate to resistance against the acute, cytopathic virus diabetogenic encephalomyocarditis virus (EMCV-D) in relatively Th1-biased, C57BL/6-based backgrounds. We show now that these results generalize to Th2-biased, hypersensitive BALB/c mice. CD1d-KO BALB/c mice were more susceptible to EMCV-D. Furthermore, alpha-galactosylceramide (alpha-GalCer), a CD1d-presented lipid antigen that specifically activates Valpha(invt) T cells, protected wild-type (WT) mice against EMCV-D-induced encephalitis, myocarditis, and diabetes. In contrast, neither CD1d-KO nor Jalpha281-KO mice were protected by alpha-GALCER: Finally, disease in Jalpha281-KO mice was comparable to WT, indicating for the first time equivalent roles for CD1d-reactive Valpha(invt) and noninvariant T cells in resistance to acute viral infection. A model for how CD1d-reactive T cells can initiate immune responses, which synthesizes current results, is presented.

Murine bone marrow (BM) NK T cells can suppress graft-vs-host disease, transplant rejection, and MLRs. Human BM contains T cells with similar potential. Human BM was enriched for NK T cells, approximately 50% of which recognized the nonpolymorphic CD1d molecule. In contrast to the well-characterized blood-derived CD1d-reactive invariant NK T cells, the majority of human BM CD1d-reactive T cells used diverse TCR. Healthy donor invariant NK T cells rapidly produce large amounts of IL-4 and IFN-gamma and can influence Th1/Th2 decision-making. Healthy donor BM CD1d-reactive T cells were Th2-biased and suppressed MLR and, unlike the former, responded preferentially to CD1d(+) lymphoid cells. These results identify a novel population of human T cells which may contribute to B cell development and/or maintain Th2 bias against autoimmune T cell responses against new B cell Ag receptors. Distinct CD1d-reactive T cell populations have the potential to suppress graft-vs-host disease and stimulate antitumor responses.

This study investigated interactions between SRY, the Y chromosome encoded male sex determining factor, and the androgen receptor (AR). Coexpression of AR and SRY caused marked repression of AR transcriptional activity on a series of androgen-responsive reporter genes. Mammalian one- and two-hybrid experiments demonstrated an AR-SRY interaction mediated by the AR DNA binding domain. Precipitations with glutathione S-transferase fusion proteins indicated that AR-SRY interactions were direct and mediated by the AR DNA binding domain and the SRY high mobility group box DNA binding domain. Transient expression of SRY in LNCaP prostate cancer cells repressed expression of an androgen-dependent prostate-specific antigen (PSA) reporter gene and stable SRY expression repressed the endogenous PSA gene. SRY protein expression was increased by proteosome inhibitors and by the androgen-liganded AR in transient and stable transfectants. AR transcriptional activity was also repressed by DAX1, and the effects of SRY and DAX1 on the AR were additive. These findings indicate that interactions between the AR, SRY, and DAX1 contribute to normal male development and function and suggest a general role for protein-protein interactions between high mobility group box proteins and steroid hormone receptors in regulating tissue-specific gene expression.

Human T cells expressing CD161 and an invariant T-cell receptor (TCR) alpha-chain (Valpha24invt T cells) specifically recognize CD1d and appear to have immunoregulatory functions. However, the physiological target cells for this T-cell population, and whether alterations in CD1d expression contribute to the regulation of Valpha24invt T-cell responses, remain to be determined. A series of antibodies were generated to assess CD1d expression, structure and regulation on human lymphoid and myeloid cells. CD1d was expressed at high levels by human cortical thymocytes and immunoprecipitation analyses showed it to be a 48 000-MW glycosylated protein. However, after solubilization, the majority of the thymocyte CD1d protein, but not CD1d expressed by transfected cells, lost reactivity with monoclonal antibodies (mAbs) against native CD1d, indicating that it was alternatively processed. Moreover, thymocytes were not recognized by CD1d-reactive Valpha24invt T-cell clones. Medullary thymocytes and resting peripheral blood T cells were CD1d-, but low-level CD1d expression was induced on activated T cells. CD1d was expressed by B cells in peripheral blood and lymph node mantle zones, but germinal centres were CD1d-. Resting monocytes were CD1d+ but, in contrast to CD1a, b and c, their surface expression of CD1d was not up-regulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) activation. These results demonstrate constitutive CD1d expression by human professional antigen-presenting cells and that post-translational processing of CD1d may contribute to regulation of the activity of CD1d-specific T cells.

The role of androgen receptor (AR) mutations in androgen-independent prostate cancer (PCa) was determined by examining AR transcripts and genes from a large series of bone marrow metastases. Mutations were found in 5 of 16 patients who received combined androgen blockade with the AR antagonist flutamide, and these mutant ARs were strongly stimulated by flutamide. In contrast, the single mutant AR found among 17 patients treated with androgen ablation monotherapy was not flutamide stimulated. Patients with flutamide-stimulated AR mutations responded to subsequent treatment with bicalutamide, an AR antagonist that blocks the mutant ARs. These findings demonstrate that AR mutations occur in response to strong selective pressure from flutamide treatment.

BACKGROUND: Functional inactivation of the tsg101 gene in mouse fibroblasts leads to cell transformation and the ability to form metastatic tumors in nude mice. Abnormal TSG101 transcripts with highly-specific deletions in the protein-coding region have been identified in human tumor samples and cancer cell lines, including prostate and breast carcinomas, and have been attributed to alternative splicing of TSG101 mRNA. The function of the TSG101 protein is not known, although its predicted sequence has suggested that it may function as a transcription factor.

METHODS: Human TSG101 N-terminal (encoding amino acids 10-240) and C-terminal (encoding amino acids 230-391) fragments were cloned and used in both transient transfection and protein binding experiments. The transient transfections were carried in CV-1 cells. Protein-protein interactions were determined by both glutathione-S-transferase fusion protein binding and co-immunoprecipitation.

RESULTS: The N-terminal region of TSG101, when fused to the GAL4 DNA binding domain, can activate transcription; whereas the C-terminal region mediates transcriptional repression. Full-length TSG101 or its separated regions repressed ligand-dependent transcriptional activation by nuclear receptors, including androgen receptor and estrogen receptor, which play central roles in prostate carcinoma and breast carcinoma, respectively. In addition, a direct association between TSG101 and the transcriptional co-factor p300 was demonstrated in vitro and in vivo.

CONCLUSIONS: These results indicate that TSG101 can function as a transcription modulator to affect nuclear receptor-mediated transcriptional activation, which raises the possibility that the tumor suppression by TSG101 observed previously may be mediated at least in part by its effects on nuclear receptor function.

An important biological feature of prostate cancer (PCa) is its marked preference for bone marrow as a metastatic site. To identify factors that may support the growth of PCa in bone marrow, expression of receptor and nonreceptor tyrosine kinases by androgen-independent PCa bone marrow metastases was assessed. Bone marrow biopsies largely replaced by PCa were analyzed using reverse transcriptase-polymerase chain reaction amplification with degenerate primers that amplified the conserved kinase domain. Sequence analyses of the cloned products demonstrated expression of multiple kinases. Expression of the receptor and nonreceptor tyrosine kinases, alpha platelet-derived growth factor receptor and Jak 1, respectively, was confirmed by immunohistochemistry. In contrast, the type 1 insulin-like growth factor receptor, thought to play a role in PCa development, was lost in metastatic PCa. These results implicate several specific growth factors and signaling pathways in metastatic androgen-independent PCa and indicate that loss of the type 1 insulin-like growth factor receptor contributes to PCa progression.

TSG101 has been identified as a candidate tumor suppressor gene and abnormal transcripts have been identified in a substantial fraction of breast cancers. To determine whether TSG101 expression is commonly altered in other tumors, a series of 15 primary and metastatic prostate cancers were analysed by reverse transcriptase-PCR amplification. Abnormal transcripts with extensive deletions in the coding region were found in nine of these tumors, while only the normal transcript was found in control and benign prostatic hypertrophy tissues. More than one abnormal transcript was found in four of these nine cases and distinct abnormal TSG101 transcripts were found in separate biopsies taken from one tumor. Importantly, the normal TSG101 transcript was undetectable in two metastatic prostate cancers, indicating the absence of TSG101 protein. Sequence analysis demonstrated that there were at least six distinct deletions, with four of these deletions found in more than one tumor sample. The most commonly identified deletion, from bp 153 to 1055, was identical to a deletion reported previously in breast cancer. These results demonstrate that TSG101 transcripts are frequently abnormal in prostate cancer and suggest that loss of TSG101 protein contributes to disease development or progression.

An increasing number of proteins which bind to hormone-dependent nuclear receptors and mediate their effects on gene expression are being identified. The human prostate-specific antigen (PSA) and kallikrein 2 (KLK2) genes are regulated by the androgen receptor (AR). Using electrophoresis mobility shift assays (EMSA), a common nuclear protein(s) which binds upstream of the androgen-responsive elements (AREs) in the PSA and KLK2 promoters was identified. Binding occurred between bp -539 and -399 and bp -349 and -224 in the PSA and KLK2 promoters respectively, which were shown previously to be necessary for AR-mediated transactivation. Glutathione S-transferase (GST)-AR fusion proteins were constructed to determine whether the AR interacted directly with this protein or protein complex. Specific interactions were observed with AR fusion proteins containing the DNA binding domain. EMSA supershift experiments and GST-AR pull-down experiments followed by Western blotting identified a Fos-related protein(s) of approximately 40 kDa as part of this complex. Competition experiments with a double-stranded oligonucleotide containing an AP-1 binding site demonstrated that DNA binding was not mediated by AP-1. These results indicate that a Fos-containing protein complex distinct from AP-1 binds upstream of the AREs in the PSA and KLK2 promoters, interacts with the AR and may participate in regulation of these two androgen-responsive genes.

TCR expression by human fetal intestinal intraepithelial lymphocytes (ilELs) and intestinal lamina propria lymphocytes was analyzed to address whether T cell development occurs in human fetal intestine, the diversity of human fetal iIELs, and whether human fetal iIELs may contribute to the adult iIEL repertoire. ilELs and intestinal lamina propria lymphocytes from second trimester human fetal intestine were analyzed for TCR-alphabeta transcripts. Rearranged TCR-alpha transcripts were undetectable at 14 wk in the intraepithelial lymphocytes (IELs), whereas multiple TCR-beta transcripts were found at this stage. The TCR-alpha repertoire remained restricted relative to TCR-beta at later stages, and the IEL repertoire was restricted relative to the lamina propria lymphocytes at all stages. A previously reported T early alpha message was the major transcript from the TCR-alpha locus early in gestation. A previously undescribed TCR-beta transcript initiating upstream of the Dbeta1 locus and spliced to Cbeta1 or Cbeta2 was also identified and may represent a T early beta message. These results provide evidence for ongoing TCR gene rearrangement in human fetal intestine and suggest that transcription from the TCR-beta locus initiates with a T early beta transcript. The TCR-alpha repertoire (and hence the repertoire of potentially functional IELs) was limited through the second trimester.