Direct evidence for a functional role of HLA-DRB1 and -DRB3 gene products in the recognition of Dermatophagoides spp. (house dust mite) by helper T lymphocytes

The contribution of the HLA-DRB1, -B3, and -BS gene products in the recognition of Dermatophagoides spp. (house dust mite) by helper T cells Isolated from an atopic individual (HLA-DRw12, DR7; DRw52b) with perennial rhinitis was investigated. Using a panel of histocompatlble and histoincompatible accessory cells, the restriction specificity obtained for a long term T cell suggested that a component of the dust mite reactive repertoire recognized antigen in association with DRB3 gene products. Ollgonucleotide DNA typing of the presenting cell panel demonstrated a correlation between the DRw52b allele and T cell responsiveness. Murine fibroblasts expressing DRw52b, but not DRw52a or -c molecules, presented antigen to both the T cell line and cloned T cells (DE26) derived from the line, Indicating that the supertypic specificity DRw52b was able to restrict recognition of dust mite antigens. Additional T cell clones (DE9 and DE41) also isolated from the line were restricted by the products of the B1 gene locus (DRw12B1) as determined by murine fibroblasts transfected with the appropriate HLA-DR genes. Clone DE9 was degenerate in Its restriction specificity, also recognizing dust mite presented by accessory cells expressing the DR2 subtypes. Presentation by fibroblasts transfected with DRw12B1, DR2Dw2B5 genes and EBV-transformed B cell lines expressing DR2Dw21B1 and -B5 indicated that the functional site restricting recognition may be associated with residues 70 and 71 of the DR/3 chain helical wall of the antigen combining site. Furthermore, we have recently demonstrated that both T cell clones DE9 and DE26 induce allergen dependent IgE synthesis in vitro. Thus these results demonstrate directly that the DRB1, -B3, and -B5 gene products are functional in the restriction of T cell recognition of dust mite antigen

HES1 in immunity and cancer

Hairy and enhancer of split homolog-1 (HES1) is a part of an extensive family of basic helix-loop-helix (bHLH) proteins and plays a crucial role in the control and regulation of cell cycle, proliferation, cell differentiation, survival and apoptosis in neuronal, endocrine, T-lymphocyte progenitors as well as various cancers. HES1 is a transcription factor which is regulated by the NOTCH, Hedgehog and Wnt signalling pathways. Aberrant expression of these pathways is a common feature of cancerous cells. There appears to be a fine and complicated crosstalk at the molecular level between the various signalling pathways and HES1, which contributes to its effects on the immune response and cancers such as leukaemia. Several mechanisms have been proposed, including an enhanced invasiveness and metastasis by inducing epithelial mesenchymal transition (EMT), in addition to its strict requirement for tumour cell survival. In this review, we summarize the current biology and molecular mechanisms as well as its use as a clinical target in cancer therapeutics

FRA2 is a STAT5 target gene regulated by IL-2 in human CD4 T cells

Signal transducers and activators of transcription 5(STAT5) are cytokine induced signaling proteins, which regulate key immunological processes, such as tolerance induction, maintenance of homeostasis, and CD4 T-effector cell differentiation. In this study, transcriptional targets of STAT5 in CD4 T cells were studied by Chromatin Immunoprecipitation (ChIP). Genomic mapping of the sites cloned and identified in this study revealed the striking observation that the majority of STAT5-binding sites mapped to intergenic (>50 kb upstream) or intronic, rather than promoter proximal regions. Of the 105 STAT5 responsive binding sites identified, 94% contained the canonical (IFN-γ activation site) GAS motifs. A number of putative target genes identified here are associated with tumor biology. Here, we identified Fos-related antigen 2 (FRA2) as a transcriptional target of IL-2 regulated STAT5. FRA2 is a basic -leucine zipper (bZIP) motif 'Fos' family transcription factor that is part of the AP-1 transcription factor complex and is also known to play a critical role in the progression of human tumours and more recently as a determinant of T cell plasticity. The binding site mapped to an internal intron within the FRA2 gene. The epigenetic architecture of FRA2, characterizes a transcriptionally active promoter as indicated by enrichment for histone methylation marks H3K4me1, H3K4me2, H3K4me3, and transcription/elongation associated marks H2BK5me1 and H4K20me1. FRA2 is regulated by IL-2 in activated CD4 T cells. Consistently, STAT5 bound to GAS sequence in the internal intron of FRA2 and reporter gene assays confirmed IL-2 induced STAT5 binding and transcriptional activation. Furthermore, addition of JAK3 inhibitor (R333) or Daclizumab inhibited the induction in TCR stimulated cells. Taken together, our data suggest that FRA2 is a novel STAT5 target gene, regulated by IL-2 in activated CD4 T cells

Intermittent Antibody-Based Combination Therapy Removes Alloantibodies and Achieves Indefinite Heart Transplant Survival in Presensitized Recipients

BACKGROUND: It is well established that primed/memory T cells play a critical role in heart transplant rejection. This contributes to the challenges faced in the transplant clinic since current treatments which are efficient in controlling naïve T cell alloresponses have limited efficacy on primed T cell responders. METHODS: Fully MHC mismatched heart transplantation was performed from BALB/c to C57BL/6 mice pre-sensitised with BALB/c splenocytes 14 days pre-transplantation. A combination therapy comprising CD70-, CD154- and CD8-specific antibodies was administered at day 0 and 4 post-transplantation with Rapamycin on days 0-4. RESULTS: The antibody combination therapy extended heart transplant survival in pre-sensitised recipients from MST 8 days (median survival time) to MST 78 days. A decrease in the number of splenic IFN-γ secreting cells measured by ELISpot assay was seen in the treated group compared to the untreated controls. However, graft-infiltrating CD8(+) and CD4(+) T cells persisted despite treatment and the number of intra-graft CD4(+) T cells increased at day 30 post-transplantation. When an additional “rescue therapy” comprising the same antibodies was re-administered at days 30, 60 and 90 post-transplantation, T cell infiltration was reduced and indefinite graft survival was observed. Furthermore, rescue therapy resulted in gradual decrease in titre and, by day 90 post-transplantation the complete loss of the pre-existing, donor-specific antibodies. CONCLUSION: We conclude that our antibody combination therapy extends allograft survival in pre-sensitised recipients. When combined with intermittent antibody-mediated rescue therapy, this results in indefinite allograft survival and a loss of the pre-existing, donor-specific antibodies from the circulation

The locations of putative STAT5 binding sites in activated CD4+ T cells.

<p>Mapping of STAT5 binding sites relative to annotated genes were categorized into four groups. Intergenic region denotes binding sites present greater than 10<10 kb upsteam denotes the region less than 10 kb upstream of the 5′ region of the nearest gene. Internal intron denotes introns other than intron 1 of the gene and intron 1 depicts the presence of a binding site within the first intron of the gene.</p

IL-2 regulates expression of FRA2.

<p>Quantitative RT-PCR was performed on PHA activated CD4+ T cells stimulated with or without IL-2 for different times to evaluate the expression of FRA2. Expression levels are presented as fold increase (logarithmic scale) and compared to the baseline levels (cells not treated with IL-2). 18 s was used as the housekeeping gene and served as the endogenous control. IL-2 stimulation strongly induced <i>FRA2</i> expression with two peaks observed at 4–6 hours and 24 hours post stimulation. This is representative of at least three independent experiments performed in triplicate.</p

STAT5 motif analysis of the mapped ChIP clones in activated CD4+ T cells.

<p>Out of the 105 binding sites for activated CD4+ T cells immunoprecipitated with anti-STAT5 Ab, 68% had TTN₅AA motifs and TTCN₃GAA sites were present in 26% of the target sites. The labels denote the category name and percentage.</p

Gene ontologies of putative STAT5 targets from activated CD4+T cells.

<p>Gene function of putative binding sites is based on the summary information provided in the PANTHER database. Out of the 105 binding sites identified, functional categorisation was done for 104 genes, out of which 99 could be classified and the remaining 5 genes were of unknown/uncategorized function. Those 99 genes were classified based on their molecular function (A) and biological processes (B). In some instances, a given gene was represented in more than one category (for example, FRA2 was incorporated under the binding (GO:0005488) as well as transcriptional regulator activity (GO: 0030528) molecular functions).</p

TCR induced FRA2 expression is dependent on IL-2 signaling.

<p>CD4+ T cells were TCR activated ±HAT (humanized anti-Tac antibody) treatment and mRNA was prepared pre- and 17 hours post-stimulation. qRT-PCR analysis for FRA2 expression was carried out. TCR activation induces FRA2 expression, which is abrogated by the addition of HAT, indicating that IL-2R function is essential for induction of FRA2. This is representative of at least three independent experiments performed in triplicate.</p

Activation of the JAK3-STAT5 pathway is essential for the induction of FRA2 gene expression by TCR activation.

<p>CD4+ T cells were stimulated via the TCR ± the JAK3 inhibitor R333, RNA was prepared four hours post treatment. qRT-PCR analysis was performed to detect the relative expression of FRA2. FRA2 gene expression was induced by TCR activation and abrogated by R333 treatment. Shown is a representative experiment performed in triplicate and repeated three times.</p