The Mycobacterium tuberculosis exported repetitive protein (RvErp) is a crucial virulence-associated factor as determined by its role in the survival and multiplication of mycobacteria in cultured macrophages and in vivo. Although attempts have been made to understand the function of Erp protein, its exact role in Mycobacterium pathogenesis is still elusive. One way to determine this is by searching for novel interactions of RvErp. Using a yeast two-hybrid assay, an adenylyl cyclase (AC), Rv2212, was found to interact with RvErp. The interaction between RvErp and Rv2212 is direct and occurs at the endogenous level. The Erp protein of Mycobacterium smegmatis (MSMEG_6405, or MsErp) interacts neither with Rv2212 nor with Ms_4279, the M. smegmatis homologue of Rv2212. Deletion mutants of Rv2212 revealed its adenylyl cyclase domain to be responsible for the interaction. RvErp enhances Rv2212-mediated cyclic AMP (cAMP) production. Also, the biological significance of the interaction between RvErp and Rv2212 was demonstrated by the enhanced survival of M. smegmatis within THP-1 macrophages. Taken together, these studies address a novel mechanism by which Erp executes its function. IMPORTANCE RvErp is one of the important virulence factors of M. tuberculosis. This study describes a novel function of RvErp protein of M. tuberculosis by identifying Rv2212 as its interacting protein.Rv2212 is an adenylyl cyclase (AC) and produces cAMP, one of the prime second messengers that regulate the intracellular survival of mycobacteria. Therefore, the significance of investigating novel interactions of RvErp is paramount in unraveling the mechanisms governing the intracellular survival of mycobacteria. Discerning the molecular mechanisms used by specific mycobacterial proteins involved in infection and virulence requires an understanding of the protein-protein interaction network. The interactions of secretory proteins of Mycobacterium with the host machinery are vital for successful infection. One such secretory protein involved in virulence of Mycobacterium tuberculosis is Erp (Rv3810).The erp gene of M. tuberculosis encodes an ϳ28.0-kDa secretory protein that migrates as a 36.0-kDa protein and is present in all species of mycobacteria. Its disruption results in a marked decrease in virulence, with lower levels of survival not only in in vitro and cell culture assays but also under in vivo conditions (1, 2). It was recently shown that the nature of the erp allele strongly affects the number and the size of the lung lesions in infected animals (3). No homologue of Erp has been found in other bacterial species, making Erp a mycobacterial signature (4).Erp has a composite structure made up of three domains. While the amino-terminal domain (amino acids 1 to 80) and the carboxyterminal domain (amino acids 176 to 284) are conserved, the central domain, consisting of tandem repeats of 5 amino acids based on a PGLTS motif, is subjected to a high level of interspecies variability (1). A signal sequence is present in the amino termi...
SummaryGene transfer to create tumour epitope-specific cytolytic T cells for adoptive immunotherapy of cancer remains an area of active inquiry. When the Mart-1 27-35 -specific DMF5 T-cell receptor (TCR) is transferred into peripheral human CD4 + T cells, the reprogrammed cells exhibit a T helper type 1 (Th1) phenotype with significant multifactorial effector capabilities. The T-bet transcription factor plays an important role in determination of the Th1 differentiation pathway. To gain a deeper understanding of how T-bet controls the outcome of human T-cell reprogramming by gene transfer, we developed a system for examining the effects of short hairpin RNA-mediated T-bet gene knockdown in sorted cell populations uniformly expressing the knockdown construct. In this system, using activated peripheral human CD4 + CD25 À and CD8 + T cells, T-bet knockdown led to attenuation of the interferon-c response to both antigen-specific and non-specific TCR stimulation. The interleukin-2 (IL-2) antigen-specific response was not attenuated by T-bet knockdown. Also, in TCR-reprogrammed CD8 + cells, the cytolytic effector response was attenuated by T-bet knockdown. T-bet knockdown did not cause redirection into a Th2 differentiation pathway, and no increased IL-4, IL-10, or IL-17 response was detected in this system. These results indicate that T-bet expression is required for maintenance of the CD4 + CD25 À and CD8 + effector phenotypes in TCR-reprogrammed human T cells. They also suggest that the activation protocol necessary for transduction with retrovectors and lentivectors may commit the reprogrammed cells to the Th1 phenotype, which cannot be altered by T-bet knockdown but that there is, nevertheless, a continuous requirement of T-bet expression for interferon-c gene activation.
Adoptive cell therapy (ACT) with in vitro expanded populations of T cells engineered to express tumor epitope specific T cell receptors (TCR) is now undergoing clinical trials for various malignancies. In this context, ACT with the melanoma epitope, , specific TCR engineered T cells has shown encouraging results in metastatic melanoma. A number of biological processes regulating T cell functions have, however, turned out to be impediments in this form of cancer therapy. As such, efforts are underway to gain a fuller understanding of the biology (functionality and constraints) of TCR-engineered (TCReng) T cells so as to extract more robust therapeutic effects from ACT. Traditional T cell-based assays are, however, somewhat inadequate for the purpose. Using Next Generation RNA-Seq (NGS) and qRT-PCR assays, we examined the transcriptome of melanoma epitope, Mart-1 (27)(28)(29)(30)(31)(32)(33)(34)(35) , specific TCReng human T cells. We found that the transcriptional profiles of the Mart-1 (27)(28)(29)(30)(31)(32)(33)(34)(35) , specific TCReng T cell (both CD8+ and CD4+) is remarkably similar when exposed to the cognate peptide. The genes responsible for T-cell activation, apoptosis, cellular proliferation, cytolytic response, and T-cell differentiation showed similar patterns of expression. Further, our analysis also revealed a number of alternate splice variants and novel isoforms related to immune response previously not associated with T cell activation, as well as expression of a number of recently discovered long non-coding RNAs (lncRNA).
Back ground: Metastatic melanoma is an aggressive tumor of the epidermal melanocytes with high mortality rates in patients. The anti- melanoma immune response has been extensively studied while immune based treatment strategies are showing promising activity in melanoma patients. A number of immune characteristics are evaluated as predictive and prognostic biomarker. Methods: With approval from Institution's regulatory board, we analyzed T cells from peripheral blood of 12 metastatic melanoma patients for baseline frequency of T cells reacting against melanoma related Mart-1 27-35 epitope. We expanded CD 8+ T lymphocytes by exposing them to the Mart-127-35 epitope pulsed on autologous mononuclear cell derived antigen presenting cells ex vivo. Using magnetic bead separation techniques, we also isolated from the peripheral blood CD4+CD25+ (naturally occurring Treg) and CD4+CD25- cells and analyzed in vitro proliferation of CD4+CD25- T cells in response to anti CD3 antibody and PHA and analyzed the effects of CD4+CD25+ cells on their proliferation. Finally, we induced CD4+ T cells into Th1 or Th2 type responses and analyzed their function in vitro. Results: There was variable degrees of CD8+effector T lymphocytes responses to the antigens in in vitro cultures from all the patients studied and their cytokine profile noted. The percentage of nTreg activity in the PBL averaged about 5% that demonstrated suppression of the CD4+CD25- T cells proliferation in response to anti CD3 antibody in about 50% of patients, but lacked suppression activity in about 50% of the patients. It was possible to generate Th1 type cells from all the patients but generation of the Th2 type cells was limited to 50%. Favorable immune characteristics were observed in majority of the patients who are responding to the treatment. Conclusion: Our findings suggest that in vitro functional T cell immune characteristics could be examined in patients diagnosed with metastatic melanoma. Examination of these characteristics in larger number of patients diagnosed with melanoma might provide predictive marker for immune intervention. Citation Format: Upendra P. Hegde, Sidharth S. Jha, Nitya G. Chakraborty. In vitro analysis of T cells effector vs regulatory function in patients with metastatic melanoma and its correlation to patient outcome. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1305. doi:10.1158/1538-7445.AM2015-1305
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