Gabriela L Cosma

Characterization of T-Bet and Eomes in Peripheral Human Immune Cells

The T-box transcription factors T-bet and Eomesodermin (Eomes) have been well defined as key drivers of immune cell development and cytolytic function. While the majority of studies have defined the roles of these factors in the context of murine T-cells, recent results have revealed that T-bet, and possibly Eomes, are expressed in other immune cell subsets. To date, the expression patterns of these factors in subsets of human peripheral blood mononuclear cells beyond T-cells remain relatively uncharacterized. In this study, we used multiparametric flow cytometry to characterize T-bet and Eomes expression in major human blood cell subsets, including total CD4+ and CD8+ T-cells, γδ T-cells, invariant NKT cells, natural killer cells, B-cells, and dendritic cells. Our studies identified novel cell subsets that express T-bet and Eomes and raise implications for their possible functions in the context of other human immune cell subsets besides their well-known roles in T-cells.

Targeting the mRNA-binding protein HuR impairs malignant characteristics of pancreatic ductal adenocarcinoma cells.

Post-transcriptional regulation is a powerful mediator of gene expression, and can rapidly alter the expression of numerous transcripts involved in tumorigenesis. We have previously shown that the mRNA-binding protein HuR (ELAVL1) is elevated in human pancreatic ductal adenocarcinoma (PDA) specimens compared to normal pancreatic tissues, and its cytoplasmic localization is associated with increased tumor stage. To gain a better insight into HuR’s role in PDA biology and to assess it as a candidate therapeutic target, we altered HuR expression in PDA cell lines and characterized the resulting phenotype in preclinical models. HuR silencing by short hairpin and small interfering RNAs significantly decreased cell proliferation and anchorage-independent growth, as well as impaired migration and invasion. In comparison, HuR overexpression increased migration and invasion, but had no significant effects on cell proliferation and anchorage-independent growth. Importantly, two distinct targeted approaches to HuR silencing showed marked impairment in tumor growth in mouse xenografts. NanoString nCounter® analyses demonstrated that HuR regulates core biological processes, highlighting that HuR inhibition likely thwarts PDA viability through post-transcriptional regulation of diverse signaling pathways (e.g. cell cycle, apoptosis, DNA repair). Taken together, our study suggests that targeted inhibition of HuR may be a novel, promising approach to the treatment of PDA.

Differentiation and Protective Capacity of Virus-Specific CD8+ T Cells Suggest Murine Norovirus Persistence in an Immune-Privileged Enteric Niche

&NA; Noroviruses can establish chronic infections with active viral shedding in healthy humans but whether persistence is associated with adaptive immune dysfunction is unknown. We used genetically engineered strains of mouse norovirus (MNV) to investigate CD8+ T cell differentiation during chronic infection. We found that chronic infection drove MNV‐specific tissue‐resident memory (Trm) CD8+ T cells to a differentiation state resembling inflationary effector responses against latent cytomegalovirus with only limited evidence of exhaustion. These MNV‐specific Trm cells remained highly functional yet appeared ignorant of ongoing viral replication. Pre‐existing MNV‐specific Trm cells provided partial protection against chronic infection but largely ceased to detect virus within 72 hours of challenge, demonstrating rapid sequestration of viral replication away from T cells. Our studies revealed a strategy of immune evasion by MNV via the induction of a CD8+ T cell program normally reserved for latent pathogens and persistence in an immune‐privileged enteric niche. Graphical Abstract Figure. No caption available. HighlightsMNV‐specific Trm cells during chronic infection are largely functionalMNV Trm cells are transcriptionally similar to inflationary T cells in latent herpesMNV‐specific CD8+ T cells are protective, but this protection wanes after ˜3 daysCD8+ T cell ignorance in chronic MNV infection is due to poor antigen presentation &NA; Chronic infections often cause T cell dysfunction, but how noroviruses (NV) evade immunity is unknown. Tomov et al. show that gut‐resident T cells against NV remain functional but ignorant of chronic viral replication, suggesting that NV persists in an immune‐privileged enteric niche.

Corrigendum: Characterization of T-bet and Eomes in Peripheral Human Immune Cells

[This corrects the article on p. 217 in vol. 5, PMID: 24860576.].

CD8 + T-cell responses in vaccination: reconsidering targets and function in the context of chronic antigen stimulation

Cytotoxic CD8 T cells play important roles in eliminating infected and transformed cells. Owing to their potential for therapeutic applications, significant efforts are dedicated toward developing CD8 T cell–based vaccines. Thus far, CD8 T-cell vaccination strategies have had limited success therapeutically in contrast to those targeting antibody-based immunity. However, if the current challenges and gaps in the understanding of T-cell biology are overcome, the full potential of rational CD8 T-cell vaccine design might be realized. Here, we review recent progress in this direction, focusing on target selection and maintenance of function in the settings of chronic infections and cancers.

Abstract 1294: Identification of tumor-specific antigens associated with RET/PTC3 expression

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Relocated in transformation/papillary thyroid carcinoma, RET/PTC3 (RP3) is a fusion oncogene that causes a form of papillary thyroid cancer (PTC). In addition to driving transformation, the constitutively active kinase precociously phosphorylates itself as well as other intracellular proteins, thereby providing tumor-specific targets for the adaptive immune system. PTCs are known to have the ability to escape the immune system driving a dysregulation of the activity of several cell populations involved in the immune response. Indeed we found that mice immunized with RP3+/MHC class II+ cells produce dramatically fewer IFNg-secreting CD4+ T cells compared to mice immunized with RP3-/MHC class II+ counterparts as measured in the spleens. We demonstrated the immunogenicity of RP3 with reactivity in both BALB/c and C57BL/6 mice to RP3/IEd- and RP3/CIITA-expressing cells. Also, we observed immunogenicity of RP3-derived phosphopeptides in ELISpot assays, supporting the hypothesis that the aberrant autophosphorylation of RP3 is a source of tumor-specific immunogenicity. Finally, utilizing a RP3-expressing vaccinia vector for immunization of C57BL/6 mice, we identified peptide sequences that appear to be immunogenic on the basis of unique conformation of the fusion protein that impacts antigen processing. Taken together these results could pave the way for better vaccine approaches without accompanying autoimmunity. Citation Format: Laura Sponton, Gabriela Cosma, Mark Mendonca, Mirella Giovarelli, Laurence Crane Eisenlohr. Identification of tumor-specific antigens associated with RET/PTC3 expression. [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 1294. doi:10.1158/1538-7445.AM2015-1294

Abstract 5133: The RNA-binding protein HuR facilitates proliferation and metastasis in human pancreatic ductal adenocarcinoma

Background: Pancreatic ductal adenocarcinoma (PDA) is the fourth most common cause of cancer-related death in the Unites States, with an overall 5-year survival rate of Methods: Stable MiaPaCa2 cell lines were generated that produce two different shRNAs against the HuR coding sequence (labeled Mia.sh290 and Mia.sh700) in response to doxycycline (DOX) induction. HuR silencing was validated by RT-qPCR and Western blotting. Similarly, a DOX-inducible MiaPaCa2 cell line that overexpresses HuR cDNA (Mia.HuR) was generated and validated. Sustained ∼50% knockdown and 1.5-2-fold overexpression were achieved, respectively. Following characterization of HuR expression in these cell lines under ±DOX conditions, we performed a 7-day cell proliferation (PicoGreen) assay and assessed anchorage-independence over a 4-week period by soft agar colony formation assay. Metastatic potential was measured by Matrigel invasion and in vitro scratch test assays. Results: Upon DOX induction, Mia.sh290 and Mia.sh700 cell lines showed significantly reduced cell proliferation over 7 days (22% and 15% reduction compared to non-induced in sh290 and sh700 respectively, p Conclusion: Taken together with our previous findings, our work demonstrates that manipulation of HuR expression affects key characteristics of tumorigenesis (i.e. proliferation, invasion, and migration). These findings, along with ongoing in vivo work, continue to support the notion that targeting HuR in PDA cells may be a promising therapeutic strategy. Citation Format: Masaya Jimbo, Fernando F. Blanco, Brad Screnci, Gabriela Cosma, Vitali Alexeev, Yu-Hung Huang, Jordan M. Winter, Charles J. Yeo, Janet A. Sawicki, Jonathan R. Brody. The RNA-binding protein HuR facilitates proliferation and metastasis in human pancreatic ductal adenocarcinoma. [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 5133. doi:10.1158/1538-7445.AM2015-5133