Disruption of IFNγ, GZMB, PRF1, or LYST Results in Reduced Suppressive Function in Human CD8+ T Cells.

An imbalance between proinflammatory and regulatory processes underlies autoimmune disease pathogenesis. We have shown that acute relapses of multiple sclerosis are characterized by a deficit in the immune suppressive ability of CD8+ T cells. These cells play an important immune regulatory role, mediated in part through cytotoxicity (perforin [PRF]/granzyme [GZM]) and IFNγ secretion. In this study, we further investigated the importance of IFNγ-, GZMB-, PRF1-, and LYST-associated pathways in CD8+ T cell-mediated suppression. Using the CRISPR-Cas9 ribonucleoprotein transfection system, we first optimized efficient gene knockout while maintaining high viability in primary bulk human CD8+ T cells. Knockout was confirmed through quantitative real-time PCR assays in all cases, combined with flow cytometry where appropriate, as well as confirmation of insertions and/or deletions at genomic target sites. We observed that the knockout of IFNγ, GZMB, PRF1, or LYST, but not the knockout of IL4 or IL5, resulted in significantly diminished in vitro suppressive ability in these cells. Collectively, these results reveal a pivotal role for these pathways in CD8+ T cell-mediated immune suppression and provide important insights into the biology of human CD8+ T cell-mediated suppression that could be targeted for immunotherapeutic intervention.

Mucin-producing urothelial-type adenocarcinoma of the prostate with sarcomatoid features and novel molecular phenotype.

Introduction: Mucin-producing urothelial-type adenocarcinoma of the prostate is a rare tumor that may not elevate serum prostate-specific antigen, creating significant diagnostic and monitoring challenges. We evaluate our case in detail and review prior studies to demonstrate that the pathologic and molecular features of this tumor are distinct from conventional prostate adenocarcinoma. Case presentation: Our patient had a remote history of radiation-treated conventional prostate adenocarcinoma and presented many years later with an abscess-like prostate mass leading to urinary obstruction and hematuria. Biopsy revealed mucin-producing urothelial-type adenocarcinoma of the prostate with concurrent sarcomatoid features. Molecular studies showed a unique phenotype involving alterations in the KRAS, PTEN, RAD21, and TP53 genes. Conclusions: To our knowledge, this is the first report that describes sarcomatoid features and molecular mutations in mucin-producing urothelial-type adenocarcinoma of the prostate.

Pathology Data-Based Risk Group Stratification Is Equivalent to That Obtained by Oncotype DX Testing in Prostatic Adenocarcinoma.

CONTEXT.­: Low-risk (Gleason score 3 + 3 = 6) and intermediate-risk (Gleason score 3 + 4 = 7) prostate carcinoma cases diagnosed on needle biopsies are frequently referred for gene expression studies such as Oncotype DX to help validate the risk. Risk assessment helps in determining prognosis and therapeutic decision making. OBJECTIVE.­: To determine if addition of molecular testing is necessary, by evaluating its correlation with risk stratification provided by pathology report (Gleason score, Grade Group, proportion of positive cores) and serum prostate-specific antigen (PSA) level. DESIGN.­: Our institutional database was searched for cases that had Oncotype DX testing after prostate biopsy. The final risk category determined by molecular testing was compared to the risk stratification predicted by the pathology report and serum PSA levels. Cases were classified as concordant if they fell under the same National Comprehensive Cancer Network risk and recommended initial therapy group. Follow-up information on discordant cases was obtained and used to determine if risk stratification by molecular testing was superior to that obtained from the clinicopathologic data. RESULTS.­: A total of 4967 prostate biopsies (2015-2020) were screened. Of these, 131 prostate carcinoma cases (2.6%) had Oncotype DX testing and 111 of 131 cases (85%) had follow-up information. There was risk stratification concordance in 93 of 111 cases (84%). All 18 of 111 cases (16%) that were discordant had a follow-up course that matched the risk predicted by pathology data and serum PSA. CONCLUSIONS.­: Risk stratification provided by information in the pathology report on routine biopsy assessment coupled with the serum PSA level is equivalent to that obtained by Oncotype DX testing.

Teaching About Intergenerational Dynamics: An Exploratory Study of Perceptions and Prevalence in US Medical Schools.

PURPOSE: Prior studies suggest a role for promoting recognition of generational differences as a part of workplace ethics. To our knowledge, there is no published comprehensive analysis demonstrating how commonly or by what methods intergenerational dynamics are taught as structured coursework in medical school curricula. To address this gap, we carried out a survey of curriculum leaders of US medical schools to assess the current practices and attitudes toward content related to generational differences in medical school coursework. METHODS: A survey consisting of 23 closed- and open-ended questions that aimed to assess the presence, characteristics, and attitudes of participants towards intergenerational dynamics in medical school coursework was disseminated via email. Curriculum deans at 154 allopathic medical schools were invited to complete the survey and sent one reminder email. Quantitative responses were descriptively analyzed, and qualitative responses were thematically analyzed. RESULTS: The response rate was 38.3%, with the majority (58%) of responding institutions stating that their curriculum did not include coursework on intergenerational dynamics. When taught, the most frequent method of instruction was small-group activities. Most stated that the educational content for intergenerational dynamics has been part of their curriculum for fewer than five years. In total, 34% of respondents agreed that some form of education about intergenerational dynamics should be required during medical education as they felt that content could improve cultural competence. Those that were less supportive of inclusion of intergenerational material stated concerns about stereotyping and the value of generational descriptions. CONCLUSION: Our findings show a heterogeneity of responses on the perceptions and practice of curriculum leaders regarding inclusion of content related to intergenerational differences in medical school education. In summary, we present the first work assessing current practices and attitudes toward content related to the inclusion of material on intergenerational dynamics in undergraduate medical education in US allopathic medical schools.

IL-12-Induced Immune Suppressive Deficit During CD8+ T-Cell Differentiation.

Autoimmune diseases are characterized by regulatory deficit in both the CD4+ and CD8+ T-cell compartments. We have shown that CD8+ T-cells associated with acute relapse of multiple sclerosis are significantly deficient in their immune suppressive ability. We hypothesized that distinct CD8+ cytotoxic T-cell (Tc) lineages, determined by cytokine milieu during naïve T-cell differentiation, may harbor differential ability to suppress effector CD4+ T-cells. We differentiated purified human naïve CD8+ T-cells in vitro toward Tc0 (media control), Tc1 and Tc17 lineages. Using in vitro flow cytometric suppression assays, we observed that Tc0 and Tc17 cells had similar suppressive ability. In contrast, Tc1 cells showed significant loss of suppressive ability against ex vivo CD4+ T-cells and in vitro-differentiated Th0, Th1 and Th17 cells. Of note, Tc1 cells were also suboptimal in suppressing CD4-induced acute xenogeneic graft versus host disease (xGVHD) in vivo. Tc subtypes derived under various cytokine combinations revealed that IL-12-containing conditions resulted in less suppressive cells exhibiting dysregulated cytotoxic degranulation. RNA sequencing transcriptome analyses indicated differential regulation of inflammatory genes and enrichment in GM-CSF-associated pathways. These studies provide insights into the role of T-cell differentiation in CD8 suppressive biology and may reveal therapeutically targetable pathways to reverse suppressive deficit during immune-mediated disease.

Altered expression of SIRPγ on the T-cells of relapsing remitting multiple sclerosis and type 1 diabetes patients could potentiate effector responses from T-cells.

Factors regulating self-antigen directed immune-responses in autoimmunity are poorly understood. Signal regulatory protein gamma (SIRPγ) is a human T-cell specific protein with genetic variants associated with type 1 diabetes (T1D). SIRPγ's function in the immune system remains unclear. We show that T1D and relapsing remitting multiple sclerosis (RRMS) subjects have significantly greater frequency of rs2281808 T genetic variant, that correlates with reduced SIRPγ-expression in T-cells. Importantly, reduced SIRPγ-expression in RRMS and T1D subjects was not restricted to T variant, suggesting SIRPγ-expression is also regulated by disease specific factors in autoimmunity. Interestingly, increased frequencies of SIRPγlow T-cells in RRMS and T1D positively correlated with proinflammatory molecules from T-cells. Finally, we show that SIRPγlow T-cells have enhanced pathogenecity in vivo in a GVHD model. These findings suggest that decreased-SIRPγ expression, either determined by genetic variants or through peripherally acquired processes, may have a mechanistic link to autoimmunity through induction of hyperactive T-cells.

CD4 T cell-intrinsic role for the T helper 17 signature cytokine IL-17: Effector resistance to immune suppression.

Untoward effector CD4+ T cell responses are kept in check by immune regulatory mechanisms mediated by CD4+ and CD8+ T cells. CD4+ T helper 17 (Th17) cells, characterized by IL-17 production, play important roles in the pathogenesis of autoimmune diseases (such as arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease, among others) and in the host response to infection and cancer. Here, we demonstrate that human CD4+ T cells cells exposed to a Th17-differentiating milieu are significantly more resistant to immune suppression by CD8+ T cells compared to control Th0 cells. This resistance is mediated, in part, through the action of IL-17A, IL-17F, and IL-17AF heterodimer through their receptors (IL-17RA and IL-17RC) on CD4+ T cells themselves, but not through their action on CD8+ T cells or APC. We further show that IL-17 can directly act on non-Th17 effector CD4+ T cells to induce suppressive resistance, and this resistance can be reversed by blockade of IL-1ß, IL-6, or STAT3. These studies reveal a role for IL-17 cytokines in mediating CD4-intrinsic immune resistance. The pathways induced in this process may serve as a critical target for future investigation and immunotherapeutic intervention.

Autoimmunity-associated intronic SNP (rs2281808) detected by a simple phenotypic assay: Unique case or broader opportunity?

Multiple genome-wide association studies have shown that the single-nucleotide polymorphism (SNP) rs2281808 TT variant, present within the signal regulatory protein gamma (SIRPG) gene, is associated with autoimmune diseases, such as type 1 diabetes. SIRPγ is the only SIRP expressed on T cells. The role of SIRPγ in human T-cells or the effect of the TT variant are poorly understood. In this short report, we demonstrate the rather unusual finding that this intronic SNP is associated with a reduction of SIRPγ expression on T cells, both in healthy subjects as well as patients with type 1 diabetes. Using this information, we propose that a simple flow cytometric detection of SIRPγ could be a potential diagnostic testing approach for the presence of SNP in the appropriate clinical context.

An autoimmune disease risk SNP, rs2281808, in SIRPG is associated with reduced expression of SIRPγ and heightened effector state in human CD8 T-cells.

Multiple GWAS studies have shown that the SNP rs2281808 TT variant, present within the SIRPG gene, is associated with autoimmune diseases, such as type 1 diabetes. However, the role of SIRPγ in human T-cells is not known, neither is the functional significance of TT variant. Here we investigated SIRPG genotypes and their effects on the fate and function of human T-cells. We found that the presence of T variant resulted in reduction of SIRPγ expression on T-cells. Functionally, SIRPγlow CD8 T-cells in CT and TT individuals existed in a heightened effector state with lower activation threshold and had greater expression of genes and molecules associated with migratory and cytotoxic potential. Further, SIRPγlow CD8 T-cells were deficient in transcription factors associated with long-term functional memory formation. Our study reveals biological consequences of the SNP rs2281808 and provides novel insights into the potential mechanisms by which SIRPγ might regulate human immune responses.