Citations

Abstract Purpose: Regulatory T-cells (Treg) are essential to Tregs homeostasis and modulate the antitumor immune response in patients with lymphoma. However, the biology and prognostic impact of Tregs in splenic marginal zone lymphoma (SMZL) have not been studied. Experimental Design: Biopsy specimens from 24 patients with SMZL and 12 reactive spleens (rSP) from individuals without lymphoma were analyzed by using CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), CyTOF (mass cytometry) analysis, and flow cytometry to explore the phenotype, transcriptomic profile, and clinical significance of intratumoral Tregs and their subsets. The biological characteristics and cell signaling pathways of intratumoral Treg subsets were confirmed by in vitro functional assays. Results: We found that Tregs are more abundant in SMZL patients' spleens than rSP, and Tregs from patients with SMZL and rSP can be separated into CD161+Treg and CD26+Treg subsets. CD161+Tregs are increased in SMZL but have dysregulated immune function. We found that CD161+Treg and CD26+Tregs have unique gene expression and phenotypic profiles and are differentially correlated with patient outcomes. Specifically, increased CD161+Tregs are significantly associated with a favorable prognosis in patients with SMZL, whereas CD26+Tregs are associated with a poor prognosis. Furthermore, activation of the IL2/STAT5 pathway contributes to the induction of CD26+Tregs and can be reversed by STAT5 inhibition. Conclusions: IL2/STAT5-mediated expansion of CD26+Tregs contributes to a poor clinical outcome in SMZL and may represent a therapeutic opportunity in this disease.

Abstract Previous studies have found that the prevalence of subtypes of T cells may be associated with patient outcomes, but a comprehensive analysis of the immune profile in the TM of FL has not been done. In the present study, we identified groups of FL patients with discretely unique TMEs and determined whether the T-cell phenotypes in the TME differed among groups. Using a cohort of 82 FL patients with biopsy specimens collected before treatment, we defined the type of TME based on the content of major lineages (T, B, monocytes/macrophages and NK cells) determined by CyTOF analysis. Hierarchical clustering of this cohort stratified patients into 4 groups with different TME: group 1 (G1) included patients with a high percentage of monocyte/macrophages/NK cells; patients from G2 and G3 were enriched for intratumoral T and B cells, respectively. Patients with intermediate numbers of T and B cells were included in G4. CITRUS analysis revealed that T-cell clusters with phenotypes expressing KLRG1, CD57, PD-1dim and rich in TEMRA cells were significantly more abundant in G1 when compared to G2, G3 or G4. In contrast, T-cell clusters with phenotypes containing CD127, CD45RA, CCR7 or PD-1high cells were significantly less abundant in G1 when compared to other groups. When compared to G3 or G4, 2 classes of T cell clusters, all from CD4+ T cells, were significantly more (CD127+KLRG1-) or less (CD57+PD-1high) abundant in G2, respectively. Clusters with a phenotype rich in short-lived effector cells (SLEC) (from CD8+) were upregulated in G3 when compared to G4. These results suggest that patient groups with distinct TME exhibited variable T-cell phenotypes. To determine the role of T-cell differentiation in predicting patient outcome in FL, we identified T-cell subsets using tSNE plots based on the expression of T-cell maturation markers. We identified 18 subsets (S1-S18) of T cells (11 from CD4, 6 from CD8 and 1 from CD4-CD8-) in each sample. Four subsets - S4 (CD4+TN), S5 (CD4+TCM), S7 (CD8+TN) and S10 (CD8+MEPCs) - were considered to be naïve cells or cells in the early stages of differentiation. Four additional subsets - S8 (CD57+TFH), S9 (CD4+TEXH), S13 (CD8+SLECs) and S17 (CD4+PD-1+Treg) - were memory cells with expression of surface markers indicting late-stage differentiation. We found that the 4 subsets (S4: p=0.08, S5: p=0.01, S7: p=0.04 and S10: p<0.01) with an early-stage phenotype were associated with a favorable clinical outcome. In contrast, subsets (S8: p=0.02, S9: p=0.04, S13: p=0.07 and S17: p=0.06) with a late-stage differentiation phenotype had an unfavorable survival. Supporting this finding, we observed that increased numbers of CD45RA+ T cells correlated with a favorable survival. These results indicate that the differentiation stage may determine the role of T cells in predicting patient outcome in FL. Citation Format: Zhi-Zhang Yang, Hyo Jin Kim, Hongyan Wu, Xinyi Tang, Jordan Krull, Patrizia Mondello, Jose Villasboas, Anne Novak, Stephen Ansell. T-cell phenotype and differentiation vary in the tumor microenvironment of follicular lymphoma and are associated with patient outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2529.

Background. Long noncoding RNAs (lncRNAs) represent an important novel class of noncoding RNA molecule greater than 200 nucleotides that play a key role in the regulation of autoimmune diseases. Previous studies have demonstrated that MAFTRR (MAF transcriptional regulator RNA) regulated Th1 cells differentiation by inhibiting the expression of MAF in activated CD4+ T cells. However, the effect of MAFTRR on the pathogenesis of Hashimoto’s thyroiditis (HT) remains unclear. This research was aimed at investigating the expression of MAFTRR in Hashimoto’s thyroiditis (HT) as well as the correlation between MAFTRR and Th1 cells. Methods. Thirty-eight HT patients and thirty-eight healthy controls were enrolled in the study. The proportion of Th1 cells and CD8+IFN-γ+ T cells in peripheral blood mononuclear cells (PBMCs) from these specimens was determined by flow cytometric analysis. The transcript levels of MAFTRR, MAF, and IFNG in PBMCs and thyroid glands were detected by quantitative real-time PCR. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the potential value of MAFTRR in the HT patients. Results. We found that the proportion of circulating Th1 cells and the transcript levels of IFNG were increased in peripheral blood of the HT patients. The transcript levels of MAFTRR were significantly increased in the HT patients and positively correlated with the percentage of Th1 cells and serum levels of antithyroglobulin antibody and antithyroperoxidase antibody. The transcript levels of MAF, a transcription factor that inhibits Th1 cells activity and IFN-γ production, were attenuated in PBMCs from the HT patients. The transcript levels of IFNG had positive and inverse correlations with MAFTRR and MAF expression in PBMCs from the HT patients, respectively. Additionally, a significantly positive correlation between upregulated MAFTRR expression and augmented IFNG expression was revealed in thyroid tissues from the HT patients. ROC curve suggested that MAFTRR could potentially differentiate the HT patients from healthy controls. Conclusion. MAFTRR is significantly augmented in the HT patients and may contribute to the pathogenic role of the Th1 cells response in HT.

Abstract The tumor microenvironment plays a crucial role in mediating the tumor immune response, thereby affecting patient outcomes in follicular lymphoma (FL). Using CyTOF, we analyzed single cell suspensions created from pre-treatment biopsies in a cohort of 82 FL patients and retrospectively extracted patient clinical parameters. Hierarchical analysis of the tumor microenvironment composition stratified these patients into 4 groups: group 1 represents patients with high percentage of monocyte/macrophages/NK cells; patients from group 2 and 3 are rich of intratumoral T and B cells, respectively. Patients with intermediate number of T and B cells are classed into group 4. Intratumoral T cells from these 4 groups exhibited distinct phenotypical characteristics. Group 1 was enriched with KLRG1 +CD8 + T cells, while group 4 showed increased number of CXCR3 - T EM and CD4 + T N cells. Patients from group 2 and 3 featured with increased number of ICOS + T reg cells and CD57 + T FH cells, respectively. We observed that higher numbers of T cells significantly correlated with early disease stage, lack of B-symptoms, event-free survival at 24 months (EFS24) and a favorable overall and EFS survival in patients with histological grade 1 and 2. However, this association was not seen in patients with histological grade 3a and b, suggesting that T cell-mediated anti-tumor immunity is preserved in low grade patients, but overridden in higher grade patients. CITRUS analysis revealed that a cluster that is CD57 + and PD-1 high was significantly more abundant in patients who had disease progression than patients who had a complete response to therapy, suggesting a role of CD57 + T FH cells in promoting malignant cell growth in FL. Consistent with this finding, we observed that higher number of CD57 + T FH cells correlated with an inferior survival in FL. Using CITE-seq technology, we found that CD57 + T FH cells exhibited a substantially different transcriptome when compared to CD57 - T FH cells. Genes that were differentially upregulated in CD57 + T FH cells when compared to CD57 - T FH cells included genes involved in cell survival, compromised inflammatory response, and metabolism activation including GZMK, CCL4, CST7, DUSP2, LGALS3, CYTOR, CHI3L2, SYNE2, CXCL13, CD27 and FABP5. Taken together, our results indicate different tumor microenvironments among FL patient groups that is associated with variable T-cell phenotype. We found that CD57 + T FH cells play an important role in predicting disease progression and patient prognosis in FL. Figure 1 Figure 1. Disclosures Novak: Celgene/BMS: Research Funding. Ansell: Bristol Myers Squibb, ADC Therapeutics, Seattle Genetics, Regeneron, Affimed, AI Therapeutics, Pfizer, Trillium and Takeda: Research Funding.

Background CD8+ T-lymphocyte subsets defined by killer lectin-like receptor G1 (KLRG1) and CD127 expression have been reported to have an important role in infection, but their role in the setting of lymphoid malignancies, specifically follicular lymphoma (FL), has not been studied. Methods To characterize the phenotype of KLRG1/CD127-defined CD8+ subsets, surface and intracellular markers were measured by flow cytometry and Cytometry by time of flight (CyTOF), and the transcriptional profile of these cells was determined by CITE-Seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing). The functional capacity of each subset was determined, as was their impact on overall survival (OS) and event-free survival (EFS) of patients with FL. Results We found that intratumoral CD8+ cells in FL are skewed toward effector cell subsets, particularly KLRG+CD127- and KLRG1-CD127- cells over memory cell subsets, such as KLRG1-CD127+ and KLRG1+CD127+ cells. While effector subsets exhibited increased capacity to produce cytokines/granules when compared with memory subsets, their proliferative capacity and viability were found to be substantially inferior. Clinically, a skewed distribution of intratumoral CD8+ T cells favoring effector subtypes was associated with an inferior outcome in patients with FL. Increased numbers of CD127+KLRG1- and CD127+KLRG1+ were significantly associated with a favorable OS and EFS, while CD127-KLRG1- correlated with a poor EFS and OS in patients with FL. Furthermore, we demonstrated that interleukin (IL)-15 promotes CD127-KLRG1+ cell development in the presence of dendritic cells via a phosphoinositide 3-kinase (PI3K)-dependent mechanism, and treatment of CD8+ T cells with a PI3K inhibitor downregulated the transcription factors responsible for CD127-KLRG1+ differentiation. Conclusions Taken together, these results reveal not only a biological and prognostic role for KLRG1/CD127-defined CD8+ subsets in FL but also a potential role for PI3K inhibitors to manipulate the differentiation of CD8+ T cells, thereby promoting a more effective antitumor immune response.

Abstract Purpose: T-cell immunoglobulin and ITIM domain (TIGIT), a member of the immune checkpoint family, is important in normal T-cell biology. However, the phenotypical profile and clinical relevance of TIGIT in follicular lymphoma is largely unknown. Experimental Design: Biopsy specimens from a cohort of 82 patients with follicular lymphoma were analyzed using mass cytometry to explore the phenotype and biological and clinical significance of TIGIT+ T cells. Results: TIGIT is highly expressed on intratumoral T cells and its expression alters T-cell phenotype in follicular lymphoma. TIGIT is abundantly expressed on Treg cells, resulting in an enhanced suppressive property. TIGIT expression on non-Treg/TFH T cells defines a population that exhibits an exhausted phenotype. Clinically, increased numbers of TIGIT+ T cells are associated with inferior patient outcomes and poor survival. We observe that anti–PD-1 therapy with pembrolizumab alters the phenotype of TIGIT+ T subsets and identifies a role for CD28 expression on TIGIT+ T cells in treatment response. Conclusions: The current study provides a comprehensive analysis of the phenotypic profile of intratumoral TIGIT+ T subsets and their prognostic relevance in follicular lymphoma. Inhibition of TIGIT signaling may be an additional mechanism to prevent T-cell suppression and exhaustion in B-cell lymphoma.