Jalali, Shahrzad

Abstract Effector CD8+ T cells play a crucial role in anti-tumor immunity. Two major subsets of effector CD8+ T cells have been identified based on expression of KLRG1 and CD127: KLRG1+CD127- short-lived effector cells (SLECs) and KLRG1-CD127+ memory precursor effector cells (MPECs). In B-cell non-Hodgkin lymphoma (NHL), the frequency and function of SLECs and MPECs are unknown. Furthermore, the underlying mechanism by which these two CD8+ subsets were regulated and differentiate in B-cell NHL remains poorly understood. The goal of the present study is therefore to phenotypically and functionally characterize SLECs and MPECs in B-cell NHL. Using patient biopsy specimens, we observed that the median percentage of SLECs was greater than MPECs and was 39.9% (range: 22.7%~ 54.7%) and 18% (range: 7.66%~31.6%), respectively. In controls, the median percentage of SLECs was lower than that of MPECs and was 13.71% (range: 5.26%~29.2%) and 51.63% (range: 22.5%~70.1%) in PBMC and 12.14% (range: 4.01%~26%) and 51.62% (range: 29.2%~70.3%) in tonsil, respectively. Using mass cytometry (CyTOF), we observed that SLECs have higher expression levels of TIGIT and PD-1 and lower expression of CCR7, CD26 and CD27 when compared to MPECs. SLECs were functionally superior to MPECs as the numbers of cytokine (IFN-γ and TNF-α) - and granule (granzyme B and perforin)-producing cells were higher in SLECs than MPECs. However, SLECs had a lower proliferative capacity and a higher apoptosis rate when compared to MPECs. We observed that a reciprocal differentiation pathway exists between SLECs and MPECs. Activation and cytokine stimulation (IL-2 and IL-15) promoted the development of SLECs and decreased the number of MPECs. This effect was reversed when cells were treated with neutralizing antibodies to block IL-2 or IL-15 signaling. We also found that these two subsets had a distinct transcription factor profile as SLECs had higher expression of Eomes and T-bet, and lower expression of Tcf-1than MPECs. IL-2 and IL-15 enhanced the expression of T-bet and decreased the expression of Tcf-1. Taken together, our results show that SLECs are more prevalent than MPECs in B-cell NHL when compared to normal control tissue. These two effector cell subsets have distinct phenotypical and function profiles and their development is controlled by cytokines that may be dysregulated in lymphoma. Despite the fact that SLECs produce more granzyme B and IFN-γ, they are less proliferative and more susceptible to apoptosis. This may compromise an effective anti-tumor immune response in B-cell NHL. Disclosures Ansell: Celldex: Research Funding; Takeda: Research Funding; Merck & Co: Research Funding; Affimed: Research Funding; Bristol-Myers Squibb: Research Funding; Trillium: Research Funding; Regeneron: Research Funding; LAM Therapeutics: Research Funding; Pfizer: Research Funding; Seattle Genetics: Research Funding.

Abstract T cell Ig and ITIM domain (TIGIT) is an immune checkpoint molecule that is expressed on a variety of cell types including NK cells, effector and memory T cells, and Treg cells. Upon ligation with CD155, TIGIT delivers an inhibitory signal and negatively regulates anti-tumor responses. While important in normal T-cell biology, the pathological significance of TIGIT expression and function in the tumor microenvironment of the patients with follicular lymphoma (FL) is largely unknown. The present study sought to phenotypically and functionally characterize TIGIT+ T cell subsets in FL. While its expression is not detected on resting T cells in peripheral blood, we found that TIGIT is highly expressed on intratumoral T cells from FL. Treatment with cytokines such as IL-4 and TGF-β downregulated TIGIT expression on T cells. We found that TIGIT is predominantly expressed on effector memory T cells (TEM) with an activation/exhausted phenotype, and TIGIT+ T cells have higher expression levels of CD69 and PD-1 when compared to TIGIT- T cells. Functionally, TIGIT+ T cells displayed reduced capacity of cell proliferation and cytokine production (IFN-γ and IL-2). Using mass cytometry (CyTOF), we observed that TIGIT is abundantly expressed on some intratumoral Treg (CD4+CD25+Foxp3+) cells, while other Treg cells lack TIGIT expression in FL, forming two subsets of Treg cells: CD4+CD25+TIGIT+ and CD4+CD25+TIGIT-. These two subsets are phenotypically distinct in that CD25+TIGIT+ cells exhibited increased expression of activation/costimulatory markers such as CD69, CD27 and CD28 compared to CD25+TIGIT-, suggesting an activated Treg subset of CD25+TIGIT+ cells. This CD25+TIGIT+ subset had increased suppressive function and could more effectively inhibit activation and proliferation of CD8+ T cells than CD25+TIGIT- cells. Furthermore, we found that lymphoma B cells promoted the development of TIGIT-expressing T cells as TGF-β-mediated downregulation of TIGIT on T cells was inhibited when CD19+ lymphoma cells were present. Using a cohort of 31 FL patients, we found that intratumoral TIGIT-expressing T cells were associated with a favorable prognosis. Patients with TIGIT+ T cell numbers greater than 50% had better overall survival than patients with TIGIT+ T cell numbers less than 50%. Taken together, our results reveal a role of TIGIT in defining Treg cell subsets with different immune function and TIGIT expression may be useful in predicting patient outcome in FL. Disclosures Ansell: Takeda: Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer: Research Funding; Seattle Genetics: Research Funding; Celldex: Research Funding; Regeneron: Research Funding; LAM Therapeutics: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Merck & Co: Research Funding.

Abstract Introduction Immunoglobulin M monoclonal gammopathy of undetermined significance (IgM MGUS) is a relatively common pre-malignant condition that may progress to Waldenstrom macroglobulinemia (WM), an incurable low-grade non-Hodgkin lymphoma. The acquisition of genomic alterations in a multistep process of progression is likely a central mechanism responsible for the progression of IgM MGUS to WM, leading to increased tumor burden and end-organ damage. The exact role of the immune system in this process is still poorly characterized. The modulation of the immune system by malignant cells leading to anti-lymphoma immunity or immune evasion is a well-stablished principle associated with lymphoma survival and progression, respectively. The understanding of the pathobiology of IgM MGUS and WM has grown significantly over the last few years; however, most of the past research has focused on genetics and flow cytometric analysis of malignant cells. The introduction of CyTOF, a novel platform coupling mass spectometry with single-cell flow cytometry using antibody-metal isotope pairs, has allowed a broader single-cell analysis and the study of functional profiles. We here report the use of CyTOF technology to comprehensively profile the immune system of patients with IgM MGUS and WM. Methods Viably-cryopreserved (DMSO 10%) single-cell suspensions containing peripheral blood mononuclear cells from 15 patients (WM n=9 and IgM MGUS n=6) were used in this study. Recovered cells (1-3 x 106 per sample) were stained with a 30-parameter surface protein CyTOF panel designed to characterize multiple cell types and profile the immune system. Nucleated cellular events were identified using a DNA intercalator conjugated to natural abundance iridium (191Ir and 193Ir). Cisplatin (195Pt) was used for dead-live cell discrimination and calibration beads containing natural abundance cerium (140/142Ce), europium (151/153Eu), holmium (165Ho), and lutetium (175/176Lu) were used for normalization of the instrument signal. CyTOF analysis of normalized files was carried out using the Astrolabe Diagnostics platform. Cell clusters were assigned using the FlowSOM package and differential abundance analysis was performed using the edgeR package. Analysis is adjusted for multiple comparisons. Results The median live single-cell count for the entire cohort was 573,300 (range 50,681 to 869,727), representing a median of 81% (range 39% to 87%) of the total event counts [median 820,800 (range 130,452 to 1,000,000]. The cell count and the proportion of event count was similar in patients with WM or IgM MGUS. T cells represented 45.8% of the total events; granulocytes represented 31.3%; NK cells represented 8.6%; monocytes represented 8.1%; B cells represented 2.4% of the total events for the entire cohort. Figure 1 shows a heat map of different immune cell sub-types proportion per patient sample. A difference was noted in the proportion of monocytes (CD14+, CD16+) and B-cells when comparing patients with IgM MGUS and WM (figure 2). Patients with WM had a higher proportion of monocytes compared to IgM MGUS patients (p=0.05). A higher proportion of B-cells was noted in patients with IgM MGUS when compared to WM (4.2% vs 0.6%, p=0.01). When B-cells were sub-classified as naïve, transitional, non-switched memory, switched memory, and plasmablasts, the proportion of non-switched memory B-cells was higher in IgM MGUS compared to WM (p=0.05). While the results of this pilot study suggest important differences between IgM MGUS and WM as regards the immune repertoire, confirmatory studies are in progress in a larger cohort of patients. Conclusion To our knowledge, this is the first study of mass cytometry in patients with WM and IgM MGUS patients. A higher proportion of monocytes and lower proportion of memory B-cells was noted in patients with WM compared to IgM MGUS. CyTOF study of the immune profile in these patients is feasible and can potentially uncover relationships between cell types not typically associated with the disease progression continuum (IgM MGUS to WM). While definitive conclusion cannot be made using this dataset due to the small sample size, CyTOF is a powerful tool in the study of the immune profile of WM patients. Disclosures Gertz: Amgen: Consultancy; Abbvie: Consultancy; Apellis: Consultancy; annexon: Consultancy; spectrum: Consultancy, Honoraria; Teva: Consultancy; Alnylam: Honoraria; Physicians Education Resource: Consultancy; Ionis: Honoraria; janssen: Consultancy; celgene: Consultancy; Prothena: Honoraria; Research to Practice: Consultancy; Medscape: Consultancy. Kapoor:Celgene: Research Funding; Takeda: Research Funding. Ailawadhi:Takeda: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Pharmacyclics: Research Funding. Reeder:Affimed: Research Funding. Ansell:Seattle Genetics: Research Funding; Regeneron: Research Funding; Takeda: Research Funding; Pfizer: Research Funding; Merck & Co: Research Funding; Trillium: Research Funding; Affimed: Research Funding; LAM Therapeutics: Research Funding; Celldex: Research Funding; Bristol-Myers Squibb: Research Funding.

Abstract Waldenstrom's Macroglobulinemia (WM) is an indolent B-cell malignancy that is characterized by bone marrow infiltration of lymphoplasmacytic cells and excessive synthesis of IgM. Despite improved treatment strategies and increased understanding of the underlying molecular mechanisms contributing to malignant disease progression, WM remains incurable. While whole exome sequencing analysis has revealed the genomic events associated with malignant B cell transformation in WM, the bone marrow (BM) microenvironment is also emerging as a major player in the pathogenesis of WM. Previously, our lab has reported that an array of cytokines, including IL-21 and IL-6 are increased in the WM BM and such increases are associated with high proliferation rates and IgM secretion by WM cells. In this study, we aimed to identify how immune checkpoint molecules in the BM microenvironment, specifically programmed cell death-1 (PD-1) and its ligands PD-L1 and PD-L2, contribute to malignant cell expansion in WM. Using Real-Time PCR analysis, we show that the expression of PD-1, PD-L1 and PD-L2 are all significantly elevated in the cells of WM BM as compared to their normal BM counterparts. Interestingly, PD-L1 expression is also upregulated in the malignant WM cells. These findings were further confirmed by flow cytometry analysis. Subsequent in vitro analysis showed that PD-L1 and PD-L2 are cleaved from the WM cells and these soluble ligands (sPD-L1 and sPD-L2) are able to bind to the cells in the BM microenvironment, including T-cells. This results in reduced activation and proliferation of T-cells, and is accompanied by a reduction in cyclin A and p-ERK levels. In validation of the in vitro data, we also found sPD-L1 and sPD-L2 in the bone marrow plasma obtained from WM patients. While sPD-L2 was found to be present in both WM and normal BM plasma (though in a reduced level in WM), sPD-L1 was exclusive to the WM samples, implying that the function of these molecules are differentially regulated in WM. Furthermore, overexpression of PD-L1 in a WM cell line, MWCL.1, resulted in an increase in IgM secretion. Interestingly, treatment with IL-21 increased the expression of PD-L1 in WM cells, indicating that elevated IL-21 in WM BM could contribute to increased expression of PD-L1 by malignant WM cells. In summary, our data support a role for PD-1 ligands as immune regulatory molecules in WM. Further studies are ongoing to explore the molecular mechanisms by which PD-1/PD-L1/PD-L2 signaling affects the pathophysiology of WM cells. Disclosures No relevant conflicts of interest to declare.

Key Points Cytokines in the BM microenvironment regulate PD-1 ligand expression and secretion in WM. Secreted PD-1 ligands modulate T-cell function in WM.