Kim, Hyojin

Splenic marginal zone lymphoma (SMZL) is a B-cell malignancy with a typically indolent course. However, some patients have more aggressive disease and initial treatment can range from observation to chemoimmunotherapy. The reason for this spectrum of disease behavior is incompletely understood but the tumor microenvironment (TME) likely plays a role as recent studies have shown that an optimized antitumor immune response in SMZL patients would allow the immune system to better target malignant B cells and irradicate the disease. Regulatory CD4+T cells (Tregs) are crucial in maintaining TME homeostasis by suppressing effector T cells and malignant B cells and in this study, we therefore explored the key factors influencing the function and development of intratumoral Tregs in SMZL patients. We identified two distinct Treg subsets in the TME of SMZL patients: CD26+Tregs and CD161+Tregs, each with unique phenotypic profiles, functions, and prognostic significance. CD161+Tregs suppress CD8+T cells and malignant B cells more effectively than CD26+Tregs, which only inhibit CD8+T cells. Clinically, patients with inferior outcomes have significantly higher numbers of CD26+Tregs and fewer CD161+Tregs. To delineate differences between these subsets, we performed Cellular Indexing of Transcriptomes and Epitopes Sequencing (CITE-seq) on biopsies from 7 SMZL patients. We found that cytokine-inducible SH2-containing protein (CISH) is the most highly expressed gene in CD26+Tregs compared to CD161+Tregs, aside from FOXP3. CISH has been reported to be a negative regulator of immune effector cell activation in tumor models. Although CISH deficiency leads to a loss of Foxp3 expression and reduced Treg suppressive function, the mechanism by which CISH modulates Treg function, especially immune suppression in hematologic malignancies, remains largely unknown. To further investigate the mechanism of CISH in Treg function in MZL patients, we conducted CITE-seq analysis on a larger cohort of MZL patients (n=12), divided into groups based on outcomes (EFS24 failed, n=4; EFS24 achieved, n=8). The CITE-seq analysis results showed significantly higher CISH expression in Tregs from patients with inferior outcomes (P=1.07E-06). We then explored factors influencing CISH expression and found that TCR engagement and treatment with cytokines (IL-15, IL-2, and IL-7) elevated CISH expression in Tregs. While we previously observed that lymphoma B cells could enhance Foxp3 expression in CD4+CD25-T cells, this study we found that B cells could also enhance CISH and CD26 expression in CD4+CD25-T cells in vitro, indicating that a high frequency of B cells in the environment may lead to increased CISH expression in Treg cells leading to an unfavorable outcome in patients. We further investigated the role of CISH in the function of Treg cells. Following CISH knockdown in Treg cells, we observed decreased CD26 expression and reduced suppressive function on CD8+ T cell proliferation. Using CRISPR-Cas9, we knocked out CISH in the Karpas 299 cell line, a human T cell lymphoma line that constitutively expresses CD25, Foxp3, and CISH. CISH-KO Karpas 299 cells showed significantly less apoptosis and Foxp3 expression compared to CISH-WT cells. Bulk RNA-seq analysis of CISH-KO and CISH-WT Karpas 299 cells revealed that CISH-KO cells showed enhanced DNA replication and cell cycle pathway activity. Taken together, our results suggest that high expression of CISH in Tregs may predict an unfavorable outcome in SMZL patients. Malignant B-cells in the TME of SMZL patients may promote expression of CD26 and CISH in Tregs, thereby modulating Treg function. Targeting CISH could decrease the immunosuppressive activity of Tregs in the TME, potentially optimizing antitumor immunity in SMZL patients.

Myeloid derived suppressor cells (MDSC) are a heterogeneous population of undifferentiated myeloid cells that are expanded and activated in pathological conditions and have the ability to potently suppress T-cell function and thereby contribute to immunosuppression and tumor progression. While there have been studies showing a role for MDSC in a variety of hematological malignancies, no data is available indicating that MDSCs contribute to the tumor progression in Waldenstrom's Macroglobulinemia (WM), an indolent lymphoma characterized by bone marrow (BM) infiltration of lymphoplasmacytic (LPL) cells and increased secretion of monoclonal IgM. In previous work, we have found increased GM-CSF and reduced arginine and cysteine in the BM microenvironment in WM. We hypothesized that this was due to the presence and activity of MDSCs in WM. BM aspirates from WM patients (n=17) were therefore processed to isolate LPL (CD19+/CD138+) cells from the rest of the BM cells (CD19-/CD138-). Sorted (CD19-/CD138-) cells from BM of patients with WM were studied with flow cytometry. Using a sequential gating strategy (lack of lineage markers, low levels of HLADR, CD33+, CD11b+) we identified a population of MDSCs that were then subdivided using CD14 and CD15 expression into total-, monocytic-, or granulocytic- MDSCs (m-MDSC, g-MDSC). We also analyzed unsorted BM cells using cytometry by time-of-flight (CyTOF) in order to further identify and phenotypically characterize the BM MDSC population in a group of WM patients with smoldering (asymptomatic) disease, symptomatic disease, or in remission post-treatment. BM samples from normal subjects were used as a control. Flow cytometry data showed significant higher numbers of MDSC subsets expressing PD-L1 and Arginase1 in WM patients when compared to the normal samples. BM cells from WM patients (n=18) then were compared to controls (n=11), and the absolute number of the total MDSC (p=0.05), m-MDSC (p=0.002), g-MDSC (p=0.02) was increased in WM specimens. When MDSCs from WM or normal monocytes from healthy controls were co-cultured with activated T-cells, the proliferation of activated T-cells in the presence of MDSCs from WM patients was impaired compared to controls, confirming the suppressive role of MDSCs. We then performed high dimensional analysis of the total BM MDSC cells using t-SNEand identified phenotypically distinct MDSC cell populations in the BM that were differentially present when healthy controls were compared to patients with smoldering WM or those with WM needing treatment. Specifically, WM patients needing treatment had increased numbers of a distinct MDSC population that was highly positive for CD163, and CD138. Moreover, conventional markers denoting m-MDSC and g-MDSC, such as CD14 and CD15, were highly expressed in all populations and their pattern of expression did not specifically define the MDSC subtypes, indicating that high dimensional phenotyping further details the MDSC sub-compartments beyond the conventional categorization of MDSC using conventional cytometry. In summary, we find that MDSCs are increased in the BM of WM patients compared to controls. MDSCs expressing CD163 and CD138 increase when WM patients become symptomatic and require therapy. Furthermore, MDSCs in the BM of WM patients suppress T-cell function and likely contribute to progression of the disease. MDSCs in the BM therefore present a therapeutic target that should be explored in WM patients. Disclosures Ansell: Bristol Myers Squibb: Other: research funding for clinical trials; Merck: Other: research funding for clinical trials; AI Therapeutics: Other: research funding for clinical trials; Affimed: Other: research funding for clinical trials; Takeda: Other: research funding for clinical trials; Pfizer: Other: research funding for clinical trials; Regeneron: Other: research funding for clinical trials; Seattle Genetics: Other: research funding for clinical trials.