Kim, Hyo Jin

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.

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 BACKGROUND Monocytes and macrophages (mo/mΦ) are a key part of the composition of peripheral blood (PB) and tissues and increased numbers of mo/mΦ have been associated with patient outcome in non-Hodgkin lymphoma (NHL). Because CD14 is abundantly expressed on the surface of human mo/mΦ, it is often used to identify or isolate human mo/mΦ, and immunosuppressive CD14+HLA-DRlow monocytes have been shown to be increased in the peripheral blood of NHL patients. However, we have previously shown that CD14 expression on mo/mΦ is substantially lower than CD68 expression suggesting that many CD68+ mo/mΦ are CD14 negative, especially in spleen and lymph node tissues. To characterize both CD14+ and CD14- mo/mΦ in PB and tissues, we isolated all mo/mΦ from B-cell NHL specimens and normal controls and assessed their phenotype and function. METHODS Human mo/mΦ were isolated by negative selection from PB and tissue biopsy specimens (B-cell NHL and normal tissues) using the immunomagnetic isolation (monocyte enrichment kit). Morphological and immunophenotypic characteristics of isolated mo/mΦ were determined by Giemsa stain and flow cytometry. Phagocytosis and migration assays were used to determine the function of isolated mo/mΦ. T cells were co-cultured with mo/mΦ and T cell proliferation was evaluated by CFSE staining and detected by flow cytometry. RESULTS Using a monocyte enrichment kit to isolate all mo/mΦ, the purity of isolated mo/mϕ was 85~99%, which was defined by the percentage of lineage-negative cells (i.e. cells without expression of CD3,CD19, CD20, and CD56). We found that these isolated mo/mΦ constituted 2 populations: a more frequent population of larger cells and a less common population of smaller cells. In contrast to PB, CD14 positive mo/mΦ constituted less than 40% of the tissue mo/mΦ from the isolated population. Furthermore, we found that the cell size from CD14+ mo/mΦ were larger than CD14- mo/mΦ. Using CD14 and SIRP-α, we could identify 3 populations of mo/mΦ: CD14+SIRP-αhigh, CD14-SIRP-αdim and CD14-SIRP-α- cells. CD14+SIRP-αhigh cells and CD14-SIRP-αdim cells typically constituted the population of larger cells, while CD14-SIRP-α- cells constituted the population of smaller cells. CD14-SIRP-α- cells lacked the typical phenotypic markers and had decreased phagocytic and migratory ability compared to CD14+SIRP-αhigh and CD14-SIRP-αdim cells. Furthermore, we found that these 3 populations of mo/mΦ had a differential effect on activated T-cells and that the CD14-SIRP-α- cells appeared increased number in biopsy specimens from NHL when compared to normal tissues. CONCLUSIONS We have identified a unique population of small CD68+ mo/mΦ that lack expression of CD14, SIRP-α, and other FcγR markers. This subset of mo/mΦ is more prevalent in NHL tissues and has limited phagocytic and migratory functions. This CD14-SIRP-α- mo/mΦ subpopulation may play an inhibitory role in anti-cancer and inflammatory responses. Disclosures No relevant conflicts of interest to declare.