CD84 regulates PD-1/PD-L1 expression and function in chronic lymphocytic leukemia
Hadas Lewinsky, Avital F. Barak, Victoria Huber, Matthias P. Kramer, Lihi Radomir, Lital Sever, Irit Orr, Vita Mirkin, Nili Dezorella, Mika Shapiro, Yosef Cohen, Lev Shvidel, Martina Seiffert, Yair Herishanu, Shirly Becker-Herman, Idit Shachar
Hadas Lewinsky, Avital F. Barak, Victoria Huber, Matthias P. Kramer, Lihi Radomir, Lital Sever, Irit Orr, Vita Mirkin, Nili Dezorella, Mika Shapiro, Yosef Cohen, Lev Shvidel, Martina Seiffert, Yair Herishanu, Shirly Becker-Herman, Idit Shachar
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Research Article Immunology Oncology

CD84 regulates PD-1/PD-L1 expression and function in chronic lymphocytic leukemia

Abstract

Chronic lymphocytic leukemia (CLL) is characterized by clonal proliferation and progressive accumulation of mature B lymphocytes in the peripheral blood, lymphoid tissues, and bone marrow. CLL is characterized by profound immune defects leading to severe infectious complications. T cells are numerically, phenotypically, and functionally highly abnormal in CLL, with only limited ability to exert antitumor immune responses. Exhaustion of T cells has also been suggested to play an important role in antitumor responses. CLL-mediated T cell exhaustion is achieved by the aberrant expression of several inhibitory molecules on CLL cells and their microenvironment, prominently the programmed cell death ligand 1/programmed cell death 1 (PD-L1/PD-1) receptors. Previously, we showed that CD84, a member of the SLAM family of receptors, bridges between CLL cells and their microenvironment. In the current study, we followed CD84 regulation of T cell function. We showed that cell-cell interaction mediated through human and mouse CD84 upregulates PD-L1 expression on CLL cells and in their microenvironment and PD-1 expression on T cells. This resulted in suppression of T cell responses and activity in vitro and in vivo. Thus, our results demonstrate a role for CD84 in the regulation of immune checkpoints by leukemia cells and identify CD84 blockade as a therapeutic strategy to reverse tumor-induced immune suppression.

Authors

Hadas Lewinsky, Avital F. Barak, Victoria Huber, Matthias P. Kramer, Lihi Radomir, Lital Sever, Irit Orr, Vita Mirkin, Nili Dezorella, Mika Shapiro, Yosef Cohen, Lev Shvidel, Martina Seiffert, Yair Herishanu, Shirly Becker-Herman, Idit Shachar

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Figure 6

Downregulation of CD84 expression in human CLL reduces PD-L1 on CLLs and stroma and induces T cell activity.

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Downregulation of CD84 expression in human CLL reduces PD-L1 on CLLs and...
(A) Primary human CLL cells were incubated for 48 hours in the presence or absence of anti-CD84–activating or control (IgG) (5 μg/ml) antibodies, IFN-γ (500 IU/ml), or both treatments together. Cells were then analyzed by FACS for PD-L1 cell-surface expression (n = 4; *P < 0.05 , **P < 0.01, and ****P < 0.0001, 1-way ANOVA with Holm-Sidak–corrected multiple comparisons). (B and C) CLL cells were treated with siCTRL or siCD84 for 24 hours, and levels of (B) CD84 (n = 4; *P < 0.05) and (C) PD-L1 (n = 4–5; ***P < 0.001, 2-tailed, paired t test) were analyzed. (D–F) The siRNA-treated cells were then incubated for 48 hours with M210B4 (D and F), or T cells (E, G, and H) derived from the same patient. (D and E) PD-L1 cell-surface expression on CLL cells derived from the coculture was analyzed by FACS. E shows a representative histogram. n = 4, *P < 0.05 (D) and n = 11, ****P < 0.0001 (E), 2-tailed, paired t test. (F) M210B4 cells were stained for PD-L1 cell-surface expression. A representative histogram is shown (n = 4; *P < 0.05, 2-tailed, paired t test). (G and H) CLL cells were treated with siCTRL or siCD84. The siRNA-treated cells were then incubated in the presence or absence T cells treated with anti–PD-1–blocking antibody (5 μg/ml) for 24 hours prior to coculture. (G) CD8+ T cells were analyzed for expression of the exhaustion markers PD-1, LAG-3, and CTLA-4 (n = 4). Representative histograms are shown in G. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, 2-tailed, paired t test for siCTRL and siCD84, and 1-way ANOVA with Fisher’s least significant difference (LSD) for the others. (H) Cultures were incubated with anti-CD3 (0.5 μg/ml) and brefeldin A for the last 2 hours. T cells were then analyzed for GZMB and IFN-γ expression. Graph show the percentage of CD8+ T cells that expressed GZMB and IFN-γ with and without PD-1 inhibition (5 μg/ml, clone EH12.2H7) (n = 4; *P < 0.05 and **P < 0.01, 2-tailed, paired t test for siCTRL and siCD84, and 1-way ANOVA with Fisher’s LSD for the others). (I) CLL cell lysis by the T cells in B was determined with the CytoTox-ONE Kit (Promega) (n = 4; *P < 0.05, 2-tailed, paired t test). (J–M) Purified human T cells were treated with anti-CD84 (5 μg/ml) and analyzed for p-JAK2 (I), p-STAT3 (K), PD-1, LAG-3, and CTLA-4 (L), and IFN-γ expression (M) (n = 4–6; **P < 0.01 and ***P < 0.001, 2-tailed, paired t test).