Immune Checkpoint CD200/CD200R Decreases T Cell-Mediated Cytotoxicity Via Dok2 and Is Regulated By P53 in Multiple Myeloma

Introduction

The membrane glycoprotein CD200 and its cognate receptor CD200R convey immunoregulatory functions in myeloid-derived immune cells. Expression of CD200 can also be detected in many hematological malignancies including multiple myeloma (MM) even though the mechanism of CD200 expression in cancer cells is largely elusive. The inhibitory receptor CD200R is expressed on T lymphocytes, yet the mechanism of inhibition is unclear. In this study, we analyze the functional role of the CD200/CD200R axis in immunoregulation and escape of MM cells and investigate the mechanism of inhibition in T cells.

Methods

Expression of CD200 on the surface of primary patient-derived MM cells and MM cell lines was assessed by flow cytometry. Cytotoxic effects in co-cultures of CD3/CD28 stimulated healthy donor T cells and CD200-/+ MM cell lines were analyzed using luciferase assay or flow cytometry. To evaluate the impact of CD200 expression by MM cells on regulatory T cells (Treg), stimulated naïve healthy donor T cells were co-cultured with CD200-/+ MM cell lines in 1:5 (E:T) ratio for 3 days and analyzed with flow cytometry. To study downstream signaling of CD200R activation, CD3/CD28 stimulated T cells were co-cultured overnight with CD200-/+ MM cell lines and re-isolated with CD3 positive isolation. Potential effector proteins were then analyzed by Western blotting or intra-cellular phospho flow cytometry. The changes of CD200 transcription in MM cell lines after p53 induction by Nutlin-3a were analyzed by real time PCR (RT-PCR).

Results

CD200 surface expression was present in 70% of patient-derived primary MM cells (n=97). In MM cell lines (n=9), however, no CD200 expression on protein or mRNA level could be detected. We therefore used a Sleeping Beauty transposon vector system for stable expression of CD200 on the MM cell lines. In co-culture with primary T cells, up to 50% increase in MM cell survival upon CD200 expression was observed by flow cytometry and luciferase-based cytotoxicity assay. We also found an increase in the population of CD4+ CD25+ FoxP3+ CD127- Tregs in presence of CD200+ MM cells. It is known that docking protein-2 (Dok2) can be recruited by CD200R signaling in myeloid-derived immune cells which leads to the downregulation of the MAPK pathway. In CD3/CD28 stimulated T cells, we observed increased levels of phospho-Dok2 in presence of CD200 along with reduced levels of phosphorylation of ERK1/2, STAT3 and ZAP70. In TP53 wildtype MM cell lines, we observed an increase in CD200 mRNA levels after treatment with Nutlin-3a. In addition, wildtype TP53 status and CD200 surface expression in patient-derived primary MM cells (n=103) correlated positively.

Conclusion

Our study demonstrates an immunoinhibitory function of the CD200/CD200R axis in MM. CD200 expression on MM cells leads to reduced primary CD3+ T cell-mediated cytotoxicity via a Dok2-mediated inhibitory mechanism which is accompanied by an increase in Tregs. Our data also indicate that CD200 expression in MM cells may be conveyed by p53.

Kortuem:Janssen: Research Funding; Janssen, BMS, GSK, Abbvie, Pfizer: Consultancy. Einsele:BMS/Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel Grants, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Grants, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grants; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grants; Sanofi: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Other: travel grants.