OMIP‐050: A 28‐color/30‐parameter Fluorescence Flow Cytometry Panel to Enumerate and Characterize Cells Expressing a Wide Array of Immune Checkpoint Molecules

This 28-color/30-parameter panel catalogs, enumerates and characterizes cells expressing molecules that regulate T-cell responses (“checkpoint” molecules). The primary purpose is to measure combinatorial expression of PD-1, CTLA-4, TIM-3, CD244/2B4, TIGIT, BTLA, CD137/4-1BB, GITR, OX40, CD27, and CD278/ICOS, many of which are targets of the immunotherapy drugs in preclinical or clinical development. This panel is optimized for cryopreserved healthy human peripheral blood mononuclear cells (PBMCs) and cryopreserved mononuclear cells isolated from tumor specimens (tissue MNCs). CD45 is used to distinguish leukocytes from stromal cells and debris often present in tissue digests. CD3, CD4, and CD8 identify T-cells, whose differentiation status is then assessed with CD45RO, CD95, CCR7, CD27, CD57, and CD28. CD25, HLA-DR, and CD69 mark activated T-cells. CD69 is also used to identify tissue-resident lymphocytes in combination with CD103. CXCR3 and CXCR6 mediate lymphocyte trafficking toward chemokines often found in the tumor microenvironment. Regulatory T-cells are defined here by antibodies against CD25 and CD127. In sum, this panel comprehensively characterizes cells expressing checkpoint markers. Immunotherapy has shown clinical benefit in certain solid tumors, although the markers predicting a successful response remain poorly defined 1. This is a consequence of the multiple immune evasion mechanisms employed by tumors and the fact that different arms of the immune system are targeted by different immunotherapy agents. Thus, biomarkers of response are likely to be complex and may differ between patients and across diseases. This OMIP uses new 28-color/30-parameter flow cytometry technology 2 to comprehensively monitor immune responses to immunotherapy, by surveying—in a single tube—multiple T-cell subsets and immunotherapy targets. Lymphocytes are regulated by an array of co-stimulatory and co-inhibitory receptors that may be targeted by immunotherapy 3. Blockade of co-inhibitory/checkpoint molecules CTLA-4 and PD-1 have provided lasting clinical benefit for a subset of patients with solid tumors 4. Other checkpoint molecules, including TIM3, 2B4, and TIGIT, are being targeted in an effort to remove the “brakes” from T-cells in the highly immunosuppressive tumor microenvironment 5-7. Agonists of co-stimulatory receptors, including ICOS, OX40, GITR, CD27, and 4–1BB, are also being developed to potentiate lymphocyte activation 8-10. The functions of cells expressing particular combinations of these markers may be unique, and could be linked to the cells’ differentiation status or tissue distribution. Notably, the frequency of certain memory subsets, such as central memory (Tcm) and T helper type 1 (Th1), has been associated with clinical benefits 11. More recently, CD103-expressing tissue-resident T cells have been shown to provide a strong, rapid effector response that recruits additional lymphocytes 12. Thus, the differentiation status and trafficking potential of leukocytes provides additional clues as to the patient's potential to mount an anti-tumor response. In this panel, we incorporate a number of T-cell differentiation and trafficking markers, deployed successfully in lower parameter OMIPs 13-15. Figure 1 shows staining patterns for each of the markers in the panel. Because many checkpoint molecules are absent or expressed at low levels in healthy individuals, PBMCs were stimulated with PMA-ionomycin 36 h to induce detectable levels of all checkpoint molecules examined. Successful staining was also observed in primary glioblastoma tumor samples (data not shown). All antibodies were titrated to achieve the greatest resolution, and checkpoint molecules were assigned the highest priority. Multiple clones were tested for molecules with unexpectedly low or high (nonspecific) staining (Supporting Information Fig. S2), and adjustments were made when spillover spreading obscured antigen detection (Supporting Information Fig. S3). The tumor microenvironment is profoundly immunosuppressive, therefore there is strong interest in combining “checkpoint” therapies to increase the frequency and quality of activated immune cells around the tumor. The flow cytometry panel presented here could be valuable in designing logical immunotherapy combinations, allowing clinical researchers to balance anti-tumor immunity against immune-related adverse events. It might also reveal immune signatures of successful responses. Moreover, because co-inhibitory, co-stimulatory, and differentiation antigens provide complementary information regarding immune function, a comprehensive analysis of these markers should provide rich context for understanding the lymphocyte landscape of tumor samples. Finally, this 28-color/30-parameter panel maximizes the information that can be collected from what is frequently limited tumor material. This panel contains markers that overlap with OMIP-036 (CD3, CD4, CD8, CCR7, CD127, CD279/PD-1, CD366/TIM-3, CD152/CTLA-4), OMIP-037 (CD127, CD8, CD279/PD-1, CD3, CD137/4–1BB, TIGIT, CD366/TIM-3, CD4), and murine OMIP-031. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.