A Novel Standardized Quantitative Suppression Assay Reveals a Diversity of Human Immune-Regulatory Cell Potency
Authors: Miner Samantha , Hensel Nancy F. , Salem Bahey , Dunavin Neil , Tanimoto Kazushi , Battiwalla Minoo , Lu Kit , Keyvanfar Keyvan , Sabatino Marianna , Stroncek David , Hanley Patrick J , Bollard Catherine M. , Rezvani Katayoun , Kortylewski Marcin , Muranski Pawel , Barrett John , Ito Sawa 
Published: November 2019

Abstract
Immunologic tolerance is a critical homeostatic function to protect self from auto-immunity. Various immune-regulatory cells, including regulatory T cell (Treg), myeloid derived suppressor cell (MDSC), tolerogenic dendritic cell, and mesenchymal stromal cell (MSC) are responsible for orchestrating this tolerance. Immune-regulatory cells play a central role in the pathophysiology of cancer immunity, autoimmune disease, and graft versus host disease and can be used in adoptive cell therapy. There is therefore a need for a standardized method to evaluate the suppressive function of these heterogeneous cell populations. We developed an in vitro standardized quantitative suppression assay utilizing the suppressor cell line KARPAS-299 as a standard by which to compare suppressive potency of human immune-regulatory cell populations.
Peripheral CD4 T-cells, used as targets in all assays, were isolated from healthy donors (n=30) using an automated cell separator or by flow sorting. After labeling with Cell Tracer Violet (CTV), CD4 T-cells were co-cultured with immune-regulatory cells such as autologous Treg, autologous or third party MDSC (CD11b+CD14+), third party bone marrow derived MSC, and primary leukemia cells, or with the reference KARPAS-299 cells. After stimulating with CD3/CD28 beads, CD154 activation of CD4 T-cells was measured at 16 hours, and CD4 T-cell proliferation was measured by CTV dilution within the viable cell population at 72 hours. Suppressive capacities of immune-regulatory cell types were represented as KARPAS-299 suppressor units (KSU), calculated using the equation b/a, where b is the percentage of suppression in the presence of a given immune-regulatory cell, and a is the percentage of suppression in the presence of KARPAS-299.
KARPAS-299 cells reproducibly suppressed healthy donor CD4 T-cells at suppressor: responder ratio of 4:1 for both CD154 activation (percentage of suppression 46.2±18.8%) and proliferation (51.0±20.7%). Immune-regulatory cells showed diverse suppressive capacities: autologous MDSC (63.2±24.5%), allogeneic MDSC (55.8±21.8%), third party MSC (51.26±23.67%), and autologous Treg, (8.7±10.4%). After standardization with KSU, MDSC and MSC showed significantly higher suppressive capacity compared to Treg (MDSC 1.18±0.6 KSU, MSC 1.3±1.11 KSU, Treg 0.15±0.3 KSU: p<0.0001). This standardized assay was also applicable to other types of proliferating targets, such as flow-sorted conventional T cells (Tcon: CD4+CD127highCD25low: 2.73±1.78 KSU with autologous MDSC, 1.6±0.75 KSU with third party MSC) and CD8 T-cells (1.49±0.96 KSU with autologous MDSC, 1.08±0.77 with third party MSC). We validated the method as a potency assay of MSC products, and showed inter-individual differences in MSC. Finally, we used the assay to demonstrate a modest suppressive capacity of acute myeloid leukemia blasts (0.35±0.28 KSU).
This method provides a platform for standardizing suppressor function to facilitate comparison between laboratories and for use in cell product release assay.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.