Flow Cytometric Phenotyping and Analysis of CD8 Regulatory and Suppressor Cells In Multiple Myeloma
Authors: Raja Karthick Raja Muthu , Kovarova Lucie , Buresova Ivana , Hajek Roman , Michalek Jaroslav 
Published: October 2019

Abstract
Abstract 2947
In solid tumors and hematological malignancies, elevated proportions of CD4 regulatory (CD4 Tregs) cells were reported and the function was also documented well. In the context of CD8 regulatory cells (CD8 Tregs) existence has been revealed in various solid tumors including colorectal, prostate and ovarian cancers. In hematological malignancies or multiple myeloma (MM) the presence of CD8 Tregs are yet to be revealed. To unveil and understand the existence of CD8 Tregs, we characterized and quantified CD8 Tregs along with CD8 suppressor cells in MM. A total of 91 monoclonal gammopathy patients were screened for CD8 Tregs and suppressor cells in peripheral blood (PB) and bone marrow (BM). For comparison, 21 healthy volunteers (HVs) PB were also analyzed. Clinical diseases of the patients were represented as monoclonal gammopathy of undetermined significance (MGUS)-14% (13/91), untreated MM- 60% (55/91) and relapsed MM (Rel MM)-26% (23/91). A simple three-color flow cytometric analysis was used to phenotype the CD8 Tregs and suppressor cells. CD8 Tregs were determined as CD8+FoxP3+ (Frisullo et al, 2010, Hum Immunol) and CD8 suppressor cells were identified by negative expression of co-stimulatory molecule CD28 (CD8+CD28-). All the results were summarized as percentage median and range. Our results showed strong significant increase in CD8 Tregs and suppressor cells in PB of patient cohort compared to HVs [CD8 Tregs- 0.39% (0.03%–2.41%) vs. 0.18% (0.04%–0.61%); P=0.002 and CD8 suppressor cells- 69.42% (10.99%–95.91%) vs. 27.58% (9.08%–70.52%); P<0.0001]. Additionally, strong significant difference was also noticed between HVs vs. MGUS, MM, and Rel MM for PB CD8 Tregs and suppressor cells; the data with statistical results has been depicted in table 1. More or less similar frequencies of PB CD8 Tregs were found between MGUS vs. MM vs. Rel MM. In term of PB CD8 suppressor cells Rel MM had insignificant increase compared to MGUS and MM. Surprisingly, MM cohort had insignificantly decreased frequency of CD8 suppressor cells compared to MGUS. As similarly to PB, BM CD8 Tregs and suppressor cells did not show any statistical significance in between patient groups. The BM CD8 Tregs frequency was observed respectively as: MGUS-0.32% (0.08%–1.04%), MM-0.44% (0.06%–1.82%) and Rel MM-0.27% (0.11%–1.16%). Respective BM CD8 suppressor cells frequency was noticed as: MGUS-63.32% (24.83%–94.43%), MM-62.15% (6.19%–93.45%) and Rel MM-67.29% (18.55%–87.61%). Subgroup of patients with < 95% of abnormal plasma cells [ANPCs- 59.21% (13.35%–91.83%)] had significantly decreased level of PB CD8 suppressor cells compared to counter group [≥95% of ANPCs- 74.23% (10.99%–95.91%)]; P=0.046. In line with PB, BM CD8 suppressor cells also showed significantly reduced frequency in <95% of ANPCs cohort [56.99% (16.48%–94.43%)] compared to ≥ 95% ANPCs cohort [69.61% (6.19%–93.45%)]; P=0.013. No relevant association was noticed between CD8 Tregs and suppressor cells with other clinical features including β2M, albumin, creatinine, lactate dehydrogenase, hemoglobulin and monoclonal protein level. In conclusion, our results disclose that as similar to CD4 Tregs, CD8 Tregs are also deregulated in MM compared to HVs; this is also in line with CD8 suppressor cells. This data suggest that in MM immune dysfunction is also enhanced by the shift in favor of CD8 immune regulatory and suppressor cells. This study was supported by the respective grants-MSM0021622434, LC06027, IGA NS10406, IGA NS10408, and GACR P304/10/1395.
Table 1: Comparison of Peripheral Blood CD8 Tregs and Suppressor Cells between Hvs versus MGUS, MM and Relapsed MM

Disclosures:
No relevant conflicts of interest to declare.