Flow Cytometric Phenotyping and Analysis of T Regulatory Cells in Monoclonal Gammopathy Patients.
Authors: Raja Karthick Raja Muthu , Zahradova Lenka , Kovarova Lucie , Mikulkova Zuzana , Buresova Ivana , Michalek Jaroslav , Hajek Roman 
Published: October 2019

Abstract
Abstract 2819
Poster Board II-795

Introduction:
T regulatory (T reg) cells play an important role in maintenance of self-tolerance, control of immune functions and regulation of homeostasis of T cell populations. T reg cells also influence immune responses against infectious agents and tumor cells. Several recent studies have demonstrated increased counts of T reg cells in patients with solid tumors and hematological malignancies. Monoclonal gammopathies (MGs) are associated with abnormal immune functions, mainly immunoparesis. Abnormal T cell functions have also been reported. We have evaluated the frequency of T reg cells in MG patients in different stages of disease.

Patients and Methods:
A total of 69 patients (pts) with MGs were enrolled in this pilot study (16% \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(1169\) \end{document} with MGUS, 7% \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(569\) \end{document} with asymptomatic myeloma (AMM), 58% \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(4069\) \end{document} with newly diagnosed symptomatic myeloma (MM), and 19% \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(1369\) \end{document} with relapsed MM). Twelve healthy individuals were also screened for the frequency of T reg cells in the peripheral blood and used as a control group. Flow cytometric phenotyping of T reg cells was carried by staining for CD4, CD25 and CD127, followed by intracellular staining for Foxp3 was performed. T reg cells were identified by the phenotype of CD4+ CD25hi+ CD127dim Foxp3+. Both peripheral blood (PB) and bone marrow (BM) samples were analyzed for the frequency of T reg cells. Subgroups of pts were compared based on the bone marrow plasma cell (BMPC) infiltration (50 pts ' 10% vs. 19 pts >10%) and serum monoclonal immunoglobulin (M-Ig) level (25 pts ' 30g/L vs. 34 pts >30g/L).

Results:
Our study showed an overall trend towards the increase of T reg cell frequency in the peripheral blood of MG pts comparing to healthy individuals. The following T reg cell percentages were observed in the PB of different groups: healthy individuals 4.7% (range: 3.4 - 6.7%), MGUS 4.9% (range: 2.1 - 7.3%), AMM 4.5% (range: 3.0 - 7.5%), MM 5.5% (range: 2.7 - 11.7%) and relapsed MM 6.8% (range: 4.8 - 8.7%). Statistically significant differences were observed between healthy individuals vs. relapsed MM (p= 0.008), MGUS vs. relapsed MM (p= 0.043), and AMM vs. relapsed MM (p= 0.035). Similarly, in the bone marrow the frequency of T reg cells was also proportional to the increasing severity of MG. The following percentages of T reg cells were observed in the BM: MGUS 3.5% (range: 2.1 - 6.6%), AMM 3.0% (range: 2.6 - 7.5%), MM 4.7% (range: 2.3 - 7.5%) and relapsed MM 5.6% (range: 3.3 - 7.0%). Differences between the groups did not reach statistical significance. We have found a significant positive correlation between BMPC infiltration and BM T reg cell percentage (r= 0.25, p= 0.030). Increased frequency in the level of T reg cells was observed in the group with >10% of BMPC infiltration comparing to the group with ' 10% of BMPC infiltration (5.2% [3.0 - 7.4%] vs. 4.7% [2.1 - 7.6%] respectively; p= 0.41). Increased number of T reg cells was also observed in both PB and BM of pts with >30g/L of serum M-Ig comparing to pts with ' 30g/L of serum M-Ig (PB: 5.9% [2.7- 11.7%] vs. 5.0% [2.8 - 8.7%]) and (BM: 5.3% [2.3 -7.5%] vs. 4.7% [2.6-7.5%]), although no statistical significant differences have been observed between the two groups.

Conclusion:
Our results suggest that increases in the frequency of T reg cells in monoclonal gammopathies are associated with advancing disease. The positive correlation between BMPC infiltration and BM T reg cells indicates that T reg cells could play a role in the pathophysiology of monoclonal plasma cell disorders.
This work was supported by MSMTLC06027, MSM0021622434 and MSMTNPVII 2B06058.

Disclosures:
Hajek: Janssen-Cilag: Honoraria.