Abstract 11: Progression from newly diagnosed multiple myeloma to relapsed refractory multiple myeloma is associated with significant alterations in the CD4+ Treg population phenotype
Authors: Cooke Rachel , Quach Hang , Harrison Simon , Prince Miles , Koldej Rachel , Ritchie David 
Published: August 2018

Abstract
Introduction: Immune compromise is a recognized complication of multiple myeloma (MM), aging, and anti-MM therapies used throughout the course of the disease. Our group have previously reported that patients with relapsed/refractory MM (RRMM) have marked CD4+ T lymphopenia that is not present in newly diagnosed MM (NDMM) and does not recover with successive cycles of lenalidomide and dexamethasone (len/dex) treatment1,2. However, the proportion of Tregs within the CD4+ T-cell population (defined as CD127dimCD25hiFoxp3+) increased with successive cycles of treatment and approached normal range by cycle 9. We hypothesized that this represented homeostatic proliferation of peripherally-derived Tregs (pTreg) as opposed to thymic production of naturally-derived Tregs (nTreg), which can be differentiated by methylation status of the T-cell specific demethylation region (TSDR) found in intron 1 of the Foxp3 gene3. This bears relevance, as (a) pTregs are likely to be derived from senescent cells that are functionally different to recent thymic emigrants, (b) pTregs may exhibit plasticity, as opposed to nTregs, and (c) the population of effector memory T (TEM) cells may be compromised.
Methods: We sought to investigate at what point this change occurred throughout the course of disease in MM by analyzing 5 paired samples from patients with NDMM (ANZCTR trial ID ACTRN12613000344796) and RRMM (ANZCTR trial ID NCT00482261), and comparing with 5 age-matched normal donors. In the NDMM cohort, we examined Tregs from samples at baseline, after 4 cycles of len/dex, and after autologous stem cell transplant (ASCT), and in the RRMM cohort samples from baseline and after 9 cycles of len/dex. Tregs (defined as CD4+CD127-CD25+) were FACS sorted from each sample and DNA was extracted and bisulfite converted. The TSDR was PCR amplified using primers previously described3, and transformed into chemically competent E.coli. Individual colonies were picked and their DNA plasmids were sequenced. Cytosine matches/mismatches at CpG sites in the TSDR were identified and % methylation was calculated.
Results: The TSDR methylation status of Tregs from baseline NDMM patient samples is largely unmethylated and similar to age-matched controls, i.e., predominantly nTregs.
(1) The TSDR methylation status of Tregs from baseline RRMM patient samples is largely methylated, i.e., predominantly pTregs.
(2) Len/dex treatment in both NDMM and RRMM patient did not alter the respective phenotypes.
(3) After ASCT, 2 of the 5 NDMM patient samples analyzed had changed to a “pTreg phenotype.” Both these patients subsequently relapsed from complete remission, compared to 2 of 3 of the patients with an “nTreg phenotype” maintaining CR 8-9 years later.
Conclusions: This study adds evidence that the character of the CD4+ T cell population is radically altered in RRMM, compared with NDMM and age-matched controls. It is likely that thymic involution is a contributor to this, which is not only a normal occurrence with immunosenescence, but may be accelerated with ASCT in some patients. In this way, methylation status of the TSDR could potentially represent a biomarker for poorer prognosis.
References
1. Hsu A, Ritchie DS, Neeson,P. Are the immuno-stimulatory properties of Lenalidomide extinguished by co-administration of Dexamethasone? Oncoimmunology 2012;1:372-4.
2. Cooke RE et al. Spontaneous onset and transplant models of the Vk*MYC mouse show immunological sequelae comparable to human multiple myeloma. J Transl Med 2016;14:259.
3. Baron U et al. DNA demethylation in the human FOXP3 locus discriminates regulatory T cells from activated FOXP3(+) conventional T cells. Eur J Immunol 2007;37:2378-89.
Citation Format: Rachel Cooke, Hang Quach, Simon Harrison, Miles Prince, Rachel Koldej, David Ritchie. Progression from newly diagnosed multiple myeloma to relapsed refractory multiple myeloma is associated with significant alterations in the CD4+ Treg population phenotype [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 11.