Th₁₇ Cells Are Increased in the Bone Marrow but Not the Peripheral Blood in Multiple Myeloma and Demonstrate Defective Functionality and Aberrant Phenotypes

Introduction

Immune dysfunction in multiple myeloma (MM) is now a well accepted, if not yet completely understood, phenomenon contributing to both an increased incidence of infection and suboptimal responses to immunotherapies. The recently described Th17 subset of T helper cells is expanded in a range of cancers, and may contribute either to the evolution or control of tumours, but its role in myeloma pathogenesis remains unclear. We therefore investigated the presence, phenotype and function of this novel immune subset in a cohort of MM patients and healthy donors.

Methods

Peripheral blood (PB) samples were taken from healthy donors (HC, n=27) and MM patients (n=31); mononuclear cells (PBMC) were isolated by density centrifugation. Bone marrow aspirate (BM) samples were taken from MM patients (n=7) and HC (n=3); mononuclear cells were isolated by red cell lysis (BioLegend). For phenotyping, cells were treated with phorbol 12-myristate 13-acetate, ionomycin and brefeldin A and assessed by flow cytometry. Where stated, cells were stimulated with candidal wall mannoprotein MP65 (Peptivator, Miltenyi Biotec). For coculture experiments, CD4⁺T cells were isolated from PBMC using magnetic bead separation (MACS, Miltenyi Biotec) and cultured with human myeloma cell lines (HMCLs: U266B, JIM3, KMS11) with or without human bone marrow stromal cell lines (BMSC: HS-5, HS-27). Statistical analysis was performed using GraphPad Prism v6.

Results

As noted in previous studies, patients with myeloma have a marked CD4⁺ T-cell lymphopenia. Within the CD4⁺ population, MM patients also had a reduced frequency of IL-17⁺ cells (Th17) compared to HC: 0.41% (95% CI 0.27-0.56) vs. 0.81% (95% CI 0.47-1.15), p=0.0241 (student’s t-test). Using the mean fluorescence intensity for IL-17 as a surrogate indicator of cytokine production, IL-17 production in Th17 from MM patients was significantly reduced compared to HC (p=0.0195). Advancing disease stage correlated significantly with a reduction in PB Th17 frequency (p<0.0001, one-way ANOVA), with those with relapsed disease having the lowest levels (0.10%). In contrast, Th17 cells were expanded in the BM of MM patients compared to HC: 0.9% (95% CI 0.22-1.58) vs. 0.2% (95% CI -0.1198-0.5192), p=0.15). Th17 frequency was not significantly related to serum paraprotein concentration, prior lines of therapy, previous exposure to immunomodulatory novel agents (thalidomide, lenalidomide, bortezomib) or high-dose therapy, frequency of residual normal plasma cells in BM, serum creatinine, or high risk cytogenetic or molecular markers (del13 by FISH or IGHrearrangement). Th17 frequency was, however, lower in patients with IgG isotype disease compared to IgA (p=0.036) or light chain disease (p=0.0264).

PB Th17 cells from myeloma patients showed a marked reduction in expression of CD161 compared with HC: 25.5% vs. 54.2%; this was again a stage-related phenomena (p=0.0019, one-way ANOVA). CCR6 expression on Th17s was also reduced in MM compared to controls in a stage-related manner (p=0.0036). Furthermore, PB Th17 cells in MM had impaired capacity to produce IL-17 in response to stimulation with a fungal antigen: 7% responded cf. 21% in HC (p=NS).

To determine what, if any, effect MM tumour cells exerted on Th17 homeostasis, we employed a coculture model. CD4⁺ T cells from HC were cultured with mitomycin C-treated HMCLs for 7 days. Presence of HMCLs resulted in an expanded Th17 population compared to control cultures of CD4⁺ alone: 2.4% (95% CI: 0.13-1.30) vs..0.7% (95% CI: -0.20-5.07), p=0.09. The expansion of Th17 cells was augmented when BMSC were included (p=0.0476). We observed higher CD161 expression on coculture-derived Th17s when stromal cells were present.

Conclusions

Multiple abnormalities are apparent in Th17 immunity in MM: their frequency is decreased in PB and increased in BM, in a stage-related manner suggestive of either a homing defect or a tumour microenvironmental expansion. Our in vitro co-culture data suggest tumour-derived Th17 expansion may be responsible. The Th17 phenotype in MM is aberrant, with the relative absence of key lineage markers and attenuated functional responses to fungal antigens. Since IL-17 has been reported to be a growth factor for MM cells, these abnormalities may contribute both to the propagation of the malignant clone and the impaired anti-infective immunity seen in this disease.