Competent immune responses require balanced effector and regulatory arms; cancer may influence either in an attempt to evade immune control. Pharmaco-immunotherapy seeks to redress this imbalance. The mode of action of lenalidomide (Len) in multiple myeloma (MM) is poorly characterized, but is often affirmed to comprise direct antitumor and immunostimulatory effects. Using a combination of in vivo and in vitro methods we analyzed the effect of Len and dexamethasone (Dex) on immune reconstitution in MM patients.
21 patients with relapsed MM were treated with LenDex, commencing at Len 25mg/day PO D1–21 and Dex either 480mg (n=5) or 160mg (n=16) PO per 28 day cycle. Dex doses were tapered after cycle 6; Len doses were adjusted for renal function and hematological toxicity. Peripheral blood (PB) samples were analyzed at baseline and after cycles 1, 4, 7 and 10. PB from age-matched healthy volunteers (n=14) was also analyzed. For in vitro work, the human MM cell line (HMCL) U266B was cultured with naive CD4+CD25− T cells at a 1:2 ratio in complete medium (no additional cytokines). Where indicated, U266B was incubated with Len for 24 hours, then washed prior to coculture. Cells were harvested after 7 days for flow cytometric analysis.
Coculture of HMCL with sorted CD4+CD25− T cells in vitro induced Treg (CD4+CD25BrightFoxP3+) differentiation (31.6% vs. 0.7% without HMCL, n=10, p<0.001), with only minimal effector T cell (Teff) generation (CD4+CD25+FoxP3−). Incubation of HMCL with Len prior to coculture selectively suppressed Treg cell generation (Len: 3.2±1.1%, DMSO (vehicle): 36±4.4%, p<0.001), giving rise to a higher Teff:Treg ratio. Apoptosis in HMCL after coculture was similar in Len- and DMSO-treated cells (Len: 38.2±0.9%, DMSO: 39.35±0.8%, p=NS). Patients with MM showed profound CD4+ lymphopenia (MM: 369±82 cells/μl, controls: 700±182 cells/μl; p=0.027), which was not corrected by LenDex, despite steroid tapering. The number of recent thymic emigrants (RTE: CD4+CD45RA+CD31+) in MM patients was significantly lower than in control subjects (MM: 37.4±9.9/μl, control: 159±25.1/μl, p=0.0002). A significant reduction in the proportion of RTE was seen after cycle 1 of LenDex (11+2.2%, p=0.028) with evidence of recovery toward normal both in terms of proportion (17.4+3.4%, p=0.48) and absolute numbers (65.13+3.5, p=0.042) by cycle 7. We examined the activation status of circulating lymphocytes and noted a significant increase in the proportion of CD3+ cells expressing HLA-DR compared with controls (50.6+4% vs. 17.7+2.8%, p<0.0001); this was unaffected by LenDex. In contrast, activated CD4+ Teff cells (CD4+CD25+FoxP3−) were significantly reduced in the PB of MM patients pre-treatment compared to controls (26.1+5.1% vs. 45.6+2.3%, p<0.001). LenDex increased the proportion of activated CD4+ T cells (1-way ANOVA p=0.021); this was predominantly seen between cycles 4 and 7. As has been reported previously, Treg cells as a proportion of CD4+ cells were expanded in MM patients (MM: 10.92±2.7%, controls: 3.24±0.3%, p=0.005), although absolute counts were not significantly lower (MM: 45.5±15/μl, controls: 67.3±8/μl, p=0.181). Treg cells rapidly fell early in LenDex therapy, but returned to baseline in association with steroid tapering. There was a non-significant correlation between the baseline level of Treg cells and time to progression (TTP; r2=0.707, p=0.08) and a significant correlation between the level of Treg cells after cycle 4 and TTP (r2=0.669, p=0.009). The Teff:Treg ratio in patients at baseline was lower than in controls (9.2+3 vs 14.8+1, p=0.05). Whilst an early reduction in the ratio was seen with higher steroid dosing, the ratio rose above baseline by cycle 4 (9.2+3 vs. 11.4+3.6; p=NS). Neither baseline (p=0.29) nor cycle 4 (p=0.22) ratios correlated with TTP.
Patients with MM exhibit marked immune dysfunction, particularly characterized by imbalance between the effector and regulatory arms of the immune response. Using an in vitro culture system we show lenalidomide abrogates the ability of MM cells to drive Teff:Treg imbalance. Furthermore, in the clinic, LenDex therapy for patients with relapsed MM leads to correction of the Teff:Treg ratio. Further appraisal is now required to allow correlation of this immunotherapeutic effect with clinical outcomes.
Cook:Celgene: Consultancy; Janssen: Consultancy.
Asterisk with author names denotes non-ASH members.