Lenalidomide Redresses the Effector T Cell:Treg Cell Imbalance in Patients with
Multiple Myeloma
Clive R Carter1, Christopher Parrish2, Sylvia Feyler3, Natuley Smalle1, Gina B Scott2,
Charlotte Kallmeyer2, Philip Wood1 & Gordon Cook2
1Department
of Immunology and Transplant Immunology, Leeds Teaching Hospitals NHS
Trust, Leeds, United Kingdom; 2Transplant Immunology Group, University of Leeds,
Leeds, United Kingdom; 3Haematology, Calderdale & Huddersfield NHS FoundationTrust,
Huddersfield, England
Rationale. Competent immune responses require balanced effector and regulatory arms;
cancer may influence either in an attempt to evade immune control. Pharmacoimmunotherapy 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.
Methods. 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.
Results. 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 T reg 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 T reg 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.
Conclusions.
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.