CD4+CD25+ Marrow Infiltrating Lymphocytes in Myeloma Patients Display an Activated Phenotype and Lack Suppressive Function.

We have previously shown that marrow infiltrating lymphocytes (MILs) from myeloma patients proliferate upon stimulation with anti-CD3/CD28 beads more efficiently and exhibit greater specificity to autologous tumor than their peripheral blood lymphocyte (PBL) counterparts. In attempting to dissect the mechanisms responsible for greater tumor specificity of MILs, we examined the frequency and function of the putative CD4⁺/CD25⁺ regulatory T cells (Tregs) in both compartments. Phenotypic and functional differences in the CD4⁺/CD25⁺ populations were examined in MILs and PBLs from 12 multiple myeloma patients and 3 normal donors. CD4⁺/CD25⁺ cells were stained for the suppressive marker: FoxP3; or the activation markers: CD40L, CD71 (transferrin receptor). CD4⁺/CD25⁺ MILs of myeloma patients are CD25^lo, FoxP3^lo and predominantly express activation markers. They expand more readily upon anti-CD3/CD28 stimulation, produce high amounts of the Th1 cytokines: IFNγ and IL-2, and low amounts of IL-10. Importantly, they fail to suppress the tumor specific T cell proliferation. These findings are all consistent with an activated T cell phenotype. In contrast, CD4⁺/CD25⁺ cells from PBLs of myeloma patients are CD25^hi, FoxP3⁺, primarily produce IL-10 and IL-4 and markedly suppress T cell expansion in a tumor specific assay, suggestive of a regulatory T cell phenotype. Although MILs possess a lower number of FoxP3⁺ cells as compared to PBLs, the higher MFI may indicate the presence of a small yet highly regulatory T cell population. Interestingly, MILs from normal donors possess a more regulatory phenotype then their myeloma counterparts as determined by high FoxP3 and low CD40L and CD71 expression as well as reduced expansion upon activation. Taken together, these data underscore major differences in the immuno-inhibitory pathways present in blood as compared to marrow. Notably, MILs of myeloma patients demonstrate a paucity of Tregs. These differences may explain the disparities seen in the tumor-specificity of T cells from these two compartments. The ability to expand a T cell population with fewer endogenous Tregs and heightened tumor-specificity may have significant implications for the implementation of adoptive immunotherapy.