Expanded and activated marrow-infiltrating lymphocytes exhibit potent antimyeloma activity against autologous multiple myeloma cells

Abstract

Adoptive immunotherapy represents a promising treatment for multiple myeloma (MM), relying on the availability of sustainable tumor-specific cytotoxic T cells. This study generated potent ex vivo expanded and activated marrow-infiltrating lymphocytes (eMILs) from MM patients and evaluated their immunologic characteristics and cytotoxic potential. MILs were expanded using anti-CD3/CD28 beads in the presence of IL-2, IL-7, and IL-15. The expansion rate, proportions of effector cells (including CD4⁺T cells, CD8⁺T cells, natural killer cells, and memory T cells), and the functional capacity of eMILs were assessed over 2 weeks of culture. Co-culturing MILs with anti-CD3/CD28 beads and cytokines resulted in substantial expansion and activation of MILs during the 14-day culture period. The eMILs displayed an increased proportion of CD8⁺T cells and a high prevalence of central memory T cells (Tcm; > 80 %), with minimal presence of myeloid-derived suppressor cells or regulatory T cells. Compared to expanded peripheral blood lymphocytes, eMILs demonstrated potent cytotoxicity against target MM cells, particularly CD138⁺ primary MM cells from autologous patients. These findings suggest that MILs derived from the bone marrow (BM) of MM patients can be expanded and activated to exhibit enhanced antigen specificity for CD138⁺ MM cells. Furthermore, eMILs may induce sustained cytotoxic effects due to their high proportion of Tcms. In conclusion, as a unique subset of T cells shaped by the BM microenvironment, MILs show promise as a novel immunotherapeutic approach for MM.

Keywords: Bone marrow; Immunotherapy; Marrow-infiltrating lymphocytes; Multiple myeloma; Treatment.

Fig. 1

Characteristics of expanded and activated marrow-infiltrating lymphocytes (eMILs). (A) Schematic representation of eMIL generation from multiple myeloma (MM) patients. (B) Representative FACS plots showing the percentage of eMILs (CD3⁺CD56⁻). (C) Fold expansion of eMILs (CD3⁺CD56⁻), NK cells (CD3⁻CD56⁺), and CIK cells (CD3⁺CD56⁺) during culture. (D) Representative FACS plots and (E) bar graphs depicting the proportions of CD8⁺T cells and CD4⁺T cells. (F) Fold expansions of CD8⁺T cells and CD4⁺T cells after 3 weeks of expansion. (G) Representative FACS plots and (H) bar graphs showing the proportions of memory T cells on days 14 and 21 of culture. *P < 0.05; **P < 0.01. Data are representative of at least six experiments.

Fig. 2

Expression of checkpoint molecules on eMILs. Representative histograms from flow cytometric analysis (A) and bar graph (B) showing the expression levels of PD-1, TIGIT, and CD73 checkpoint molecules on eMILs on days 0 and 14 of culture. ***P < 0.001; ****P < 0.0001. Representative FACS plots (C) and bar graph (D) showing the expression levels of PD-1, TIGIT, and CD73 checkpoint molecules on CD8⁺T cells and CD4⁺T cells on day 14 of eMIL culture. Data are representative of at least three experiments.

Fig. 3

Decreased proportions of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) in eMILs. The proportions of CD4⁺CD25⁺Foxp3⁺ Tregs (A and B) and CD3-CD11b⁺CD33⁺ MDSCs (C and D) were measured by flow cytometry (left panel) and compared using quantitative bar graphs (right panel). On days 14 and 21 of culture, a significant reduction in the proportions of both Tregs and MDSCs was observed in eMILs (*P < 0.05). For comparison, we also generated ePBLs from the PBMCs of three MM patients used for MIL generation; on day 14 of culture, ePBLs exhibited higher percentages of Tregs (E and F) and MDSCs (G and H) compared to eMILs. Data are representative of at least three experiments. eMILs and matched ePBLs were derived from the same MM patients to ensure paired comparison.

Fig. 4

eMILs exhibit potent cytotoxic activity against autologous CD138+ primary MM cells. (A) The production of IFN-γ, (B) TNF-α, and (C) IL-10 in response to MM cells, including U266, RPMI8226, and CD138⁺ primary MM cells was quantified using an ELISA. These assays were performed both in the presence and absence of an anti-MHC class I monoclonal antibody. (D) FACS plots and (E) bar graphs illustrating the cytotoxic activity of eMILs against CFSE-labeled MM cells, including U266, RPMI8226, and CD138⁺ primary MM cells, in the presence or absence of an anti-MHC class I monoclonal antibody. (F) FACS analysis and (G) bar graph showing the percentage of CD107a⁺ eMILs following coculture with target cells, including K562, RPMI8226, and autologous CD138⁺ primary MM cells. The cytotoxic potential of eMILs was assessed at culture days 14 (H) and 21 (I) against a range of cancer cell lines (K562, U266, RPMI8226, ARH77, and IM9) and autologous CD138⁺ primary MM cells, utilizing an LDH-release cytotoxicity assay, with and without the addition of an anti-MHC class I monoclonal antibody. Data are presented as mean ± SEM (n = 3). Statistical significance was determined using a two-tailed t-test, with *P < 0.05, **P < 0.01, and ***P < 0.001. All experiments were conducted in biological triplicates, and representative data from at least five independent experiments are shown. eMILs and ePBLs used in cytotoxicity assays were generated from the same MM patients to ensure valid paired comparison.

Fig. 5

Long-term cytotoxicity of eMILs against cancer target cells with or without anti-MHC class I mAb. Tumor cell growth was measured using the IncuCyte Zoom System. The killing capacity of eMILs on day 14 of culture against U266 cells (A). The killing capacity of eMILs on day 21 of culture against U266 (B) and RPMI8226 cell lines (C). *P < 0.05; ****P < 0.0001. Data are representative of at least three independent experiments for each tumor cell line, shown as means ± SD (n = 3 technical replicates).