Human CD4+ iNKT cell adoptive immunotherapy induces anti-tumour responses against CD1d-negative EBV-driven B lymphoma

Abstract

Invariant natural killer T (iNKT) cells are a conserved population of innate T lymphocytes that are uniquely suitable as off-the-shelf cellular immunotherapies due to their lack of alloreactivity. Two major subpopulations of human iNKT cells have been delineated, a CD4^- subset that has a T_H1/cytolytic profile, and a CD4⁺ subset that appears polyfunctional and can produce both regulatory and immunostimulatory cytokines. Whether these two subsets differ in anti-tumour effects is not known. Using live cell imaging, we found that CD4^- iNKT cells limited growth of CD1d⁺ Epstein-Barr virus (EBV)-infected B-lymphoblastoid spheroids in vitro, whereas CD4⁺ iNKT cells showed little or no direct anti-tumour activity. However, the effects of the two subsets were reversed when we tested them as adoptive immunotherapies in vivo using a xenograft model of EBV-driven human B cell lymphoma. We found that EBV-infected B cells down-regulated CD1d in vivo, and administering CD4^- iNKT cells had no discernable impact on tumour mass. In contrast, xenotransplanted mice bearing lymphomas showed rapid reduction in tumour mass after administering CD4⁺ iNKT cells. Immunotherapeutic CD4⁺ iNKT cells trafficked to both spleen and tumour and were associated with subsequently enhanced responses of xenotransplanted human T cells against EBV. CD4⁺ iNKT cells also had adjuvant-like effects on monocyte-derived DCs and promoted antigen-dependent responses of human T cells in vitro. These results show that allogeneic CD4⁺ iNKT cellular immunotherapy leads to marked anti-tumour activity through indirect pathways that do not require tumour cell CD1d expression and that are associated with enhanced activity of antigen-specific T cells.

Keywords: CD1d; EBV; adjuvancy; iNKT cells; lymphoma; tumour immunotherapy.

Figure 1.. EBV-lymphoma killing by iNKT and γδ T cells in vitro.

A) Live cell imaging was performed to assess in vitro responses of γδ T cells, CD4⁻ and CD4⁺ iNKT cells to EBV-transformed lymphoblastoid cells. Left plot shows relative specific lysis mediated over time, with symbols representing means + SEM of 3 replicates. Middle panels show microscopic images at 4X magnification of lymphoblastoid spheroids at indicated time points, with live lymphoblastoid cells masked in green and dead in red. Right plot shows percent change in spheroid size (mean + SEM) from 8–48h of culture. B) NSG mice were injected with EBV-treated human CBMCs, and after 25 days were given 5–10×10⁶ allogeneic γδ T cells or CD4⁻ iNKT cells or mock-treated (control), then tumor mass was assessed 4–6 days later. C) Flow cytometric analysis of CD1d expression (shaded histograms) compared to isotype control staining (dotted line).

Figure 2.. In vivo anti-tumor effects of CD4⁺ iNKT immunotherapy.

A) Left plot shows normalized tumor mass of control mice compared to mice given 5–10×10⁶ allogeneic CD4⁺ iNKT cells at day 25, with tumors assessed at day 29–31; right plot shows tumor incidence. B) Dose-response comparison for CD4⁺ iNKT and γδ T cell immunotherapy given at d25. Symbols show means and SEM of results from 3–24 mice with asterisks indicating significance (*=P<0.05; ***=P<0.001) compared to no immunotherapy. C) Mice were given 5×10⁶ CD4⁺ iNKT cells or mock-treated at day 25, and tumors assessed 2–4 days later.

Figure 3.. CD4⁺ iNKT cells mediate adjuvant-like activation of other immune cells.

A) Human PBMCs were incubated alone, or with 2% allogeneic CD4⁺ iNKT cells, or in medium containing 200 ng/ml α-GalCer, and after 48h CD69 expression by B, T, and NK cells from the PBMC sample was determined by flow cytometry (left plot) and levels of secreted IFN-γ were quantitated by ELISA (right plot). B) CD4⁺ iNKT cells were incubated in parallel with autologous or allogeneic PBMCs (filled symbols), or the PBMCs were incubated alone (open symbols). Left axis shows CD69 expression, right axis shows secreted IFN-γ. C) Monocyte-derived DCs were incubated with CD4⁺ iNKT cells, or treated with synthetic GLA, or kept in medium alone, and after 24h were stained for the indicated markers. Top row histograms show DCs that were autologous to the iNKT cells, bottom row DCs were allogeneic. Plot on right shows fold-increase of each marker for DCs incubated with iNKT cells compared to medium alone; each symbol shows results from an independent analysis, with asterisks indicating statistical significance of fold-increase as determined by a two-tailed 1-sample t-test. D) Monocyte-derived DCs were pulsed with tetanus toxoid (TT Ag) or mock-treated (No Ag) and incubated for 24–48h with autologous T cells in the presence or absence of 0.5% allogeneic iNKT cells. IFN-γ expression by CD4⁺ T cells (excluding iNKT cells) was determined by flow cytometry.

Figure 4.. CD4⁺ iNKT immunotherapy does not mediate early immunosurveillance.

A) Tumor mass results from control mice or mice given CD4⁺ iNKT cells within the first 3 days after injection of EBV-treated CBMCs. B) Mice were given CD4⁺ iNKT cells at the indicated times after injection of EBV-treated CBMCs and tumor mass was determined at day 29.

Figure 5.. Impact of CD4⁺ iNKT immunotherapy on splenic T cells.

A) Lymphoma-bearing mice were injected with fluorescently labeled CD4⁺ iNKT cells. Spleen and tumor tissue was collected after 24h. Left images show fluorescence microscopy of spleen and tumor sections at 20X magnification, with iNKT cells shown in green and cell nuclei in blue; right plot shows quantitation of labeled iNKT cells per mm² (means and SEM from 5–6 sections). B) Flow cytometric analysis of splenocytes from lymphoma-bearing mice (29–31 days post-EBV infection) showing identification of a small population of human CD33⁺ myeloid cells that express cell surface CD1d. Samples were first gated by light scatter and DAPI to exclude dead cells, then gated specifically on human cells using antibodies against murine CD45, human CD45, and pan-HLA class I. CD33⁺ cells shown in left plot were further gated to exclude CD3⁺ or CD19⁺ events, then staining for CD1d (filled histogram) was compared to isotype control (dotted line). Plot on right shows paired results from 3 different mice. C) Mice were given CD4⁺ iNKT cells or mock-treated (control) at day 21 after injection of EBV-treated CBMCs. Plot shows frequency of non-iNKT CD4⁺ T cells out of total human cells in spleen 8 days later. D) Human T cells from spleens of iNKT-treated or control mice were depleted of iNKT cells then cultured alone (No stim) or with autologous EBV-infected splenocytes, and secreted IFN-γ was quantitated by ELISA. E) T cells were cultured alone (No stim) or with uninfected autologous CBMCs treated with vehicle or synthetic EBV or CMV peptides.