IL-21-treated naive CD45RA+ CD8+ T cells represent a reliable source for producing leukemia-reactive cytotoxic T lymphocytes with high proliferative potential and early differentiation phenotype

Abstract

Clinical tumor remissions after adoptive T-cell therapy are frequently not durable due to limited survival and homing of transfused tumor-reactive T cells, what can be mainly attributed to the long-term culture necessary for in vitro expansion. Here, we introduce an approach allowing the reliable in vitro generation of leukemia-reactive cytotoxic T lymphocytes (CTLs) from naive CD8+ T cells of healthy donors, leading to high cell numbers within a relatively short culture period. The protocol includes the stimulation of purified CD45RA+ CD8+ T cells with primary acute myeloid leukemia blasts of patient origin in HLA-class I-matched allogeneic mixed lymphocyte-leukemia cultures. The procedure allowed the isolation of a large diversity of HLA-A/-B/-C-restricted leukemia-reactive CTL clones and oligoclonal lines. CTLs showed reactivity to either leukemia blasts exclusively, or to leukemia blasts as well as patient-derived B lymphoblastoid-cell lines (LCLs). In contrast, LCLs of donor origin were not lysed. This reactivity pattern suggested that CTLs recognized leukemia-associated antigens or hematopoietic minor histocompatibility antigens. Consistent with this hypothesis, most CTLs did not react with patient-derived fibroblasts. The efficiency of the protocol could be further increased by addition of interleukin-21 during primary in vitro stimulation. Most importantly, leukemia-reactive CTLs retained the expression of early T-cell differentiation markers CD27, CD28, CD62L and CD127 for several weeks during culture. The effective in vitro expansion of leukemia-reactive CD8+ CTLs from naive CD45RA+ precursors of healthy donors can accelerate the molecular definition of candidate leukemia antigens and might be of potential use for the development of adoptive CTL therapy in leukemia.

Fig. 1

IL-21 promotes the generation of AML-reactive CD8⁺ T cells from naive CD45RA⁺ precursors. a Numbers of AML-reactive cultures per 96 replicate wells obtained with purified CD45RA⁺ CD8⁺ T cells or unseparated CD8⁺ T cells in three AML patient/donor pairs with full HLA-class I match. Allogeneic mini-MLLCs were initiated with (10 or 30 ng/mL) or without IL-21, respectively, and were analyzed 5 days after the second (d14) or third (d21) antigen-specific restimulation for reactivity to primary AML blasts in split-well IFN-γ ELISpot assay. Wells considered as “positive” for AML recognition had ≥5-fold higher spot numbers compared to background level (i.e. spontaneous IFN-γ production). b Numbers of AML-reactive mini-cultures per 96 replicate wells generated from naive CD45RA⁺ CD8⁺ T cells from seven HLA-class I-matched AML patient/donor pairs

Fig. 2

HLA-class I restriction, cross-reactivity, in vitro expansion, and TCR Vβ chains of AML-reactive CD8⁺ T-cell populations. Shown are three representative CTL clones from three different AML patient/donor pairs. a CTL 2D8 (MZ201-AML/Don.168) recognizing exclusively patient’s AML blasts, b CTL 5H11 (MZ580-AML/Don.931) reacting to patient’s AML blasts as well as patient-derived lymphocytes, c CTL 7H1 (MZ529-AML/Don.730) recognizing patient’s hematopoietic as well as non-hematopoietic cells. Left panel cross-reactivity patterns to patient’s hematopoietic cells and stromal fibroblasts as well as donor’s hematopoietic cells and the natural killer cell-target K562 determined by IFN-γ ELISpot assay. Note that anti-HLA-class I mAb W6/32 inhibited recognition of AML blasts. Cross-reactivity to fibroblasts, PHA-activated T-cell blasts, and donor PBMCs was defined as significant if values were >10% of AML reactivity. Middle panel mini-MLLCs were initiated with 10⁴ naive CD45RA⁺ CD8⁺ T cells and were stimulated once a week with irradiated AML blasts. Numbers of expanding cells were weekly determined as soon as cell counting was feasible (mostly d28–d35). Right panel flow cytometric characterization of the TCR Vβ chain used by the CTLs that grew out of mini-MLLCs. CTL was defined as monoclonal if >90% of cells expressed a single TCR Vβ chain. *CTL 5H11 cross-reacts with AML blasts from patient MZ946. Because bone marrow aspirate from patient MZ580 (whose AML cells were used for stimulation) was not available, stromal fibroblasts isolated from patient MZ946 were used

Fig. 3

IL-21 does not impact on cytolytic activity of AML-reactive CTLs. CD8⁺ T-cell populations were restimulated with AML blasts over 2 weeks in the presence (gray 10 ng/mL, black 30 ng/mL) or absence (white) of IL-21, and were subsequently tested in 5 h ⁵¹Chromium-release assays for cytolytic activity against patient’s AML cells, patient-derived LCL cells, donor-derived LCL cells, and K562 cells. a CTL 2D8 (MZ201-AML/Donor168) originally generated with IL-21 and recognizing AML blasts. b CTL 7H1 (MZ529-AML/Donor730) originally generated with IL-21 and recognizing AML blasts as well as patient-derived LCL cells. c CTL 1E3 (MZ653-AML/Donor069) originally generated without IL-21 and recognizing AML blasts. d CTL 2E8 (MZ529-AML/Donor730) originally generated without IL-21 and recognizing AML blasts as well as patient-derived LCL cells. Cytokines were not added to the assay medium

Fig. 4

CD45RA⁺-derived leukemia-reactive CTLs retain markers of an early differentiation phenotype, which is further promoted by IL-21. a Flow cytometric characterization of CTL clone 4B2 (MZ653-AML/Donor069) at d35 of culture. Shown are the stainings for several T-cell differentiation markers (black lines) and the respective isotype control (gray shadings), as well as the co-stainings for CD8 and TCR Vβ 11. b Box plots showing the median expression level for CD28, CD127, CD45RO, CD45RA, CD62L, and CXCR4 on 5–11 CTLs per group (without/with IL-21) in three AML patient/donor pairs analyzed at d35 of culture. Expression level is shown as relative fluorescence intensity calculated from the median fluorescence intensity (MFI) value of the relevant staining divided by the MFI value of the respective IgG isotype control staining. All control stainings had absolute MFI values in the range 0.2–0.8 depending on the used fluorochrome, analyzed with EXPO32™ software. P values were calculated with the Mann–Whitney U test