Alloreactive and leukemia-reactive T cells are preferentially derived from naive precursors in healthy donors: implications for immunotherapy with memory T cells

Abstract

Background: HLA mismatch antigens are major targets of alloreactive T cells in HLA-incompatible stem-cell transplantation, which can trigger severe graft-versus-host disease and reduce survival in transplant recipients. Our objective was to identify T-cell subsets with reduced in vitro reactivity to allogeneic HLA antigens.

Design and methods: We sorted CD4 and CD8 T-cell subsets from peripheral blood by flow cytometry according to their expression of naive and memory markers CD45RA, CD45RO, CD62L, and CCR7. Subsets were defined by a single marker to facilitate future establishment of a clinical-grade procedure for reducing alloreactive T-cell precursors and graft-versus-host disease. T cells were stimulated in mixed lymphocyte reactions against HLA-deficient K562 cells transfected with single HLA-A/-B/-C/-DR/-DQ mismatch alleles. Alloreactivity was measured by interferon-γ spot production and cell proliferation.

Results: We observed that allogeneic HLA-reactivity was preferentially derived from subsets enriched for naïve T cells rather than memory T cells in healthy donors, irrespective of the HLA mismatch allele. This separation was most efficient if CD45RA (versus other markers) was used for sorting. The numbers of allogeneic HLA-reactive effector cells were in median 7.2-fold and 16.6-fold lower in CD45RA(neg) memory CD8 and CD4 T cells than in entire CD8 and CD4 T cells, respectively. In contrast, proliferation of memory T cells in response to allogeneic HLA was more variably reduced (CD8) or equivalent (CD4) when compared to that of naïve T cells. We also demonstrated in HLA-matched donor-patient pairs that leukemia-reactive CD8 cytotoxic T-lymphocytes were mainly derived from subsets enriched for naïve T cells compared to memory T cells.

Conclusions: Memory T-cell subsets of most healthy individuals showed decreased allogeneic HLA-reactivity, but lacked significant anti-leukemia responses in vitro. The clinical use of memory or naïve-depleted T cells might be beneficial for HLA-mismatched patients at high risk of graft-versus-host disease and low risk of leukemia relapse. Preferred allografts are those which contain leukemia-reactive memory T cells. Alternatively, replenishment with leukemia-reactive T cells isolated from naïve subsets is desirable.

Figures

Figure 1.

Flow cytometric staining and sorting of T-cell subsets. PBMC from healthy donors were stained with monoclonal antibodies for CD3 and CD8 together with monoclonal antibodies for T-cell differentiation markers (i.e. CD45RA, CD45RO, CD62L, CCR7). (A, B) Gating of CD3 CD8 T cells (A) and CD3 CD4 T cells (B) with subsequent analysis of CD45RA and CD45RO as well as CD62L and CCR7 co-expression. Note the reciprocal expression of CD45RA and CD45RO, and the unequal distribution of CD62L and CCR7 expression, indicating that CD62L and CCR7 identify different T-cell subsets. Representative results with PBMC of donor SIB 369 are shown. (C) Gating strategy for cell sorting. After gating of CD3 CD8 T cells or CD3 CD4 T cells, the gates for a single T-cell differentiation marker were set according to strong or absent expression of this marker (>0.5 log difference in fluorescence intensity). The sorting gates and resulting fractions for CD45RA in CD4 T cells of donor 372 are shown here. For sorting gates of CD45RO, CD62L and CCR7 see Online Supplementary Figure S1.

Figure 2.

HLA class I allele-specific allorecognition of CD8 T-cell subsets. MLR cultures were tested for allo-HLA reactivity in an IFN-γ ELISpot assay on d12 (i.e. 5 days after first allo-HLA restimulation on d7). (A) Reactivity to original K562-HLA mismatch stimulator cells, as well as to parental K562 cells and K562 transfectant cells carrying an irrelevant HLA allele. To demonstrate HLA-restriction of detected reactivity, monoclonal antibodies blocking T-cell receptor-HLA interactions (and IgG isotype controls) were used. Representative results with naïve-enriched CD8 T-cell subsets of donor 898 for allo-HLA-A*02:01 are shown. (B) Numbers of spot-forming cells from d12 cultures initiated with sorted subsets (CD45RA, CD45RO, CD62L, CCR7) or entire CD8 T cells from six healthy donors. Allo-HLA mismatch alleles used for in vitro stimulation were HLA-A*02:01 (upper panels), HLA-B*35:03 and HLA-C*03:03 (lower panels). (C) Box plots and P values of data presented in (B). Median (line), 25th to 75th percentile (box), minimum and maximum values (error bars) are indicated. If PBMC numbers were limited, MLR stimulations were restricted to fewer allo-HLA alleles or T-cell subsets.

Figure 3.

Parallel analysis of CD8 T-cell subsets for reactivity to HLA-matched AML blasts and to K562-HLA mismatch cells. MLLC and MLR were initiated with sorted CCR7pos and CCR7neg CD8 T-cell subsets from healthy individuals. Stimulator cells were primary AML blasts with complete HLA class I-match (4 digits) in MLLC and K562-HLA mismatch cells in MLR. (A) Representative functional data from MLLC of a sibling donor/patient pair SIB 369/MZ369-AML (left panels) and an unrelated donor/patient pair Don 069/MZ653-AML (right panels) are shown. Cultures were analyzed on d19 (i.e. 5 days after second AML restimulation on d14) in IFN-γ ELISpot (upper panel) and 51Cr-cytotoxicity assays (lower panel). Targets of 51Cr-assays were AML blasts (■), patient-derived LCL (▴), donor-derived LCL (▵), and K562 cells (○). (B) Results from MLR of donors SIB 369 and Don 069, in which HLA-A*02:01 was used as the mismatch allele (for HLA types see Online Supplementary Table S1) are shown. Cultures were screened on d19 for allo-HLA-A*02:01-reactive CD8 T cells in IFN-γ ELISpot assays. LCL from HLA-A*02:01-positive and -negative third-party donors were included as control targets.

Figure 4.

HLA class II allele-specific allorecognition of CD4 T-cell subsets. MLR cultures were tested for allo-HLA reactivity in IFN-γ ELISpot assays on d12 (i.e. 5 days after first allo-HLA restimulation on d7). (A) Reactivity to original K562-HLA mismatch stimulator cells, as well as to parental K562 cells and K562 transfectant cells carrying an irrelevant HLA allele. To demonstrate HLA-restriction of detected reactivity, monoclonal antibodies blocking T-cell receptor-HLA interactions (and IgG isotype controls) were used. Representative results with naïve-enriched CD4 T-cell subsets of donor 372 for allo-HLA-DQB1*06:02 are shown (B) Numbers of IFN-γ spot-forming cells from d12 cultures of MLR started with sorted subsets (CD45RA, CD45RO, CD62L, CCR7) or total CD4 T cells from six healthy donors. HLA class II mismatch alleles used for allostimulation were HLA-DRB1*07:01 (upper panel) and HLA-DQB1*06:02 (lower panel). (C) Box plots and P values of data presented in (B); for explanation see legend to Figure 2C. If PBMC numbers were limited, MLR stimulations were restricted to fewer allo-HLA alleles.