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A. Laghmouchi et al.
tion.3,8,13 Therefore, scheduled donor lymphocyte infu-
sions are often given to patients following T-cell-depleted alloSCT.4,14-17
The effect of the level of HLA matching on clinical out- come after alloSCT has been extensively studied and has led to a standardized donor-patient matching procedure in which HLA-DP is frequently not taken into account.18- 20 The 10/10 HLA-matched stem cell grafts from unrelat- ed donors are, therefore, often mismatched for one or two HLA-DP alleles. HLA-DP mismatches are in theory acceptable, as under non-inflammatory conditions expression of HLA-class II molecules is mainly restricted to hematopoietic cells. The balance between the induc- tion of GvL and GvHD is one of the remaining chal- lenges in treatment with (T-cell-depleted) alloSCT or donor lymphocyte infusion.14 Whether or not donor T cells targeting the mismatched HLA-DP allele(s) con- tribute to GvL and/or GvHD may depend on the magni- tude, specificity and diversity of the allo-HLA-DP immune response as was illustrated for HLA-class I- restricted T-cell responses,21 and the tissue-specific pat- tern of HLA-DP expression.3,14,22-25 The magnitude of the T-cell response mounted against allo-HLA-DP molecules has been shown to be different depending on the specific HLA-DP allele(s) expressed in the donor and the patient.25,26 Differences at the amino acid sequence level have been found to influence the immunogenicity of spe- cific HLA-DP molecules, probably caused by a different landscape of peptides (peptidome) presented in these HLA-DP molecules.26-28 Based on these insights, an algo- rithm has been proposed to predict the risk on an immune reaction in the host versus graft direction (rejec- tion) and the graft versus host direction (GvL and/or GvHD), based on the immunogenicity of specific HLA- DP molecules and the differences between specific HLA- DP alleles.29 This has led to the distinction of two groups of HLA-DP mismatches, called the more tolerable, per- missive HLA-DP mismatches that are predicted to induce T-cell responses with a lower amplitude, and the non- permissive mismatches that induce stronger T-cell responses.29-32 In addition to the specificity and magni- tude of the allo-HLA-DP T-cell response, the pattern of expression of HLA-DP on patients’ tissues is decisive in the induction of GvL and/or GvHD. In some patients, profound CD4 T-cell responses targeting the mis- matched allo-HLA-DP allele(s) have been found to be associated with the induction of different types of GvHD (e.g. skin GvHD, gut GvHD) mediated by recognition of inflamed HLA-class II-expressing non-hematopoietic tis- sues.23 In other patients specific GvL reactivity without coinciding GvHD mediated by allo-HLA-DP-reactive CD4 donor T cells was demonstrated. In these patients the allo-HLA-DP response appeared to be restricted to hematopoietic cells without cross-reactivity against non- hematopoietic tissues.22,24
To initiate the allo-HLA-DP-specific immune response in vivo, donor T cells most likely first have to encounter patient-derived HLA-DP-expressing antigen-presenting cells, such as dendritic cells (DC).33,34 Since DC are of hematopoietic origin, allo-HLA-DP T cells activated by DC will probably recognize antigens expressed by hematopoietic cells presented in the mismatched HLA- DP molecule.14 When the allo-HLA-DP-specific immune response is initiated by DC residing in inflamed HLA-DP- expressing non-hematopoietic tissue, the immune
response is likely to be also directed against antigens expressed by non-hematopoietic cells presented in the mismatched HLA-DP molecule of the DC.35 The level of overlap between the antigens expressed by hematopoiet- ic versus non-hematopoietic cells, will dictate the induc- tion of a specific GvL response, a specific GvHD response, or a combination of both.3,14
In this study we analyzed the tissue/cell-lineage-specif- ic recognition patterns within the allo-HLA-DP-specific T-cell repertoire provoked by stimulation with allogeneic HLA-DP-mismatched monocyte-derived DC. We observed that the allo-HLA-restricted T-cell repertoire contains T cells with a diverse spectrum of cell-lineage- specific recognition profiles, including T cells that show restricted recognition of hematopoietic cells, including primary malignant cells, or even T cells with myeloid-lin- eage-restricted recognition, including recognition of pri- mary acute myeloid leukemia blasts.
Methods
Cell collection and preparation
The collection and preparation of cells is described in the Online Supplementary Appendix.
Induction of allo-HLA-DP-specific immune responses
CD14-depleted donor peripheral blood mononuclear cells were stimulated with irradiated (25 Gy) HLA-DP mismatched allogeneic DC (alloDC) in a 10:1, responder T-cell to stimulator- cell ratio. The cells were cultured in Iscove modified Dulbecco medium (IMDM) containing 10% heat-inactivated ABOS sup- plemented with interleukin-7 (10 ng/mL, Miltenyi Biotec), inter- leukin-15 (0.1 ng/mL, Miltenyi Biotec) and interleukin-2 (50 IU/mL, Novartis Sandoz Pharmaceuticals). To activate and remove auto-reactive T cells, cell cultures were stimulated after 14 days with autologous DC (autoDC), followed by depletion of these reactive T cells around 36 h later based on activation mark- er (CD137) expression using CD137-allophycocyanin (BD Pharmingen, San Diego, CA, USA), APC-beads (Miltenyi Biotec) and MACS LD columns (Miltenyi Biotec) according to the man- ufacturer’s instructions. To activate allo-HLA-DP-reactive T cells, the negative fraction was subsequently specifically restim- ulated with the allogeneic HLA-DP-mismatched DC. After approximately 36 h, HLA-DP-mismatched-DC-reactive CD4 T cells were quantified and clonally isolated by single cell flow cytometric cell sorting based on CD137 expression using a FACSAria (BD Biosciences, San Jose, CA, USA). The time-point for CD137 isolation was chosen based on our previous work, including an unpublished clinical trial [administration of leukemia-reactive donor T cells after alloSCT or donor lympho- cyte infusion to patients with persistent or relapsed mature B- cell neoplasms with blood and/or bone marrow involvement (EudraCT number 2012-003691-40)] and our experience with the isolation of virus-specific CD4 and CD8 T cells.36,37 Unstimulated peripheral blood mononuclear cells and autoDC- stimulated peripheral blood mononuclear cells from the same responder cells were used as controls to determine the gating strategy. The T-cell clones were expanded using allogeneic feed- er mixture consisting of IMDM containing 5% heat-inactivated ABOS and 5% heat-inactivated fetal calf serum supplemented with 5x irradiated (35 Gy) allogeneic feeder cells, 0.5x irradiated (60 Gy) allogeneic Epstein-Barr virus-transformed lymphoblas- toid cell line (EBV-LCL), 100 IU/mL interleukin-2, and 800 ng/mL phytohemagglutinin (PHA-HA16, Oxoid, Altrincham, UK).
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haematologica | 2019; 104(1)