Page 209 - 2019_01-Haematologica-web

HLA-DP-restricted cell-lineage recognition patterns
Generation and culture of stimulator cells for functional analyses
The method of generating and culturing stimulator cells is described in the Online Supplementary Appendix.
Recognition assay
The procedure for testing the recognition profiles of expanded T-cell clones is described in the Online Supplementary Appendix.
Flow cytometric analyses
Information on the flow cytometric analyses performed is pro- vided in the Online Supplementary Appendix.
Approval by ethics committee
Donors and patients had given written informed consent to the storage of biomaterials in the LUMC Biobank and the use of these materials was approved by the institutional medical ethical com- mittee (protocol number B 16.039).
Results
Depletion of reactivity against autologous dendritic cells allows efficient identification and enrichment of allo-reactive T cells upon stimulation with HLA-DP-mismatched dendritic cells
To study the composition of the allo-HLA-DP-specific T-cell repertoire, HLA-DP-mismatched alloDC were used to provoke allo-HLA-DP-reactive T-cell responses from healthy donor peripheral blood mononuclear cells. Responder/stimulator pairs with various HLA-DP mis- matches were selected (Table 1): two pairs with a targeted HLA-DP*04:01 or HLA-DP*04:02 mismatch using HLA- DP*04:02- or HLA-DP*04:01-expressing responder cells, respectively, classified as minimal, permissive mismatch- es31 (pairs 1 and 2), one pair with a targeted HLA-DP mis- match classified as permissive (pair 3), one pair with a tar- geted HLA-DP mismatch classified as non-permissive (pair 4), and one 9/10 HLA-matched pair with an HLA-DP mismatch classified as permissive and an additional HLA- DQB1 mismatch (pair 5).
Since we previously illustrated that stimulation with alloDC results in coinciding stimulation of reactivity against autoDC,38 we first investigated whether this would hamper the isolation of allo-HLA-DP reactive T cells. Donor peripheral blood mononuclear cells were stimulated with HLA-DP-mismatched alloDC, cultured for 2 weeks, and then restimulated with either autoDC or
Table 1. HLA-DP typing of the responder/stimulator pairs.
alloDC. Flow cytometric analysis of the expression of the activation marker CD137 on CD4 T cells showed that upon (re)stimulation with autoDC and alloDC similar fre- quencies of CD4 T cells were activated (Figure 1A), indi- cating that a depletion step for the autoDC reactivity could allow more efficient identification and enrichment of allo-HLA-DP reactive T cells.
Therefore, a strategy was developed using initial stimu- lation with HLA-DP-mismatched alloDC and subsequent culture for 2 weeks, followed by a stimulation step with autoDC and a depletion step using magnetic bead separa- tion (MACS) based on the expression of CD137 on autoDC reactive T cells. The depleted fraction (CD137-) was subsequently restimulated with alloDC to specifically activate allo-reactive T cells (Online Supplementary Figure S1). For four of the five pairs the frequencies of CD137- expressing CD4 T cells were consistently higher upon spe- cific restimulation with the HLA-DP-mismatched alloDC compared to the non-restimulated and autoDC-stimulat- ed controls, although there was biological variability in the magnitude of the immune responses (Figure 1B). These data indicate that this strategy using a depletion step for autoDC reactivity allows more specific identifica- tion of allo-reactive T cells.
Next, CD4 T cells expressing CD137 upon restimulation with HLA-DP-mismatched alloDC were sorted as single cells per well, expanded and screened (n=1679) for allo- reactivity by interferon-γ enzyme-linked immunosorbent assay upon stimulation with alloDC, using autoDC as controls (response 4, as a representative example, is shown in Online Supplementary Figure S2). The clonality of expanded T-cell clones was confirmed by T-cell receptor Vβ repertoire analysis (data not shown). For responses 3-5 the majority of expanded T-cell clones were found to be allo-reactive (79-94%, n=1235). For responses 1 and 2, in which minimal, permissive HLA-DP mismatches were tar- geted resulting in less profound immune responses, only a limited proportion of the expanded clones were found to be allo-reactive (21-29%, n=68). A small proportion of the clones showed no reactivity or showed coinciding reactiv- ity against the autoDC (Figure 1C).
The allo-HLA-DP-specific T-cell repertoire provoked by in vitro stimulation with HLA-DP-mismatched dendritic cells contains T cells that selectively recognize dendritic cells, but not Epstein-Barr-transformed lymphoblastoid cell lines
To investigate the HLA-DP restriction of the allo-reac- tive CD4 T-cell clones, clones (n=1303) were tested in a
In vitro
response
1.
2.
3.
4.
5.
Responder
DPB1*03:01 & DPB1*04:02
DPB1*04:01:01
DPB1*04:01:01
DPB1*04:01:01
DPB1*03:01 &
Stimulator
DPB1*04:01:01
DPB1*04:01 &
DPB1*04:02
DPB1*02:01:01 & DPB1*04:01:01
DPB1*03:01:01 &
DPB1*04:01:01 DPB1*04:01
HLA-DP disparity
Permissive
Permissive
Permissive
Non-permissive
Permissive
Level of HLA-matching
10/10
10/10
10/10
10/10
HLA-class II mismatch
DPB1*04:01:01
DPB1*04:02
DPB1*02:01:02
DPB1*03:01:01
DPB1*04:01 &
DPB1*09:01
DQB1*06:03
9/10
haematologica | 2019; 104(1)
199