CD4 Donor Lymphocyte Infusion Can Cause Conversion of Chimerism Without GVHD by Inducing Immune Responses Targeting Minor Histocompatibility Antigens in HLA Class II

Abstract

Under non-inflammatory conditions HLA class II is predominantly expressed on hematopoietic cells. Therefore, donor CD4 T-cells after allogeneic stem cell transplantation (alloSCT) may mediate graft-vs.-leukemia reactivity without graft-vs.-host disease (GVHD). We analyzed immune responses in four patients converting from mixed to full donor chimerism without developing GVHD upon purified CD4 donor lymphocyte infusion (DLI) from their HLA-identical sibling donor after T-cell depleted alloSCT. In vivo activated T-cells were clonally isolated after CD4 DLI. Of the alloreactive T-cell clones, 96% were CD4 positive, illustrating the dominant role of CD4 T-cells in the immune responses. We identified 9 minor histocompatibility antigens (MiHA) as targets for alloreactivity, of which 8 were novel HLA class II restricted MiHA. In all patients, MiHA specific CD4 T-cells were found that were capable to lyse hematopoietic cells and to recognize normal and malignant cells. No GVHD was induced in these patients. Skin fibroblasts forced to express HLA class II, were recognized by only two MiHA specific CD4 T-cell clones. Of the 7 clones that failed to recognize fibroblasts, two targeted MiHA were encoded by genes not expressed in fibroblasts, presentation of one MiHA was dependent on HLA-DO, which is absent in fibroblasts, and T-cells recognizing the remaining 4 MiHA had an avidity that was apparently too low to recognize fibroblasts, despite clear recognition of hematopoietic cells. In conclusion, purified CD4 DLI from HLA-identical sibling donors can induce conversion from mixed to full donor chimerism with graft-vs.-malignancy reactivity, but without GVHD, by targeting HLA class II restricted MiHA.

Keywords: CD4 donor lymphocyte infusion; HLA class II; allogeneic stem cell transplantation; graft-vs.-tumor reactivity; minor histocompatibility antigen.

Figure 1

Sorted cell populations. Marked cell populations, being in vivo activated HLA-DR positive CD4 or CD8 T-cells from four patients were single cell sorted using flowcytometry using HLA-DR PE, CD4 FITC, and CD8 AF700 monoclonal antibodies.

Figure 2

IFNγ production of alloreactive CD4 T-cell clones. Recognition of patient and donor derived EBV-LCL by CD4 T-cells measured by INFγ production after overnight incubation. Each dot represents the INFγ release by one alloreactive CD4 T-cell clone after overnight incubation in a responder to stimulator ratio of 1:10. All CD4 T-cell clones did recognize patient derived EBV-LCL and did not recognize donor derived EBV-LCL and are therefore defined as alloreactive.

Figure 3

Representative example of the identification of a MiHA by WGAS. (A) IFNγ production of clone P07-066 from patient C upon incubation with a panel of 71 SNP genotyped EBV-LCL which were transduced with HLA-DRB1*15:01. (B) Identification of associating SNPs on chromosome 11 that are present in EBV-LCL that were recognized by clone P07-066 and absent in EBV-LCL that were not recognized. (C) Identification of missense SNP rs2271001 encoding the possible MiHA recognized by clone P07-066. (D) IFNγ production by clone P07-066 after incubation with donor EBV-LCL loaded with donor or patient derived allelic peptide variants at titrated concentrations. The patient, but not donor, peptide was recognized by the T-cells, thereby validating the peptide as MiHA.

Figure 4

Cytotoxic capacities of MiHA specific CD4 T-cells. Cell lysis calculated after measurement of target cell survival using flowcytometric cell counting after 24 h of incubation with the MiHA specific CD4 T-cells in an effector to target ratio of 10:1. Target cells were patient EBV-LCL and effector cells were MiHA specific CD4 T-cells. CD4 T-cells with irrelevant specificity were used as negative controls. EBV-LCL from patient A, B, C and D are represented by black, dark gray, light gray, and blocked bars, respectively. For all MiHA, individual MiHA specific CD4 T-cell clones were identified that exerted cytotoxic activity against EBV-LCL, resulting in 19–51% specific lysis, although not all differences of the mean lysis by MiHA specific CD4 T-cell clones with background lysis were statistically significant (Mann-Whitney U-test) due to low number of experiments.

Figure 5

Recognition of PHA blasts and malignant cells by MiHA specific T-cells. Recognition of PHA blasts and malignant cells by MiHA specific CD4 T-cells was measured by IFNγ production, represented as percentage of production after incubation with patient EBV-LCL. Surface expression of HLA class II was confirmed by flow cytometry (data not shown). (A) Recognition of patient derived PHA blasts. CD4 T-cells specific for LB-RPS4Y, LB-ABCA5-1R, and LB-LY75-2R as well as CD8 T-cells specific for LB-NADK-1K produced high amounts of IFNγ upon incubation with patient derived PHA blasts. SLC19A1 specific CD4 T-cells produced less IFNγ after incubation with PHA blasts than with EBV-LCL and the other MiHA specific CD4 T-cells did not recognize PHA blasts. T-cell clones directed against the HLA restriction molecule were used as positive controls (B) Recognition of MiHA and HLA restriction molecule expressing malignant cells by MiHA specific T-cells. LB-LILRB1-1I specific T-cells were tested against myeloma cell-lines UM-6, UM-3, RPMI8226, and U266. LB-RPS4Y specific T-cells against myeloma cell-lines RPMI8226 and U266 and LB-ACBA5-1R against myeloma cell-lines UM-6 and RPMI8226. T-cells derived from patient C were tested against third party CML cells and T-cells derived from patient D against patient derived AML cells. MiHA specific CD4 T-cells recognizing malignant cells could be detected in all four patients.

Figure 6

Recognition of fibroblasts by MiHA specific T-cells. T-cell recognition of EBV-LCL, fibroblasts and fibroblasts forced to express HLA class II molecules using interferon gamma (IFN) pretreatment. (A) CD8 T-cells specific for LB-NADK-1K recognized fibroblasts already without IFNγ pretreatment. (B) CD4 T-cells specific for LB-RPS4Y and LB-ZDHHC13-1K recognized fibroblasts only after IFNγ pretreatment. (C) CD4 T-cells specific for LB-LILRB1-1I and LB-LY75-2R failed to recognize fibroblasts due to lack of expression of the MiHA encoding genes. (D) CD4 T-cells specific for the remaining 5 MiHA failed to recognize fibroblasts despite detectable gene expression and pretreatment with IFNγ. After transduction with HLA-DOα/β, fibroblasts were recognized by LB-LGALS8-1C specific CD4 T-cells, indicating that presentation of this MiHA was HLA-DO dependent.*, not tested.