CD4+ T-cell alloreactivity after haploidentical hematopoietic stem cell transplantation

Allogeneic hematopoietic stem cell transplantation (HSCT) is an important, potentially curative therapeutic modality in many hematopoietic disorders. A fully human leukocyte antigens (HLA)-matched sibling or unrelated donor is the preferred source for stem cells. However, finding such a donor may pose a problem, especially for non-Caucasian patients. 1 Stem cells from a haploidentical donor or umbilical cord blood can serve as an alternative for those lacking a fully matched donor. In the past, haploidentical HSCT was associated with two problems; a higher rate of graft failure and, a higher incidence of graft-versus-host disease (GvHD), both necessitating intensive immunosuppressive measures. The development of conditioning regimens with intensified immunosuppressive therapy, including high-dose posttransplant cyclophosphamide (PT-Cy) therapy, improved results after haploindentical HSCT by ensuring engraftment, less GvHD, and a more favorable restoration of immune recovery. We earlier showed the strong presence of alloreactive CD4 T cells in most patients early after double umbilical cord blood transplantation (dUCBT) and their potential role in enhancing the graftversus-leukemia effect. In this study, we addressed whether such HLA-class II specific CD4 T cells are present early after haploidentical HSCT. The trial was approved by the ethics committees of the participating institutions and was conducted in accordance with the Declaration of Helsinki. All participants had given written informed consent. A total of 22 patients with high-risk hematologic diseases, eligible for haploidentical HSCT were retrospectively included in this study. The primary endpoint was the proportion of evaluable patients with activated class-II specific CD4 T cells. Patients received myeloablative conditioning, consisting of thiotepa (10 mg/kg), busulfan 6.4 or 9.6 mg/kg and fludarabine 150 mg/m, followed by transplantation of an unmanipulated bone marrow graft from a haploidentical family member, as previously described. GvHD prophylaxis consisted of PT-Cy, tacrolimus, and mycophenolate mofetil. Peripheral blood from patients was collected 1 month after transplantation for isolation of peripheral blood mononuclear cells (PBMC). Engraftment was achieved in all patients at the time of sampling. A library of stimulator cells expressing a single HLA-class II allele (HLA-DRA1/B1, HLA-DQA1/B1 or HLA-DPA1/B1) was generated by retroviral transduction of HLA-class II negative HeLa cells. Post-transplantation PBMC were assayed for the presence of alloreactive T cells. The very few alloreactive T cells were propagated in a co-culture with irradiated HeLa cells expressing a single mismatched HLA-class II allele derived from the recipient with addition of a cytokine mixture. Subsequently, alloreactivity was measured by flow cytometry and expressed as fold increase (%CD137/CD4) of reactivity toward HeLa cells transduced with mismatched HLA-class II alleles relative to reactivity towards the not-transduced ‘empty’ HeLa cells, as described previously. In addition, alloreactivity towards matched ‘control’ alleles were included (Online Supplementary Figure S1). The characteristics of the 22 patients and their grafts are presented in Table 1A and 1B, respectively. The majority of the patients were diagnosed with acute myeloid or lymphoblastic leukemia. Figure 1 shows immune recovery of natural killer (NK) cells, B cells, and CD4 and CD8 T cells after haploidentical HSCT. Normal values were not reached during the follow-up of 3 months. High resolution typing was performed for all 22 patients. In vitro tests could be performed for 11 out of the 22 patients for whom HLA-class II transduced HeLa cells were available (Figure 2). T-cell numbers increased a median of 1.6-fold (range, 0.3-36.2) in the HLA-class II allele-specific propagation cultures of the post-haploidentical HSCT PBMC samples. In four out of 11 (36%) patients tested, CD4 T-cell alloreactivity towards HLAclass II mismatched alleles of the recipient was detected, in three cases towards DR alleles and in one case towards a DQ allele (Figure 2). The median CD4 T-cell alloreactivity towards DR, DQ and DP alleles was increased 19.7-fold (range, 1.3-200.0), 1.6-fold (range, 1.0-3.8) and 1.4-fold (range, 1.0-1.6), respectively, as compared to reactivity towards the not transduced ‘empty’ HeLa cells. In the present study, a positive CD4 T-cell response towards ‘control’ matched alleles was not observed for any of the tested alleles. The magnitude of the CD4 Tcell response was significantly higher towards mismatched alleles than towards matched alleles, with the median fold increases being 1.7 (range, 1.0-200.0) versus 1.0 (range, 0.9-1.8), respectively (P=0.03; Mann-Whitney test). It should be noted that alloreactivity towards controls could not be determined for three patients because of an insufficient number of expanded T cells (Figure 2). Both haploidentical HSCT and dUCBT are important alternative treatment modalities for patients in need of allogeneic HSCT but lacking a matched sibling or unrelated donor. The early challenges of transplant complications related to graft failure and GvHD have been overcome with new strategies such as using two cord blood units in dUCBT and intensified immunosuppressive therapy including high dose PT-Cy in haploidentical HSCT. Haploidentical HSCT has a number of advantages in comparison to dUCBT, including unlimited donor availability and accessibility of post-transplant donor-derived immune cells. We earlier showed the presence of donorderived CD4 T cells specific for mismatched HLA class II