Leukemia Escape From HLA-Specific T Lymphocyte Pressure in a Recipient of HLA One Locus-Mismatched Bone Marrow Transplantation

Abstract 1899

Allogeneic HSCT is curative for leukemia by virtue of the immune reaction mediated by donor T lymphocytes, but unfortunately, some patients relapse after transplantation. It is reasonable to assume that the selective pressure exerted by donor T lymphocytes can lead to the outgrowth of pre-existing leukemia variants that have lost expression of gene products such as HLA molecules. However, the mechanisms of leukemia relapse in HLA-mismatched HSCT recipients remain largely uninvestigated. A case of leukemia escape from an HLA-specific cytotoxic T lymphocyte (CTL) response in a recipient of HLA one locus-mismatched BMT is presented.

A 24-year-old man with primary refractory T lymphoblastic leukemia/lymphoma received BMT from his HLA-B*51:01-mismatched mother with a T lymphocyte-repleted graft. The preparative regimen consisted of L-PAM and TBI. GVHD prophylaxis consisted of tacrolimus and methotrexate. The patient developed acute GVHD involving skin, gut, and liver on day 46 (maximum grade: III on day 53). Acute GVHD was incurable and transitioned to chronic GVHD. The patient relapsed with ascites, a hydrocele, and a subpapillary tumor on day 261. Immunosuppressant therapy was required to control GVHD until his death on day 279. Flow cytometric analysis for each HLA-A, B, or DR locus in the leukemia blasts was performed, and it showed that only the expression of B51 was down-regulated in post-transplant leukemia blasts compared with that in pre-transplant blasts. Ten CTL clones were isolated from the patient's blood on day 56. In a Cr release assay, all isolated CTL clones lysed recipient B-LCL but failed to lyse donor B-LCL. The nucleotide sequences of the uniquely rearranged TCR Vβ gene of each clone indicated that 10 clones had been derived from six independent clones. COS cells transfected with the HLA-B*51:01 cDNA construct clearly stimulated IFN-γ production by six independent CTL clones (ELISA), and the donor B-LCL transfected with HLA-B*51:01 cDNA were lysed by six CTL clones (Cr release assay), indicating that all clones recognized the B*51:01 molecule as an alloantigen. On the other hand, these data suggest that the CTL response toward the B*51:01 molecule accounted for a majority of the recipient's CTL alloresponse during acute GVHD. The recipient B*51:01 and the donor B*52:01 differed in two amino acids at positions 63 and 67, both of which constitute peptide-binding pockets. We generated four mutated B*51:01 cDNA constructs, Asn63Glu and Phe67Ser, in which individual amino acids were substituted with the corresponding amino acid in B*52:01, and Val194Ile and Ala199Val, in which both amino acids localize outside the positions constituting peptide binding pockets. Stimulation by COS cells transfected with Asn63Glu or Phe67Ser, but not with Val194Ile or Ala199Val, significantly decreased IFN-γ production by all CTL clones, suggesting that recognition of the HLA-B*51:01 molecule by CTL clones was peptide-dependent. Additionally, CTL clones should recognize certain peptides other than leukemia antigens, because B*51:01-transfected “COS cells” stimulated IFN-γ production of CTLs, and B*51:01-transfected “donor B-LCL” were lysed by CTLs. We then attempted to determine that the leukemia blasts escaped from the cytotoxicity of B*51:01-specific CTL clones. The pre-transplant leukemia blasts (purity, ∼62%) were weakly but clearly lysed by CTL clone, whereas the post-transplant leukemia blasts (∼99%) were not lysed by any CTL clones at all. In addition, the IFN-γ ELISPOT assay was performed to detect the B*51:01-reactive T lymphocytes in patient blood on day 232, one month before clinical leukemia relapse. IFN-γ-producing B*51:01-reactive T lymphocytes were detected at a level nearly equal to the level of recipient B-LCL-reactive T lymphocytes, that is, the total of the T lymphocyte alloresponse.

The mechanism of leukemia relapse in this patient can be explained as follows: CTLs specific for HLA-B*51:01 molecule/non-leukemia peptide complexes were generated in the patient blood during acute GVHD, and these CTLs continued to produce immunological pressure on leukemia blasts for at least 8 months after transplantation, but B*51:01-down-regulated leukemia blasts escaped from the pressure of B*51:01-specific CTLs, and then the leukemia clinically relapsed. These findings can explain, at least in part, the mechanism of how leukemia relapse occurs during persistent GVHD after HSCT.