Freke M. Kloosterboer

Hematopoiesis-restricted minor histocompatibility antigens HA-1- or HA-2-specific T cells can induce complete remissions of relapsed leukemia

Donor lymphocyte infusion (DLI) into patients with a relapse of their leukemia or multiple myeloma after allogeneic stem cell transplantation (alloSCT) has been shown to be a successful treatment approach. The hematopoiesis-restricted minor histocompatibility antigens (mHAgs) HA-1 or HA-2 expressed on malignant cells of the recipient may serve as target antigens for alloreactive donor T cells. Recently we treated three mHAg HA-1- and/or HA-2-positive patients with a relapse of their disease after alloSCT with DLI from their mHAg HA-1- and/or HA-2-negative donors. Using HLA-A2/HA-1 and HA-2 peptide tetrameric complexes we showed the emergence of HA-1- and HA-2-specific CD8+ T cells in the blood of the recipients 5–7 weeks after DLI. The appearance of these tetramer-positive cells was followed immediately by a complete remission of the disease and restoration of 100% donor chimerism in each of the patients. Furthermore, cloned tetramer-positive T cells isolated during the clinical response specifically recognized HA-1 and HA-2 expressing malignant progenitor cells of the recipient and inhibited the growth of leukemic precursor cells in vitro. Thus, HA-1- and HA-2-specific cytotoxic T lymphocytes emerging in the blood of patients after DLI demonstrate graft-versus-leukemia or myeloma reactivity resulting in a durable remission. This finding implies that in vitro generated HA-1- and HA-2-specific cytotoxic T lymphocytes could be used as adoptive immunotherapy to treat hematological malignances relapsing after alloSCT.

Direct cloning of leukemia-reactive T cells from patients treated with donor lymphocyte infusion shows a relative dominance of hematopoiesis-restricted minor histocompatibility antigen HA-1 and HA-2 specific T cells

Donor T cells recognizing hematopoiesis-restricted minor histocompatibility antigens (mHags) HA-1 and HA-2 on malignant cells play a role in the antileukemia effect of donor lymphocyte infusion (DLI) in patients with relapsed leukemia after allogeneic stem cell transplantation. We quantified the contribution of HA-1 and HA-2 specific T cells to the total number of leukemia-reactive T cells in three HA-2 and/or HA-1 positive patients responding to DLI from their mHag negative donors. Clinical responses occurring 5–7 weeks after DLI were accompanied by an increase in percentages HLA-DR expressing T cells within the CD8+ T cell population. To clonally analyze the leukemia-reactive immune response, T cells responding to the malignancy by secreting IFNγ were isolated from peripheral blood, directly cloned, and expanded. Tetramer analysis and specific lysis of peptide-pulsed target cells showed that 3–35% of cytotoxic T lymphocyte (CTL) clones isolated were specific for HA-1 or HA-2. TCR VB analysis showed oligoclonal origin of the HA-1 and HA-2 specific CTL clones. The HA-1 and HA-2 specific CTL clones inhibited leukemic progenitor cell growth in vitro. The relatively high frequency of HA-1 and HA-2 specific T cells within the total number of tumor-reactive T cells illustrates relative immunodominance of mHags HA-1 and HA-2.

Minor histocompatibility antigen-specific T cells with multiple distinct specificities can be isolated by direct cloning of IFNγ-secreting T cells from patients with relapsed leukemia responding to donor lymphocyte infusion

Graft-vs-leukemia reactivity after donor lymphocyte infusion (DLI) can be mediated by donor T cells recognizing minor histocompatibility antigens (mHags) on recipient hematopoietic cells. To study the diversity of cells involved in this immune response, hematopoietic cell reactive T cells were directly clonally isolated from peripheral blood of patients entering complete remission after DLI. T cells were briefly stimulated with bone marrow cells from patients pretransplant, and IFNγ-secreting T cells were directly clonally isolated, and expanded. Cytotoxic T-lymphocyte (CTL) clones from individual patients used multiple distinct HLA-restricting molecules and varied in reactivity against patient-derived normal and/or malignant hematopoietic cells. For each patient, CTL clones specific for known immunodominant mHags as well as distinct unknown mHags were found. Within individual patients, CTL clones using the same HLA-restricting element could show differential recognition patterns, indicating further diversity in mHag reactivity. CTL clones from individual patients exhibiting identical specificities could show oligoclonal origin. In conclusion, the direct cloning technique shows that the response to hematopoietic cells after DLI is directed against multiple distinct mHags, including but not limited to known immunodominant mHags, implying that immunotherapy with T cells against multiple mHag specificities may be more effective in eradicating malignant cells.