J.H.F. Falkenburg

Recognition of clonogenic leukemic cells, remission bone marrow and HLA-identical donor bone marrow by CD8+ or CD4+ minor histocompatibility antigen-specific cytotoxic T lymphocytes.

We investigated whether minor histocompatibility (mH) antigen-specific cytotoxic T lymphocytes (CTL) can discriminate between leukemic hematopoietic progenitor cells (leukemic-HPC) from AML or CML patients, the HPC from their remission bone marrow (remission-HPC), and normal HPC from their HLA-identical sibling bone marrow donor (donor-HPC). Specific lysis by CD8+ CTL clones was observed not only of the leukemic-HPC but also of the donor-HPC in 3/4 patient/donor combinations expressing mH antigen HA-1, 3/5 combinations expressing mH antigen HA-2, 2/3 combinations expressing mH antigen HA-3, and 2/2 combinations expressing mH antigen HY-A1. In four patient/donor combinations the recognition of the donor-HPC was clearly less than of the leukemic-HPC, indicating differential susceptibility to lysis by these mH CTL clones. In addition, differential recognition of leukemic-HPC and remission-HPC within seven patients was analyzed. In one patient expressing the HA-2 antigen on the leukemic cells the recognition of the remission-HPC was clearly less than of the leukemic-HPC. One CD4+ CTL clone showed specific lysis of the leukemic-HPC from an AML patient and a CML patient as well as of normal remission-HPC and donor-HPC. These results illustrate that in general CD8+ and CD4+ mH antigen specific CTL clones do not differentially recognize leukemic-HPC and normal-HPC. However, differences in susceptibility to lysis of malignant versus normal cells may contribute to a differential GVL effect.

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.

Generation of donor-derived antileukemic cytotoxic T-lymphocyte responses for treatment of relapsed leukemia after allogeneic HLA-identical bone marrow transplantation

Allogeneic bone marrow transplantation (BMT) has been associated with an antileukemic effect, the graft-versus-leukemia (GVL) reactivity. Since T-cell depletion of the bone marrow graft performed to reduce the incidence and severity of graft-versus-host disease (GVHD) after BMT has been associated with an increase risk of relapse, the GVL reactivity has been attributed to the T lymphocytes from the graft. Previously, we demonstrated that leukemia-reactive cytotoxic T-lymphocyte (CTL) lines and clones could be generated from the peripheral blood of HLA-genotypically identical siblings of patients with leukemia by stimulation of the donor cells with irradiated leukemic cells from the patients. HLA class I as well as class II restricted CTL clones could be generated that recognized the leukemic cells. Some clones recognized the leukemic cells from the patient, but not the interleukin (IL)-2-stimulated lymphocytes from the same individual. To explore the possibility of clinically using donor-derived CTL lines directed against the leukemic cells from patients who relapsed after allogeneic BMT, a pilot study was performed using eight donor-recipient combinations. In seven of eight combinations donor-derived CTL lines could be generated that showed specific lysis of the leukemic cells from the patient. In five of these cases, the CTL lines showed reactivity with the leukemic cells, but not with the IL-2-stimulated lymphocytes from the same individual. In two cases, the CTL lines were cytotoxic for the IL-2-stimulated lymphoblasts as well as the leukemic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

HLA-DP as specific target for cellular immunotherapy in HLA class II-expressing B-cell leukemia

Mismatching for human leukocyte antigen (HLA)-DPB1 in unrelated donor hematopoietic stem cell transplantation (URD-SCT) has been associated with a decreased risk of disease relapse, indicating that HLA-DP may represent a target for graft-versus-leukemia (GVL) reactivity in HLA class II-expressing hematological malignancies. To investigate whether HLA-DP-specific T cells could mediate GVL reactivity following HLA-DPB1-mismatched URD-SCT and donor lymphocyte infusion (DLI), we analyzed the immune response in a patient with leukemic lymphoplasmacytic lymphoma responding to DLI without graft-versus-host disease. The emergence of leukemia-reactive CD4+ T cells during the clinical immune response was demonstrated by interferon-γ (IFN-γ) enzyme-linked immunosorbent spot(ELISPOT)analysis. Following clonal isolation of these leukemia-reactive CD4+ T cells, blocking studies, panel studies and retroviral transduction experiments of both mismatched HLA-DPB1 alleles identified HLA-DPB1*0201 and HLA-DPB1*0301 as the targets of this immune response. The HLA-DPB1-specific CD4+ T-cell clones were capable of recognizing and lysing several HLA-DP-expressing myeloid and lymphoid hematological malignant cells. Since HLA-DP expression is mainly restricted to hematopoietic cells, HLA-DP may be used as a specific target for immunotherapy following T-cell-depleted URD-SCT. Therefore, in patients with HLA class II-expressing hematological malignancies HLA-DP-mismatched SCT may be preferable over fully matched SCT allowing DLI to induce a GVL effect.

The number of nucleated cells reflects the hematopoietic content of umbilical cord blood for transplantation.

A single umbilical cord blood (UCB) collection may contain sufficient hematopoietic stem cells to achieve engraftment and repopulation of the hematopoietic system of children and adults after myeloablative therapy. The hematopoietic potential of a UCB unit is often defined by the number of CD34+ cells or the number of colony-forming units as measured in semisolid hematopoietic progenitor cell (HPC) cultures. However, these assays are relatively difficult to standardize between UCB banks. The number of nucleated cells infused per kilogram body weight of the recipient is also reported to be a significant factor in the speed of recovery of neutrophils and platelets after transplantation. To analyze which parameters could be used to evaluate the hematopoietic potential of a UCB graft, we evaluated almost 300 UCB units that were collected for banking for unrelated transplantation. A strong correlation was found between the frequencies of CD34+ cells and the HPC as measured in semi-solid medium cultures. From the various leukocyte subpopulations, the concentration and total numbers of nucleated cells correlated best with both the HPC content and the number of CD34+ cells. Differentiation of these nucleated cells into subsets of leukocytes offered no advantage for better prediction of HPC or CD34+ cells. These results indicate that the nucleated cell count probably reflects the hematopoietic potential of a UCB graft, and may for that reason correlate with the speed of engraftment after transplantation.

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.

High susceptibility of human leukemic cells to Fas-induced apoptosis is restricted to G1 phase of the cell cycle and can be increased by interferon treatment.

In this study, we analyzed the influence of cell cycle status manipulations of leukemic cells on Fas-mediated apoptosis using the GM-CSF-dependent human myeloid leukemia cell line AML-193 as a model. GM-CSF and long-term treatment with interferon-gamma (IFN-γ) or interferon-alpha (IFN-α) were used to manipulate the cell cycle status. Control cells were GM-CSF deprived, nonproliferating cells. IFN-γ or IFN-α treatment did not induce proliferation in control cells, but resulted in recruitment of cells from resting G0 phase into activated G1 phase. Using agonistic anti-Fas antibodies (FAS18), we demonstrated that this shift from G0 to G1 was accompanied by a 2.5-fold increase in Fas sensitivity. A similar increase in sensitivity to FAS18 could be obtained by induction of proliferation with GM-CSF. Quantitative FACS analysis of surviving cells after FAS18-induced apoptosis showed deletion of the G1 compartment, but complete protection of resting G0 cells. Cells in S or G2/M phase were relatively protected against Fas induction. In conclusion, sensitivity to Fas-mediated apoptosis was restricted to cells in G1 phase of the cell cycle, and can be increased by treatment of cells with interferons. By this mechanism, interferon treatment may render leukemic cells more susceptible to lysis by T cells during immunotherapeutic interventions.

Successful generation of primary virus-specific and anti-tumor T-cell responses from the naïve donor T-cell repertoire is determined by the balance between antigen-specific precursor T cells and regulatory T cells

Background One of the major challenges in allogeneic stem cell transplantation is to find a balance between the harmful induction of graft-versus-host disease and the beneficial graft-versus-leukemia and pathogen-specific immune responses. Adoptive transfer of in-vitro generated donor T cells with specific anti-leukemic or pathogen-specific activity may be effective. However, in many cases this requires the in-vitro priming and expansion of antigen-specific precursor T cells from the naïve donor T-cell repertoire. Design and Methods Antigen-specific CD8 T cells were generated by co-culture of CD45RO-depleted, regulatory T cell-depleted donor peripheral blood mononuclear cells with autologous peptide-loaded dendritic cells, followed by two re-stimulations with peptide-loaded autologous monocytes. Responding T cells were isolated based on CD137 expression and further purified using peptide/major histocompatibility complex tetramers. Results Using this method we were able to reproducibly generate functionally high avidity T cells directed against multiple viral antigens and minor histocompatibility antigens from the naïve T-cell repertoire of seronegative, minor histocompatibility antigen-negative donors. Furthermore, we demonstrated that reduction of the regulatory T-cell frequency by depletion of CD45RO+ responder cells resulted in improved priming and expansion of antigen-specific precursor T cells. Conclusions In conclusion, we present a robust method for the in-vitro induction and isolation of antigen-specific T cells from the naïve repertoire. We demonstrate that the likelihood of successful generation of primary immune responses is determined by a delicate balance between the numbers of antigen-specific precursor T cells and the numbers and activation state of regulatory T cells locally at the site of priming of the immune response.

Myeloablative T cell-depleted alloSCT with early sequential prophylactic donor lymphocyte infusion is an efficient and safe post-remission treatment for adult ALL

The prognosis of adult patients with ALL remains unsatisfactory. AlloSCT is associated with a beneficial GVL response mediated by donor T cells. However, GVHD results in substantial mortality and long-term morbidity. T-cell depletion (TCD) of the graft reduces the severity of GVHD, but is associated with an increased relapse rate after alloSCT. Therefore, early sequential donor lymphocyte infusion (DLI) is likely to be necessary for a successful GVL reaction. Twenty-five adult ALL patients (10 Ph+ALL) were eligible for early DLI after initial disease control with myeloablative TCD-alloSCT in first CR (CR1), if active GVHD was absent at 3–6 months after alloSCT. Patients with a sibling donor or an unrelated donor were scheduled for 3.0 × 106 CD3+ cells/kg or 1.5 × 106 CD3+ cells/kg, respectively, at 6 months after alloSCT. Three patients died before evaluation (one early relapse). Five patients had active GVHD. Fourteen of the remaining seventeen patients received DLI (median time-to-DLI: 185 days). Overall, only 17% required long-term systemic immunosuppression for GVHD. With a median follow-up after TCD-alloSCT of 50 months, 2-year survival probability was 68% (95% confidence interval (CI) 49–87%). In conclusion, myeloablative TCD-alloSCT with early sequential DLI is an efficient and safe post-remission treatment for adult ALL patients in CR1.

Discovery of T Cell Epitopes Implementing HLA-Peptidomics into a Reverse Immunology Approach

T cell recognition of minor histocompatibility Ags (MiHA) plays an important role in the graft-versus-tumor effect of allogeneic stem cell transplantation. Selective infusion of T cells reactive for hematopoiesis-restricted MiHA presented in the context of HLA class I or II molecules may help to separate the graft-versus-tumor effects from graft-versus-host disease effects after allogeneic stem cell transplantation. Over the years, increasing numbers of MiHA have been identified by forward immunology approaches, and the relevance of these MiHA has been illustrated by correlation with clinical outcome. As the tissue distribution of MiHA affects the clinical outcome of T cell responses against these Ags, it would be beneficial to identify additional predefined MiHA that are exclusively expressed on hematopoietic cells. Therefore, several reverse immunology approaches have been explored for the prediction of MiHA. Thus far, these approaches frequently resulted in the identification of T cells directed against epitopes that are not naturally processed and presented. In this study we established a method for the identification of biologically relevant MiHA, implementing mass spectrometry–based HLA-peptidomics into a reverse immunology approach. For this purpose, HLA class I binding peptides were eluted from transformed B cells, analyzed by mass spectrometry, and matched with a database dedicated to identifying polymorphic peptides. This process resulted in a set of 40 MiHA candidates that were evaluated in multiple selection steps. The identification of LB-NISCH-1A demonstrated the technical feasibility of our approach. On the basis of these results, we present an approach that can be of value for the efficient identification of MiHA or other T cell epitopes.