ALBERT

Description: Recently, we have demonstrated that antibodies that block the function of the β2-integrin leukocyte function-associated antigen-1 (LFA-1) completely abrogate the rapid mobilization of hematopoietic progenitor cells (HPC) with colony-forming and radioprotective capacity induced by interleukin-8 (IL-8) in mice. These findings suggested a direct inhibitory effect of these antibodies on LFA-1–mediated transmigration of stem cells through the bone marrow endothelium. Therefore, we studied the expression and functional role of LFA-1 on murine HPC in vitro and in vivo. In steady state bone marrow ± 50% of the mononuclear cells (MNC) were LFA-1neg. Cultures of sorted cells, supplemented with granulocyte colony-stimulating factor (G-CSF)/granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-1/IL-3/IL-6/stem cell factor (SCF) and erythropoietin (EPO) indicated that the LFA-1neg fraction contained the majority of the colony-forming cells (CFCs) (LFA-1neg 183 ± 62/7,500 cells v LFA-1pos 29 ± 17/7,500 cells,P 〈 .001). We found that the radioprotective capacity resided almost exclusively in the LFA-1neg cell fraction, the radioprotection rate after transplantation of 103, 3 × 103, 104, and 3 × 104 cells being 63%, 90%, 100%, and 100% respectively. Hardly any radioprotection was obtained from LFA-1pos cells. Similarly, in cytokine (IL-8 and G-CSF)–mobilized blood, the LFA-1neg fraction, which comprised 5% to 10% of the MNC, contained the majority of the colony-forming cells, as well as almost all cells with radioprotective capacity. Subsequently, primitive bone marrow-derived HPC, represented by Wheat-germ-agglutinin (WGA)+/Lineage (Lin)−/Rhodamine (Rho)− sorted cells, were examined. More than 95% of the Rho− cells were LFA-1neg. Cultures of sorted cells showed that the LFA-1neg fraction contained all CFU. Transplantation of 150 Rho− LFA-1neg or up to 600 Rho−LFA-1pos cells protected 100% and 0% of lethally irradiated recipient mice, respectively. These results show that primitive murine HPC in steady-state bone marrow and of cytokine-mobilized blood do not express LFA-1.

Description: Patients with the B-cell malignancy hairy cell leukemia (HCL) exhibit a skewed T-cell repertoire with oligoclonal expression or absence of many members of the T-cell receptor (TCR) BV gene families. To evaluate whether interferon-α (IFN-α) therapy would not only restore normal hematopoiesis, but also the abnormal T-cell repertoire, we studied T lymphocytes from a cohort of HCL patients treated by IFN-α in the past, at initiation, and at several intervals up to 6 years of IFN-α treatment. The junctional regions from 22 TCRBV gene families were analyzed after polymerase chain reaction amplification of cDNA (RT-PCR) using family specific primers. In all seven patients improvement of the skewed T-cell repertoire was not seen until 2 years of treatment. It consisted of disappearance of oligoclonal subpopulations and (polyclonal) reappearance of absent TCRBV gene families. The RT-PCR results were correlated with the TCRBV protein expression using TCRBV-specific monoclonal antibodies. T lymphocytes from four patients with active HCL contained large expansions of particular TCRBV-expressing cells (up to 25% of the CD3+cells; 600 to 700/μL whole blood), which decreased during IFN-α therapy in both patients tested. Finally, restoration of the TCR repertoire matched normalization of the functional immune repertoire as measured by proliferative, helper, and cytotoxic T-lymphocyte precursor frequencies against major histocompatibility complex–unrelated individuals. In conclusion, oligoclonal bands of TCRBV gene families found by RT-PCR correspond with a dramatic increase in circulating T lymphocytes expressing the same TCRBV family. Moreover, IFN-α can restore the skewed T-cell repertoire and suppress persistent T-cell clones upon treatment of the accompanying malignancy.

Description: In allogeneic stem cell transplantation (SCT) T-cell depletion reduces transplant related mortality by diminishing GVHD. We have investigated a myeloablative regimen for matched unrelated donor SCT using both in vivo and in vitro CAMPATH-1H for effective T-cell depletion, utilising DLI at a later time point for graft versus tumor effect if necessary. Thirty patients (median age 33 years, range 18–48) were transplanted from January 1997 to June 2002. Diagnoses were: CML CP (n=9), CML AP (n=2), AML/MDS (n=9), ALL (n=8), NHL (n=1) and Fanconi anemia (n=1). Six patients had one HLA mismatch, the others were identical for HLA A, B, C, DR and DQ. Conditioning consisted of CAMPATH-1H 5mg/d on days −8 to −4, TBI 6 Gy on days −8 and −7 and cyclofosfamide 60 mg/kg on days −6 and −5. T-cell depletion was performed by in vitro incubation of the graft with 20 mg CAMPATH-1H for 30 minutes (Campath “in the bag”). Post-transplant GVHD prophylaxis consisted of cyclosporine A and methotrexate. The stem cell source was bone marrow in 19 patients (63%) and peripheral blood in 11 patients. One graft failure was observed, all other patients had sustained engraftment of donor cells. Acute GVHD was observed in 12 patients (40%), maximally grade I-II skin. No severe acute GVHD (grade III-IV) was experienced. Limited chronic GVHD developed in 2 patients, resolving after treatment. Only in one patient extensive chronic GVHD developed, which did not resolve. CMV reactivation occurred in 23% of patients, one patient developed CMV disease. No EBV disease was observed. Ten patients received donor lymphocyte infusion (DLI) at a median of 17.4 months after SCT (8 patients with relapsed CML, one patient with relapsed ALL, one patient with autoimmune hemolytic anemia). After DLI acute GVHD grade I-II developed in 4 patients, and GVHD grade III-IV in 3. Chronic GVHD developed in 5 patients, of which 2 extensive, resolving in all except one patient. With a median follow up of 37 (range 21–84) months 17 patients are alive (57%). One of the CML patients shows persistence of molecular disease not responding to increasing doses of DLI. All other patients are in CR with the CML patients in molecular remission. Five patients (17%) died because of relapsed disease (2 AML/MDS and 3 ALL). Treatment related mortality was 26% (1 rejection, 2 GVHD, 1 myocardial infarction, 4 infections). In conclusion, matched unrelated donor SCT following myeloablative conditioning using T-cell depletion with CAMPATH-1H in vivo as well as in vitro results in good engraftment, minimal grade I-II GVHD and an overall survival of 57%. Relapse rate was not increased with this strategy. This regimen appears to be successful for young adults with high-risk malignancies.

Description: Allogeneic stem cell transplantation can be successfully applied in the treatment of hematological malignancies and relies on the graft versus leukemia (GVL) effect mediated by donor T cells directed against minor histocompatibility antigens (mHag) selectively expressed on malignant hematopoietic cells of the patient. However, due to insufficient in-vivo priming of donor T cells the GVL response may not be adequately initiated or amplified. Vaccination strategies using immunogenic peptides derived from hematopoeisis specific mHag like HA-1 may form a strategy to initiate or boost the in-vivo GVL response. However, it has been reported that repetitive vaccination with HLA-class I binding 9-mer peptides can lead to the induction of T cell anergy. We hypothesized that repetitive strong priming of the mHag specific T cells may also lead to prolonged downregulation of the T cell receptor (TCR) resulting in inability of the T cells to subsequently attack tumor cells expressing the mHag, allowing tumor escape despite the presence of potentially effective T cells. We tested this hypothesis in an in-vitro model using CFSE-labeled HA-1+ CD34+ chronic myeloid leukemia (CML) cells as target/stimulator cells, and HA-1 specific T cells as effector cells. In previous studies we have demonstrated the resistance of a small population of quiescent CML stem cells to all high avidity T cells, allowing the subsequent outgrowth of malignant progeny from this population. To mimick a peptide vaccination strategy, we loaded CD34+ CML cells from an HA-1+ patient with various concentrations (E-12-E-6M) of the 9-mer HA-1 peptide, and investigated the direct and residual functional cytotoxic capacity of an HA-1 specific CD8+ T cell clone. In accordance with our previous results, we observed complete deletion of all proliferating CML precursor cells after 24–48 hours of exposure to the CTLs, whereas a small subpopulation of quiescent CD34+ cells was resistant to T cell attack. The exogenous peptide loading resulted in more rapid lysis and also attack of part of the quiescent stem cell population. However, in the next days malignant progeny was formed from the quiescent stem cell population in the conditions of high peptide stimulation despite the continuous presence of the T cells, suggesting impaired residual cytotoxic function of these T cells. Therefore, we analyzed the level of TCR downregulation after exposure to the HA-1 positive CML CD34+ cells in the absence or presence of E-12-E-6 M HA-1 peptide loaded to the target cells. We observed strong dose-dependent TCR downregulation as measured by specific tetramer staining (20%–78% decrease in fluorescence intensity after 24 hours of exposure to targets loaded with 0-E-6M HA-1 peptide). At high peptide concentrations it took 6–9 days before proper functional TCR expression could be again demonstrated. In conclusion, we here demonstrate that high affinity T cells show a prolonged TCR downregulation after vigorous stimulation by peptide loaded target cells. In this period the T cells showed a dramatic loss of function and allowed the outgrowth of a leukemic subpopulation expressing the HA-1 antigen. Milder vaccination strategies using longer peptides requiring uptake and processing by the target cells may lead to expression of more physiological levels of the mHag and less vigorous priming of the mHag specific T cells, thereby preserving their functional capacity and responsiveness.

Description: Donor lymphocyte infusion (DLI) induces complete remissions in 70–80% of patients with a relapse of CML after allogeneic stem cell transplantation (SCT). However, in some patients molecular persistence of bcr-abl positive stem cells can be found. To determine possible mechanisms of this persistence we studied in detail a patient who received DLI for a hematological relapse after HLA identical sibling SCT. After obtaining a hematological and cytogenetic remission in the absence of any graft versus host disease persistence of molecular disease was observed despite multiple additional escalating doses of DLI. To determine the nature of this T cell response, donor T cells isolated from bone marrow of the patient at the time of clinical response were stimulated with CML cells from the patient. T cells responding to the leukemia by secreting IFN-γ were isolated, directly cloned by cell sorting and expanded. Four different types of CD8+ HLA class I restricted CTL clones were obtained. All clones were cytolytic against EBV-LCL of the patient, but did not lyse EBV-LCL of the donor, indicating recognition of a minor histocompatibility antigen (mHag). PHA blasts of patient and donor were not lysed, indicating that the clones recognized an antigen present on B-cells but not on T-cells. To further study the lineage restriction of the clones, bone marrow and peripheral blood CML cells were stained with CFSE and incubated with the clones. The survival of different cell populations was measured by counterstaining with different monoclonal antibodies (Jedema, Blood2004; 103: 2677). Two clones only lysed CD14 positive monocytic cells, two other clones lysed CD14 positive monocytic cells as well as CD13–33 positive myeloid cells. Interestingly, immature myeloid cells were not lysed, whereas recognition by the T cells did occur after maturation of these cells in vitro by growth factors. By morphological examination it was demonstrated that the cell clones are capable of lysing myelocytes but not myeloblasts. These results indicate that the clones recognize a mHag expressed during myeloid maturation. Subsequently, we investigated whether the CTL clones could lyse CML progenitor cells using a hematopoietic progenitor cell inhibition assay (van der Hoorn, Methods2003; 31: 113). In the presence of the CTL clones persistent proliferation of 20–40% was found, in contrast to the complete proliferation inhibition of CML progenitors observed when mHag clones HY-A1 and HA-1 were used as control. Secondly, survival studies showed that the CTL clones were not able to lyse CFSE labelled CD34 positive CML cells, in contrast to the control clones HY-A1 and HA-1. In conclusion, different types of CD8+ mHag-specific CTL clones recognizing lineage specific antigens have been isolated from a relapsed CML patient with molecular persistence of bcr-abl after repeated administrations of DLI. These clones recognize mHag’s present on B-cells, monocytic cells and mature myeloid CML cells, but not on CML progenitor cells. The isolation of these CTL’s in the absence of T cells against CML progenitor cells explains the clinical hematological remission but molecular disease persistence of this patient after DLI. These findings suggest that for adequate eradication of CML a cytotoxic T cell response against leukemic progenitor cells is needed.

Description: For the clinical evaluation of the efficacy of cellular immunotherapy it is necessary to analyze the effector functions of T cells against primary leukemic target cell populations which are usually considerably heterogeneous caused by differential maturation stages of the leukemic cells. An appropriate assay should not only allow the quantitative analysis of rapid cell death induction as measured by the conventional 51Cr release assay but also of the more slowly executing pathways of T-cell-induced apoptosis occurring within days instead of hours which cannot be measured using this method. Furthermore, it should dissect the differential susceptibility to T-cell-induced cell death of various target cell subpopulations and characterize the malignant precursor cells capable of producing malignant progeny. To fulfill these requirements we developed a new assay based on carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling of the target cell population combined with antibody staining of specific cell populations and addition of fluorescent microbeads to quantitatively monitor target cell death occurring within a longer time frame up to at least 5 days. This new assay facilitates the analysis of differential recognition of distinct cell types within a heterogeneous target cell population and allows simultaneously evaluation of the proliferative status of surviving target cells in response to relevant cytokines. (Blood. 2004;103: 2677-2682)

Description: We perform reduced intensity allogeneic stem cell transplantation (SCT) with HLA identical sibling and matched unrelated donors using a conditioning regimen consisting of fludarabine, busulphan and ATG combined with alemtuzumab added to the stem cell graft for extensive donor T cell depletion. With this regimen hardly any acute and chronic graft versus host disease (GVHD) are observed after SCT and mixed chimerism is induced in most patients (Barge et al, Exp Hematol 2003). In patients transplanted for malignant disease, donor lymphocytes (DLI) are administered in escalating doses from 6 to 9 months to convert mixed into complete donor chimerism and to induce graft versus tumor (GVT) responses. We report results of chimerism analysis, clinical antitumor response and GVHD after DLI. 41 patients (median age 54 years, range 37–65) transplanted with stem cells from sibling donors (29) or matched unrelated donors (12) for various malignancies received DLI with a median dose of 5 × 106 CD3+ cells/kg (range 1–5) at a median time point of 7 months after SCT (range 5.1–15). After DLI administration 35 of 38 evaluable patients (92%) developed an immune response as defined as a major decrease in patient chimerism. In 26 patients full donor chimerism was achieved, in 9 patients a low patient signal remained present (1–2%). Patient chimerism decreased from median 12% (range 1 to 86%) to 0% (range 0–2%). In most patients conversion occurred after the first DLI, in 4 patients after the second and in 1 patient after the third DLI. In 31 of 38 patients measurable disease was present before DLI. In 22 of these patients conversion from mixed to full donor chimerism coincided with clinical GVT responses. Seven patients with active myeloid disease all achieved complete remission (4 AML/MDS patients with 8–65% blasts, 2 patients with progressive CML and 1 with active CMMOL). All these patients are still in CR after a median time of 22 months. In 8 myeloma patients with measurable disease 5 complete and 1 partial response were observed. However, most responding myeloma patients subsequently developed bone or extramedullary relapses without evidence of bone marrow involvement. In 13 patients with lymphoid disease 9 responses were observed. One patient with an immunocytoma and one with T-PLL achieved CR. In 7 CLL patients 3 CR and 1 PR were observed, notably two CR occurred in patients with massive bone marrow involvement. Two of four patients with aggressive NHL showed a CR to DLI in combination with rituximab and one patient a PR to DLI. Although no regression of tumor was observed in three patients with renal cell carcinoma after DLI, disease progression was halted for several years. Grade 3–4 acute GVHD developed in 22% of patients, grade 1–2 in 39%. Limited or extensive chronic GVHD was observed in 30 and 23% of patients, respectively. GVHD responded to therapy in most patients, only in 9% of patients chronic GVHD did not resolve. Mortality due to acute and chronic GVHD was 10%. In conclusion, after T cell depleted reduced intensity SCT a state of mixed chimerism is induced in most patients, which can be converted into full donor chimerism with DLI in more than 90% of patients. Conversion to full donor chimerism is accompanied with clinical GVT responses in a high percentage of patients with acceptable GVHD.

Description: The genetic engineering of T lymphocytes is an attractive strategy to specifically redirect T cell immunity towards viral infections and malignancies. Transfer of virus- or tumor-specific TCRs has demonstrated to endow T cells with redirected antigen specificity. We demonstrated redirected anti-leukemic reactivity of CMV specific T cells using gene transfer of minor histocompatibility antigen HA-2 specific TCRs. The HA-2-TCR-modified T cells exerted high cytolytic activity against HA-2 expressing target cells, including leukemic cells, and not against target cells negative for the HA-2 mHag. After cloning of the TCR-transferred T cells, we demonstrated that the HA-2-TCR cell surface expression, measured by HA-2-tetramer staining, was variable on the transduced T cell clones, and that the cytolytic capacity of the T cells correlated with the level of HA-2-TCR expression. Since we could demonstrate that this variation in HA-2-TCR expression was not due to differences in transgene expression, we investigated whether the endogenous TCRs influenced the expression of the introduced TCR. CMV-A2 specific T cells were isolated from peripheral blood and transduced with the HA-2-TCR. In control transduced CMV specific T cells we observed 5 different high affinity CMV specific TCRs. CMV specific T cells transduced with the HA-2-TCR that expressed predominantly the HA-2-TCR, expressed only one of these types of CMV-TCR, and in CMV specific T cells with low HA-2-TCR expression two different types of CMV-TCRs were found. These data indicated that the level of expression of the introduced TCR is strongly influenced by the endogenous TCR. To investigate whether this was due to differences in promotor activity of the endogenous and retrovirally introduced TCR, the three CMV-TCRs were characterized and transferred into unselected peripheral T cells. T cells transferred with the weak competitior CMV-TCR that was strongly downregulated in CMV specific T cells by introduction of the HA-2-TCR, showed low CMV specific cytotoxicity and no tetramer staining. In contrast, T cells transferred with the strong competitor CMV-TCR that was modestly downregulated in CMV specific T cells by introduction of the HA-2-TCR, revealed strong CMV specific cytotoxic activity and tetramer staining. These data demonstrate that the introduced and endogenous TCRs compete for cell surface expression, and that this competition is dependent on characteristics of the different TCRs and independent of whether the TCR is retrovirally introduced or naturally expressed. To investigate whether the cell surface expression of the different TCRs was determined by preferential pairing properties of the individual TCR chains, TCR α and β deficient Jurkat 76 cells were transduced with the three CMV-specific TCRs or with chimeric TCRs consisting of the TCR α chain of one TCR with the TCR β chain of another TCR. TCRαβ membrane expression revealed that TCRs with a strong competitor phenotype expressed higher levels of TCRαβ than the TCR that was a weak competitor. TCRαβ expression of Jurkat cells transduced with chimeric TCRs indicated that the expression level of the different TCRs was determined by the pairing properties of the individual TCR α and β chains and not by differences in protein expression. In conclusion these data demonstrated that introduced and endogenous TCRs compete for cell surface expression in favor of the TCR that has the highest intrinsic pairing properties.