In allogeneic bone marrow transplantation (allo-BMT), donor lymphocytes play a central therapeutic role in both graft-versus-leukemia (GvL) and immune reconstitution. However, their use is limited by the risk of severe graft-versus-host disease (GvHD). Eight patients who relapsed or developed Epstein-Barr virus-induced lymphoma after T cell-depleted BMT were then treated with donor lymphocytes transduced with the herpes simplex virus thymidine kinase (HSV-TK) suicide gene. The transduced lymphocytes survived for up to 12 months, resulting in antitumor activity in five patients. Three patients developed GvHD, which could be effectively controlled by ganciclovir-induced elimination of the transduced cells. These data show that genetic manipulation of donor lymphocytes may increase the efficacy and safety of allo-BMT and expand its application to a larger number of patients.

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T-cell therapy after hematopoietic stem cell transplantation (HSCT) has been used alone or in combination with immunosuppression to cure hematologic malignancies and to prevent disease recurrence. Here, we describe the outcome of patients with high-risk/advanced stage hematologic malignancies, who received T-cell depleted (TCD) haploidentical-HSCT (haplo-HSCT) combined with donor T lymphocytes pretreated with IL-10 (ALT-TEN trial). IL-10-anergized donor T cells (IL-10-DLI) contained T regulatory type 1 (Tr1) cells specific for the host alloantigens, limiting donor-vs.-host-reactivity, and memory T cells able to respond to pathogens. IL-10-DLI were infused in 12 patients with the goal of improving immune reconstitution after haplo-HSCT without increasing the risk of graft-versus-host-disease (GvHD). IL-10-DLI led to fast immune reconstitution in five patients. In four out of the five patients, total T-cell counts, TCR-Vβ repertoire and T-cell functions progressively normalized after IL-10-DLI. These four patients are alive, in complete disease remission and immunosuppression-free at 7.2 years (median follow-up) after haplo-HSCT. Transient GvHD was observed in the immune reconstituted (IR) patients, despite persistent host-specific hypo-responsiveness of donor T cells in vitro and enrichment of cells with Tr1-specific biomarkers in vivo. Gene-expression profiles of IR patients showed a common signature of tolerance. This study provides the first indication of the feasibility of Tr1 cell-based therapy and paves way for the use of these Tr1 cells as adjuvant treatment for malignancies and immune-mediated disorders.

Stromal cell-derived factor-1 (SDF-1) is a CXC chemokine produced by stromal cells that acts as a chemoattractant for human CD34 + progenitor cells. We investigated the expression of CXCR4, the receptor for SDF-1, on CD34 + cells from different hematopoietic sites and developmental stages. CXCR4 was detected by flow cytometry on 37 % of fetal bone marrow (BM) [gestation weeks (gw) 14-23] and 40 % of adult BM CD34 + cells. Interestingly, in fetal liver CD34 + cells, CXCR4 was expressed at lower levels at later stages (9 %, gw 20-23) compared to early stages of development (39 %, gw 7.5-18), suggesting a development-related change in the migratory capacity of progenitors. CXCR4 was detected at similar levels on both phenotypically primitive and committed progenitors from fetal and adult sites. However, B cell lineage progenitor and precursor cells expressed CXCR4 at the highest density (80 % of BM CD34 + /CD10 + pro-B cells are CXCR4 +). CXCR4 was also expressed in the fetal thymus in early T cell precursors and found to be down-regulated during T cell matura-tion. Finally, we found that stem cell factor, alone or in combination with other cytokines, can up-modulate CXCR4 expression on CD34 + cells by three-to fourfold. In conclusion, our results suggest that CXCR4 may play an important role in the local and systemic trafficking of human CD34 + cells as well as in human B lymphopoiesis and that its expression can be modulated by cytokines.

The infusion of donor lymphocytes after allogeneic bone marrow transplantation is a promising therapeutic tool for achieving a graft versus leukemia (GvL) effect in case of leukemic relapse (1-7), and for the treatment of other complications related to the severe immunosuppressive status of transplanted patients, such as Epstein Barr virus-induced lymphoproliferative disorders (EBV-BLPD) (8) or reactivation of CMV infection (9). Although the delay in the administration of T lymphocytes is expected to reduce the risk of severe GvHD, this risk is still present at higher doses of donor T-cells. The transfer of a suicide gene into donor lymphocytes could allow the in vivo selective elimination of cells responsible for severe GvHD. Additionally, under appropriate conditions, it may allow in vivo modulation of donor anti-tumor responses, and to separate GvL from GvHD. Finally, crucial questions concerning survival and function of donor lymphocytes could be answered by their gene marking. Previous studies documented that T lymphocytes are suitable targets for gene transfer through retroviral vectors (10, 11). This protocol has been designed to evaluate in the contest of allogeneic BMT: 1--the safety of increasing doses of donor lymphocytes transduced with a suicide retroviral vector; 2--the efficacy in terms of survival and immunologic potential of donor lymphocytes after in vitro activation, gene transduction, and immunoselection; 3--the possibility of in vivo down regulation of GvHD by the administration of ganciclovir to patients treated by tk-transduced donor lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Little is known about the molecular characteristics of alloantigens recognized by alloreactive T cells mediating hematologic stem cell graft rejection. In particular, it has never been shown that such alloantigens can be encoded by HLA-DP␤ alleles. Indeed, matching for HLA-DP antigens is generally not considered to be of functional importance for the outcome of allogeneic bone marrow or peripheral blood stem cell transplantation. In this study, a case of peripheral blood stem

Human CD34+ cells lacking detectable levels of HLA-DR antigens (CD34+ DR-) are highly enriched in hematopoietic pluripotent progenitors with long-term marrow repopulating ability. We investigated the feasibility of transducing and marking CD34+ DR- progenitor cells from bone marrow (BM) or mobilized peripheral blood samples (MPB) of 13 patients undergoing BM transplantation with the purpose of developing a protocol for a large-scale clinical application. A new retroviral vector coding for the truncated form (delta) of the low-affinity nerve growth factor receptor (LNGFR) was used to quantitate the level of gene transfer into CD34+ cells and their progeny by multiparameter cytofluorimetry and immunocytochemistry. Light-density mononuclear cells as well as purified CD34+ cells were transduced either by direct incubation with retroviral supernatants or prestimulated in vitro with various combinations of growth factors prior to transduction. Transduction efficiency, assessed as G418-resistant growth of granulocyte-macrophage colony-forming units (CFU-GM) progenitors from MPB, was 1.7-fold higher (14.9% +/- 4.5%) than those from BM (8.5% +/- 3.9%) and it was further improved (26.9% +/- 3.1%) using a purified CD34+ population as target cells. Three-color fluorescence-activated cell sorting (FACS) analysis demonstrated the presence of transduced delta LNGFR+ cells within the CD34+ DR- subpopulation. In the absence of growth factors, gene transfer into BM or MPB CD34+ DR- cells was generally poor, but following a 72-hr prestimulation it peaked at 38% of total CD34+ DR- bone marrow (BM) cells in the presence of the c-kit ligand (KL) and at 31% in the presence of IL-3. Furthermore, KL gave, compared to the other cytokines, the highest absolute yield of BM delta LNGFR+ CD34+ DR- cells recovered after transduction (p = 0.05 compared to 24 hr). Gene transfer into in vitro primitive progenitor cells was further confirmed by expression of the delta LNGFR marker on CD34+ cells and CFU-GM derived from 5-week long-term culture on stroma.

Serum ferritin has been suggested as a tumor marker in the diagnosis of certain malignancies and for following the activity or dissemination of the malignant process. Since neoplastic tissues generally contain more acidic isoferritins than their normal tissue counterparts, it has also been suggested that the specific assay of such isoferritins in serum may be of particular value in the diagnosis of malignancy. In this work, we have evaluated ferritin concentration in the serum of normal subjects and patients with acute nonlymphocytic leukemia, Hodgkin's disease, breast cancer and lung cancer by simultaneously using three different immunoassays: an immunoradiometric assay based on polyclonal antibodies against human liver (basic, L-subunit rich) ferritin, a radioimmunoassay based on polyclonad antibodies against HeLa cell (acidic, H-subunit rich) ferritin, and an immunoradiometric assay based on the monoclonal antibody 2A4 raised against human heart (acidic, H-subunit rich) ferritin. Most of the patients studied had increased values for liver-type ferritin in the absence of increased iron stores. Binding of serum ferritin to concanavalin A did not prove to be useful in distinguishing a tumor-specific basic isoferritin. The HeLa ferritin assay was found to be less specific than the heart ferritin assay in the detection of acidic isoferritins, and did not provide any advantage over the liver assay in detecting the increased levels of serum ferritin associated with malignant disease. Heart-type ferritin was found in one-fifth of normal sera and 64% of sera from patients with malignancy. Values were very low compared with those for basic ferritin, ranging from less than 0.1 to 17% of total serum ferritin (geometric mean value 1.3%) in patients with malignancy. These findings indicate that at present there is little application for serum ferritin immunoassays based on antibodies to HeLa cell or heart ferritin in the diagnosis or monitoring of malignant disease. This seems to be due to the presence in human serum of biding factors which are responsible for the rapid clearance of acidic isoferritins from the circulation. The serum concentration of basic ferritin, however, can be useful in the diagnosis and management of some malignancies, and it is possible that studies on cell isoferritins can be important in biologic monitoring of neoplastic disorders. It should also be noted that the increased levels of serum ferritin found in patients with malignancy can exert adverse effects on the host immune response and perhaps an inhibitory effect on hematopoiesis.