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Administration of herpes simplex thymidine kinase (HSV-tk)-expressing, gene-modified T cells (GMCs) with T cell-depleted bone marrow transplantation (TCD-BMT) can allow modulation of posttransplantation alloreactivity. Twelve patients received 2 x 10(5) to 2 x 10(6) CD3+ donor GMCs per kilogram with HLA-identical sibling TCD-BMT. Despite extensive T cell depletion of bone marrow, an intensive conditioning regimen, and immunosuppressive graft-versus-host disease (GvHD) prophylaxis, infusion at the time of TCD-BMT of this low number of GMCs sufficed to induce a rapid GMC-specific immune response, as detected by interferon- enzyme- linked immunospot assay in six of eight patients, preferentially targeting HSV-tk. Maximal responses were reached early (median time, 49 [35-68] days post-BMT), with a subsequent rapid and significant decrease in five of six evaluable patients. Immune responses were negatively correlated with the maximal circulating GMC counts. However, such immune response did not result in the elimination of circulating GMCs and was not associated with measurable ex vivo cytotoxic activity against GMCs. Furthermore, alloreactive GMCs still could induce GCV-sensitive GvHD in one patient despite an ongoing immune response. Overall, infusion of HSV-tk-expressing GMCs at the time of BMT results in an early immune response. Such immune response may be altered and may not prevent persistent GCV-sensitive alloreactivity.

In our previous phase 1/2 study aimed at controlling graft-versus-host disease, 12 patients received Herpes simplex virus thymidine kinase (HSV-tk(+))/neomycin phosphotransferase (NeoR(+))-expressing donor gene-modified T cells (GMCs) and underwent an HLA-identical sibling T-cell-depleted bone marrow transplantation (BMT). This study's objective was to follow up, to quantify, and to characterize persistently circulating GMCs more than 10 years after BMT. Circulating GMCs remain detectable in all 4 evaluable patients. However, NeoR- and HSV-tk-polymerase chain reaction (PCR) differently quantified in vivo counts, suggesting deletions within the HSV-tk gene. Further experiments, including a novel "transgene walking" PCR method, confirmed the presence of deletions. The deletions were unique, patient-specific, present in most circulating GMCs expressing NeoR, and shown to occur at time of GMC production. Unique patient-specific retroviral insertion sites (ISs) were found in all GMCs capable of in vitro expansion/cloning as well. These findings suggest a rare initial gene deletion event and an in vivo survival advantage of rare GMC clones resulting from an anti-HSV-tk immune response and/or ganciclovir treatment. In conclusion, we show that donor mature T cells infused with a T-cell-depleted graft persist in vivo for more than a decade. These cells, containing transgene deletions and subjected to significant in vivo selection, represent a small fraction of T cells infused at transplantation.

The monitoring of immune reconstitution in murine models of HC transplantation, using accurate and automated methods, is necessary in view of the recent developments of hematopoietic cell (HC) transplantation (including reduced intensity conditioning regimens) as well as emerging immunological concepts (such as the involvement of dendritic cells or regulatory T cells). Here, we describe the use of a single-platform approach based on flow cytometry and tubes that contain a defined number of microbeads to evaluate absolute blood cell counts in mice. This method, previously used in humans to quantify CD34+ stem cells or CD4+ T cells in HIV infected patients, was adapted for mouse blood samples. A CD45 gating strategy in this "lyse no wash" protocol makes it possible to discriminate erythroblasts or red blood cell debris from CD45+ leukocytes, thus avoiding cell loss. Tubes contain a lyophilized brightly fluorescent microbead pellet permitting the acquisition of absolute counts of leukocytes after flow cytometric analysis. We compared this method to determine absolute counts of circulating cells with another method combining Unopette reservoir diluted blood samples, hemocytometer, microscopic examination and flow cytometry. The sensitivity of this single-platform approach was evaluated in different situations encountered in allogeneic HC transplantation, including immune cell depletion after different conditioning regimens, activation status of circulating cells after transplantation, evaluation of in vivo cell depletion and hematopoietic progenitor mobilization in the periphery. This single-platform flow cytometric assay can also be proposed to standardize murine (or other mammalian species) leukocyte count determination for physiological, pharmacological/toxicological and diagnostic applications in veterinary practice.

We have recently shown that the use of allogeneic granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood hematopoietic stem cell transplantation (PBHSCT), as compared with bone marrow transplantation (BMT), is associated with increased titers of antibodies (Abs) directed against red blood cell ABO antigens. To further evaluate the influence of a G-CSF-mobilized PBHSCT graft on alloimmune Ab responses, we examined the frequency of anti-HLA Abs after transplantation in the setting of the same randomized study, comparing PBHSCT with BMT in adults. Anti-HLA Ab presence was determined by complement-dependent cytotoxicity assay (CDC) and flow cytometry in the recipient before and 30 days after transplantation as well as in the donor before graft donation. The use of PBHSCT was significantly associated with increased detection of anti-HLA immunoglobulin G (IgG) Abs early after transplantation as evidenced by flow cytometry (11 of 24 versus 4 of 27 transplant recipients, P =.03) and, less so, by CDC (5 of 24 versus 1 of 27 transplant recipients, P =.09). The difference between PBHSCT and BMT was further heightened when analysis was restricted to anti-HLA IgG Ab-negative donor/recipient pairs. In such a setting, early anti-HLA Ab was never detected after BMT but was repeatedly detected after PBHSCT (flow cytometry, 6 of 18 versus 0 of 17 transplant recipients, P =.02; CDC, 4 of 23 versus 0 of 26 transplant recipients, P =.04). Importantly, the PBHSCT-associated increase in anti-HLA Ab detection was observed despite a reduction in the median number of platelet-transfusion episodes per patient in PBHSC transplant versus BM transplant recipients (3 platelet-transfusion episodes [range, 1-21] in PBHSCT group vs 6 platelet-transfusion episodes [range, 3-33] in the BMT group; P =.02). In conclusion, this study strongly suggests that G-CSF-mobilized PBHSCT results in an increased incidence of circulating anti-HLA Abs and further confirms that the use of such a graft alters alloimmune Ab responses.

Several acute hemolysis episodes, sometimes lethal, have been recently described after transplantation of allogeneic peripheral blood hematopoietic stem cells (PBHSCs). Hemolysis resulted from the production of donor-derived antibodies (Abs) directed at ABO antigens (Ags) present on recipient red blood cells (RBCs). A multicenter randomized phase III clinical study comparing allogeneic PBHSC transplantation (PBHSCT) versus bone marrow hematopoietic stem cell transplantation (BMHSCT) has been conducted in France. In the course of this study, serum anti-A and/or anti-B Ab titers were compared before the conditioning regimen and on day +30 after transplantation in 49 consecutive evaluable PBHSCT (n = 21) or BMHSCT (n = 28) recipients. PBHSCT resulted in a higher frequency of increased anti-A and/or anti-B Ab titers 30 days after transplantation as compared to BMHSCT: 8 (38%) of 21 versus 3 (11%) of 28 (P =.04). In PBHSCT recipients, increased titers were observed mostly after receiving a minor ABO mismatch transplant: 5 of 7 versus 3 of 14 in the absence of any minor ABO mismatch (P =.05), whereas this was not the case after BMHSCT: 1 of 8 versus 2 of 20. Anti-A and/or anti-B serum Abs detectable at day +30 after PBHSCT were always directed against A and/or B Ags absent both on donor and recipient RBCs. Finally, 3 of 21 PBHSCT versus 0 of 28 BMHSCT recipients developed anti-allogeneic RBC Abs other than ABO (P =.07). Overall, the data strongly suggest that immunohematologic reconstitution differs significantly after granulocyte colony-stimulating factor-mobilized PBHSCT when compared to BMHSCT. Such a difference could contribute to the acute hemolysis described after PBHSCT as well as to distinct alloreactivity after PBHSCT.

Administration of donor T cells expressing the herpes simplex-thymidine kinase (HS-tk) with a hematopoietic stem cell (HSC) transplantation could allow, if graft-versus-host disease (GVHD) was to occur, a selective in vivo depletion of these T cells by the use of ganciclovir (GCV). The study evaluates the feasibility of such an approach. Escalating numbers of donor HS-tk-expressing CD3(+) gene-modified cells (GMCs) are infused with a T-cell-depleted bone marrow transplantation (BMT). Twelve patients with hematological malignancies received 2 x 10(5) (n = 5), 6 x 10(5) (n = 5), or 20 x 10(5) (n = 2) donor CD3(+) GMCs/kg with a BMT from a human leukocyte antigen (HLA)-identical sibling. No acute toxicity was associated with GMC administration. An early increase of circulating GMCs followed by a progressive decrease and long-lasting circulation of GMCs was documented. GCV treatment resulted in significant rapid decrease in circulating GMCs. Three patients developed acute GVHD, with a grade of at least II, while one patient developed chronic GVHD. Treatment with GCV alone was associated with a complete remission (CR) in 2 patients with acute GVHD, while the addition of glucocorticoids was necessary to achieve a CR in the last case. Long-lasting CR occurred with GCV treatment in the patient with chronic GVHD. Unfortunately, Epstein-Barr virus-lymphoproliferative disease occurred in 3 patients. Overall, the administration of low numbers of HS-tk-expressing T cells early following an HLA-identical BMT is associated with no acute toxicity, persistent circulation of the GMCs, and GCV-sensitive GVHD. Such findings open the way to the infusion of higher numbers of gene-modified donor T cells to enhance post-BMT immune competence while preserving GCV-sensitive alloreactivity.

Since low T cell counts evaluated 1 month after allogeneic bone marrow transplantation (BMT) are associated with an increased risk of leukemia relapse (Powles et al., Blood 1998; 91: 3481-3486), we compared, in a randomized multicentric clinical study, the peripheral blood cells obtained 30 days after allogeneic BMT vs allogeneic G-CSF-mobilized peripheral blood stem cell transplantation (BCT) in an HLA-identical setting. T cell counts were higher 30 days after BCT (718+/-142 cells/microl, n = 20) than after BMT (271+/-53 cells/microl, n = 26, P = 0.006). However, T cells were less activated after BCT than after BMT, as demonstrated by a lower expression level of CD25 and a lower percentage of HLA-DR+ and CD95+ T cells. Furthermore, CD4+, CD8+ and CD45RA+ post-BCT T cell counts correlated with the number of cells infused with the PBSC graft, while such a correlation was not observed between post-BMT counts and BM graft cell numbers, suggesting that the intensity of post-transplant peripheral lymphoid expansion and/or deletion differed between BCT and BMT. A comparison of the input of T cells expressing different CD45 isoforms with the post-transplant cell recovery further confirmed that, within the CD4+ T cell subset, post-transplant expansions occurred at a higher level after BMT than after BCT, affecting mainly the CD4+ CD45RO+ subset. Altogether, our data demonstrate for the first time in a randomized setting that homeostasis of the T cell pool is less altered early after BCT than after BMT. This may have a strong impact on the graft-versus-leukemia (GVL) effect and subsequent relapse rate.

Background: We are presently investigating the therapeutic potential of herpes simplex-thymidine kinase-expressing donor T cells in the setting of a T cell-depleted allogeneic bone marrow transplantation. The generation, expansion, and selection of the gene-modified T cells require a 12-day ex vivo culture period in high-dose interleukin (IL)-2 that could significantly alter their in vivo alloreactivity., Methods: We evaluated the alloreactive potential of such cultured cells in a murine allogeneic bone marrow transplantation model., Results: The present studies demonstrate that ex vivo-expanded cultured T cells are capable of strong alloreactivity as evidenced by the occurrence of lethal acute graft-versus-host disease (GVHD). However, GVHD mortality after administration of the cultured T cells occurred later than after the administration of a same number of fresh T cells. Similar kinetics of GVHD-induced mortality between cultured and fresh T cells required a 10-fold increase in the number of cultured T cells, indicating a reduced alloreactive potential of these cells. The addition of a 2-day "resting" period in low-dose IL-2 resulted in T cells with enhanced alloreactive potential identical to the alloreactivity observed with fresh T cells., Conclusion: Ex vivo IL-2-expanded T cells are capable of significant in vivo alloreactivity. However, an increase in the number of cultured T cells administered or the introduction of a short resting culture period prior to infusion is necessary in order to achieve in vivo alloreactivity identical to the alloreactivity observed with fresh T cells.

Cytokines may be helpful in promoting hematopoietic reconstitution but have also an impact on the cellular interactions that contribute to GvHD and immunologic graft rejection. Because IL-12 is emerging as a central cytokine in immune response, we have investigated its levels in serum samples of patients undergoing bone marrow transplantation and transplant-related events. A double-antibody radioimmunoassay method for monitoring levels of endogenous IL-12, before and after allogeneic (27 patients) or autologous (19 patients) bone marrow transplantation, was used. The serum levels of IL-12 after allogeneic BMT were found to be relatively low (140-300 pg/ml) and similar to the IL-12 levels in the healthy donors (183 pg/ml). Seric IL-12 levels following autologous BMT (350 pg/ml) were higher than those observed in patients receiving an allogeneic BMT and in healthy donors. Our data indicate that the occurrence of GvHD and the development of infection after allogeneic BMT are not associated with IL-12 induction which suggests a possible down-regulation due to immunosuppressive treatment.

We have investigated tumor necrosis factor-alpha levels in serum samples of patients before and after allogenic (16 patients) or autologous (8 patients) bone marrow transplantation. A sensitive immunoradiometric assay for monitoring levels of endogenous tumor necrosis factor-alpha was used. The serum levels of tumor necrosis factor-alpha were found to be relatively low (ranging from less than 15 to 77 pg/ml). Among 13 patients having graft-versus-host disease following allogeneic bone marrow transplantation 8 patients did not have detectable tumor necrosis factor-alpha (less than 15 pg/ml) while 4 out of 8 patients undergoing autologous bone marrow transplantation had detectable tumor necrosis factor-alpha levels (15 pg/ml), indicating a lack of correlation between tumor necrosis factor-alpha serum levels and the occurrence of graft-versus-host disease. Because the tumor necrosis factor-alpha levels detected in patient sera could be regulated by TNF-receptor expression, the presence of TNF-receptor on patients' peripheral blood mononuclear cells was also studied using fluorescent liposome-conjugated tumor necrosis factor-alpha and immunofluorescence analysis. Our data indicate that peripheral blood mononuclear cells of some patients receiving either autologous or allogeneic bone marrow transplantation expressed significant levels of TNF-receptors, suggesting a lack of correlation between TNF-receptor expression and graft-versus-host disease development.