2: Donor-Recipient Host-Versus-Graft Human Leukocyte Antigen Mismatches and Outcome of Cord Blood Transplants

Digitalitzat per Artypla

Phase I Clinical Trial of Systemically Administered TUSC2(FUS1)-Nanoparticles Mediating Functional Gene Transfer in Humans

Background: Tumor suppressor gene TUSC2/FUS1 (TUSC2) is frequently inactivated early in lung cancer development. TUSC2 mediates apoptosis in cancer cells but not normal cells by upregulation of the intrinsic apoptotic pathway. No drug strategies currently exist targeting loss-of–function genetic abnormalities. We report the first in-human systemic gene therapy clinical trial of tumor suppressor gene TUSC2. Methods: Patients with recurrent and/or metastatic lung cancer previously treated with platinum-based chemotherapy were treated with escalating doses of intravenous N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP):cholesterol nanoparticles encapsulating a TUSC2 expression plasmid (DOTAP:chol-TUSC2) every 3 weeks. Results: Thirty-one patients were treated at 6 dose levels (range 0.01 to 0.09 milligrams per kilogram). The MTD was determined to be 0.06 mg/kg. Five patients achieved stable disease (2.6–10.8 months, including 2 minor responses). One patient had a metabolic response on positron emission tomography (PET) imaging. RT-PCR analysis detected TUSC2 plasmid expression in 7 of 8 post-treatment tumor specimens but not in pretreatment specimens and peripheral blood lymphocyte controls. Proximity ligation assay, performed on paired biopsies from 3 patients, demonstrated low background TUSC2 protein staining in pretreatment tissues compared with intense (10–25 fold increase) TUSC2 protein staining in posttreatment tissues. RT-PCR gene expression profiling analysis of apoptotic pathway genes in two patients with high posttreatmen

Functional assessment and specific depletion of alloreactive human T cells using flow cytometry

Human T-cell alloreactivity plays an important role in many disease processes, including the rejection of solid organ grafts and graft-versus-host disease (GVHD) following allogeneic stem cell transplantation. To develop a better understanding of the T cells involved in alloreactivity in humans, we developed a cytokine flow cytometry (CFC) assay that enabled us to characterize the phenotypic and functional characteristic of T cells responding to allogeneic stimuli. Using this approach, we determined that most T-cell alloreactivity resided within the CD4+ T-cell subset, as assessed by activation marker expression and the production of effector cytokines (eg, tumor necrosis factor α [TNF]α) implicated in human GVHD. Following prolonged stimulation in vitro using either allogeneic stimulator cells or viral antigens, we found that coexpression of activation markers within the CD4+ T-cell subset occurred exclusively within a subpopulation of T cells that significantly increased their surface expression of CD4. We then developed a simple sorting strategy that exploited these phenotypic characteristics to specifically deplete alloreactive T cells while retaining broad specificity for other stimuli, including viral antigens and third-party alloantigens. This approach also was applied to specifically enrich or deplete human virus-specific T cells

Long-Term Follow-Up of Patients Who Experienced Graft Failure Postallogeneic Progenitor Cell Transplantation. Results of a Single Institution Analysis

AbstractThe long-term outcome of graft failure after allogeneic stem cell transplantation (SCT) has not been well described. To fill this knowledge gap we performed a retrospective analysis of patients with graft failure over a 10-year time period in a single institution. Cases were included for analysis if they had failed to achieve an absolute neutrophil count (ANC) of 500/μL or more by 28 days post-SCT or 42 days after cord blood transplantation (primary graft failure); had a decrease in their ANC to <500/mL for 3 consecutive days after having achieved neutrophil engraftment (secondary graft failure); or failed to have evidence of at least 5% or more donor cell engraftment (primary graft failure with autologous reconstitution). Among 1726 patients who underwent allografts from January 1, 1990, through December 31, 2000, we identified 68 patients with graft failure. The 1-, 2-, and 5-year overall survival (OS) for all patients was 31%, 24%, and 15%. A diagnosis of acute leukemia was a significant predictor for poor survival on multivariate analysis. We conclude that graft failure is an uncommon complication postallogeneic SCT, and is associated with poor outcomes. Collection of autologous stem cells prior to high-risk allografting can salvage a fraction of patients and lead to prolonged survivals

Cell-Autonomous Upregulation of Dendritic Cell Immunocompetence Is Antigen-Dependent

Abstract Introduction: Dendritic cells (DCs) are the master regulators of the adaptive immune response. In previous experiments, the double-loading of DCs with matched sets of MHC class I and class II repertoires (i.e. mRNA and cell lysate derived from the same source) appeared to enhance T-cell responses by maximizing the availability of T-cell help. T-cell help, provided by accessory CD4+ lymphocytes, elevates DC immunocompetence in response to antigen-specific interactions between the T-cell receptor and the peptide antigen/MHC class II complex. While T-cell help is specific and aids in the priming of only cognate CD8+ effectors, the mechanism governing this specificity is not understood. Here, we have explored the hypothesis that the specificity of T-cell help may be initiated by the DC itself upon detection of an antigen-dependent signal: the loading of the DC with matched sets of class I and class II determinants. Methods: To examine this hypothesis, we doubly-loaded DCs with either matched sets of antigenic determinants derived from acute myelogenous leukemia (AML) mRNA and lysate preparations, or, mismatched sets of antigenic preparations derived from AML products, the human TF-1a erythroblastic cell line, or the murine FBMD-1 stromal cell line. Following loading and maturation, we analyzed DCs for markers of immunocompetence such as IL-12 secretion and CD83 surface expression. Using specific siRNA oligonucleotides, we also examined a possible role for the intracellular CD63 tetraspanin in this process. DCs were generated by CD14 magnetic selection of apheresis products and six days of culture in GM-CSF and IL-4. Analysis of immature DCs at this time point by flow cytometry typically showed a CD3+ content equivalent to or less than that of the isotype control. DCs were then loaded with either matched or mismatched sets of determinants (as described above) and matured. Results: Matched-loaded DCs exhibited a 4-fold increase in IL-12 secretion over unloaded DCs while mismatch-loaded DCs showed only a 0.7-fold increase (similar to controls) (p = 0.0086). Moreover, matched-loaded DCs demonstrated a 21% increase in CD83 surface expression over unloaded/singly-loaded controls while mismatch-loaded DCs showed only a 6% increase (statistically identical to controls) (p = 0.009). In a single-antigen model system, electroporation of CD63 siRNA could reduce IL-12 secretion from matched-loaded DCs by 60% in comparison to matched-loaded DCs electroporated with non-targeting siRNAs. Secretion of non-Th-1 cytokines (i.e. IL-10) was unaffected. Conclusions: It appears that DC immunocompetence may be upregulated in a cell-autonomous, antigen-dependent fashion. Such upregulation is induced by the loading of DCs with matched sets of MHC class I and class II antigenic determinants and does not occur if DCs are loaded with mismatched determinants. CD63 appears to be involved in this process. CD63 is a member of the tetraspanin family of integral membrane proteins, molecules that facilitate the interaction of membrane and intracellular signaling complexes. In DCs, CD63 is localized exclusively to lysosomal exosomes, sites at which MHC class II, MHC class I, and phagocytosed antigens also co-localize. The data are suggestive of a cross-licensing model by which T-cell help might first be solicited by DCs loaded with matched antigenic determinants. Permission granted by a receptive DC, the helper T-cell might then license DC priming of CD8+ responses. Confirmatory studies are in progress

CD25 expression on donor CD4+ or CD8+ T cells is associated with an increased risk for graft-versus-host disease after HLA-identical stem cell transplantation in humans

AbstractGraft-versus-host disease (GVHD) occurs in an unpredictable fashion after 30% to 50% of matched-related transplantations. The presence of increased frequencies of CD4+CD25+ regulatory T cells in donor grafts has been shown to ameliorate GVHD after allogeneic transplantation in murine models. To determine whether a similar relationship exists in humans, we quantitated the coexpression of CD25 on CD4+ and CD8+ T cells within 60 donor grafts infused into matched siblings and examined GVHD incidence in the respective recipients. Recipients in whom GVHD developed received donor grafts containing significantly higher frequencies of CD4+ T cells coexpressing CD25 than those who did not (median, 9.26% vs 2.22%; P = .004). Frequencies of donor graft CD8+ T cells coexpressing CD25 were also higher (0.65% vs 0.14%; P = .002). Furthermore, transplant recipients who received grafts containing fewer CD4+CD25+ and CD8+CD25+ T cells were less likely to acquire acute GVHD, even though these donor-recipient pairs were similar to others with respect to relevant clinical variables. These data suggest that the coexpression of CD4 and CD25 may be insufficient to identify regulatory T cells in humans and that increased frequencies and numbers of CD25+ T cells in donor grafts is associated with GVHD in transplant recipients. (Blood. 2004;103:1140-1146

Characterization of optimal T Cell/Dendritic Cell (DC) Co-Culture Conditions for Ex Vivo Expansion of Antigen-Specific Human T Cells

Abstract Adoptive T cell immunotherapy has demonstrated clinical efficacy in controlling reactivation of human herpesviruses (e.g., cytomegalovirus, CMV and Epstein-Barr Virus, EBV) that are known etiologic agents of life-threatening illness in immunocompromised populations. Early approaches utilized in the expansion of large numbers of human virus-specific CD4+ and CD8+ T cell often involved laborious culture techniques impractical for widespread clinical use in populations at risk, including stem cell transplant (SCT) recipients. Advances in our understanding of the biology of myeloid dendritic cells (DC) can now facilitate more practical approaches for ex vivo antigen-specific T cell expansion. These advances have included novel approaches to isolate human peripheral blood monocytes (e.g., CD14 selection) and the optimization of cytokine cocktails inducing DC maturation. To define the optimal culture conditions for human antigen-specific T cell expansion, we conducted an iterative series of pairwise CMV-specific T cell expansions, in which important determinants of the expansion process could be directly compared. T cells, derived from apheresis products obtained from CMV-seropositive healthy donors, were expanded under GLP-compliant conditions, following incubation with autologous myeloid DC pulsed with overlapping pentadecapeptide pools spanning the CMV pp65 protein. In each case, we assessed the expanded CMV-specific T cell populations by immunophenotyping of the generated DC, and by phenotypic analysis, cytokine flow cytometry (CFC), ELISPOT analysis and HLA-peptide tetramer staining of the expanded T cells. Specifically, we compared:DC generated from monocytes isolated via plastic adherence to those isolated via positive selection of CD14+ T cells using magnetic beads;DC matured using tumor necrosis factor-α (TNFα) alone or in combination with other cytokines (ITIP, containing TNFα, IL-1β, IL-6 and PGE2);the quality of expansions derived from a starting lymphocyte population selected by non-adherence to plastic vs. the CD14-negative fraction in the monocyte selection process.From this series of experiments, we can conclude that while CD14 selection of monocytes results in a more phenotypically homogeneous population of DC, that this does not improve the quantity or quality of expanded virus-specific T cells. When we compared TNFα to ITIP maturation of DC, we found that while ITIP induced significantly higher CD83 expression on DC, TNFα-matured DC favored the maintenance of CMV-specific CD4+ T cells in culture resulting in ultimately greater numbers and functional proportions of tetramer-stained CD8+ T cells. Finally, we consistently found that the use of non-adherent PBMC resulted in dramatically better quantitative expansions of CMV-specific CD4+ and CD8+ T cells, vs. a lymphocyte population obtained following depletion of monocytes using CD14 positive selection. These results suggest a set of optimal T cell/DC co-culture conditions that lead to the generation of the largest numbers of expanded, functional CD4+ and CD8+ T cells, and are likely to have relevance for human trials wherein adoptive immunotherapy and/or DC immunization is planned