CD83 Antibody Inhibits Human B Cell Responses to Antigen as well as Dendritic Cell-Mediated CD4 T Cell Responses.

Anti-CD83 Ab capable of Ab-dependent cellular cytotoxicity can deplete activated CD83+ human dendritic cells, thereby inhibiting CD4 T cell-mediated acute graft-versus-host disease. As CD83 is also expressed on the surface of activated B lymphocytes, we hypothesized that anti-CD83 would also inhibit B cell responses to stimulation. We found that anti-CD83 inhibited total IgM and IgG production in vitro by allostimulated human PBMC. Also, Ag-specific Ab responses to immunization of SCID mice xenografted with human PBMC were inhibited by anti-CD83 treatment. This inhibition occurred without depletion of all human B cells because anti-CD83 lysed activated CD83+ B cells by Ab-dependent cellular cytotoxicity and spared resting (CD83-) B cells. In cultured human PBMC, anti-CD83 inhibited tetanus toxoid-stimulated B cell proliferation and concomitant dendritic cell-mediated CD4 T cell proliferation and expression of IFN-γ and IL-17A, with minimal losses of B cells (<20%). In contrast, the anti-CD20 mAb rituximab depleted >80% of B cells but had no effect on CD4 T cell proliferation and cytokine expression. By virtue of the ability of anti-CD83 to selectively deplete activated, but not resting, B cells and dendritic cells, with the latter reducing CD4 T cell responses, anti-CD83 may be clinically useful in autoimmunity and transplantation. Advantages might include inhibited expansion of autoantigen- or alloantigen-specific B cells and CD4 T cells, thus preventing further production of pathogenic Abs and inflammatory cytokines while preserving protective memory and regulatory cells.

Myeloma-induced alloreactive T cells arising in myeloma-infiltrated bones include double-positive CD8+CD4+ T cells: evidence from myeloma-bearing mouse model.

The graft-versus-myeloma (GVM) effect represents a powerful form of immune attack exerted by alloreactive T cells against multiple myeloma cells, which leads to clinical responses in multiple myeloma transplant recipients. Whether myeloma cells are themselves able to induce alloreactive T cells capable of the GVM effect is not defined. Using adoptive transfer of T naive cells into myeloma-bearing mice (established by transplantation of human RPMI8226-TGL myeloma cells into CD122(+) cell-depleted NOD/SCID hosts), we found that myeloma cells induced alloreactive T cells that suppressed myeloma growth and prolonged survival of T cell recipients. Myeloma-induced alloreactive T cells arising in the myeloma-infiltrated bones exerted cytotoxic activity against resident myeloma cells, but limited activity against control myeloma cells obtained from myeloma-bearing mice that did not receive T naive cells. These myeloma-induced alloreactive T cells were derived through multiple CD8(+) T cell divisions and enriched in double-positive (DP) T cells coexpressing the CD8αα and CD4 coreceptors. MHC class I expression on myeloma cells and contact with T cells were required for CD8(+) T cell divisions and DP-T cell development. DP-T cells present in myeloma-infiltrated bones contained a higher proportion of cells expressing cytotoxic mediators IFN-γ and/or perforin compared with single-positive CD8(+) T cells, acquired the capacity to degranulate as measured by CD107 expression, and contributed to an elevated perforin level seen in the myeloma-infiltrated bones. These observations suggest that myeloma-induced alloreactive T cells arising in myeloma-infiltrated bones are enriched with DP-T cells equipped with cytotoxic effector functions that are likely to be involved in the GVM effect.

Mesenchymal stromal cells transiently alter the inflammatory milieu post-transplant to delay graft-versus-host disease.

BACKGROUND: Multipotent mesenchymal stromal cells suppress T-cell function in vitro, a property that has underpinned their use in treating clinical steroid-refractory graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. However the potential of mesenchymal stromal cells to resolve graft-versus-host disease is confounded by a paucity of pre-clinical data delineating their immunomodulatory effects in vivo. DESIGN AND METHODS: We examined the influence of timing and dose of donor-derived mesenchymal stromal cells on the kinetics of graft-versus-host disease in two murine models of graft-versus-host disease (major histocompatibility complex-mismatched: UBI-GFP/BL6 [H-2(b)]→BALB/c [H-2(d)] and the sibling transplant mimic, UBI-GFP/BL6 [H-2(b)]→BALB.B [H-2(b)]) using clinically relevant conditioning regimens. We also examined the effect of mesenchymal stromal cell infusion on bone marrow and spleen cellular composition and cytokine secretion in transplant recipients. RESULTS: Despite T-cell suppression in vitro, mesenchymal stromal cells delayed but did not prevent graft-versus-host disease in the major histocompatibility complex-mismatched model. In the sibling transplant model, however, 30% of mesenchymal stromal cell-treated mice did not develop graft-versus-host disease. The timing of administration and dose of the mesenchymal stromal cells influenced their effectiveness in attenuating graft-versus-host disease, such that a low dose of mesenchymal stromal cells administered early was more effective than a high dose of mesenchymal stromal cells given late. Compared to control-treated mice, mesenchymal stromal cell-treated mice had significant reductions in serum and splenic interferon-γ, an important mediator of graft-versus-host disease. CONCLUSIONS: Mesenchymal stromal cells appear to delay death from graft-versus-host disease by transiently altering the inflammatory milieu and reducing levels of interferon-γ. Our data suggest that both the timing of infusion and the dose of mesenchymal stromal cells likely influence these cells' effectiveness in attenuating graft-versus-host disease.

Reduced intensity conditioning for hematopoietic stem cell transplantation: has it achieved all it set out to?

At its inception, reduced intensity conditioning (RIC) was heralded as a means to limit toxicity after hematopoietic stem cell transplantation (HSCT), especially for the older patient demographic. The aim was to promote the inherent anti-leukemic activity of the transplant whilst reducing toxicity and transplant-related mortality (TRM). More than 10 years on, much has been learnt about the role of conditioning in determining outcomes after transplantation. The use of RIC as a preparative regimen has increased the number of patients that can benefit from HSCT because the initial therapy is less toxic. However, many of the early pioneers of RIC quickly realized that the toxicity from graft-versus-host disease (GvHD) was equally as potent as that from conditioning. Furthermore, questions remain concerning the efficacy of RIC regimens in retaining anti-leukemic immunity, especially in cases of aggressive disease. The undoubted synergy between chemotherapeutic and immunologic treatment of malignancy means that reduction of conditioning intensity to minimal levels may not be entirely logical.

GSK3 inhibition prevents lethal GVHD in mice.

Graft-versus-host disease (GVHD) is a major contributor to transplant-related mortality and morbidity after allogeneic stem cell transplantation. Despite advancements in tissue-typing techniques, conditioning regimens, and therapeutic intervention, the incidence rate of GVHD remains high. GVHD is caused by alloreactive donor T cells that infiltrate and destroy host tissues (e.g., skin, liver, and gut). Therefore, GVHD is prevented and treated with therapeutics that suppress proinflammatory cytokines and T-cell function (e.g., cyclosporine, glucocorticoids). Here we report that the small molecule inhibitor of glycogen synthase kinase 3, 6-bromoindirubin 3'-oxime (BIO), prevents lethal GVHD in a humanized xenograft model in mice. BIO treatment did not affect donor T-cell engraftment, but suppressed their activation and attenuated bone marrow and liver destruction mediated by activated donor T cells. Glycogen synthase kinase 3 inhibition modulated the Th1/Th2 cytokine profile in vitro and suppressed activation of signal transducers and activators of transcription 1 and 3 signaling pathways both in vitro and in vivo. Importantly, human T cells derived from BIO-treated mice were able to mediate anti-tumor effects in vitro, and BIO did not affect stem cell engraftment and multilineage reconstitution in a mouse model of transplantation. These data demonstrate that inhibition of glycogen synthase kinase 3 can potentially abrogate GVHD without compromising the efficacy of transplantation.

Unraveling the "known unknowns": lessons and reflections from the new directions in leukemia research 2012 conference.

Patients diagnosed with leukemia approach their treatment with the hope of cure despite the effect on their quality of life. Some patients will be cured, others will die from treatment, and some will die of their disease. A common theme at the New Directions in Leukemia Research (NDLR 2012) meeting was that cure will come if the drivers of the disease are better understood. Key messages included the power of combination platforms to understand the genetic and epigenetic modifications in leukemia to enable development of rational therapies, which can be tested via new clinical trial designs ensuring rapid clinical implementation.

Comprehensive transcriptome and immunophenotype analysis of renal and cardiac MSC-like populations supports strong congruence with bone marrow MSC despite maintenance of distinct identities.

Cells resembling bone marrow mesenchymal stem cells (MSC) have been isolated from many organs but their functional relationships have not been thoroughly examined. Here we compared the immunophenotype, gene expression, multipotency and immunosuppressive potential of MSC-like colony-forming cells from adult murine bone marrow (bmMSC), kidney (kCFU-F) and heart (cCFU-F), cultured under uniform conditions. All populations showed classic MSC morphology and in vitro mesodermal multipotency. Of the two solid organ-specific CFU-F, only kCFU-F displayed suppression of T-cell alloreactivity in vitro, albeit to a lesser extent than bmMSC. Quantitative immunophenotyping using 81 phycoerythrin-conjugated CD antibodies demonstrated that all populations contained high percentages of cells expressing diagnostic MSC surface markers (Sca1, CD90.2, CD29, CD44), as well as others noted previously on murine MSC (CD24, CD49e, CD51, CD80, CD81, CD105). Illumina microarray expression profiling and bioinformatic analysis indicated a correlation of gene expression of 0.88-0.92 between pairwise comparisons. All populations expressed approximately 66% of genes in the pluripotency network (Plurinet), presumably reflecting their stem-like character. Furthermore, all populations expressed genes involved in immunomodulation, homing and tissue repair, suggesting these as conserved functions for MSC-like cells in solid organs. Despite this molecular congruence, strong biases in gene and protein expression and pathway activity were seen, suggesting organ-specific functions. Hence, tissue-derived MSC may also retain unique properties potentially rendering them more appropriate as cellular therapeutic agents for their organ of origin.

CD34+ cord blood DC-induced antitumor lymphoid cells have efficacy in a murine xenograft model of human ALL.

Acute lymphocytic leukemia (ALL) patients who relapse after transplantation have few therapeutic options. An immunotherapeutic approach that enhances the graft versus leukemia effect may improve their survival. We postulate that cytotoxic T lymphocytes (CTLs) generated from total RNA loaded cord blood CD34+-derived dendritic cells can control the kinetics of leukemic growth in a nonobese diabetic/severe combined immunodeficient (NOD-SCID) mouse model of human ALL. CD34+-derived dendritic cells electroporated with total RNA from an ALL xenograft generate antileukemic CTL with specificity for the ALL xenograft while sparing autologous cord blood mononuclear cells. The CD3+ T-cell compartment of the CTL was dominated by CD4+ T cells, although CD8+ T cells accounted for an average of 30% of the CD3+ T cells present. Expansion of both CD4+ and CD8+ memory and terminal effector memory subsets from predominantly naive cells was evident. Natural killer (NK) cells accounted for an average of 13% of the final antitumor lymphoid cells produced. Blocking experiments confirmed that the CD8+ T-cell compartment was responsible for the antileukemic activity of the polyclonal CTL pool. Administration of antileukemic CTL to NOD-SCID mice bearing ALL xenograft cells was able to delay, but not prevent the growth of ALL in vivo. Coadministration of antigen-loaded antigen-presenting cells did not further improve upon the delay in ALL engraftment kinetics observed with CTL alone. The efficacy of adoptively transferred polyclonal CTL can be improved with coadministration of recombinant human interleukin-2. However, in NOD-SCID mice, the efficacy of these adoptively transferred cells is masked by interleukin-2 stimulation of murine NK cells, which facilitate killing of ALL cells. Our data highlights the role for NK cells in antileukemic responses posttransplant. Collectively, our results support the notion that ALL-specific adoptive immunotherapy could be used clinically and provide an alternative strategy for preventing and treating disease relapse posttransplant and that the success of this therapy is likely to be maximized if given in the setting of minimal residual disease.

Mobilisation strategies for normal and malignant cells.

This review evaluates the latest information on the mobilisation of haemopoietic stem cells for transplantation, with the focus on what is the current best practice and how new understanding of the bone marrow stem cell niche provides new insights into optimising mobilisation regimens. The review then looks at the mobilisation of mesenchymal stromal cells, immune cells as well as malignant cells and what clinical implications there are.

Antibody to the dendritic cell surface activation antigen CD83 prevents acute graft-versus-host disease.

Allogeneic (allo) hematopoietic stem cell transplantation is an effective therapy for hematological malignancies but it is limited by acute graft-versus-host disease (GVHD). Dendritic cells (DC) play a major role in the allo T cell stimulation causing GVHD. Current immunosuppressive measures to control GVHD target T cells but compromise posttransplant immunity in the patient, particularly to cytomegalovirus (CMV) and residual malignant cells. We showed that treatment of allo mixed lymphocyte cultures with activated human DC-depleting CD83 antibody suppressed alloproliferation but preserved T cell numbers, including those specific for CMV. We also tested CD83 antibody in the human T cell-dependent peripheral blood mononuclear cell transplanted SCID (hu-SCID) mouse model of GVHD. We showed that this model requires human DC and that CD83 antibody treatment prevented GVHD but, unlike conventional immunosuppressants, did not prevent engraftment of human T cells, including cytotoxic T lymphocytes (CTL) responsive to viruses and malignant cells. Immunization of CD83 antibody-treated hu-SCID mice with irradiated human leukemic cell lines induced allo antileukemic CTL effectors in vivo that lysed (51)Cr-labeled leukemic target cells in vitro without further stimulation. Antibodies that target activated DC are a promising new therapeutic approach to the control of GVHD.

Reduced intensity conditioning for allogeneic hematopoietic stem-cell transplant determines the kinetics of acute graft-versus-host disease.

BACKGROUND: Preparative myeloablative conditioning regimens for allogeneic hematopoietic stem-cell transplantation (HSCT) may control malignancy and facilitate engraftment but also contribute to transplant related mortality, cytokine release, and acute graft-versus-host disease (GVHD). Reduced intensity conditioning (RIC) regimens have decreased transplant related mortality but the incidence of acute GVHD, while delayed, remains unchanged. There are currently no in vivo allogeneic models of RIC HSCT, limiting studies into the mechanism behind RIC-associated GVHD. METHODS: We developed two RIC HSCT models that result in delayed onset GVHD (major histocompatibility complex mismatched (UBI-GFP/BL6 [H-2]-->BALB/c [H-2]) and major histocompatibility complex matched, minor histocompatibility mismatched (UBI-GFP/BL6 [H-2]-->BALB.B [H-2])) enabling the effect of RIC on chimerism, dendritic cell (DC) chimerism, and GVHD to be investigated. RESULTS: In contrast with myeloablative conditioning, we observed that RIC-associated delayed-onset GVHD is characterized by low production of tumor necrosis factor-alpha, maintenance of host DC, phenotypic DC activation, increased T-regulatory cell numbers, and a delayed emergence of activated donor DC. Furthermore, changes to the peritransplant milieu in the recipient after RIC lead to the altered activation of DC and the induction of T-regulatory responses. Reduced intensity conditioning recipients suffer less early damage to GVHD target organs. However, as donor cells engraft, activated donor DC and rising levels of tumor necrosis factor-alpha are associated with a later onset of severe GVHD. CONCLUSIONS: Delineating the mechanisms underlying delayed onset GVHD in RIC HSCT recipients is vital to improve the prediction of disease onset and allow more targeted interventions for acute GVHD.

RNA loading of leukemic antigens into cord blood-derived dendritic cells for immunotherapy.

The manipulation of dendritic cells (DCs) ex vivo to present tumor-associated antigens for the activation and expansion of tumor-specific cytotoxic T lymphocytes (CTLs) attempts to exploit these cells' pivotal role in immunity. However, significant improvements are needed if this approach is to have wider clinical application. We optimized a gene delivery protocol via electroporation for cord blood (CB) CD34(+) DCs using in vitro-transcribed (IVT) mRNA. We achieved > 90% transfection of DCs with IVT-enhanced green fluorescent protein mRNA with > 90% viability. Electroporation of IVT-mRNA up-regulated DC costimulatory molecules. DC processing and presentation of mRNA-encoded proteins, as major histocompatibility complex/peptide complexes, was established by CTL assays using transfected DCs as targets. Along with this, we also generated specific antileukemic CTLs using DCs electroporated with total RNA from the Nalm-6 leukemic cell line and an acute lymphocytic leukemia xenograft. This significant improvement in DC transfection represents an important step forward in the development of immunotherapy protocols for the treatment of malignancy.

Characterization of childhood acute lymphoblastic leukemia xenograft models for the preclinical evaluation of new therapies.

Continuous xenografts from 10 children with acute lymphoblastic leukemia (ALL) were established in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Relative to primary engrafted cells, negligible changes in growth rates and immunophenotype were observed at second and third passage. Analysis of clonal antigen receptor gene rearrangements in 2 xenografts from patients at diagnosis showed that the pattern of clonal variation observed following tertiary transplantation in mice exactly reflected that in bone marrow samples at the time of clinical relapse. Patients experienced diverse treatment outcomes, including 5 who died of disease (median, 13 months; range, 11-76 months, from date of diagnosis), and 5 who remain alive (median, 103 months; range, 56-131 months, following diagnosis). When stratified according to patient outcome, the in vivo sensitivity of xenografts to vincristine and dexamethasone, but not methotrexate, differed significantly (P =.028, P =.029, and P =.56, respectively). The in vitro sensitivity of xenografts to dexamethasone, but not vincristine, correlated significantly with in vivo responses and patient outcome. This study shows, for the first time, that the biologic and genetic characteristics, and patterns of chemosensitivity, of childhood ALL xenografts accurately reflect the clinical disease. As such, they provide powerful experimental models to prioritize new therapeutic strategies for future clinical trials.

The nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of childhood acute lymphoblastic leukemia reveals intrinsic differences in biologic characteristics at diagnosis and relapse.

Acute lymphoblastic leukemia cells from 19 children, including 7 who remain in first complete remission (CR1), were engrafted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. High-level infiltration of bone marrow, spleen, and liver was observed, with variable infiltration of other organs. The immunophenotypes of xenografts were essentially unaltered compared with the original patient sample. In addition, sequencing of the entire p53 coding region revealed no mutations in 14 of 14 xenografts (10 from patients at diagnosis and 4 at relapse). Cells harvested from the spleens of engrafted mice readily transferred the leukemia to secondary and tertiary recipients. To correlate biologic characteristics of xenografts with clinical and prognostic features of the patients, the rates at which individual leukemia samples engrafted in NOD/SCID mice were analyzed. Differences in biologic correlates were encountered depending on stage of disease: a direct correlation was observed between the rate of engraftment and length of CR1 for samples harvested at relapse (r = 0.96; P =.002), but not diagnosis (r = 0.38; P =.40). In contrast, the in vivo responses of 6 xenografts to vincristine showed a direct correlation (r = 0.96; P =.002) between the length of CR1 and the rate at which the leukemia cell population recovered following vincristine treatment, regardless of whether the xenografts were derived from patients at diagnosis or relapse. This study supports previous findings that the NOD/SCID model of childhood ALL provides an accurate representation of the human disease and indicates that it may be of value to predict relapse and design alternative treatment strategies in a patient-specific manner.