Normal Donor Cells Research Articles

Comparison of Certolizumab Pegol, Etanercept, Adalimumab and Infliximab

Purpose: Proinflammatory cytokines, such as interleukin-1ββ (IL-1ββ) are over-expressed by monocytes/macrophages in patients with Crohn's disease (CD). It has been suggested that anti-tumor necrosis factor αα (TNFαα) agents mediate an inhibitory effect on lipopolysaccharide (LPS)-induced cytokine production via membrane TNFαα signalling. This study compared LPS-stimulated production of IL-1ββ in the presence of certolizumab pegol (an antibody Fab' fragment conjugated with polyethylene glycol) and other anti-TNF agents. Methods: Monocytes were positively selected using CD14+ magnetic microbead-associated cell sorting (MACS) from peripheral blood mononuclear cells of normal human donors. Purified monocytes were pre-incubated for 1 hr with certolizumab pegol, etanercept, adalimumab, infliximab (100 μg/mL to 100 pg/mL) or a relevant control. After extensive washing, the monocytes were incubated — with or without LPS (100 ng/mL) — for 4 hours at 37°C. Supernatants were assessed for IL-1ββ (by enzyme-linked immunosorbent assay) and a range of chemokines, cytokines and other proteins (by Luminex). Results: LPS-stimulated production of IL-1ββ by monocytes appeared to be completely inhibited in a dose-dependent manner by certolizumab pegol, infliximab and adalimumab. In contrast, etanercept was much less efficient at mediating this activity, causing only partial inhibition of cytokine production. Certolizumab pegol was approximately 100-fold more potent than infliximab and adalimumab at inhibiting the release of IL-1ββ by monocytes. The effects of infliximab and adalimumab were similar. The Luminex analysis of a panel of cytokines and chemokines showed a range of effects with inhibition of IL-10 and IL-12 being the most profound. Again etanercept was not as potent as the other anti-TNFs agents at inhibiting these cytokines. Conclusions: Effective inhibition of IL-1ββ production was seen with certolizumab pegol; inhibition was more potent than with adalimumab or infliximab. Even at high concentrations of etanercept, inhibition of IL-1ββ production was only partial. These are in vitro data, however, the comparative trends in inhibition of cytokine production stimulated by bacterial products appear to reflect the clinical efficacy of these anti-TNF agents in CD. The potent inhibition by certolizumab pegol of cytokine production by monocytes may represent an important mechanism of action in CD.

Allogeneic mesoangioblasts give rise to alpha-sarcoglycan expressing fibers when transplanted into dystrophic mice

Cell therapy for muscular dystrophy involves transplantation of either genetically modified autologous cells or normal donor cells that will be rejected unless the host is adequately immune suppressed. The extent of the immune response appears to be mitigated in this case of stem cells, by immune-suppressive and tolerogenic molecules that they release. We previously reported significant morphological and functional amelioration of a mouse model of limb–girdle muscular dystrophy by transplantation of mesoangioblasts. These are vessel-associated stem cells that can be propagated in vitro and differentiate into several types of mesoderm including skeletal muscle. In these experiments, both donor cells and host were syngeneic (C57Bl/6J) and thus possible immune reaction to the donor cells could not be appreciated. To address this question, we transplanted H2-mismatched mesoangioblasts (BalbC) in the same dystrophic mice, and in addition, we treated the host with different pharmacological drugs (rapamycin, IL-10 or both). The results showed that donor cells give rise to fibers that express the mutated gene product (alpha-sarcoglycan) even in the absence of immune suppression; however, the combined action of rapamycin and IL-10 increases the number of alpha-sarcoglycan expressing fibers while reducing the levels of inflammatory cytokines. These results indicate that transplantation of mesoangioblasts into immunologically unrelated host leads to long-term survival of donor cells and this may be further enhanced by appropriate protocols of immune modulation, thus setting the stage for experimentation in large animals and in patients.

Phagocytosis and Production of H2O2 by Human Peripheral Blood Mononuclear Cells from Patients with Obstructive Jaundice

Background: The immune function is altered in jaundiced patients; here, the ability of their peripheral blood mononuclear cells to perform phagocytosis and to release H₂O₂ was analyzed. Methods: Cells from 53 patients before surgery for relief of cholestasis, from 38 patients 1 week and from 15 patients 2 weeks after surgery were separated and cultured for 1 h in the presence of phorbol myristate acetate. H₂O₂ release was evaluated colorimetrically and phagocytosis by the ingestion of Escherichia coli in vitro. Results: Before surgery for relief of cholestasis, the cells of the patients were unable to release H₂O₂, but, after surgery, an increasing percentage of patients had cells that were able to produce H₂O₂ (13% after 1 week; 33% after 2 weeks). This recovery did not correlate with bilirubinemia. When cultured for 1 week in the presence of normal or jaundiced plasma, regardless of collection time, cells of 12/12 patients released H₂O₂, but in lower levels if in the presence of jaundiced plasma. In contrast, H₂O₂ release by normal donor cells was enhanced in the presence of jaundiced plasma. Phagocytosis by cells of the patients was lower, but when present was associated with a significantly higher bactericidal activity. Conclusion: These significant, but reversible alterations of monocyte function in jaundiced patients might contribute to their enhanced susceptibility to surgical complications.

A Randomized Controlled Trial to Compare Once- versus Twice-Daily Filgrastim for Mobilization of Peripheral Blood Stem Cells from Healthy Donors

Although the mobilization of peripheral blood stem cells from normal donors using granulocyte colony-stimulating factor is widely used, the ideal method for the administration of filgrastim has not been determined. Therefore, we compared the efficacy of peripheral blood stem cell mobilization on day 4 of filgrastim between once-daily (group O) and twice-daily (group T) administration of filgrastim at 400 μg/m 2/d. In all, 38 and 34 donors were randomly assigned to groups O and T, respectively. The number of CD34 + cells collected on day 4 was not significantly different (1.74 × 10 6 cells/kg in group O and 2.08 × 10 6 cells/kg in group T, P = .37). The incidence and severity of adverse events were similar in the two groups. The baseline white blood cell count was the strongest predictor of poor mobilization. Donor age, sex, and serum concentrations of several cytokines did not significantly affect the CD34 + cell yield. In conclusion, once-daily administration of filgrastim at 400 μg/m 2/d appeared to be appropriate for the mobilization of CD34 + cells in normal donors when apheresis is planned on day 4 of filgrastim. Selection of a donor with a steady-state white blood cell count of 5.0 × 10 9/L or more may lead to a lower incidence of poor mobilization.

Detection of Chlamydia in the peripheral blood cells of normal donors using in vitro culture, immunofluorescence microscopy and flow cytometry techniques.

BackgroundChlamydia trachomatis (Ct) and Chlamydia pneumoniae (Cp) are medically significant infectious agents associated with various chronic human pathologies. Nevertheless, specific roles in disease progression or initiation are incompletely defined. Both pathogens infect established cell lines in vitro and polymerase chain reaction (PCR) has detected Chlamydia DNA in various clinical specimens as well as in normal donor peripheral blood monocytes (PBMC). However, Chlamydia infection of other blood cell types, quantification of Chlamydia infected cells in peripheral blood and transmission of this infection in vitro have not been examined.MethodsCp specific titers were assessed for sera from 459 normal human donor blood (NBD) samples. Isolated white blood cells (WBC) were assayed by in vitro culture to evaluate infection transmission of blood cell borne chlamydiae. Smears of fresh blood samples (FB) were dual immunostained for microscopic identification of Chlamydia-infected cell types and aliquots also assessed using Flow Cytometry (FC).ResultsELISA demonstrated that 219 (47.7%) of the NBD samples exhibit elevated anti-Cp antibody titers. Imunofluorescence microscopy of smears demonstrated 113 (24.6%) of samples contained intracellular Chlamydia and monoclonals to specific CD markers showed that in vivo infection of neutrophil and eosinophil/basophil cells as well as monocytes occurs. In vitro culture established WBCs of 114 (24.8%) of the NBD samples harbored infectious chlamydiae, clinically a potentially source of transmission, FC demonstrated both Chlamydia infected and uninfected cells can be readily identified and quantified.ConclusionNBD can harbor infected neutrophils, eosinophil/basophils and monocytes. The chlamydiae are infectious in vitro, and both total, and cell type specific Chlamydia carriage is quantifiable by FC.

730. T Cell Responses to AAV Vector Capsid in Normal Donors and Subjects Who Have Undergone Liver-Directed AAV-Mediated Gene Transfer

In a Phase I/II study of gene transfer for hemophilia B, an adeno-associated viral vector serotype 2 (AAV-2) was introduced into the liver of human subjects and therapeutic levels of transgene expression (up to 11% of normal) were reached. However, the duration of gene expression was limited; four weeks after infusion, levels of circulating factor IX (F.IX) began to decline, and returned to baseline by week 10. This phenomenon was accompanied by a mild, self-limited, increase in liver enzymes. After a similar phenomenon was observed in another patient, the clinical trial was halted. We hypothesized that T cells directed towards AAV capsid antigens harbored by transduced hepatocytes were activated and these mediated destruction of the transduced hepaotocytes, thereby causing loss of transgene expression and a transient transaminitis. We used IFN|[gamma]| ELISpot analysis of peripheral blood mononuclear cells (PBMCs) from a subject in the clinical study, combined with bioinformatics tools for the prediction of MHC class I binders, to define an MHC Class I-restricted T cell epitope for an HLA type common in the general population (B|[ast]|0702). We then designed pentamers in which five peptide-loaded MHC class I molecules are linked to a fluorochrome to detect and study AAV-specific B|[ast]|0702-restricted CD8+ T cells. Indeed, in the HLA B|[ast]|0702 subject who developed transaminitis after vector infusion, AAV-specific CD8+ T cells were detected by pentamer staining of PBMCs up to two years later. In contrast, we have not detected AAV-specific CD8+ T cells in the peripheral blood of normal donors, though after several rounds of ex vivo stimulation with AAV capsid or peptide epitopes, we have been able to enrich a population of AAV-specific T cells, suggesting that AAV-specific T cells exist in the T cell memory pool. Functional assays on expanded AAV-specific T cells have demonstrated that these T cells specifically produced IFN|[gamma]| upon detection of AAV antigen and specifically lyse target cells presenting antigen in the context of the HLA B|[ast]|0702. These data provide direct evidence that humans mount a cytotoxic immune response to AAV capsid proteins, and that a contracted pool of AAV-specific T cells can remain as memory T cells in normal donors. Thus, re-activation of an AAV-specific T cell response may account for the limited duration of transgene expression that we observed in our clinical study. Moreover, the use of alternate serotypes may not easily avoid this immune response, as T cells from subjects infused with AAV-2 showed functional responses to AAV-1 and AAV-8-derived peptides by both cytokine production and killing assays, and T cells from normal donors that were expanded with an AAV-2 derived epitope also cross-react with the homologous epitope from AAV-5 by pentamer staining. We conclude that the use of immunomodulatory therapy may be a better approach to achieving durable transgene expression in the setting of AAV-mediated gene therapy.

673. Threshold of Normal Stem Cells for Successful Correction of Murine β-Thalassemia

To achieve a permanent correction of the |[beta]|-thalassemia phenotype, it is necessary to establish basic criteria to serve as reference. Using the hemi-|[beta]|thal mouse deleted from the two adult genes in the |[beta]|-globin locus on a homogenous genetic background, we have shown that not only red blood cells (RBCs) have a shortened half-life but also that the late erythroid precursors have cellular and structural anomalies resulting in severe ineffective erythropoiesis. Based on this characterization, we hypothesized that bone marrow transplantation of normal donor cells into a thalassemic recipient would have a strong selective advantage and predominate upon erythroid differentiation and maturation. Our aim was to determine the minimal percent of normal bone marrow cells that is necessary to engraft for a future curative gene therapy approach. We have used a Competitive Repopulating Assay by transplanting lethally irradiated recipient mice with a fixed number of bone marrow cells containing reciprocal proportions of normal and hemi-|[beta]|thal cells ranging from 5 to 100%. The assays were designed to generate 11 chimeric groups of hemi-|[beta]|thal mice with at least 5 mice per group transplanted with normal and hemi-|[beta]|thal bone marrow cells. Analysis of 95 hemi-|[beta]|thal recipient mice followed for short and long-term post-transplantation (2 months to >1 year) were carried out by various cellular, molecular and physio-pathologic evaluation. These analyses showed that a chimerism of 19|[ndash]|24 % normal engrafted cells, determined by the Gpi1a marker, produced |[sim]|40|[ndash]|50% of normal RBCs in the peripheral blood. Interestingly, the osmotic fragility of the RBCs from the chimeric hemi-|[beta]|thal groups displayed a two-step lysis corresponding to the chimerism of the two RBC populations. The hematological parameters of the 19|[ndash]|24% chimeric hemi-|[beta]|thal mice were significantly improved with increase hemoglobin of |[sim]|3|[ndash]|4 g/dl and mean cell volume (MCV) reaching normal range. With 19% chimerism, RBC half-life was increased by at least 60% relative to control and their morphology was significantly ameliorated. Similarly, bone marrow and splenic erythropoiesis was improved with increasing chimerism. Consequent to the normalization of RBC half-life and splenic erythropoiesis, the pathology of chimeric mice showed a significant decrease in splenomegaly and in extramedullary splenic hematopoiesis/erythropoiesis. Furthermore, the lifespan of the chimeric mice was increased to normal life expectancy even with 24% chimerism, suggesting a general improvement of the mice physiology. Hence these results determined a two-fold selective advantage of normal RBC relative to thalassemic cells. Altogether, these results established a therapeutic range of 19|[ndash]|24% of engrafted normal/modified bone marrow cells for a physiologic impact and for a significant long-term correction of hemi-|[beta]|thal mouse condition in vivo.

Expression of Angiogenin in Normal B Lymphocytes and B-CLL Cells.

Angiogenesis is critical for the clinical progression of hematopoietic malignancies and depends on a series of angiogenic factors. Angiogenin is a potent angiogenic factor produced by the host microenvironment and certain neoplastic cells. The association between angiogenin, cancer progression and poor outcome in solid tumors has been documented, but its significance in B-CLL has not been defined. A previous study suggests that B-CLL patients in Rai stage O with higher angiogenin levels have a better clinical outcome. This is surprising in light of the association of angiogenin levels with progression in solid tumors. Therefore we analyzed angiogenin mRNA and protein expression by the leukemic cells of B-CLL patients in order to correlate the production of this molecule with disease progression in B-CLL.Angiogenin was expressed in B-CLL cells as well as in B cells of normal donors, although the expression in the former was much higher than in the latter. Q PCR revealed a 7-fold induction in angiogenin-specific mRNA transcription in B-CLL cells (n=10) in comparison to normal B cells (n=13). In addition, angiogenin protein was identified by confocal microscopy both within and on the cell surface of B-CLL cells. All B-CLL cases, regardless of IgV gene mutation status, express angiogenin as defined by FACS. Approximately 85% of the cells that comprise B-CLL clones (n=28) display angiogenin on their surface membranes (range: 55-98%). Furthermore approximately 13% of polyclonal B cells from normal subjects (n=12) also produce angiogenin (range: 1–33%). Using enzyme immunoassay, we also measured serum angiogenin levels and detected significant differences in 66 B-CLL patients (median: 381 ng/mL; range: 185–875) versus 24 age- and sex-matched healthy controls (median: 301 ng/mL; range: 192–536) (P = 0.0056). Surprisingly, there was no correlation between surface and serum angiogenin levels and the different V gene-defined B-CLL subgroups.Our results show for the first time that a small fraction of normal B cells and all cases of B-CLL express angiogenin transcripts as well as intracellular and surface membrane protein. Angiogenin levels do not correlate with IgV gene mutations status or expression of surface membrane CD38. The role of angiogenin in the pathogenesis and progression of B-CLL needs further study.

NK Cell KIR Reconstitution after Allogeneic Hematopoietic Cell Transplant (HCT) Is Affected by T Cell Number and Function.

NK cell KIR interactions are among the variables known to affect clinical outcomes including relapse, graft versus host disease (GVHD) and survival after HCT. We hypothesized that T cells in graft sources available for HCT may affect KIR recovery and the therapeutic potential of KIR alloreactivity. We studied KIR reconstitution (the percentage of KIR+ NK cells measured by flow cytometry) in blood collected from recipients at day +100 after T cell deplete (TCD-BMT) and unmanipulated (U-BMT) unrelated BM transplants. We found that KIR reconstitution was suppressed compared to the healthy donors, significantly more so after U-BMT transplants (donor: 48.42 ± 2.35% KIR+ NK cells versus recipient: 26.74 ± 1.94, n = 36; P < .001) than after TCD-BMT transplants (donor: 53.34 ± 3.25% versus recipient: 42.68 ± 3.32%, n = 38; P = .017), with P = .001 between the recipient groups. Additionally, multivariate Cox proportional hazards models showed that improved KIR recovery independently correlated with improved survival and that higher NK cell IFN-γ production independently correlated with more frequent acute GVHD in that patient cohort. These data suggested that T cell number in the graft affects KIR reconstitution and transplant outcome. We next examined other sources of hematopoietic cells in which T cell function may be suppressed either by growth factor mobilization (sibling donor unmanipulated peripheral blood: SibU-PB) or the innate naivety of the T cells (umbilical cord blood: UCB). KIR+ NK reconstitution on recovering cells at day +100 after all HCT graft types was significantly less than that on normal donor cells (normals 55.33 ± 1.73%, n = 124; all P < .0006). U-BMT recipients had significantly lower KIR+ NK recovery (27.31 ± 2.06%, n = 36 vs. SibU-PB: 37.58 ± 3.29%, n = 29; TCD-BMT: 42.68 ± 3.32%, n = 38; or UCB, 37.99 ± 2.54%, n = 49) when compared to all other transplant types. The highest absolute T cell inoculum, found in SibU-PB, showed KIR reconstitution similar to that of TCD-BMT, which had the lowest T cell content (p=0.29), perhaps due to the lower alloreactivity of the Sib grafts and to the G-CSF-priming which preferentially mobilizes T cells with a suppressive phenotype. Similarly, KIR reconstitution was better after UCB compared to U-BMT (P = .0027), possibly due to the more permissive interactions with naive T cells. These results suggest that reduced T cell number after T cell depletion, suppressed T cells found after growth factor mobilization, or naive T cells present in UCB grafts enhance in vivo KIR reconstitution after allogeneic HCT when compared to unmanipulated marrow grafts. Such enhanced KIR reconstitution may have clinical consequences. Graft T cells may directly compete for cytokines and growth factors, or may be a surrogate marker for other transplant factors such as the development of GVHD and the requirement for intensive post-transplant immunosuppression. Understanding these interactions will allow judicious selection of hematopoietic cell source to select for enhanced KIR recovery. For example, among unrelated unmanipulated donor grafts, KIR+ NK recovery was significantly better using UCB than adult donors and further investigation may show that this is advantageous to improve clinical outcomes.

Stimulation with K562 Cells Transfected with 4-1BBL and IL-15 Expands and Activates Natural Killer (NK) Cells with Specific Cytotoxicity for Multiple Myeloma (MM).

Introduction: Although stem cell transplant (PBSCT) supported high dose chemotherapy can induce remission in MM patients with high risk features (e.g. high CKS1-B expression and abnormal cytogenetics) there is a high rate of relapse. Immunologic therapies can augment high dose chemotherapy by eradicating chemo-resistant disease. We have previously established that KIR-ligand mismatched NK cells from haplo-identical donors are cytotoxic to primary MM cells. In this study we investigate whether co-incubation of NK cells with irradiated K562 cells transfected with 41BBL and membrane-bound IL15 (K562-41BBL-mIL15) could expand NK cells and augment cytotoxicity to primary MM cells.Methods: Peripheral blood mononuclear cells (PBMC) of normal donors were co-cultured with irradiated K562-41BBL -mIL15 at a 1.5:1 ratio with varying concentrations of IL2 and re-stimulated on day 7. The cultures were then analyzed for proliferation, expansion, immunophenotype and cytotoxicity.Results: By testing a range of IL2 concentrations (10–300 IU/ml) we first established that 100IU of IL2/ml induced optimal proliferation in H3 thymidine uptake assays and this concentration was used in all subsequent experiments. After two weeks PBMC co-cultured with K562-41BBL-mIL15 were enriched for NK cells (93%) and contained few T cells (0.8%) compared to PBMC cultured with IL2 alone (NK cells 11.9% and T cells 58.7% respectively). In absolute numbers there was as 60-fold (±0.6) expansion of NK cells in the K562-41BBL -IL15 cultures compared to only a 1.5-fold expansion of NK cells cultured with IL2 alone. NK cells were analyzed by flow cytometry for expression of KIR2DL1, KIR2DL2/3, and KIR3DL1 pre- and post-incubation with K562-41BBL-mIL15 and we observed no change in NK immunophenotype. 51Cr release assays of expanded NK cells showed specific killing of K562 cells (84±4%), the MM cell line U266 (88±3%), and primary MM cells (58±9%), without killing of non-myeloma patient cells (PHA-blasts) or autologous cells at a ratio of 7 NK effectors to 1 target cell. Expanded NK cells achieved much higher specific lysis and at lower E:T ratios of primary MM cells when compared side-by-side with non-expanded NK cells.Conclusion: K562-41BBL-mIL15 transfectants stimulated vigorous expansion of NK cells without expanding T lymphocytes. Analysis of the KIR repertoire showed that there was no change in NK cell subpopulations after expansion. The expanded NK cells were highly active and killed patient MM cells better than non-expanded NK cells without significant lysis of normal cells. Expanded/activated NK cells may prove especially helpful in treating patients with relapsed/refractory MM who have a high tumor burden.

Dysfunctional T Regulatory Cells in Myeloma: Molecular Mechanisms of Dysregulation.

Multiple myeloma (MM) is characterized by production of monoclonal immunoglobulin, associated with suppressed uninvolved immunoglobulins and dysfunctional T cell responses. The biological basis of this dysfunction remains ill defined. Since T regulatory (Treg) cells play an important role in suppressing normal immune responses, we have here evaluated the potential role of Treg cells in immune dysfunction in MM. We observed a significant increase in CD4+CD25+ T cells in individuals with monoclonal gammopathy of undetermined significance (MGUS) and patients with MM compared to normal donors (25% and 26% versus 14%, respectively); however, Treg cells as measured by Foxp3 expression are significantly decreased in both MGUS (1.6±0.5%, p<0.01) and MM (1.6±0.5%, p<0.01) compared to normal donors (6.0±0.8%). Additionally, these Treg cells also do not function normally. Treg cells from patients with MM and MGUS even when added in higher proportions are unable to suppress anti-CD3-mediated T cell proliferation. This decreased number and function of Treg cells in MGUS and in MM may account, at least in part, for the non-specific increase in CD4+CD25+ T cells, thereby contributing to dysfunctional T cell responses. We have further analyzed the molecular basis for the Treg cell dysfunction in myeloma. Based on the preliminary results suggesting a role of IL-6 in Treg cell function and since both serum IL-6 and soluble IL-6 receptor levels are significantly elevated in MGUS and MM, we evaluated the role of IL-6 and its soluble receptor on Treg cell function. We observed that the addition of IL-6 and/or sIL-6 receptor to the culture leads to loss of Treg cell activity in normal donor cells similar to one observed in myeloma patients; and conversely, when Treg cells from MM patients are treated with the anti-IL-6 antibody or IL-6 receptor super antagonist, sant 7, the suppressive activity of Treg cells is restored. Additionally, we have preliminary evidence of expansion of Foxp3+ cell numbers in PBMC from MM patients following in vitro treatment with anti-IL-6 antibody. This data suggests a role of IL-6 and bone marrow microenvironment in dysfunctional Treg cells in MM and that inhibition of IL-6 signaling results in beneficial effects on T cell activity by increasing Treg cell activity. A blockade of IL-6 signaling thus emerges as a potential approach to establish immune homeostasis to improve immune function in MM.

Exposure to myeloma cell lysates affects the immune competence of dendritic cells and favors the induction of Tr1‐like regulatory T cells

The aim of this work was to investigate the interactions of tumor cells with dendritic cells (DC) in normal donors and patients with multiple myeloma (MM). Normal and MM DC internalized necrotic lysates derived from myeloma cell lines (MCL) with high efficiency, whereas necrotic lysates from primary myeloma cells (PMC) were internalized with significantly lower efficiency. A positive correlation was found between susceptibility to internalization and the ability to induce DC maturation. After PMC exposure, DC produced large amounts of IL-10 and measurable amounts of IL-4 but no detectable IL-12. Two rounds of exposure to PMC-treated DC generated autologous T cells with low proliferative capacity, decreased IFN-gamma production and increased IL-10 production in the absence of IL-4 production. These data indicate that myeloma cells can affect host immunity by priming DC towards a maturation state favoring the generation of T cells with a regulatory rather than an effector phenotype.

T cells armed with anti-CD3 × anti-CD20 bispecific antibody enhance killing of CD20 + malignant B cells and bypass complement-mediated rituximab resistance in vitro

Resistance to rituximab, a chimeric monoclonal antibody that binds to CD20, is a major limitation for the successful treatment of patients with non-Hodgkin lymphoma and other CD20+ B-cell malignancies. To circumvent rituximab resistance in these patient populations, we have constructed a bispecific antibody (BiAb), anti-CD3 x anti-CD20 (CD20Bi), that combines rituximab targeting with non-major histocompatibility complex (non-MHC)-restricted cytotoxicity mediated by activated T cells (ATC). Activated T cells were obtained from anti-CD3 activated peripheral blood mononuclear cells (PBMC) of normal donors or the leukapheresis products of patients by culturing in the presence of interleukin-2 for 6-14 days. After ATC expansion, the cells were armed with CD20Bi. Killing activity was evaluated by 51Cr-release assay. Arming ATC with as little as 5 ng CD20Bi/10(6) cells significantly increased cytotoxicity above unarmed ATC. CD20Bi-armed ATC (50 ng/10(6) cells) efficiently lysed CD20+ cell lines at E:T of 6.25-50, but not the nonhematologic, CD20- SK-BR-3 cell line. High levels of cytotoxicity mediated by CD20Bi-armed ATC (p < 0.05) could not be blocked by an 8000-fold excess of soluble rituximab. CD20Bi-armed ATC in the presence of complement killed ARH-77 cells, a rituximab-complement pathway-resistant multiple myeloma, significantly (p < 0.05) better than rituximab or unarmed ATC, suggesting that CD20Bi-armed ATC may be clinically effective for treatment of rituximab-resistant CD20+ hematologic malignancies. Our findings demonstrate that CD20Bi-armed ATC enhance cytotoxicity against CD20+ B-cell lines and circumvent complement-mediated rituximab resistance, providing a strong rationale for this immune-based strategy for the treatment of rituximab-refractory CD20+ B-cell malignancies.

Effector γδ T cells and tumor cells as immune targets of zoledronic acid in multiple myeloma

The aim of this study was to investigate the in vitro immunomodulatory effects of zoledronic acid (Zol) on peripheral blood Vgamma9/Vdelta2 (gammadelta) T cells of normal donors and multiple myeloma (MM) patients. gammadelta T cells were stimulated with Zol and low doses of interleukin-2 (IL-2), and then analyzed for proliferation, cytokine production, and generation of effector activity against myeloma cell lines and primary myeloma cells. Proliferation of gammadelta T cells was observed in 100% of normal donors and 50% of MM patients. gammadelta T cells produced IFN-gamma, surface mobilized the CD107a and CD107b antigens, and exerted direct cell-to-cell antimyeloma activity irrespective of the ability to proliferate to Zol and IL-2. The memory phenotype was predominant in the MM gammadelta T cells that proliferated in response to Zol (responders), whereas effector cells were predominant in those that did not (nonresponders). Zol induced antimyeloma activity through the monocyte-dependent activation of gammadelta T cells and by enhancing the immunosensitivity of myeloma cells to gammadelta T cells. Mevastatin, a specific inhibitor of hydroxy-methylglutaryl-CoA reductase, completely abrogated this antimyeloma activity.

148 INDUCTION OF HYPOMETHYLATION BY EPIGENETIC ALTERATION OF SOMATIC NUCLEI IN CLONED BOVINE BLASTOCYSTS

Epigenetic reprogramming such as DNA methylation is incomplete in cloned embryos during early development as compared with normal embryos. The increased methylation levels of cloned bovine blastocysts are showed in centromeric heterochromatin. The aim of the present study was to investigate the change of methylation state by treatment of trichostatin A (TSA), a specific inhibitor of histone deacetylase in somatic donor nuclei and cloned blastocyst reconstructed with TSA-treated cells or nontreated cells. Bovine ear skin fibroblast cells (bESF) were used as donor cell and treated with TSA for 60 h at a final concentration of 1 �M. To methylation analysis of satellite I as specific DNA sequence, genomic DNA from 7 � 104 cells and a blastocyst were isolated, and then the genomic DNA was analyzed by bisulfite sequencing. The reduction of HDAC1, 2 and Dnmt family such as Dnmt1, Dnmt3a, Dnmt3b, and Dnmt3L after TSA treatment were shown by Western blot in bESF, but histone acetyltransferases such as Tip60 and HAT1 were not changed. Satellite I DNA in nontreated cells was highly methylated in CpG sequences, whereas methylation level of TSA-treated cells was significantly decreased (64 vs. 48%, P &lt; 0.05). After nuclear transfer using normal or altered donor cells, methylation levels of satellite were measured at the blastocyst stage of NT and TSA-NT embryos as compared with IVF embryos. In nontreated NT blastocysts, methylation levels were significantly higher than IVF blastocysts (66 vs. 29%, P &lt; 0.05) and were similar to that of nontreated bESF cells. The reduction of methylation levels in TSA-NT blastocysts were showed and were significantly lower than NT blastocyst derived with nontreated cells (37 vs. 66%, P &lt; 0.05), but no significant differences were found between TSA-NT and IVF blastocysts. Also, the levels of methylation were similar to that of TSA-treated donor cells. In blatocyst formation, TSA-NT embryos were improved significantly compared with NT or IVF embryos (45.9 vs. 31.7 or 28%, P &lt; 0.05). These results demonstrated that somatic methylation status after epigenetic alteration affect in early cloned embryo development, suggesting epigenetic control may help to solve of inherent problems in cloning.

Comparison between G-CSF and GM-CSF for Mobilization of Dendritic Cells, CD34+ Cells, and CD3+ Cells in Normal Donors.

Donor lymphocyte infusions (DLI) can induce an allo-immune mediated graft-versus-malignancy (GVM) effect in patients (pts) that relapse after allogeneic stem cell transplantation (ASCT). Dendritic cells (DC) are effective antigen presenting cells and an important regulatory element of T-cell mediated immune responses. Previously, we have shown that NHL pts given GM-CSF mobilized up to 5 fold more immature DC (CD14+CD80+ cells) compared to G-CSF mobilization (Gazitt et al. J. Hematother. Stem Cell Res. 2001. 10:177). We hypothesized that increasing the DC populations in DLI may augment the GVM effect, particularly if GM-CSF mobilization resulted in increased DC1 cells. We designed a pilot study to treat pts with relapsed disease after ASCT with GM-CSF mobilized DLI. Total DC, DC1, DC2, and CD34+cells from the same donor were compared from G-CSF mobilized peripheral blood stem cell (PBSC) collections and GM-CSF mobilized DLI collections. Donors received G-CSF 10 ug/kg/day for 5 days prior to PBSCH, and GM-CSF 500 ug/day for 5 days prior to DLI collection. All donors were HLA matched related siblings and DLI was collected when pts demonstrated relapse or persistent disease after ASCT. Total DCs were defined as CD80+CD14- cells, DC1 cells as CD11c+HLADR+lin-, and DC2 cells as CD 123+HLADR+lin-. Analysis was done by flow cytometry using a 3 color assay. No unexpected adverse events occured to the donors who received GM-CSF prior to DLI collection or to the pts during DLI administration. Diseases of patients prior to ASCT were CML accelerated phase (n=1), high risk acute leukemia (n=4), chemotherapy refractory NHL (n=2) and refractory multiple myeloma (n=1). Four of 8 pts developed aGVHD (grade II: n=3, and grade IV: n=1). Three patient had remissions post DLI for 1 to 6 months. Subsequently, seven pts died of progressive malignacy and 1 pt died of sepsis with no active GVHD or malignancy. The mean number of cell types collected for the G-CSF and GM-CSF apheresis collections are described in the table below. Mobilization with G-CSF resulted in up to a 10 fold larger number of CD34+ cells/kg and 3–5 fold larger number of total DC, DC1 and DC2 cells within the same donor compared to GM-CSF. The ratio of DC1 to DC2 in each donor remained constant in both the G-CSF and GM-CSF mobilized apheresis products. We conclude that GM-CSF mobilization results in adequate collection of CD3+ cells for DLI, and infusion of these cells is tolerated well. In this small sample of normal donors it appears that G-CSF mobilizes more CD34+, total DC, DC1 and DC2 cells within the same donor compared to GM-CSF, with no polarization by G-CSF or GM-CSF for either DC1 or DC2 cells.Mean cell counts/kg x 106 (range)CD3CD34Total DCDC1DC2ratio DC1/DC2G-CSFN/A4.6 (1.2–7.5)8.9 (2.6–13.1)5.7 (1.2–10.6)5.8 (0.6–10.1)1.1GM-CSF81.2 (50.6–133.7)0.4 (0.09–0.6)3.5 (1.4–7.2)1.8 (0.4–3.8)2.1 (0.5–3.9)0.9p value *N/A0.010.050.010.05N/A* student t-test

Human CD4 Negative NKT Cells Potently Suppress Alloreactive Responses in a TGF-β and IL-10-Independent Manner.

Acute graft versus host disease (aGVHD) remains a major source of morbidity and mortality in allogeneic stem cell transplantation. There is a need for therapeutic interventions to ameliorate its severity. NKT cells, a novel class of regulatory T cells, are restricted by the glycolipid presenting MHC-like molecule CD1d. They constitute <0.1% of T cells and modulate responses of the innate and adaptive immune system in autoreactivity and against pathogens and tumours through interactions with antigen presenting cells (APC). The regulation of alloreactivity and aGVHD by NKT cells has not been studied. We tested the effect of expanded human NKT cells on the mixed lymphocyte reaction (MLR). First, NKT cells were expanded ex vivo from peripheral blood mononuclear cells (PBMC) of normal donors in the presence of the CD1d-presented glycolipid α-galactosylceramide. Under these conditions NKT cells (n=7), identified by staining with mAbs specific to their TCR α and β chains, expanded by 238-fold±102 by day 10. Total, CD4+ (69%±17 of total) and CD4- (30%±17 of total) NKT were purified to >95% and tested for their effect on the MLR using 3H incorporation assays. Total and NKT subsets from a panel of donors were added to the MLR at a ratio of NKT:Responder (consisting of autologous T cells) of 1:10 and a panel of allogeneic irradiated PBMC were used as stimulators. Co-culture with total or CD4+ NKT had a variable effect on the MLR. By contrast, in all MLR tested using 7 different responders against a panel of 3–5 stimulators, the presence of CD4- NKT had a profound suppressive effect (mean suppression from baseline MLR: 67%±18%). Titration of the NKT numbers revealed that the magnitude of the suppressive effect was inversely proportional to the NKT:R ratio, suggesting that NKT can exert opposing effects of differential magnitude and the maximum suppression depends on an optimal NKT:R ratio. In addition we demonstrated that CD4- NKT cell lines exert linked suppression, i.e., that they have suppressive effect (mean 52%±16.5, n=2) in MLR when the responding T cells are allogeneic to the NKT themselves. In transwell experiments we determined that the suppressive effect is at least partially contact-dependent and it is not mediated by either IL-10 or TGFβ as these anti-proliferative cytokines were not detected in the supernatants of the MLR and the presence of the corresponding neutralising mAbs did not reverse the suppressive effect. These findings indicate that the suppressive effect of NKT is not due to induction of anergy on the alloresponsive T cells. Instead, it is likely that it is mediated by a modulatory effect on the APC. The mechanisms of this modulation are the subject of current investigation. Finally, all CD4- NKT cells lines tested (n=4) had markers of activation (>90% were CD25+ CD69+), demonstrated potential for homing to peripheral tissues (mean expression 52%±23.9 were CD62L−CCR7−), especially to mucosal tissues (mean expression of integrin β7 84%±8) but not to the skin (mean expression of CLA <1%). In summary, we have identified a subset of NKT cells that when expanded ex vivo are very efficient in suppressing the alloreactive response. These findings form the basis for the use of such cells at a clinical level for the treatment of aGVHD, especially of mucosal localisation, through their adoptive transfer. The procedure of expanding suppressive NKT can be easily adapted for clinical purposes as using autologous PBMC as APC can result in a >200-fold expansion after just one round of stimulation.

Antineutrophil autoantibodies and their target antigens in systemic lupus erythematosus.

Various autoantibodies have been identified in sera from patients with systemic lupus erythematosus (SLE) and autoantibodies against neutrophil have been reported. It was suggested that antineutrophil autoantibodies might be involved in the pathogenesis of neutrocytopenia; however, the role of autoantibodies against neutrophil precursors and their specific target autoantigen(s) remained further characterized. The objective was to investigate the target antigens and clinical associations of autoantibodies against neutrophils and neutrophil precursors in patients with SLE. Sera were collected from 92 patients with SLE and renal biopsy proven lupus nephritis. Cell lysates of peripheral neutrophils (as mature neutrophils) from a normal blood donor and white blood cells from a patient with blast crisis of chronic granulocytic leukemia (CGL) (as neutrophil precursors) were used as antigens in Western blot analysis to detect autoantibodies in sera from patients with SLE. The clinical significance of antineutrophil autoantibodies that recognized different antigens were further analysed. Using normal peripheral neutrophils as antigens, two bands could be blotted: 64 kD (33/92, 35.9%) and 50 kD (13/92, 14.1%). The prevalence of anti-64 kD autoantibody in patients with positive rheumatic factor was significantly higher than that in patients without (54.5 versus 18.8%, P < 0.05). Using CGL white cells as antigen, five bands could be blotted: 60 kD (34/92, 37.0%), 50 kD (32/92, 34.8%), 29 kD (27/92, 29.3%), 42 kD (19/92, 20.7%) and 18 kD (16/92, 17.4%). The prevalence of anti-60 kD autoantibody was significantly higher in patients with neutrocytopenia than that in patients without neutrocytopenia (100 versus 48.3%, P < 0.01). The prevalence of anti-29 kD autoantibody was significantly higher in patients with alopecia than that in patients without alopecia (45.8 versus 20.8%, P < 0.05). Furthermore, the prevalences of anti-60 kD, anti-50 kD and anti-42 kD autoantibodies were significantly higher in patients with anti-Ro autoantibody than those in patients without; the prevalences of anti-29 kD and anti-18 kD autoantibodies were significantly higher in patients with anti-Sm autoantibody than those in patients without. We conclude that there are heterogeneous autoantibodies against both neutrophils and their precursors in sera from patients with SLE. Different autoantibodies may have different clinical significance.

Efficient PRNP gene targeting in bovine fibroblasts by adeno-associated virus vectors.

Gene-targeted livestock can be created by combining ex vivo manipulation of cultured nuclear donor cells with cloning by nuclear transfer. However, this process can be limited by the low gene targeting frequencies obtained by transfection methods, and the limited ex vivo life span of the normal nuclear donor cells. We have developed an alternative gene targeting method based on the delivery of linear, single-stranded DNA molecules by adeno-associated virus (AAV) vectors, which can be used to introduce a variety of different mutations at single copy loci in normal human cells. Here we show that AAV vectors can efficiently target the PRNP gene encoding the prion protein PrP in bovine fetal fibroblasts, which can be used as nuclear donors to clone cattle. Cattle with both PRNP genes disrupted should be resistant to bovine spongiform encephalopathy.

Assessment of G-CSF stimulated BM hematopoietic stem cells in normal donors

The clinical use of G-CSF has recently been expanded to include mobilization of stem cells for both autologous and allogeneic transplantation. Most of the published studies have focused on stem cells released into the peripheral blood (PB) after G-CSF treatment. However, little is known about the effects of G-CSF on BM. This study evaluated the concurrent effects of short-term G-CSF on both BM and PB stem and progenitor cells in normal individuals. Volunteers received 5 or 10 microg/kg of G-CSF for 5 consecutive days (Days 1-5). On Days 0, 3, 6, 9 and 15, BM and PB samples were obtained. Flow cytometry and functional assay were performed to analyze stem cells, subpopulations, adhesion molecules, colony-forming units and LTCIC. The total nucleated cells and absolute numbers of CD34(+)/mL showed a similar response pattern in both BM and PB, with a peak around Day 6 that returned to baseline levels by Day 15. However, there was a reciprocal change in the percentage of CD34(+) cells between BM and PB compartments. The expressions of adhesion molecule showed an up- and down-regulation of alpha4 and alpha5 integrin subunits, respectively, also correlated with the CD34(+) mobilization patterns. The functional characterization of integrins, and further clinical examination of G-CSF-stimulated BM is warranted. G-CSF-stimulated BM maybe considered as an alternative source of stem cells in transplantation.