Transfer Of Peripheral Blood Mononuclear Cells Research Articles

Myeloid-derived MIF drives RIPK1-mediated cerebromicrovascular endothelial cell death to exacerbate ischemic brain injury

Macrophage migration inhibitory factor (MIF) is a multifaced protein that plays important roles in multiple inflammatory conditions. However, the role of MIF in endothelial cell (EC) death under inflammatory condition remains largely unknown. Here we show that MIF actively promotes receptor-interacting protein kinase 1 (RIPK1)-mediated cell death under oxygen-glucose deprivation condition. MIF expression is induced by surgical trauma in peripheral myeloid cells both in perioperative humans and mice. We demonstrate that MIF-loaded myeloid cells induced by peripheral surgery adhere to the brain ECs after distal middle cerebral artery occlusion (dMCAO) and exacerbate the blood-brain barrier (BBB) disruption. Genetic depletion of myeloid-derived MIF in perioperative ischemic stroke (PIS) mice with MCAO following a surgical insult leads to significant reduction in ECs apoptosis and necroptosis and the associated BBB disruption. The adoptive transfer of peripheral blood mononuclear cells (PBMC) from surgical MIFΔLyz2 mice to wild-type (WT) MCAO mice also shows reduced ECs apoptosis and necroptosis compared to the transfer of PBMC from surgical MIFf l/f l mice to MCAO recipients. The genetic inhibition of RIPK1 also attenuates BBB disruption and ECs death compared to that of WT mice in PIS. The administration of MIF inhibitor (ISO-1) and RIPK1 inhibitor (Nec-1s) can both reduce the brain EC death and neurological deficits following PIS. We conclude that myeloid-derived MIF promotes ECs apoptosis and necroptosis through RIPK1kinase-dependent pathway. The above findings may provide insights into the mechanism as how peripheral inflammation promotes the pathology in central nervous system.

Ruxolitinib, a JAK1/2 Inhibitor, Is Efficacious in a Novel Humanized GvHD Model Characterized By Enhanced NK, NK-T and T-Cell Engraftment

Background: Graft-versus-host disease (GvHD) is defined as a syndrome in which cells originating from a donor recognize and mount a deleterious immune response against antigens found in the immunocompromised recipient. Most humanized pre-clinical studies of models of GvHD rely on adoptive transfer of human peripheral blood mononuclear cells (PBMC) into severely immunodeficient recipient animals. Transferred PBMC can recognize murine xeno-antigens presented by major histocompatibility complex (MHC) and develop GvHD. Only limited numbers of transferred human T-cells survive, and tissue damage is generally observed in only a few tissues (i.e. gastrointestinal (GI) tract and liver). There is a need to develop refined humanized models that better recapitulate the clinical settings of allogeneic hematopoietic cell transplantation. Aims: a) To develop a refined model of pre-clinical GvHD where NOG animals transgenic for human IL-15 (hIL-15 NOG) are able to support long-term engraftment of T-cells as well as NK cells. b) To characterize the role of JAK1/2 inhibition on steroid refractory GvHD in the hIL-15 NOG model. Materials and Methods: Xenogeneic GvHD was induced in hIL-15 NOG receiving 3 different doses of human PBMCs, as well as in control NOG animals. Engraftment was weekly assessed by flow cytometry. Ruxolitinib, prednisolone or vehicle was orally administered twice daily. Efficacy was assessed by GvHD symptom scores, cachexia, and histological analysis of end organ damage. Results: hIL-15 NOG animals develop a rapid onset, highly aggressive, form of GvHD after adoptive transfer of 5x106 PBMC, including a significant engraftment of NK, NK-T and T-cells. Significant PBMC infiltration and inflammation was found in GI tract, lung, liver, skin, eyes and pancreas. Ruxolitinib treatment, but not prednisolone, ameliorated GvHD severity scores with concomitant reduction of GI tract infiltration and tissue damage. Overall GvHD mice survival was significantly improved with JAK1/2 inhibition. Therapeutic ruxolitinib treatment initiated after steroid failure significantly inhibited the chronic-progressive disease and reduced tissue markers of inflammation and fibrosis. Conclusion: hIL-15 NOG animals develop a rapid onset, highly reproducible, form of GvHD after PBMC transfer, with successful engraftment of NK, NK-T and T-cells. Ruxolitinib, a potent and selective JAK1/2 inhibitor, significantly ameliorated GvHD scores, reduced tissue-inflammation and improved survival. Ruxolitinib was also able to improve clinical parameters in mice unresponsive to prednisolone treatment, underscoring the relevance of the new model. Ruxolitinib has demonstrated statistically significant improvement in in patients with steroid-refractory GvHD (REACH3, NCT03112603). Disclosures Huarte: Incyte corporation: Current Employment. Peel:Incyte corporation: Current Employment. Dube:Taconic: Current Employment. Stephens:Incyte: Current Employment. Stewart:Incyte: Current Employment. Long:Incyte: Current Employment. Czerniak:Incyte: Current Employment. Oliver:Incyte: Current Employment. Smith:Incyte corporation: Current Employment.

Endometrium immunomodulation by intrauterine insemination administration of treated peripheral blood mononuclear cell prior frozen/thawed embryos in patients with repeated implantation failure.

In assisted reproductive technology (ART) programmes, approximately 10% of infertile patients have at least two or three repeated implantation failures (RIFs) after an in vitro fertilization (IVF) protocol. Successful implantation mainly depends on local immune tolerance mechanisms involving a spectrum of cytokines, interleukins and growth factors. The latter have played pivotal roles in the recruitment of immune cells (and notably T-lymphocyte cells). In total, 250 couples participating in frozen-thawed embryo transfer programme were incorporated in a randomized clinical trial (peripheral blood mononuclear cells (PBMC) subgroup: n=122; control subgroup: n=128). In the PBMC group, a blood sample was collected 5 days before the scheduled frozen-thawed embryo transfer; PBMCs were isolated using Ficoll separation and then cultured for 72 h. Two days prior to embryo transfer, 0.4 ml of cultured PBMCs were transferred into the patient's uterus. Although the clinical pregnancy rate was higher in the PBMC group (34.4%) than in the control group (23.4%), this difference was not statistically significant (P=0.05 in a chi-squared test). Nevertheless, when we limited the analysis to patients with ≥3 RIFs (n=138), there was a significant difference in the clinical pregnancy rate between the PBMC group (38.6%) and the control group (19.7%; P=0.01). Our results imply that PBMC transfer can be part of effective fertility treatment for patients with RIF.

PDL241, a novel humanized monoclonal antibody, reveals CD319 as a therapeutic target for rheumatoid arthritis

IntroductionTargeting the CD20 antigen has been a successful therapeutic intervention in the treatment of rheumatoid arthritis (RA). However, in some patients with an inadequate response to anti-CD20 therapy, a persistence of CD20- plasmablasts is noted. The strong expression of CD319 on CD20- plasmablast and plasma cell populations in RA synovium led to the investigation of the potential of CD319 as a therapeutic target.MethodsPDL241, a novel humanized IgG1 monoclonal antibody (mAb) to CD319, was generated and examined for its ability to inhibit immunoglobulin production from plasmablasts and plasma cells generated from peripheral blood mononuclear cells (PBMC) in the presence and absence of RA synovial fibroblasts (RA-SF). The in vivo activity of PDL241 was determined in a human PBMC transfer into NOD scid IL-2 gamma chain knockout (NSG) mouse model. Finally, the ability of PDL241 to ameliorate experimental arthritis was evaluated in a collagen-induced arthritis (CIA) model in rhesus monkeys.ResultsPDL241 bound to plasmablasts and plasma cells but not naïve B cells. Consistent with the binding profile, PDL241 inhibited the production of IgM from in vitro PBMC cultures by the depletion of CD319+ plasmablasts and plasma cells but not B cells. The activity of PDL241 was dependent on an intact Fc portion of the IgG1 and mediated predominantly by natural killer cells. Inhibition of IgM production was also observed in the human PBMC transfer to NSG mouse model. Treatment of rhesus monkeys in a CIA model with PDL241 led to a significant inhibition of anti-collagen IgG and IgM antibodies. A beneficial effect on joint related parameters, including bone remodeling, histopathology, and joint swelling was also observed.ConclusionsThe activity of PDL241 in both in vitro and in vivo models highlights the potential of CD319 as a therapeutic target in RA.

LY6Chi MONOCYTES MIGRATE TO THE PANCREAS DURING PANCREATITIS AND REGULATE ITS SEVERITY

Introduction: Monocyte depletion reduces pancreatitis severity but the cell sub-type and mechanisms responsible for this effect are unknown. Methods: We gave diphtheria toxin to mice expressing the human diphtheria toxin receptor coupled to the CD11b promoter to deplete monocytes. Caerulein was given 16 hrs later to induce pancreatitis. Adoptive transfer of peripheral blood mononuclear cells (PBMC) or bone marrow cells (BMC) was performed 4-6 hrs after the start of pancreatitis induction. We monitored monocyte trafficking by pre-labelling donor cells with CFSE, 111In, or by transferring CD45.1 cells into CD45.2 recipients. Results: Monocyte depletion reduced pancreatitis severity but adoptive transfer of PBMCs or BMCs restored that severity unless Ly6Chi cells had been removed from the transferred cell pool. Restoration of severity was directly proportional to the number of adoptively transferred Ly6Chi cells. Only 3-4% of the donor-derived, transferred BMCs trafficked to the pancreas but, by triggering an increase in the pancreatic content of recipient-derived Ly6Chi cells, adoptive transfer of Ly-6Chi cells led to a large increase in the total number of intrapancreatic Ly6Chi cells. Conclusions: Ly6Chi monocytes regulate pancreatitis severity by migrating to the pancreas and/or triggering the phenotypic transformation, into the Ly-6Chi subset, of other cells that are already in the pancreas at the time of pancreatitis induction.

The intraspleen huPBL NOD/SCID model to study the human HIV-specific antibody response selected in the course of natural infection

The intrasplenic injection of human peripheral blood mononuclear cells (PBMCs) into severely immune deficient NOD/SCID mice, causes a massive and transient dominant expansion of human B cells in the spleen. This permits the easy isolation of human monoclonal antibodies specific for different antigens by a Kohler and Milstein-based method. Here we studied the human HIV-specific antibody response in the circulation of mice after intrasplenic transfer of PBMC from untreated HIV-infected patients with detectable to high viral load as well as from HAART-treated and from untreated patients, who kept an undetectable viral load (the latter referred to as “natural suppressors”). Excellent B cell expansion was obtained for all PBMC. High level replication of virus was observed after transfer of PBMC of untreated viremic patients only. A strong and multispecific HIV-specific antibody response was observed after transfer of PBMC of untreated viremic patients and natural suppressors. In contrast, only a weak and pauci-specific antibody response was detected in mice reconstituted with PBMC from successfully treated patients. Based on these observations we conclude that the use of the intraspleen mouse model confirmed a) the presence of HIV-specific circulating memory B cells in untreated patients and natural suppressors; b) the nearly complete absence of circulating memory B cells in patients receiving highly active antiretroviral therapy. Using the intraspleen model we generated large numbers of immortalized B cells and isolated two anti-p24 human monoclonal antibodies. We further conclude that the intraspleen huPBL NOD/SCID model is a small animal model useful for the analysis of the antibody response against HIV found in patients.

Chemokine receptor expression in EBV-associated lymphoproliferation in hu/SCID mice: implications for CXCL12/CXCR4 axis in lymphoma generation

AbstractThe mechanisms by which intraperitoneal injection of peripheral blood mono-nuclear cells (PBMCs) from Epstein-Barr virus (EBV)–seropositive donors into severe combined immunodeficient (SCID) mice gives rise to lymphomas (hu/SCID tumors) are far from clear. This study addressed whether chemokine receptors and their ligands could be implicated in this experimental model. CXCR4 was found to be highly expressed in hu/SCID tumors; surface expression of CXCR4 was prevalently limited to a tumor cell subset poorly expressing CD23, whereas the CXCR4 ligand, CXCL12, was predominantly expressed by the tumor subpopulation expressing CD23. In vitro inhibition of this autocrine/paracrine CXCL12/CXCR4 axis significantly inhibited lymphoma proliferation and survival. Furthermore, CXCL12 was expressed in cells recovered from the mouse peritoneal cavity early after PBMC transfer as well as by EBV-transformed B cells but not by resting or activated B lymphocytes; also, lymphoma development was associated with a dramatic increase in the levels of murine CXCL12 present in the peritoneal cavity. Finally, antagonizing the CXCL12/CXCR4 axis in vivo strongly counteracted lymphoma development. These studies demonstrate that CXCL12 expression may be associated with EBV infection and suggest that the CXCR4/CXCL12 axis may participate in the EBV-associated lymphomagenesis process in immunodeficient hosts.

Tumor outgrowth in peripheral blood mononuclear cell-injected SCID mice is not associated with early Epstein-Barr virus reactivation.

Epstein-Barr virus (EBV)-positive B-cell lymphoproliferative disease develops in severe combined immunodeficient (SCID) mice inoculated with peripheral blood mononuclear cells (PBMC) from EBV(+) individuals (SCID/hu mice). In this study, we investigated the contribution of EBV reactivation and de novo infection of B lymphocytes to tumor outgrowth in SCID/hu mice. Evaluation of BZLF-1, an early EBV activation transcript, in cells recovered from the mouse peritoneal cavity within 16 days following PBMC transfer did not reveal EBV reactivation, while BZLF-1 expression was only detected in tumor masses or in vitro established lymphoblastoid cell lines. To confirm these data by a different strategy, we coinjected PBMC from seropositive donors with purified B cells from seronegative donors of different sex. Fluorescence in situ hydridization analysis of the resulting tumor masses disclosed that the overwhelming majority of lymphoma cells originated from the seropositive donor, implying that no substantial in vivo production and transmission of virus had occurred. Further, treatment of SCID/hu mice with ganciclovir did not prevent lymphoma development. Our results suggest that in the SCID/hu mouse, early EBV replication and secondary infection of bystander B cells does not occur, and that the direct outgrowth of the transformed B lymphocytes present within the PBMC inoculum is the predominant mechanism, which leads to lymphoma generation in this experimental model.

Antigen-specific B and T cells in human/mouse radiation chimera following immunization in vivo.

Adoptive transfer of human peripheral blood mononuclear cells (PBMC) into mice with severe combined immunodeficiency (SCID) or into lethally irradiated BALB/c mice radioprotected with SCID bone marrow, leads to marked engraftment of human T and B cells. In such chimeras, human serum antibody responses can be stimulated readily by vaccination with recall antigens, but the detection of antigen-specific functional T or B cells has been extremely difficult. In the present study, we were able to detect by Elispot analysis high frequencies of immunoglobulin G (IgG)-secreting B cells and mitogen-responsive interferon-gamma (IFN-gamma) or interleukin-4 (IL-4)-secreting T cells in peritoneum and spleen of human/BALB/c chimeric mice during the first 3 weeks after PBMC transfer. Moreover, specific memory responses were elicited by vaccination with tetanus toxoid (TT) or hepatitis B virus (HBV) surface (HBs) antigen of chimeric mice transplanted with PBMC derived from TT- or HBV-immune donors. Substantially higher TT-specific B-cell frequencies were found during the first 3 weeks after vaccination in mice challenged with the specific antigen compared to the levels found in control animals. High numbers of TT-specific IFN-gamma-secreting T cells persisted in the peritoneum of vaccinated, but not of unvaccinated, animals during the entire observation period, but only low numbers of specific IL-4-secreting T cells were found in vaccinated mice. Similar results were achieved following vaccination with HBs antigen of chimeric mice, transplanted with PBMC of HBV immunized donors. Thus, TT or HBsAg-specific antibody responses in our model correlate closely with the existence of specific IFN-gamma-secreting T helper 1/0 cells. Furthermore, these results show that adoptive transfer of human PBMC into lethally irradiated mice provides an efficient approach to generate specific B-cell fusion partners for the production of human monoclonal antibodies and specific T-cell lines for adoptive cell therapy of malignant or infectious diseases.

Transplantation of systemic sclerosis (SSc) skin grafts into severe combined immunodeficient mice: increased presence of SSc leukocytes in SSc skin grafts and autoantibody production following autologous leukocyte transfer.

This study was performed to evaluate the potential of the severe combined immunodeficient (Scid) mouse as a model to study the pathogenesis of systemic sclerosis (SSc). Scid mice were divided into three treatment groups: group 1 received skin grafts and autologous peripheral blood mononuclear cells (PBMC) from either SSc patients or normal individuals, group 2 received only SSc or normal skin grafts, and group 3 received only SSc or normal PBMC transfer. Antinuclear antibodies with a similar expression pattern as in the donor patients were detected in SSc-Scid group 1. Increased numbers of human PBMC were detected after autologous PBMC transfer in normal or SSc skin grafts without crossover into the adjacent mouse tissue. The CD4/CD8 ratio in these infiltrates was approximately 1.0. Although SSc skin transplantations and autologous PBMC transfer into Scid mice did not reproduce the inflammatory events found in the original SSc skin biopsies, these mice could be useful to study the expression of SSc-specific autoantibodies.

Cloned human T lymphocytes with lymphostimulatory capacity preferentially activate suppressor cells.

A proportion of cloned T cells derived from allogeneic mixed leukocyte cultures (MLC) was found to stimulate rapid primary, but not secondary, lymphoproliferative responses of autologous as well as allogeneic peripheral blood mononuclear cells (PBMC). Of eight major histocompatibility complex (MHC) class II-specific monoclonal antibodies (mAb), the reagent TU39 (which preferentially inhibits allostimulation by SB- rather than DR- or DC-associated determinants) most strongly inhibited stimulation by these clones. MAb specific for DC or DR molecules inhibited weakly or not at all. Stimulatory, but not nonstimulatory, clones were found to be strongly suppressive when titrated directly into MLC. Suppression was no MHC restricted, was radioresistant (20 Gy) and was not abrogated by the addition of partially purified interleukin 2 to the test cultures. Transfer of PBMC cocultured with stimulatory, but not with nonstimulatory, clones into a second MLC resulted in its strong suppression, suggesting that a suppressor effector population had been "induced" by the clones. These results are consistent with the hypothesis that SB-like, rather than DR or DC, determinants present on the surface of certain activated T cells are intimately involved in the regulation of cellular immune responses by rapidly inducing suppressor effector cells in normal lymphocyte populations.