Allogeneic Peripheral Blood Mononuclear Cells Research Articles

KIR3DL2 (CD158k) is a potential therapeutic target in primary cutaneous anaplastic large-cell lymphoma.

KIR3DL2, an inhibitory receptor expressed by natural killer cells and a subset of normal CD8(+) T cells, is aberrantly expressed in neoplastic cells in transformed mycosis fungoides and Sézary syndrome. Anti-KIR3DL2 targeted antibody therapy has shown potent activity in preclinical models for these diseases. To examine the expression of KIR3DL2 and its potential use as a therapeutic target in patients with primary cutaneous anaplastic large-cell lymphoma (pcALCL), the most aggressive cutaneous CD30(+) lymphoproliferative disease. Samples from 11 patients with pcALCL and three CD30(+) lymphoproliferative disease cell lines - Mac1, Mac2a and Mac2b - were used in KIR3DL2 expression studies using immunohistochemistry, flow cytometry and reverse-transcriptase quantitative polymerase chain reaction. The effect of IPH4102, a monoclonal humanized IgG1 targeting KIR3DL2, was assessed by invitro cytotoxicity assays against Mac1, Mac2a and Mac2b using allogeneic peripheral blood mononuclear cells as effectors. KIR3DL2 mRNA and protein were found in all human samples of pcALCL, and in the Mac2a and Mac2b cell lines. KIR3DL2 protein expression was present on 85·8±14·0% of CD30(+) skin-infiltrating tumour cells. In vitro functional studies showed that KIR3DL2(+) Mac2a and Mac2b pcALCL lines are sensitive to antibody-derived cytotoxicity mediated by IPH4102, through activation of natural killer cells, in a concentration-dependent manner. pcALCL tumour cells express KIR3DL2, and we provide preclinical proof of concept for the use of IPH4102, a humanized anti-KIR3DL2 antibody, to treat patients with primary cutaneous CD30(+) ALCL.

Cryopreserved Mesenchymal Stromal Cells Are Susceptible to T-Cell Mediated Apoptosis Which Is Partly Rescued by IFNγ Licensing.

We have previously demonstrated that cryopreservation and thawing lead to altered Mesenchymal stromal cells (MSC) functionalities. Here, we further analyzed MSC's fitness post freeze-thaw. We have observed that thawed MSC can suppress T-cell proliferation when separated from them by transwell membrane and the effect is lost in a MSC:T-cell coculture system. Unlike actively growing MSCs, thawed MSCs were lysed upon coculture with activated autologous Peripheral Blood Mononuclear Cells (PBMCs) and the lysing effect was further enhanced with allogeneic PBMCs. The use of DMSO-free cryoprotectants or substitution of Human Serum Albumin (HSA) with human platelet lysate in freezing media and use of autophagy or caspase inhibitors did not prevent thaw defects. We tested the hypothesis that IFNγ prelicensing before cryobanking can enhance MSC fitness post thaw. Post thawing, IFNγ licensed MSCs inhibit T cell proliferation as well as fresh MSCs and this effect can be blocked by 1-methyl Tryptophan, an Indoleamine 2,3-dioxygenase (IDO) inhibitor. In addition, IFNγ prelicensed thawed MSCs inhibit the degranulation of cytotoxic T cells while IFNγ unlicensed thawed MSCs failed to do so. However, IFNγ prelicensed thawed MSCs do not deploy lung tropism in vivo following intravenous injection as well as fresh MSCs suggesting that IFNγ prelicensing does not fully rescue thaw-induced lung homing defect. We identified reversible and irreversible cryoinjury mechanisms that result in susceptibility to host T-cell cytolysis and affect MSC's cell survival and tissue distribution. The susceptibility of MSC to negative effects of cryopreservation and the potential to mitigate the effects with IFNγ prelicensing may inform strategies to enhance the therapeutic efficacy of MSC in clinical use. Stem Cells 2016;34:2429-2442.

(107) - Leukocytes Transfer More Severe Rejection from Lung Transplant Recipients with Severe PGD into Humanized Mice and Are Sensitive to T Cell Regulation

Primary graft dysfunction (PGD) is the main cause of early morbidity and mortality after lung transplantation. Here, we studied correlations between PGD after clinical lung transplantation and leukocyte-mediated development of transplant arteriosclerosis in a humanized mouse model. The pericardiophrenic artery was procured from donor lungs transplanted in our clinical program and was implanted into the abdominal aorta of immune deficient mice. Of 17 lung recipients, 10 (43.47%) developed severe PGD (grade 2-3) and 13 (56.52%) did not develop PGD (grade 0-1) at 24 h. Negative control mice received no human leukocyte reconstitution (neg. co). PBMC PGD+ group mice received 5x106 allogeneic human peripheral blood mononuclear cells (PBMC) from recipients with severe PGD, whereas PBMC PGD- group mice received PBMC from patients without PGD. Two groups of animals were reconstituted with the respective PBMC additionally enriched with autologous CD4+CD25 high cells (putative regulatory T cells, Treg) from recipients with severe PGD (PBMC+Treg PGD+ group) or without PGD (PBMC+Treg PGD- group). Transplant arteriosclerosis was histologically assessed 28 days after PBMC reconstitution. The neg. co group showed only mild thickening of the intima (13.86±14.22%). In the PBMC PGD+ group, intimal thickening obliterating the vessel lumen was significantly more severe than in the PBMC PGD- group (41.71 ± 15.01% vs. 10.52 ± 10.22%, p= 0.001). Then, intimal thickening was significantly inhibited in the PBMC+Treg PGD+ group as compared to the PBMC PGD+ group (3,42 ± 5,41% vs. 41.71 ± 15.01%, p=0.016). In the experiments using PBMC from lung recipients without PGD, enriching Treg also further suppressed the development of transplant arteriosclerosis (3.88 ± 5.54% PBMC PGD- vs. 10.52 ± 10.22%, PBMC+Treg PGD-, p=0.010). Leukocytes from lung transplant recipients with PGD transfer pro-inflammatory properties eliciting transplant arteriosclerosis into a humanized mouse model. This indicates a link between early graft dysfunction and the later development of chronic rejection. Importantly, however, transplant arteriosclerosis remains sensitive to inhibition by autologous Treg, suggesting a novel cell therapy-based approach for the treatment of patients with severe PGD.

(105) - The Real Time Visualization of Neutrophil Extracellular Trap Formation in Lung Transplants Reveals a Critical Role in Tolerance

Methods: The pericardiophrenic artery was procured from donor lungs transplanted in our clinical program and was implanted into the abdominal aorta of immune deficient mice. Of 17 lung recipients, 10 (43.47%) developed severe PGD (grade 2-3) and 13 (56.52%) did not develop PGD (grade 0-1) at 24 h. Negative control mice received no human leukocyte reconstitution (neg. co). PBMC PGD+ group mice received 5x106 allogeneic human peripheral blood mononuclear cells (PBMC) from recipients with severe PGD, whereas PBMC PGDgroup mice received PBMC from patients without PGD. Two groups of animals were reconstituted with the respective PBMC additionally enriched with autologous CD4+CD25 high cells (putative regulatory T cells, Treg) from recipients with severe PGD (PBMC+Treg PGD+ group) or without PGD (PBMC+Treg PGDgroup). Transplant arteriosclerosis was histologically assessed 28 days after PBMC reconstitution. Results: The neg. co group showed only mild thickening of the intima (13.86±14.22%). In the PBMC PGD+ group, intimal thickening obliterating the vessel lumen was significantly more severe than in the PBMC PGDgroup (41.71 ± 15.01% vs. 10.52 ± 10.22%, p= 0.001). Then, intimal thickening was significantly inhibited in the PBMC+Treg PGD+ group as compared to the PBMC PGD+ group (3,42 ± 5,41% vs. 41.71 ± 15.01%, p= 0.016). In the experiments using PBMC from lung recipients without PGD, enriching Treg also further suppressed the development of transplant arteriosclerosis (3.88 ± 5.54% PBMC PGDvs. 10.52 ± 10.22%, PBMC+Treg PGD-, p= 0.010). Conclusion: Leukocytes from lung transplant recipients with PGD transfer proinflammatory properties eliciting transplant arteriosclerosis into a humanized mouse model. This indicates a link between early graft dysfunction and the later development of chronic rejection. Importantly, however, transplant arteriosclerosis remains sensitive to inhibition by autologous Treg, suggesting a novel cell therapy-based approach for the treatment of patients with severe PGD.

HLA Class II Antibody Activation of Endothelial Cells Promotes Th17 and Disrupts Regulatory T Lymphocyte Expansion.

Kidney transplantation is the most successful treatment option for patients with end-stage renal disease, and chronic antibody-mediated rejection is the principal cause of allograft loss. Predictive factors for chronic rejection include high levels of HLA alloantibodies (particularly HLA class II) and activation of graft endothelial cells (ECs). The mechanistic basis for this association is unresolved. We used an experimental model of HLA-DR antibody stimulation of microvascular ECs to examine the mechanisms underlying the association between HLA class II antibodies, EC activation and allograft damage. Activation of ECs with the F(Ab')2 fragment of HLA-DR antibody led to phosphorylation of Akt, ERK and MEK and increased IL-6 production by ECs cocultured with allogeneic peripheral blood mononuclear cells (PBMCs) in an Akt-dependent manner. We previously showed that HLA-DR-expressing ECs induce polarization of Th17 and FoxP3(bright) regulatory T cell (Treg) subsets. Preactivation of ECs with anti-HLA-DR antibody redirected EC allogenicity toward a proinflammatory response by decreasing amplification of functional Treg and by further increasing IL-6-dependent Th17 expansion. Alloimmunized patient serum containing relevant HLA-DR alloantibodies selectively bound and increased EC secretion of IL-6 in cocultures with PBMCs. These data contribute to understanding of potential mechanisms of antibody-mediated endothelial damage independent of complement activation and FcR-expressing effector cells.

Expansion of Tumor-reactive T Cells From Patients With Pancreatic Cancer.

Generation of T lymphocytes with reactivity against cancer is a prerequisite for effective adoptive cellular therapies. We established a protocol for tumor-infiltrating lymphocytes (TILs) from patients with pancreatic ductal adenocarcinoma. Tumor samples from 17 pancreatic cancer specimens were cultured with cytokines (IL-2, IL-15, and IL-21) to expand TILs. After 10 days of culture, TILs were stimulated with an anti-CD3 antibody (OKT3) and irradiated allogeneic peripheral blood mononuclear cells. Reactivity of TILs against tumor-associated antigens (mesothelin, survivin, or NY-ESO-1) was detected by intracellular cytokine production by flow cytometry. Cytotoxicity was measured using a Chromium 51 release assay, and reactivity of TILs against autologous tumor cells was detected by INF-[gamma] production (ELISA). TIL composition was tested by CD45RA, CCR7, 4-1BB, LAG-3, PD-1, TIM3, and CTLA-4 marker analysis. TCR V[beta] was determined by flow cytometry and TCR clonality was gauged measuring the CDR3 region length by PCR analysis and subsequent sequencing. We could reliably obtain TILs from 17/17 patients with a majority of CD8(+) T cells. CD3(+)CD8(+), CD3(+)CD4(+), and CD3(+)CD4(-)CD8(-)[double-negative (DN) T cells] resided predominantly in central (CD45RA(-)CCR7(+)) and effector (CD45RA-CCR7-) memory subsets. CD8(+) TILs tested uniformly positive for LAG-3 (about 100%), whereas CD4(+) TILs showed only up to 12% LAG-3(+) staining and PD-1 showed a broad expression pattern in TILs from different patients. TILs from individual patients recognized strongly (up to 11.9% and 8.2% in CD8(+)) NY-ESO-1, determined by ICS, or mesothelin, determined respectively by TNF-[alpha] and IFN-[gamma] production. Twelve of 17 of CD8(+) TILs showed preferential expansion of certain TCR V[beta] families (eg, 99.2% V[beta]13.2 in CD8(+) TILs, 77% in the V[beta]1, 65.9% in the V[beta]22, and 63.3% in the V[beta]14 family). TCR CDR3 analysis exhibited monoclonal or oligoclonal TCRs, some of them (eg, CD8(+) V[beta]13.2) reacting strongly against autologous tumor defined by INF-[gamma] production or by cytotoxicity. We have optimized methods for generating pancreatic cancer–specific TILs that can be used for adoptive cellular therapy of patients with pancreatic cancer.

Abstract A176: Efficacy and safety of allogeneic double negative T cell as anti-AML therapy and its underlying mechanism

Abstract Acute myeloid leukemia (AML) is the most common form of adult acute leukemia that is associated with a low long-term survival rate. While chemotherapy achieves remission in the majority of AML patients, many relapse due to residual chemotherapy-resistant AML populations. Allogeneic hematopoetic stem cell transplantation is a potential curative treatment for AML that demonstrates the efficacy of a cell-mediated treatment for chemotherapy-resistant disease. However, its wide application is limited by suitable donor availability and associated toxicity such as graft-versus-host disease (GvHD). Hence, there is a need for a new treatment approach that targets chemotherapy-resistant AML blasts with minimal side effects. The goals of this study were to characterize allogeneic CD3+CD4-CD8- double negative T (DNT) cells as a potential new target of treatment for AML patients, including ones that are resistant to chemotherapy, and to dissect its underlying mechanisms. Using two-hour flow cytometry-based in vitro killing assay, we demonstrated that the allogeneic DNT cells expanded from healthy volunteers were cytotoxic against 23/29 primary AML patient blasts in a dose-dependent manner. Of those, 13 blasts were obtained from chemotherapy refractory or relapsing AML patients and nine of them were susceptible to DNT cells. The anti-leukemia activity of DNT cells was further validated in a AML-NSG xenograft model: a single infusion of DNT cells into mice pre-engrafted with primary AML blasts from chemoresponsive, chemorefractory, and relapsed patients significantly reduced the leukemia burden. Although residual blasts were observed from DNT cell-treated group, they did not develop resistance to DNT cells, as they remained susceptible to DNT cells in ex vivo killing assay. Further, we demonstrated the safety of allogeneic DNT cells, as DNT cells did not target allogeneic peripheral blood mononuclear cells (PBMC) and hematopoietic stem/progenitor cells (HSPC) in vitro. In addition, administration of allogeneic DNT cells into NSG mice, engrafted with human HSPC, had no effect on the engraftment level of human hematopoetic cells and their differentiation into different lineages. Further, infusion of human DNT cells did not cause xenogeneic GvHD in mice, collectively demonstrating the safety of allogeneic DNT cells. Using blocking assays, we showed the roles of HLA-class I, NKG2D, and DNAM-1 in DNT cell-mediated cytotoxicity against AML, whereas HLA-class II and T cell receptor were not involved. While IFNγ release correlated with cytotoxicity of DNT cells, IFNγ treatment alone did not induce AML cell death. Neutralizing IFNγ reduced susceptibility to DNT cell-mediated cytotoxicity while pretreating AML with recombinant IFNγ increased their susceptibility by inducing higher expression of NKG2D and DNAM-1 ligands, where NKG2D- and DNAM-1-blocking antibodies abrogated the effect of IFNγ pretreatment. Cytolytic activity of DNT cells was mediated in perforin-granzyme-dependent fashion, which subsequently activated caspase-8 and caspase-9 pathways in AML cells to induce AML cell death. Collectively, these studies demonstrated the safety and efficacy of allogeneic DNT cell therapy as a potential treatment for AML patients, including those with chemotherapy-resistant leukemia, and revealed important molecules for the anti-leukemia activity of DNT cells. Citation Format: Jong Bok Lee, Claire Weihsu Chen, Mark D. Minden, John E. Dick, Li Zhang. Efficacy and safety of allogeneic double negative T cell as anti-AML therapy and its underlying mechanism. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A176.

Tissue-Related Hypoxia Attenuates Proinflammatory Effects of Allogeneic PBMCs on Adipose-Derived Stromal Cells In Vitro.

Human adipose tissue-stromal derived cells (ASCs) are considered a perspective tool for regenerative medicine. Depending on the application mode ASC/allogeneic immune cell interaction can occur in the systemic circulation under plenty high concentrations of O2 and in target tissues at lower O2 levels. Here we examined the effects of allogeneic PHA-stimulated peripheral blood mononuclear cells (PBMCs) on ASCs under ambient (20%) oxygen and “physiological” hypoxia (5% O2). As revealed with microarray analysis ASCs under 20% O2 were more affected by activated PBMCs, which was manifested in differential expression of more than 300 genes, whereas under 5% O2 only 140 genes were changed. Altered gene pattern was only partly overlapped at different O2 conditions. Under O2 ASCs retained their proliferative and differentiative capacities, mesenchymal phenotype, and intracellular organelle' state. ASCs were proinflammatory activated on transcription level that was confirmed by their ability to suppress activation and proliferation of mitogen-stimulated PBMCs. ASC/PBMCs interaction resulted in anti-inflammatory shift of paracrine mediators in conditioning medium with significant increase of immunosuppressive LIF level. Our data indicated that under both ambient and tissue-related O2 ASCs possessed immunosuppressive potential and maintained functional activity. Under “physiological” hypoxia ASCs were less susceptible to “priming” by allogeneic mitogen-activated PBMCs.

Targeting JAK2 By Gene Knockout or Pacritinib Treatment Reduces Gvhd and Xenograft Rejection By Promoting Induced Treg Differentiation

Janus kinase 2 (JAK2) signal transduction is a critical mediator of immune response. JAK2 activation promotes T-cell allosensitization, as well as Th1 and Th17 differentiation. JAK2-mediated STAT3 phosphorylation limits the generation of induced regulatory T cells (iTregs) by disrupting interactions between STAT5 and Foxp3. JAK2 is implicated in the onset of graft-versus-host disease (GVHD), which is a significant cause of transplant-related mortality after allogeneic hematopoietic cell transplantation (allo-HCT). Using murine models of allo-HCT we show here that transfer of donor JAK2-/- T cells is associated with significantly less GVHD, compared with wild-type or JAK2 replete donors (Figure 1, P =.003 and 0.01, respectively). Th1 differentiation among JAK2-/- T-cells is dramatically decreased, compared with controls. Conversely, iTreg polarization and stability are significantly increased among the JAK2 deficient T cells. To investigate the effect of pharmacologic JAK2 inhibition on T-cell alloresponses, pacritinib (supplied by CTI BioPharma), was chosen as it does not induce myelosuppression or increase risk for opportunistic infections in myelofibrosis patients - unlike JAK1/JAK2 inhibitors. Pacritinib potently inhibits JAK2, but also has suppressive activity toward JAK3, CSF1R, and IRAK1. Pacritinib was administered at 100mg/kg twice a day by oral gavage for 4 weeks beginning on the day of MHC-disparate allo-HCT, significantly reducing GVHD in recipient mice compared with methylcellulose vehicle control. In allogeneic mixed leukocyte reactions using human cells, pacritinib (2.5μM) significantly reduces T-cell proliferation after 5 days of culture (P <.0001). In vitro studies verified pacritinib eliminates STAT3 activity by IL-6 in human CD4+ T-cells, while permitting IL-2 induced STAT5 phosphorylation despite its effects on JAK3. This offers a platform to reduce Th17 development, while promoting iTreg populations. As such, pacritinib dramatically suppresses Th17 differentiation reflected by RORgammaT expression among naïve CD4+ T cells stimulated by allogeneic dendritic cells (DC), compared with DMSO (P <.0001). iTregs were generated with allogeneic DCs in the presence of pacritinib or DMSO for 5 days, and then cultured with self T-cell responders and fresh DCs without additional drug exposure. The suppressive potency of either pacritinib- or DMSO-treated iTregs was similar, suggesting JAK2 is not required for iTreg function. We then evaluated whether JAK2 inhibition could prevent human skin graft rejection in an NSG mouse xenograft model. A 1x1 cm split-thickness human skin graft was transplanted onto the animal dorsally. The mice rested for 30 days after surgery to permit engraftment, then received 5x106 allogeneic peripheral blood mononuclear cells (PBMC) by intraperitoneal injection. Pacritinib was administered at the same dose and schedule for 2 weeks beginning at time of PBMC injection as tolerated by the NSG mice. The treatment significantly delayed allograft rejection by the human donor PBMCs, compared with vehicle control (Figure 2: Median graft survival 32.5 v 51 days, Day +60 survival 0% v 33.3%, pooled data from 2 experiments, n=5-6 mice per arm, P =.0011). Collectively, these data clearly identify JAK2 as a therapeutic target to control donor alloreactivity and promote iTreg responses after HCT or solid organ transplantation.*BCB and DB contributed equally to this work. [Display omitted] [Display omitted] DisclosuresSinger:CTI BioPharma, Corp: Employment, Equity Ownership.

Efficacy and Safety of Allogeneic Double Negative T Cell As a Cellular Therapy for AML and Its Underlying Mechanism

Acute myeloid leukemia (AML) is the most common form of adult acute leukemia that is associated with a low long-term survival rate. While chemotherapy achieves remission in more than 70% of the treated AML patients, most relapse due to residual chemotherapy-resistant AML populations. Allogeneic hematopoietic stem cell transplantation reduces the relapse rate and demonstrates the efficacy of a cell-mediated treatment for the chemotherapy-resistant disease. However, its wide application is limited by donor availability and associated toxicity such as graft-versus-host disease (GvHD). Hence, there is a need for a new treatment approach that targets chemotherapy-resistant AML blasts with minimal side effects. The goals of this study were to characterize allogeneic CD3+ CD4-CD8- double negative T cells (DNTs) as a potential new therapy for AML patients, and to dissect its underlying mechanisms.Using a flow cytometry-based in vitro killing assay, we demonstrated that the allogeneic DNTs expanded from healthy volunteers were cytotoxic against 23/29 primary AML patient blasts in a dose-dependent manner. Thirteen blast samples were obtained from chemotherapy refractory or relapsing AML patients and, of those, 9 were susceptible to DNTs. Further, the average level of specific killing mediated by DNTs against chemotherapy-susceptible and -resistant blasts were comparable (19.30% ± 3.34% and 15.91% ± 3.63%, respectively). The anti-leukemia activity of DNTs was further validated in AML-NSG xenograft models. A single infusion of DNTs into mice pre-engrafted with primary AML blasts from chemoresponsive, chemorefractory, and relapsed patients significantly reduced the leukemia burden. While a single injection of DNTs resulted in a significant reduction of leukemia burden from 30% to 12.8%, it was further reduced to 2.6% after three injections of DNTs. Furthermore, the survival of NSG mice administered with a lethal dose of AML cell line, MV4-11, in DNT-treated group was significantly superior to the untreated group (57% vs. 0% survival on day 34, respectively). Although residual blasts were observed from the DNT -treated group, their susceptibility to DNT cell-mediated cytotoxicity remained comparable to blasts obtained from the PBS-treated group and primary AML blasts initially used for engraftment, suggesting that AMLs do not develop resistance to DNT-mediated cytotoxicity. In contrast to its potent cytolytic activity against leukemic cells, DNTs did not target allogeneic peripheral blood mononuclear cells (PBMC) and hematopoietic stem/progenitor cells (HSPC) in vitro. Administration of allogeneic DNTs into NSG mice engrafted with human HSPC had no effect on the engraftment level of human hematopoietic cells and their differentiation into different lineages. Further, in contrast to human PBMC, infusion of human DNTs did not cause xenogeneic GvHD in mice, collectively demonstrating the selective cytotoxic activity of allogeneic DNTs against leukemic cells. Using blocking assays, we showed that HLA-class I, NKG2D, and DNAM-1 were involved in DNT cell-mediated cytotoxicity against AML, whereas HLA-class II and T cell receptor did not play a significant role. We detected high levels of IFNγ release by DNTs upon encounter of susceptible AML targets. While IFNγ treatment alone did not induce AML cell death, neutralizing IFNγ reduced and pretreating AML cells with recombinant IFNγ increased their susceptibility to DNT cell killing. IFNγ treatment induced higher expressions of NKG2D and DNAM-1 ligands and blocking of NKG2D and DNAM-1 partially abrogated the effect of IFNγ on the AML-DNT interaction. Collectively, these studies demonstrated the safety and efficacy of allogeneic DNT therapy as a potential treatment for AML patients, including those with chemotherapy-resistant leukemia, and revealed the important molecules for the anti-leukemic activity of DNTs. DisclosuresNo relevant conflicts of interest to declare.

IMPS-38CETUXIMAB THERAPY REQUIRES IMMUNE STIMULATION FOR EFFICACY IN PEDIATRIC GLIOBLASTOMA

Pediatric glioblastoma (GBM) patients have <10% 5-year survival rate, and numerous experimental therapeutic approaches have failed to significantly improve survival. Effective therapies will therefore require a better understanding of the tumor and its environment. Myeloid cells and t-cells are increased in GBM compared to normal brain tissue, and have been shown to be immunosuppressive. This immunosuppressive tumor microenvironment will negatively impact immunotherapeutic approaches, such as EGFR-targeted antibody treatments, notably EGFR-targeted cetuximab and nimotuzumab. This class of antibodies has shown modest activity in pediatric GBM and we propose that adjuvant immune stimulatory treatment, to reverse inherent immunosuppression, will be required for improved efficacy. Here we investigate the effect of cetuximab in GBM cell line/immune effector cell co-cultures with and without immune stimulating adjuvants, IL-2 and GM-CSF. Different GBM cell lines were co-cultured with HLA-matched allogenic peripheral blood mononuclear cells and treated with cetuximab, GM-CSF, IL-2, or combinations of these. Cetuximab alone had no effect on cytotoxicity. However, addition of immune stimulants to cetuximab resulted in cytotoxicity in GBM cells as measured by accumulation of cleaved caspase 3/7. Combination of cetuximab with both GM-CSF and IL-2 resulted in the greatest cytotoxicity. Additionally, and ex-vivo pediatric GBM organotypic slice culture model was used to better evaluate immunotherapeutic strategies. Organotypic slice cultures contain patient, therefore autologous, infiltrating myeloid cells and t-cells, providing a clinically relevant model of GBM. The slices were treated for 10 days and followed by flow cytometric measurement of proliferation and cell death. Cetuximab alone reduced growth of tumor cells and also reduced cell death. Combination therapy of GM-CSF, IL-2, and cetuximab, however, increased immune cell proliferation, and reduced tumor cell proliferation and increased tumor cell cytotoxicity. We suggest that cetuximab, along with any other therapeutic antibody approach, requires combination immune stimulation in the treatment of GBM.

Dendritic Cells Differentiated from Human Umbilical Cord Blood-Derived Monocytes Exhibit Tolerogenic Characteristics.

Human umbilical cord blood (UCB) is rich in diverse hematopoietic stem cells that are competent to differentiate into various cell types with immunological compatibility at transplantation. Thus, UCB is a potential source for the preparation of dendritic cells (DCs) to be used for cell therapy against inflammatory disorders or cancers. However, the immunological properties of UCB-derived DCs are not fully characterized. In this study, we investigated the phenotypes and functions of UCB monocyte-derived DCs (UCB-DCs) in comparison with those of adult peripheral blood (APB) monocyte-derived DCs (APB-DCs). UCB-DCs contained less CD1a(+) DCs, which is known as immunostimulatory DCs, than APB-DCs. UCB-DCs exhibited lower expression of CD80, MHC proteins, and DC-SIGN, but higher endocytic activity, than APB-DCs. Lipopolysaccharide stimulation of UCB-DCs minimally augmented the expression of maturation markers and production of interleukin (IL)-12 and tumor necrosis factor (TNF)-α, but potently expressed IL-10. When UCB-DCs were cocultured with CD14(+) cell-depleted allogeneic peripheral blood mononuclear cells, they weakly induced the proliferation, surface expression of activation markers, and interferon (IFN)-γ production of T lymphocytes compared with APB-DCs. UCB possessed higher levels of prostaglandin E2 (PGE2) than APB, which might be responsible for tolerogenic phenotypes and functions of UCB-DCs. Indeed, APB-DCs prepared in the presence of PGE2 exhibited CD1a(-)CD14(+) phenotypes with tolerogenic properties, including weak maturation, impaired IL-12 production, and negligible T lymphocyte activation as UCB-DCs did. Taken together, we suggest that UCB-DCs have tolerogenic properties, which might be due to PGE2 highly sustained in UCB.

Nivolumab and Urelumab Enhance Antitumor Activity of Human T Lymphocytes Engrafted in Rag2-/-IL2Rγnull Immunodeficient Mice.

A current pressing need in cancer immunology is the development of preclinical model systems that are immunocompetent for the study of human tumors. Here, we report the development of a humanized murine model that can be used to analyze the pharmacodynamics and antitumor properties of immunostimulatory monoclonal antibodies (mAb) in settings where the receptors targeted by the mAbs are expressed. Human lymphocytes transferred into immunodeficient mice underwent activation and redistribution to murine organs, where they exhibited cell-surface expression of hCD137 and hPD-1. Systemic lymphocyte infiltrations resulted in a lethal CD4(+) T cell-mediated disease (xenograft-versus-host disease), which was aggravated when murine subjects were administered clinical-grade anti-hCD137 (urelumab) and anti-hPD-1 (nivolumab). In mice engrafted with human colorectal HT-29 carcinoma cells and allogeneic human peripheral blood mononuclear cells (PBMC), or with a patient-derived gastric carcinoma and PBMCs from the same patient, we found that coadministration of urelumab and nivolumab was sufficient to significantly slow tumor growth. Correlated with this result were increased numbers of activated human T lymphocytes producing IFNγ and decreased numbers of human regulatory T lymphocytes in the tumor xenografts, possibly explaining the efficacy of the therapeutic regimen. Our results offer a proof of concept for the use of humanized mouse models for surrogate efficacy and histology investigations of immune checkpoint drugs and their combinations.

Human anti-CAIX antibodies mediate immune cell inhibition of renal cell carcinoma in vitro and in a humanized mouse model in vivo.

BackgroundCarbonic anhydrase (CA) IX is a surface-expressed protein that is upregulated by the hypoxia inducible factor (HIF) and represents a prototypic tumor-associated antigen that is overexpressed on renal cell carcinoma (RCC). Therapeutic approaches targeting CAIX have focused on the development of CAIX inhibitors and specific immunotherapies including monoclonal antibodies (mAbs). However, current in vivo mouse models used to characterize the anti-tumor properties of fully human anti-CAIX mAbs have significant limitations since the role of human effector cells in tumor cell killing in vivo is not directly evaluated.MethodsThe role of human anti-CAIX mAbs on CAIX+ RCC tumor cell killing by immunocytes or complement was tested in vitro by antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) as well as on CAIX+ RCC cellular motility, wound healing, migration and proliferation. The in vivo therapeutic activity mediated by anti-CAIX mAbs was determined by using a novel orthotopic RCC xenograft humanized animal model and analyzed by histology and FACS staining.ResultsOur studies demonstrate the capacity of human anti-CAIX mAbs that inhibit CA enzymatic activity to result in immune-mediated killing of RCC, including nature killer (NK) cell-mediated ADCC, CDC, and macrophage-mediated ADCP. The killing activity correlated positively with the level of CAIX expression on RCC tumor cell lines. In addition, Fc engineering of anti-CAIX mAbs was shown to enhance the ADCC activity against RCC. We also demonstrate that these anti-CAIX mAbs inhibit migration of RCC cells in vitro. Finally, through the implementation of a novel orthotopic RCC model utilizing allogeneic human peripheral blood mononuclear cells in NOD/SCID/IL2Rγ−/− mice, we show that anti-CAIX mAbs are capable of mediating human immune response in vivo including tumor infiltration of NK cells and activation of T cells, resulting in inhibition of CAIX+ tumor growth.ConclusionsOur findings demonstrate that these novel human anti-CAIX mAbs have therapeutic potential in the unmet medical need of targeted killing of HIF-driven CAIX+RCC. The orthotopic tumor xenografted humanized mouse provides an improved model to evaluate the in vivo anti-tumor capabilities of fully human mAbs for RCC therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0384-3) contains supplementary material, which is available to authorized users.

Regenerative and immunogenic characteristics of cultured nucleus pulposus cells from human cervical intervertebral discs.

Cell-based regenerative approaches have been suggested as primary or adjuvant procedures for the treatment of degenerated intervertebral disc (IVD) diseases. Our aim was to evaluate the regenerative and immunogenic properties of mildly and severely degenerated cervical nucleus pulposus (NP) cells with regard to cell isolation, proliferation and differentiation, as well as to cell surface markers and co-cultures with autologous or allogeneic peripheral blood mononuclear cells (PBMC) including changes in their immunogenic properties after 3-dimensional (3D)-culture. Tissue from the NP compartment of 10 patients with mild or severe grades of IVD degeneration was collected. Cells were isolated, expanded with and without basic fibroblast growth factor and cultured in 3D fibrin/poly (lactic-co-glycolic) acid transplants for 21 days. Real-time reverse-transcription polymerase chain reaction (RT-PCR) showed the expression of characteristic NP markers ACAN, COL1A1 and COL2A1 in 2D- and 3D-culture with degeneration- and culture-dependent differences. In a 5,6-carboxyfluorescein diacetate N-succinimidyl ester-based proliferation assay, NP cells in monolayer, regardless of their grade of degeneration, did not provoke a significant proliferation response in T cells, natural killer (NK) cells or B cells, not only with donor PBMC, but also with allogeneic PBMC. In conjunction with low inflammatory cytokine expression, analyzed by Cytometric Bead Array and fluorescence-activated cell sorting (FACS), a low immunogenicity can be assumed, facilitating possible therapeutic approaches. In 3D-culture, however, we found elevated immune cell proliferation levels, and there was a general trend to higher responses for NP cells from severely degenerated IVD tissue. This emphasizes the importance of considering the specific immunological alterations when including biomaterials in a therapeutic concept. The overall expression of Fas receptor, found on cultured NP cells, could have disadvantageous implications on their potential therapeutic applications because they could be the targets of cytotoxic T-cell activity acting by Fas ligand-induced apoptosis.

Cysteamine treatment restores the in vitro ability to differentiate along the osteoblastic lineage of mesenchymal stromal cells isolated from bone marrow of a cystinotic patient.

BackgroundCystinosis is a rare autosomal recessive disease caused by mutations of the CTNS gene, which encodes for a lysosomal cystine/H+ symporter. In mice, inactivation of the CTNS gene causes intralysosomal cystine accumulation and progressive organ damage that can be reversed, at least in part, by infusion of mesenchymal stromal cells (MSCs). Little is known on the mesenchymal compartment of cystinotic patients. The aim of the study was to test the phenotypical and functional properties of cystinotic MSCs (Cys-MSCs) isolated from bone marrow (BM) aspirate of a patient with nephropathic cystinosis.MethodsMorphology, proliferative capacity (measured as population doublings), immunophenotype (by flow-cytometry) and immunomodulatory properties (as phytohemagglutinin-induced peripheral blood mononuclear cell proliferation) were analyzed. The osteogenic differentiation potential of Cys-MSCs was evaluated by histological staining (alkaline phosphatase activity, Alzarin Red and von Kossa staining) spectrophotometry and Quantitative Reverse Transcriptase Polymerase Chain Reaction for osteigenic markers in the presence and in the absence of cysteamine. Cys-MSCs were compared with those isolated and expanded ex vivo from three healthy donors (HD-MSCs).ResultsDespite a slightly lower proliferative capacity, Cys-MSCs displayed a characteristic spindle-shaped morphology and similar immunephenotype as HD-MSCs. Cys-MSCs and HD-MSCs prevented proliferation of PHA-stimulated allogeneic peripheral blood mononuclear cells to the same extent. After in vitro induction into osteoblasts, Cys-MSCs showed reduced alkaline phosphatase (ALP) activity, calcium depositions and expression of ALP and collagen type 1. When Cys-MSCs were treated in vitro with increasing doses of cysteamine (50-100-200 μM/L) during the differentiation assay, recovery of Cys-MSCs differentiation capacity into osteoblasts was observed. No difference in adipogenic differentiation was found between Cys-MSCs and HD-MSCs.ConclusionsOur results indicate that, as compared to HD-MSCs, Cys-MSCs show reduced ability to differentiate into osteoblasts, which can be reverted after cysteamine treatment.

Expression of genes involved in immune response and in vitro immunosuppressive effect of equine MSCs

The immunomodulatory capacities of mesenchymal stem cells (MSCs) have made them the subject of increased clinical interest for tissue regeneration and repair. We have studied the immunomodulatory capacity of equine MSCs derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) in cocultures with allogeneic peripheral blood mononuclear cells (PBMCs). Different isoforms and concentrations of phytohaemaglutinin (PHA) were tested to determine the best stimulation conditions for PBMC proliferation and a proliferation assay was performed for 7 days to determine the optimal day of stimulation of PBMCs. The effect of the dose and source of MSCs was evaluated in cocultures of 105 PBMCs with different ratios of AT- and BM-MSCs (1:1, 1:10, 1:20 and 1:50). Proliferation rates of the PBMCs were evaluated using BrdU ELISA colorimetric assay. PHA stimulated equine PBMCs reached their peak of growth after 3 days of culture. The immunoassay showed a decrease of the PBMCs growth at high ratio cocultures (1:1 and 1:10). Equine BM-MSCs and AT-MSCs demonstrated an ability to suppress the proliferation of stimulated PBMCs. Although MSCs derived from both sources displayed immunosuppressive effects, AT-MSCs were slightly more potent than BM-MSCs. In addition, the expression of 26 genes coding for different molecules implicated in the immune response was analyzed in cocultures of BM-MSCs and PHA stimulated PBMSCs by reverse transcriptase real time quantitative PCR (RT-qPCR). An upregulation in genes associated with the production of interleukins and cytokines such as TNF-α and TGF-β1 was observed except for IFN-γ whose expression significantly decreased. The variations of interleukins and cytokine receptors showed no clear patterns. COX-1 and COX-2 showed similar expression patterns while INOs expression significantly decreased in the two cell types present in the coculture. Cyclin D2 and IDO-1 showed an increased expression and CD90, ITG-β1 and CD44 expression decreased significantly in BM-MSCs cocultured with PHA stimulated PBMCs. On the contrary, CD6 and VCAM1 expression increased in these cells. With regard to the expression of the five genes involved in antigen presentation, an upregulation was observed in both cocultured MSCs and stimulated PBMCs. This study contributes to the knowledge of the immunoregulatory properties of equine MSCs, which are notably important for the treatment of inflammation processes, such as tendinitis and osteoarthritis.

Negligible immunogenicity of induced pluripotent stem cells derived from human skin fibroblasts.

Human induced pluripotent stem cells (hiPSCs) have potential applications in cell replacement therapy and regenerative medicine. However, limited information is available regarding the immunologic features of iPSCs. In this study, expression of MHC and T cell co-stimulatory molecules in hiPSCs, and the effects on activation, proliferation and cytokine production in allogeneic human peripheral blood mononuclear cells were examined. We found that no-integrate hiPSCs had no MHC-II and T cell co-stimulatory molecules expressions but had moderate level of MHC-I and HLA-G expressions. In contrast to human skin fibroblasts (HSFs) which significantly induced allogeneic T cell activation and proliferation, hiPSCs failed to induce allogeneic CD45+ lymphocyte and CD8+ T cell activation and proliferation but could induce a low level of allogeneic CD4+ T cell proliferation. Unlike HSFs which induced allogeneic lymphocytes to produce high levels of IFN-γ, TNF-α and IL-17, hiPSCs only induced allogeneic lymphocytes to produce IL-2 and IL-10, and promote IL-10-secreting regulatory T cell (Treg) generation. Our study suggests that the integration-free hiPSCs had low or negligible immunogenicity, which may result from their induction of IL-10-secreting Treg.

Immunosuppression By Mesenchymal Stromal Cells Derived from Human Induced Pluripotent Stem Cells : Evaluation in an aGVHD Model

▪BackgroundOne major problem of allogeneic hematopoietic stem cell transplantation (HSCT) is acute Graft versus Host Disease (aGVHD). aGVHD has been managed until now with HLA matching and a constant evolving repertoire of immunosuppressive drugs. One alternative would be to generate in the host a permanent tolerance state toward the graft. Tolerant inducing cell therapy has been proposed with adult mesenchymal stromal (MSCs) cells. Ex vivo isolated somatic MSCs have been implicated in immunoregulatory functions on cells from both the innate and adaptive immune system. They were proposed for cell therapies for the treatment of aGVHD,Nevertheless, their use is restricted because of the few number that can be recovered from adult tissues, their limited in vitro expansion, and the absence of a full phenotypic characterization. Therefore other sources of well-defined and unlimited number of MSCs are needed, and MSCs derived in vitro from human Induced pluripotent stem cell (huIPS) would be a valuable tool for therapeutic approaches.AimsBecause of our expertise in pluripotent stem cell differentiation, we generated huiPS-MSCs that present a strong immunosuppressive activity on allogeneic T cell responses. Our objectives are: 1/ To evaluate and characterize in vitro this immunosuppression. 2/ To validate in vivo these results using a xenoGVHD model.MethodsTo characterize the huIPS-MSCs in vitro, FACS phenotyping and multipotency were tested. Their immunogenicity in vitro was monitored in co-cultures with allogenic peripheral blood mononuclear cells (PBMC). The in vivo immunosuppressive activity of huiPS-MSCs was evaluated using a xenoGVHD model in immunodeficient NSG (NOD/SCID/IL2rγKO) mice in which human PBMC were injected intra-peritonally. We established 3 groups : 1) huIPS-MSCs (control) 2) PBMC 3) PBMC + IPS-MSCs. We repeated huIPS-MSCs injection weekly with median number of injection n=3 (range 2-3). The activation state of human allogeneic T lymphocytes recovered from mice between 5 to 8 weeks after initial injection was evaluated and indicated the level of the xenoGVHD process and the efficiency of huiPS-MSCs to prevent it.Resultsa) In vitro characterization of huIPS-MSCsAs expected, the huiPS-MSCs were positive for CD73, CD90, CD105, HLA-I Ags and negative for CD45, CD34, HLA-II Ags and they were capable of differentiation into the classical mesenchymal-derived cells (osteoblast, chondrocytes and adipocytes). To test their immunosuppressive properties, we analyzed their action on the proliferation of human T lymphocytes stimulated in an allogeneic manner (Fig 1). The stimulation of PBMC in mixed lymphocyte reaction resulted in CD4 and CD8 T cell proliferation (28 ± 7% and 47 ± 8%, respectively), which was significantly reduced in co-culture with huiPS-MSCs (4 ± 2% and 10 ± 2, respectively, n=3 p<0,05). We were able to demonstrate using blocking antibodies that part of the inhibition exerted by the iPS-MSCs is due to a) B7H1, a membrane receptor for the B7 family, known for its inhibitory action on the activation of T lymphocytes b) and B7H3 (from the same family) whose role remains controversial.b) In vivo characterization of huIPS-MSCsAfter sacrifice of mice, human circulating cells, those present in the peritoneal cavity and in the spleen were analysed by FACS. Mostly T lymphocytes were detected, and their number was significantly reduced in mice treated with huIPS-MSCs p<0,05 (Fig 2).Intracytoplasmic labelling of recovered T cells showed that untreated mice displayed high percentages of human differentiated T cells producing IFN # and TNF # (typical of a inflammatory Th1 cytokine polarization profile), while little or none produced low inflammatory (IL-4) or anti-inflammatory (IL-10) cytokines. In contrast, in mice treated with the huiPS-MSCs, the proportion of T cells of the Th1 type was substantially reduced, while that of T cells producing IL-4 and / or IL-10 was slightly increased (Fig 3). In parallel, T cells expressing FoxP3 appeared.ConclusionWe were able to generate immune-modulatory huiPS-MSCs that can be used to reduce activation of T cells in a xeno-aGVHD model through a switch from a Th1 inflammatory differentiation pathway to a T cell regulatory pathway. Our results may favor the development of new tools and strategies based on the use of pluripotent stem cells and their derivatives to prevent aGVHD but also for the induction of specific tolerance. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Marrow Environment Post-Transplant Day (+) 7-9 Following Cyclophosphamide and Fludarabine Minimal Intensity Allotransplantation

▪Introduction: Cyclophosphamide/Fludarabine (Cy/Flu) nonmyeloablative conditioning for the treatment of acute myeloid leukemia (AML) in remission and myelodysplastic syndrome (MDS) permits engraftment with minimal tissue injury. Unrelated donor (URD) recipients experience rapid neutrophil engraftment, quicker rates to full donor T cell and peripheral blood mononuclear cell (PBMC) chimerism and significantly more effective disease control. Following allogeneic PBMC infusion on day 0, peripheral blood leukocyte counts on day (+) 1 are uniformly less < 200/mm3. Mixed chimeric, predominately donor T and NK lymphocytes are detectable on day (+) 6-8 prior to neutrophil engraftment. The lack of respiratory symptoms or splenic enlargement post-infusion suggested that cells may be detectable in the marrow at a much earlier time point post-transplant than is classically recognized after myeloablative allotransplant. We hypothesized that mature lymphocytes would be detectable in a cellular marrow environment by day (+) 8. To begin to test this hypothesis, we collected marrow between day (+) 7-9 (avoiding weekends) from 6 consenting participants with AML, MDS or chronic lymphocytic leukemia (CLL) who participated in a single institution, phase II clinical trial at Indiana University and the IU Melvin and Bren Simon Cancer Center.Methods: Marrow aspirate H and E stains, biopsies, and flow cytometric analyses (including CD2, CD3, CD56 and CD19) were obtained pre-transplant and on day (+) 7-9 following PBMC infusion in the setting of minimal intensity cyclophosphamide/fludarabine conditioning. PMBC chimerism was analyzed on day (+) 7-9. Clinical characteristics and peripheral blood cell counts were recorded.Results: Three patients had AML or MDS and 3 patients had CLL. All patients achieved full engraftment. In the AML/MDS subgroup day (+) 7-9 cellularity ranged from less than 5 to 70% with PBMC chimerism from 39.2 to 93.4% donor. In the CLL subgroup day (+) 7-9 cellularity ranged from 50 to 70% with PBMC chimerism from 0.9 to 5.6% donor. Baseline cellularity, corresponding peripheral cell counts, and marrow flow cytometric analyses from day (+) 7-9 are included in Table 1.Conclusion: Cy/Flu conditioning appears to be associated with early marrow cellularity at a time when peripheral blood chimerism is mixed. Day (+) 7-9 marrow examinations after Cy/Flu are generally quite cellular. Patients with AML/MDS and CLL appear to differ substantially in histological appearance, cell differentials and flow cytometric analyses. This suggests a unique engraftment kinetic that has yet to be appreciated and may be a useful platform for the study of cellular immune anti-cancer activity.Abstract 5851 Table 1DiseaseDisease stateBaseline cellularityCellularity Day 7-9WBC Day 7-9BM CD2%BM CD3%BM CD19%BM CD56%PBMC chimerism (donor%)AMLCR16020300751218693.4MDSactive707090061731739.2Secondary AMLactive80<54009794<1380CLLactive90705200049325.6CLLactive90704400029513.8CLLactive8050700039330.9 DisclosuresNo relevant conflicts of interest to declare.