Allogeneic Mouse Model Research Articles

CD45-Targeted Antibody Drug Conjugate Plus Post Transplant Cytoxan Is Sufficient to Enable Allogeneic Bone Marrow Transplant in a Minor Mismatch Mouse Model

Bone Marrow Transplant (BMT) is a potentially curative treatment for malignant and non-malignant blood disorders. Current regimens for patient preparation, or conditioning, prior to BMT limit the use of this curative procedure due to regimen-related mortality and morbidities, including risks of organ toxicity, infertility and secondary malignancies. Targeted preparation using antibody drug conjugates (ADCs) to mouse CD45 has previously been shown to be sufficient to enable bone marrow transplant (BMT) in syngeneic immune competent mice (Palchaudhuri et al. Nature Biotech 2016 34:738–745), and this approach to preparation has the potential to expand the utility of BMT if it can be successfully translated to patients. To further investigate the utility of this tool ADC in murine transplant models, we explored anti-CD45-saporin (CD45-SAP) in an allogeneic minor mismatch transplant model (Balb/c donor into DBA/2 recipients). The goal of the work was to identify the level of immune suppression, if any, that needs to be used in combination with CD45-SAP to enable high donor chimerism in the allogeneic setting. CD45-SAP (1.9 mg/kg, iv) was evaluated alone or in combination with additional immune modulating agents: clone 30F11 (25 mg/kg, IP), a naked anti-CD45 antibody that mimics ATG by relying on effector function to enable potent peripheral B- and T -cell depletion; pre-transplant Cytoxan (PreTCy, 200 mg/kg, IP), 2 Gy total body irradiation (TBI), and post-transplant Cytoxan (PTCy, 200 mg/kg, IP) to prevent graft versus host disease as well as block host versus graft rejection. 9 Gy TBI was used as the conventional conditioning positive control. Conditioned mice were transplanted with 2 × 107 whole bone marrow cells, and chimerism assessed over 12 weeks. CD45-SAP in combination with PTCy achieved comparable peripheral myeloid chimerism, a readout of stem cell engraftment, to 9 Gy TBI (>90%) at 8 weeks post-transplantation (Fig 1). The addition of 30F11 to the CD45-SAP/PTCy protocol had no effect on peripheral donor chimerism (59% vs 61%), suggesting additional lymphodepletion is not required. In contrast, the single agents alone, 2 Gy TBI in combination with 30F11 and PTCy resulted in These results indicate CD45-SAP in combination with PTCy is sufficient to enable high levels of donor chimerism in the minor mismatched setting without the need for additional immune suppression. CD45-SAP was more effective at conditioning than 2Gy TBI or PreTCy. Future experiments will investigate the CD45-SAP/PTCy protocol in haploidentical and fully mismatched allogeneic mouse models.

Lower Abundance and Impaired Function of CD71+ Erythroid Cells in Inflammatory Bowel Disease Patients During Pregnancy.

CD71+ erythroid cells are enriched during pregnancy with immuno suppressive properties. We investigated the frequency and functionality of CD71+ erythroid cells in peripheral blood, cord blood, and placenta of inflammatory bowel disease [IBD] patients versus healthy controls [HCs]. We aimed to determine their role in IBD pathogenesis during pregnancy. Peripheral blood was collected at preconception, the first, second and third trimesters, and postpartum. Cord blood and placental tissues were collected at the time of birth. Cells from different specimens were subjected to immune-phenotyping and functional assays. CD71+ erythroid cells were purified for quantitative polymerase chain reaction [qPCR] analysis. Using an allogeneic mouse model of pregnancy, the effects of CD71+ erythroid cells depletion on intestinal homeostasis and dysbiosis was studied. IBD patients had lower CD71+ erythroid cells during pregnancy compared with HCs. Placenta and cord blood CD71+ erythroid cells from IBD patients exhibited impaired functionality and expressed lower inhibitory molecules including VISTA, TGF-β, and reactive oxygen species [ROS]. Lower CD71+ erythroid cells were correlated with reduced regulatory T cells and increased immune-activation in IBD patients. Depletion of CD71+ erythroid cells in an allogeneic pregnancy model resulted in upregulation of TLRs, IL-6, and CXCL-1, and enhanced production of TNF-α, in intestinal tissues. In contrast, TGF-β gene expression was reduced. Excessive inflammatory response in the gut [e.g. TNF-α] affects intestinal integrity and CD71+ erythroid cells impact on the gut's bacterial composition. Reduced frequency and/or impaired functionality of CD71+ erythroid cells during pregnancy may predispose IBD patients to a more pro-inflammatory milieu in their gastrointestinal tract, characterised by lower Tregs, higher IL-6, and TNF-α, and dysbiosis.

Dual use of hematopoietic and mesenchymal stem cells enhances engraftment and immune cell trafficking in an allogeneic humanized mouse model of head and neck cancer.

In this report, we describe in detail the evolving procedures to optimize humanized mouse cohort generation, including optimal conditioning, choice of lineage for engraftment, threshold for successful engraftment, HNSCC tumor implantation, and immune and stroma cell analyses. We developed a dual infusion protocol of human hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stem cells (MSCs), leading to incremental human bone marrow engraftment, and exponential increase in mature peripheral human immune cells, and intratumor homing that includes a more complete lineage reconstitution. Additionally, we have identified practical rules to predict successful HSPC/MSC expansion, and a peripheral human cell threshold associated with bone marrow engraftment, both of which will optimize cohort generation and management. The tremendous advances in immune therapy in cancer have made the need for appropriate and standardized models more acute than ever, and therefore, we anticipate that this manuscript will have an immediate impact in cancer-related research. The need for more representative tools to investigate the human tumor microenvironment (TME) has led to the development of humanized mouse models. However, the difficulty of immune system engraftment and minimal human immune cell infiltration into implanted xenografts are major challenges. We have developed an improved method for generating mismatched humanized mice (mHM), using a dual infusion of human HSPCs and MSCs, isolated from cord blood and expanded in vitro. Engraftment with both HSPCs and MSCs produces mice with almost twice the percentage of human immune cells in their bone marrow, compared to mice engrafted with HSPCs alone, and yields 9- to 38-fold higher levels of mature peripheral human immune cells. We identified a peripheral mHM blood human B cell threshold that predicts an optimal degree of mouse bone marrow humanization. When head and neck squamous cell carcinoma (HNSCC) tumors are implanted on the flanks of HSPC-MSC engrafted mice, human T cells, B cells, and macrophages infiltrate the stroma of these tumors at 2- to 8-fold higher ratios. In dually HSPC-MSC engrafted mice we also more frequently observed additional types of immune cells, including regulatory T cells, cytotoxic T cells, and MDSCs. Higher humanization was associated with in vivo response to immune-directed therapy. The complex immune environment arising in tumors from dually HSPC-MSC engrafted mice better resembles that of the originating patient's tumor, suggesting an enhanced capability to accurately recapitulate a human TME.

In Vivo Depletion of Dividing Donor Cells Using Post-Transplant Cyclophosphamide can Reduce the Development of Graft-Versus-Host Disease in a Humanised Mouse Model

Introduction Graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic bone marrow transplantation (BMT), a curative therapy for haematological malignancies. GVHD develops when donor immune cells in the graft become reactive against the host, damaging host tissues (liver, skin and gut). Depletion of dividing donor cells reduces GVHD severity in allogeneic mouse models, but there is limited data on the effects of depletion in humanised mouse models of GVHD. This study aimed to determine the effect of depleting dividing donor immune cells using post-transplant cyclophosphamide (PTCy) on the development of disease in a well-established humanised mouse model of GVHD. Materials and Methods Isolated human peripheral blood mononuclear cells (hPBMCs) were injected (20 x 106 cells i.p) into NOD-SCID-IL2Rγnull (NSG) mice, with PTCy (33 mg/kg), or saline injected on days 3 and 4 post-hPBMC injection. Mice were monitored for 10 weeks for GVHD development using a clinical scoring system, with the engraftment of human immune cells checked by flow cytometry at 3 weeks (blood) and end-point (spleen). At end-point the relative expression of inflammatory cytokines in GVHD target organs were analysed by qPCR. Results At 3 weeks post-hPBMC injection, similar percentages of human leukocytes were found in saline- (41.18 ± 5.58%) and PTCy-injected mice (31.27 ± 4.43%) (P = 0.198, n = 9 mice per treatment group), with the majority of these cells being T cells in both groups (Saline, 97.34 ± 0.91%; PTCy, 96.01 ± 0.84%) (P = 0.332). PTCy significantly increased the human CD4:CD8 T cell ratio compared to saline-injected mice (Saline, 0.7245 ± 0.07; PTCy, 1.633 ± 0.40) (P = 0.031). Furthermore, compared to saline, PTCy decreased the percentages of human CD4+CD25+CD127lo regulatory T cells (Saline, 6.69 ± 0.64%; PTCy, 3.30 ± 0.52%) (P = 0.001) and human CD38+CD8+CD45RA-CCR7- memory T cells (Saline, 81.46 ± 2.30%; PTCy, 64.62 ± 7.80%). PTCy reduced development of GVHD, with significantly reduced weight loss (P < 0.0001) and clinical score (P < 0.0001), and increased survival (MST = 52 days) compared to saline-injected mice (MST = 26 days) (P < 0.0001). Whilst both mouse groups demonstrated similar relative human IFNγ expression in the gut (P = 0.125) and liver (P = 0.162), PTCy significantly reduced relative human IL-17 expression in both tissues (P = 0.044 and 0.048, respectively). Discussion This data indicates that the depletion of dividing donor cells using PTCy can reduce GVHD in this humanised mouse model, whilst allowing human cell engraftment. Further, PTCy reduces percentages of cells implicated in GVHD development. However, mice treated with PTCy still develop GVHD, so further strategies are required. Conclusion This humanised mouse model of GVHD will allow investigation of combinatorial therapies that use depletion strategies targeting dividing human donor immune cells as potential GVHD therapeutics.

Donor Conditioning with Recipient-Derived PD-L1/PD-L2-Expressing B Cells Prevents Lethal Acute GVHD in a Fully Allogeneic Mouse Model

Aim We recently found that the inoculation with Balb/c (H-2d) PD-L1/PD-L2-expressing B cells lead to persistent suppression of anti-Balb/c T cell-responses, and significantly prolonged the survival of subsequently grafted Balb/c hearts in C57BL/6 (B6) (H-2b) mouse recipients. Given the effectiveness of those B cells as regulatory antigen-presenting cells targeting alloreactive T cells, we investigated if donor pre-conditioning with recipient-derived PD-L1/PD-L2 B cells could prevent acute GVHD after fully allogeneic bone marrow transplantation (BMT)/donor lymphocyte infusion (DLI) in mice. Methods To induce acute GVHD, lethally irradiated BALB/c mice were transplanted with 5 × 106 bone marrow cells and 1 × 106 splenic T cells from B6 mice. Twelve days prior to the BMT/DLI, B6 donors were intravenously injected with 10 × 106 BALB/c peritoneal cavity (PerC) B cells. Results Mixed-lymphocyte reaction (MLR) assays using splenocytes from the B6 donor mice at 12 days after the B cell injection revealed the significantly reduced anti-Balb/c T cell-responses in the recipients of PerC B cells, as compared to those in untreated control mice. Since PerC B cells contained MHC class II+ CD80+ CD86+ PD-L1+ PD-L2+ cells among the CD5+ B-1a cell subset, PerC B cells from Balb/c mice were pre-incubated with anti-PD-L1/PD-L2 mAbs prior to injection. This treatment abrogated their immune-regulatory effects on anti-Balb/c T cells in the MLR assays, indicating that the expression of both PD-L1 and PD-L2 on allogeneic PerC B cells is required to inhibit T cell-responses to the cognate allostimulation through engaging PD-1. The difference of T cell repertoire between the conditioned and untreated B6 was investigated using mAbs directed against T cell receptor Vβ regions. The proportion of Vβ5.1/5.2+ and Vβ11+ CD8+ T cells in the spleen of the conditioned B6 mice was significantly diminished, while that of Vβ8.1/8.2+ CD8+ T cells was not, indicating the specificity of the deletion for super-antigens presented by the BALB/c B cells. The donor pre-conditioning with recipient-derived PerC B cells markedly improved clinicopathological manifestations and survival in the lethal acute GVHD model, i.e. the mean survival of recipients of BMT/DLI from the conditioned donor was 27.9 ± 16.0 days (n=8), while that of recipients of BMT/DLI from the untreated donor was 6.6 ± 0.9 days (n=5). Conclusions These findings demonstrate that the PerC B cells, including PD-L1/PD-L2 B-1a cells, suppress T cells responding to allostimulation by clonal deletion. Thus, our study presents what we believe to be the reversed paradigm for the prevention of lethal acute GVHD by donor pre-conditioning with those immune-regulatory B cells derived from recipients.

2543 High concentrations of CXCL12 decrease pancreatic adenocarcinoma growth

OBJECTIVES/SPECIFIC AIMS: We hypothesized that CXCL12, as a biased dimer variant or secreted at dimer-dominant concentrations, would influence PDAC growth and progression. METHODS/STUDY POPULATION: PDAC cells were genetically manipulated to express dimer-promoting levels of CXCL12. These cells were studied in vitro or orthotopically implanted into the mouse pancreas for in vivo studies. As a second approach, recombinant wild-type or engineered CXCL12 monomer or dimer proteins were applied to cells in culture or administered intra-peritoneal to study the effects on tumor growth. RESULTS/ANTICIPATED RESULTS: Mice engrafted with CXCL12-expressing cells had a better survival rate, delayed tumor growth and smaller tumors. Tumors from these mice had significantly less proliferation, measured by Ki-67 staining. In vitro analysis of CXCL12-expressing cells showed decreased viability and growth rates. Percent of cells in the cell cycle G2 phase was also decreased, suggesting cell cycle progression blockade. Viability of human PDAC cells dose-dependently declined upon wild-type CXCL12 treatment, with the non-motile dimer-dominant dose (1000 nM) exhibiting maximal effect. Treatment in an allogeneic mouse model of PDAC with locked-dimer CXCL12, but not wild-type, reduced tumor burden. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results support the notion that biased CXCL12 signaling may be therapeutically exploited to limit pancreatic cancer progression.

The P2X7 receptor antagonist Brilliant Blue G reduces serum human interferon-γ in a humanized mouse model of graft-versus-host disease.

Graft-versus-host disease (GVHD) remains a major problem after allogeneic haematopoietic stem cell transplantation, a curative therapy for haematological malignancies. Previous studies have demonstrated a role for the adenosine triphosphate (ATP)-gated P2X7 receptor channel in allogeneic mouse models of GVHD. In this study, injection of human peripheral blood mononuclear cells (PBMCs) into immunodeficient non-obese diabetic-severe combined immunodeficiency-interleukin (NOD-SCID-IL)-2Rγnull (NSG) mice established a humanized mouse model of GVHD. This model was used to study the effect of P2X7 blockade in this disease. From five weeks post-PBMC injection, humanized mice exhibited clinical signs and histopathology characteristic of GVHD. The P2X7 antagonist, Brilliant Blue G (BBG), blocked ATP-induced cation uptake into both murine and human cells in vitro. Injection of BBG (50 mg/kg) into NSG mice did not affect engraftment of human leucocytes (predominantly T cells), or the clinical score and survival of mice. In contrast, BBG injection reduced circulating human interferon (IFN)-γ significantly, which was produced by human CD4+ and CD8+ T cells. BBG also reduced human T cell infiltration and apoptosis in target organs of GVHD. In conclusion, the P2X7 antagonist BBG reduced circulating IFN-γ in a humanized mouse model of GVHD supporting a potential role for P2X7 to alter the pathology of this disease in humans.

Temporally controlled B cell depletion with universal chimeric antigen receptor (CAR) T cells for pemphigus vulgaris (PV) therapy

Abstract Therapy of PV relies on chronic immunosuppression, which results in significant morbidity. Complete, transient B cell depletion should cure PV, since autoreactive clones do not recur upon regeneration of the B cell repertoire. Engineered CAR T cells (CARTs) are the most potent means for total B cell depletion, but for therapy of autoimmunity, temporal control of CART activity is necessary to prevent lasting immunosuppression. Here we validate 3 novel strategies to control CART survival and function. We combined a B cell targeting CAR with an inducible caspase 9 suicide gene (sCAR), a reverse (constitutively active) suicide gene (revCAR), or a molecular on-switch that permits CAR surface expression (onCAR). sCAR, revCAR and onCARTs showed potent in vitro killing equivalent to conventional CARTs. Activation of the respective regulatory system resulted in in vitro depletion of &amp;gt;95% of sCART and revCARTs and loss of CAR expression in onCARTs. In an in vivo leukemia model, &amp;gt;90% of sCARTs were eliminated (p&amp;lt;0.01) as compared to vehicle treated mice, while preserving their efficacy before suicide gene activation (p&amp;lt;0.001). RevCARTs showed complete loss of leukemia control in vivo in the absence of a suicide-preventing compound (p&amp;lt;0.01), indicating their functional depletion in vivo. Finally, to allow universal (allogeneic) CART therapy, we used CRISPR genome editing to disrupt endogenous T cell receptor and MHCI expression. Universal sCARTs caused total B cell depletion in a humanized allogeneic BLT mouse model (p&amp;lt;0.05) with complete in vivo depletion of sCARTs. In summary, we present 3 novel strategies to regulate CARTs, which provide a platform for curative, universal, and large-scale applications in PV and other autoimmune diseases.

Parainfluenza virus spread and clearance in a hematopoietic cell transplant model

Abstract Parainfluenza viruses (PIV) are significant causes of morbidity and mortality in patients receiving hematopoietic cell transplant (HCT), particularly in the first 100 days post an allogeneic transplant. In the absence of effective therapeutic or preventive measures, it is critical to identify the immunological determinants that facilitate viral clearance that could be exploited for intervention, especially with respect to lower respiratory involvement. To study the dynamics of PIV infection in HCT host, we developed an allogeneic mouse model that we infect with a murine PIV. Specifically, we use a reporter Sendai virus that allows a daily, non-invasive bioluminescence imaging in living mice. HCT mice were infected shortly before transplant to study viral progression and clearance in the most critical period after transplant. Our data showed a marked prolongation of infection with a significant delay in clearance (21 days). The initiation of clearance correlated with a slight rebound in lymphocytes, which were mostly of the B type with more than 90% chimerism. Changing the initial site of infection by changing the infecting volume from 5 to 30 μL dramatically increased the lung signal, morbidity, and mortality. Treating the mice with Sendai-specific monoclonal antibodies significantly reduced the lung bioluminescence, morbidity, and prevented mortality. Overall, we developed a system that allows studying the correlation between different aspects of the immune system and PIV progression and clearance. Ongoing studies will determine the effect of manipulating the graft, including T cell enrichment versus depletion and the role of natural killer cells on Sendai virus infection.

Enhanced in utero allogeneic engraftment in mice after mobilizing fetal HSCs by α4β1/7 inhibition

AbstractIn utero hematopoietic cell transplantation (IUHCT) is a novel nonmyeloablative approach that results in donor-specific tolerance and mixed allogeneic chimerism. Clinical application is limited by low levels of donor cell engraftment. Competition from endogenous hematopoietic stem cells (HSCs) for limited “space” in fetal hematopoietic organs remains a significant barrier to successful IUHCT. AMD3100, a CXCR4 inhibitor, and firategrast, an α4β1 and α4β7 integrin inhibitor (α4β1/7), have been shown to disrupt HSC retention in the postnatal hematopoietic niche. We hypothesized that maternal administration of AMD3100 and/or firategrast prior to IUHCT would mobilize endogenous HSCs from the fetal liver (FL) and result in preferential FL homing of donor HSCs and enhanced long-term engraftment following IUHCT in an allogeneic mouse model. We demonstrate that (1) both agents cross the placenta with rapidly detectable fetal serum concentrations following maternal administration; (2) firategrast treatment alone or with AMD3100 mobilizes endogenous HSCs from the FL and results in increased FL homing of donor HSCs following IUHCT; and (3) enhanced donor HSC homing following firategrast treatment translates into increased long-term multilineage donor cell engraftment. This approach highlights the potential of mobilization strategies to overcome barriers to successful engraftment and increase the clinical promise of IUHCT.

Abstract IA14: PDX models in head and neck cancers: Present status and future application for immune-oriented biology and therapy development

Abstract Targeted therapy development in head and neck squamous cell carcinoma (HNSCC) is challenging given the rarity of activating mutations. Additionally, HNSCC incidence is dramatically increasing related to human papillomavirus (HPV). We sought to develop an in vivo model derived from patients reflecting the evolving HNSCC epidemiologic landscape, and use it to identify new therapies. Primary and relapsed tumors from over 100 HNSCC patients, both HPV+ and HPV-, have been implanted on mice, giving rise to over 50 strains. Resulting xenografts were characterized by detecting key mutations, measuring protein expression by IHC and gene expression/pathway analysis by mRNA-sequencing. The genetic makeup of these tumors was consistent with the frequencies of TP53, PI3KCA, NOTCH1, and NOTCH2 mutations found in landmark genomic characterization of large HNSCC patient cohorts. Tumors maintained their original morphology, genetic profiles, and drug susceptibilities through serial passaging. Cancer cells deceive the host to create an advantageous microenvironment and evade immune surveillance. A limitation to studying human cancer is the limited availability of in vivo models with appropriate human stroma and immunity. To address this unmet need, we generated an allogeneic humanized mouse (aHM) model where HNSCC were implanted on NOD/SCID/IL2rg-/- (NSG) mice reconstituted with human hematopoietic stem cells (HSCs) and other precursors from an unrelated donor. In aHM, bone marrow-derived human immune and mesenchymal cells homed into the growing tumor and interacted with human cancer cells as they would in a patient, partially reversing the gene expression drift that occurs after tumor passage on immune compromised mice. Further work is aimed at improving upon this platform. In summary, we have developed a HNSCC model covering its clinical spectrum whose major genetic alterations and susceptibility to anticancer agents represent contemporary HNSCC. Then, we have generated an aHM model to enable immune biology and therapy exploration in HNSCC. These models will enable prospectively testing therapeutic-oriented hypotheses leading to enhanced precision medicine, especially in the field of immune therapy. Citation Format: Antonio Jimeno. PDX models in head and neck cancers: Present status and future application for immune-oriented biology and therapy development. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr IA14.

Lysosomal acid lipase in mesenchymal stem cell stimulation of tumor growth and metastasis.

Bone marrow mesenchymal stem cells (MSCs) are an important participant in the tumor microenvironment, in which they promote tumor growth and progression. Here we report for the first time that depletion of lysosomal acid lipase (LAL) in MSCs impairs their abilities to stimulate tumor growth and metastasis both in allogeneic and syngeneic mouse models. Reduced cell viability was observed in LAL-deficient (lal−/−) MSCs, which was a result of both increased apoptosis and decreased proliferation due to cell cycle arrest. The synthesis and secretion of cytokines and chemokines that are known to mediate MSCs' tumor-stimulating and immunosuppressive effects, i.e., IL-6, MCP-1 and IL-10, were down-regulated in lal−/− MSCs. When tumor cells were treated with the conditioned medium from lal−/− MSCs, decreased proliferation was observed, accompanied by reduced activation of oncogenic intracellular signaling molecules in tumor cells. Co-injection of lal−/− MSCs and B16 melanoma cells into wild type mice not only induced CD8+ cytotoxic T cells, but also decreased accumulation of tumor-promoting Ly6G+CD11b+ myeloid-derived suppressor cells (MDSCs), which may synergistically contribute to the impairment of tumor progression. Furthermore, lal−/− MSCs showed impaired differentiation towards tumor-associated fibroblasts. In addition, MDSCs facilitated MSC proliferation, which was mediated by MDSC-secreted cytokines and chemokines. Our results indicate that LAL plays a critical role in regulating MSCs' ability to stimulate tumor growth and metastasis, which provides a mechanistic basis for targeting LAL in MSCs to reduce the risk of cancer metastasis.

Human tonsil‑derived mesenchymal stromal cells enhanced myelopoiesis in a mouse model of allogeneic bone marrow transplantation.

Mesenchymal stromal cells (MSCs) have therapeutic potential for repairing tissue damage and are involved in immune regulation. MSCs are predominantly isolated from bone marrow (BM), adipose tissue or placental tissue. Further to these well-known sources, the isolation of MSCs from human tonsils was previously reported. The aim of the present study was to investigate a potential role for tonsil-derived MSCs (T-MSCs) in BM reconstitution and application towards supplementing hematopoiesis in a mouse model of BM transplantation (BMT). Eight-week-old BALB/c female mice received 80 mg/kg busulfan (Bu)/200 mg/kg cyclophosphamide (Cy) conditioning chemotherapy for BM ablation. Subsequently, human T-MSCs were injected into the Bu/Cy-treated mice with or without BM cells (BMCs) obtained from allogeneic C57BL/6 male mice. After 3 weeks, peripheral blood and BM was collected for analysis. The red blood cell count in the group that received BMCs had almost returned to normal, whereas mononuclear cell counts and BM cellularity were most improved in the T-MSCs + BMCs group. These results indicate that the T-MSCs enhanced myelopoiesis in the allogeneic BMT mouse model, as evidenced by the restoration of BM with hematopoietic cells, as well as increased myeloid colony formation in vitro. Therefore, T-MSCs may provide a source of MSCs to facilitate myelopoiesis and megakaryocytosis following BMT.

Patient-individualized CD8⁺ cytolytic T-cell therapy effectively combats minimal residual leukemia in immunodeficient mice.

Adoptive transfer of donor-derived cytolytic T-lymphocytes (CTL) has evolved as a promising strategy to improve graft-versus-leukemia (GvL) effects in allogeneic hematopoietic stem-cell transplantation. However, durable clinical responses are often hampered by limited capability of transferred T cells to establish effective and sustained antitumor immunity in vivo. We therefore analyzed GvL responses of acute myeloid leukemia (AML)-reactive CD8(+) CTL with central and effector memory phenotype in a new allogeneic donor-patient specific humanized mouse model. CTL lines and clones obtained upon stimulation of naive CD45RA(+) donor CD8(+) T cells with either single HLA antigen-mismatched or HLA-matched primary AML blasts, respectively, elicited strong leukemia reactivity during cytokine-optimized short to intermediate (i.e., 2-8 weeks) culture periods. Single doses of CTL were intravenously infused into NOD/scidIL2Rcg(null) mice when engraftment with patient AML reached bone marrow infiltration of 1-5%, clinically defining minimal residual disease status. This treatment resulted in complete regression of HLA-mismatched and strong reduction of HLA-matched AML infiltration, respectively. Most importantly, mice receiving AML-reactive CTL showed significantly prolonged survival. Transferred CTL were detectable in murine bone marrow and spleen and demonstrated sustained AML-reactivity ex vivo. Moreover, injections with human IL-15 clearly promoted CTL persistence. In summary, we show that naive donor-derived CD8(+) CTL effectively combat patient AML blasts in immunodeficient mice. The donor-patient specific humanized mouse model appears suitable to evaluate therapeutic efficacy of AML-reactive CTL before adoptive transfer into patients. It may further help to identify powerful leukemia rejection antigens and T-cell receptors for redirecting immunity to leukemias even in a patient-individualized manner.

Sphingosine 1-Phosphate Receptor 3-Deficient Dendritic Cells Modulate Splenic Responses to Ischemia-Reperfusion Injury.

The plasticity of dendritic cells (DCs) permits phenotypic modulation ex vivo by gene expression or pharmacologic agents, and these modified DCs can exert therapeutic immunosuppressive effects in vivo through direct interactions with T cells, either inducing T regulatory cells (T(REG)s) or causing anergy. Sphingosine 1-phosphate (S1P) is a sphingolipid and the natural ligand for five G protein-coupled receptors (S1P1, S1P2, S1P3, S1P4, and S1P5), and S1PR agonists reduce kidney ischemia-reperfusion injury (IRI) in mice. S1pr3(-/-)mice are protected from kidney IRI, because DCs do not mature. We tested the therapeutic advantage of S1pr3(-/-) bone marrow-derived dendritic cell (BMDC) transfers in kidney IRI. IRI produced a rise in plasma creatinine (PCr) levels in mice receiving no cells (NCs) and mice pretreated with wild-type (WT) BMDCs. However, S1pr3(-/-) BMDC-pretreated mice were protected from kidney IRI. S1pr3(-/-) BMDC-pretreated mice had significantly higher numbers of splenic T(REG)s compared with NC and WT BMDC-pretreated mice. S1pr3(-/-) BMDCs did not attenuate IRI in splenectomized, Rag-1(-/-), or CD11c(+) DC-depleted mice. Additionally, S1pr3(-/-) BMDC-dependent protection required CD169(+)marginal zone macrophages and the macrophage-derived chemokine CCL22 to increase splenic CD4(+)Foxp3(+) T(REG)s. Pretreatment with S1pr3(-/-) BMDCs also induced T(REG)-dependent protection against IRI in an allogeneic mouse model. In summary, adoptively transferred S1pr3(-/-) BMDCs prevent kidney IRI through interactions within the spleen and expansion of splenic CD4(+)Foxp3(+) T(REG)s. We conclude that genetically induced deficiency of S1pr3 in allogenic BMDCs could serve as a therapeutic approach to prevent IRI-induced AKI.

Polycaprolactone Thin-Film Micro- and Nanoporous Cell-Encapsulation Devices.

Cell-encapsulating devices can play an important role in advancing the types of tissue available for transplantation and further improving transplant success rates. To have an effective device, encapsulated cells must remain viable, respond to external stimulus, and be protected from immune responses, and the device itself must elicit a minimal foreign body response. To address these challenges, we developed a micro- and a nanoporous thin-film cell encapsulation device from polycaprolactone (PCL), a material previously used in FDA-approved biomedical devices. The thin-film device construct allows long-term bioluminescent transfer imaging, which can be used for monitoring cell viability and device tracking. The ability to tune the microporous and nanoporous membrane allows selective protection from immune cell invasion and cytokine-mediated cell death in vitro, all while maintaining typical cell function, as demonstrated by encapsulated cells' insulin production in response to glucose stimulation. To demonstrate the ability to track, visualize, and monitor the viability of cells encapsulated in implanted thin-film devices, we encapsulated and implanted luciferase-positive MIN6 cells in allogeneic mouse models for up to 90 days. Lack of foreign body response in combination with rapid neovascularization around the device shows promise in using this technology for cell encapsulation. These devices can help elucidate the metrics required for cell encapsulation success and direct future immune-isolation therapies.

Differential Development of Donor Bone Marrow-Derived Thymocytes after Allogeneic in Utero Hematopoietic Cell Transplantation in the Murine Model

IntroductionIn Utero Hematopoietic Cell Transplantation (IUHCT) is a promising therapeutic strategy for congenital hematopoietic disorders. While mixed allogeneic hematopoietic chimerism with associated donor specific tolerance is routinely achieved by a predominant mechanism of central deletion, the critical events of donor and host thymocyte development have not been determined. In this study, we utilized the murine model of allogeneic IUHCT to analyze donor and host thymocyte development in the context of normal immune ontogeny.MethodsBone marrow (BM) cells (10x106) from C57/BL6 (B6, H2kb) mice were injected intravenously into Balb/c (H2kd) fetuses at embryonic day 14 (E14). E14 B6 fetuses injected with GFP B6 BM were used as congeneic controls. At indicated postnatal ages the thymocytes were delineated by multi-color flow cytometry. Cell apoptosis and proliferation were determined by Annexin V staining and in vivo BrdU incorporation, respectively. T cell alloreactivity was assessed by in vitro and in vivo MLR.ResultsOur findings demonstrate that the thymic processing of donor BM-derived thymocytes differs significantly from host thymocyte processing and from thymocyte development in normal control mice. While the phenotypic development of host thymocytes remained comparable with that of normal control Balb/c mice, the four major subsets of donor thymocytes showed altered distribution, with significantly higher proportions of single positive (SP) cells, and a dramatically lower proportion of CD4+CD8+ double positive (DP) cells, compared to their host-derived counterparts and B6 controls. The extent of the alteration is directly related to both BM chimerism levels and age. Higher levels of chimerism and/or older age are associated with more profound alterations in donor thymocyte distribution. Moreover, DP cells of donor origin show higher apoptosis and lower proliferation than those of the host. Donor TCR gamma/delta cells in DN cells which do not require positive selection based on MHC recognition are relatively increased compared to the host. Moreover, compared with the naive mice and congeneic chimeric mice, the donor BM-derived thymocytes in the allogeneic chimeric mice show increased proportion of DN3 and decreased proportion of DN4, but increased TCRβ+ proportions in both DN3 and DN4 cells, indicating that donor BM-derived thymocyte development is impeded during DN to DP transition, resulting from a MHC-restriction associated mechanism. In addition, in allogeneic chimeric mice, both host and donor BM-derived T cells are tolerant to allogeneic antigens in in vivo and in vitro MLR.ConclusionOur data suggests that in an allogeneic IUHCT system the immune reconstitution of the donor bone marrow-derived thymocytes differs from that of the host cells and that of normal mice. The data supports a mechanism of impaired MHC based positive selection of donor cells by the predominantly host thymic stroma resulting in lack of progression of a higher proportion of donor cells from the DN to DP stage of thymocyte development.Taken together, although donor BM-derived T cells undergo differential thymic development, permanent host-donor two-way tolerance could be achieved in the allogeneic IUHCT mouse model. These findings add to our understanding of the requirements for tolerance induction after IUHCT and have important clinical implications in choosing an optimal donor for IUHCT. DisclosuresNo relevant conflicts of interest to declare.

Synergism between bosutinib (SKI-606) and the Chk1 inhibitor (PF-00477736) in highly imatinib-resistant BCR/ABL+ leukemia cells

Interactions between the dual BCR/ABL and Src inhibitor bosutinib and the Chk1 inhibitor PF-00477736 were examined in BCR/ABL+ leukemia cells, particularly imatinib-resistant cells, including those with the T315I mutation. Bosutinib blocked PF-00477736-induced ERK1/2 activation and sharply increased apoptosis in association with Mcl-1 inhibition, p34(cdc2) dephosphorylation, BimEL up-regulation, and DNA damage in imatinib-resistant CML or Ph+ ALL cell lines. Inhibition of Src or MEK1 by shRNA significantly enhanced PF-0047736 lethality. Bosutinib/PF-00477736 co-treatment also potentiated cell death in CD34+ CML patient samples, including dasatinib-resistant blast crisis cells exhibiting both T315I and E355G mutations, but was minimally toxic to normal CD34+ cells. Finally, combined in vivo treatment significantly suppressed BaF3/T315I tumor growth and prolonged survival in an allogeneic mouse model. Together, these findings suggest that this targeted combination strategy warrants attention in IM-resistant CML or Ph+ ALL.

Immunogenicity of undifferentiated and differentiated allogeneic mouse mesenchymal stem cells

Mesenchymal stem cells (MSC) are multipotential cells with utility in tissue engineering and regenerative medicine. However, the immunological properties and immunogenicity of allogeneic MSC remain poorly defined. Recent studies investigating their immunogenicity remain inconclusive and this has hampered their clinical application. This study investigated the (1) immunogenicity and (2) immunomodulatory properties of bone marrow-derived MSC using an allogeneic mouse model involving Balb/c (responder) and C3H (stimulator) mice. Dermal fibroblasts (DF) were used as controls for cells of mesenchymal origin. Adaptations of the lymphocyte transformation assay (LTA) and mixed lymphocyte reaction (MLR) were used to investigate the immunogenicity and immunomodulatory properties of allogeneic undifferentiated and chondrogenic-differentiated MSC and DF. Both MSC and DF displayed a similar phenotypic profile with the exception of lower expression of CD44 and CD105 in DF. Tri-lineage differentiation of MSC and DF into adipocytes, chondrocytes and osteocytes confirmed their multipotency. In LTA, both undifferentiated and chondrogenic-differentiated allogeneic MSC stimulated lymphocyte proliferation. Allogeneic DF were non-stimulatory but chondrogenic-differentiated DF triggered responder lymphocyte proliferation. In one-way MLR, both allogeneic MSC and DF significantly suppressed Balb/c lymphocyte proliferation. The current challenges in distinguishing between MSC and fibroblasts were apparent throughout the work. These findings support the notion that although MSC possess immunosuppressive properties, they may not be immunoprivileged. Thus, clinical application of allogeneic MSC should be taken with due consideration of their potential immunogenicity.

The graft survival protection of subcutaneous allogeneic islets with hydrogel grafting and encapsulated by CTLA4Ig and IL1ra

In this study, we examined the combined effect of two recombinant immunomodulator proteins, cytotoxic T-lymphocyte antigen-4-Ig (CTLA4Ig) and interleukin-1 receptor antagonist (IL1ra), on the survival of subcutaneous mouse islet grafts with temperature-sensitive hydrogel, poly(N-isopropylacrylamide) (pNIPAAm)-chitosan-hyaluronic acid (CPNHA), and gelatin microspheres in an allogeneic mouse model. The phase-transition temperature for the CTLA4Ig-grafted CPNHA (CPNCHA) was 28 °C. An in vitro cytotoxicity assay and in vivo tissue reactions showed that both CPNCHA and the gelatin microspheres were biocompatible, biodegradable and nontoxic. The survival of the allogeneic islets was prolonged when the grafts were transplanted into a subcutaneous matrix composed of CPNCHA and gelatin microspheres containing IL1ra relative to those implanted in CPNHA containing gelatin microspheres without an immunomodulator. In conclusion, our results suggest that the temperature-sensitive hydrogel, CPNCHA, is a suitable subcutaneous matrix for islet transplantation, and grafted CTLA4Ig and encapsulated IL1ra reduce immune rejection and prolong the survival of the functioning allogeneic islets. The combined effect of cytotoxic T-lymphocyte antigen-4-Ig (CTLA4Ig) and interleukin-1 receptor antagonist (IL1ra) on the survival of subcutaneous mouse islet grafts with temperature-sensitive hydrogel, pNIPAAm-chitosan-hyaluronic acid (CPNHA), and gelatin microspheres in an allogeneic mouse model is examined. The survival of the allogeneic islets was prolonged when the grafts were transplanted into a subcutaneous matrix composed of CPN-CTLA4Ig-HA (CPNCHA) and gelatin microspheres containing IL1ra relative to those implanted in CPNHA containing gelatin microspheres without an immunomodulator.