B6 Bone Marrow Research Articles

The Combination of Anti-Tigit and Lenalidomide Promotes Synergistic Myeloma-Specific Immunity after ASCT

Multiple myeloma is a largely incurable bone marrow (BM) resident plasma cell malignancy that is increasing in incidence. Autologous stem cell transplantation (ASCT) is the current standard consolidation therapy and a subset of patients achieve durable progression free survival that is suggestive of long-term immune control. Utilizing novel preclinical models, we have provided definitive evidence that this is largely mediated by T cell-dependent myeloma-specific immunity. In both patients and preclinical models, myeloma progression is associated with T cell dysfunction and expression of multiple inhibitory receptors suggesting a loss of immunosurveillance. In mice, we have demonstrated potent anti-myeloma efficacy of TIGIT blockade in both ASCT and non-transplant settings. Here we utilized identical TIGIT Abs that do or do not Fc bind to demonstrate that immunological efficacy after ASCT was absolutely dependent on ADCC (median survival was unreached (>110 days) in Fc-binding vs 73 days in Fc-dead and 71 days in control Ig (cIg)-treated mice). Since TIGIT inhibition does not protect against myeloma relapse in all mice, it is apparent that combinational approaches are required to target non-responders. Therefore, we hypothesized that TIGIT blockade could be combined with immunomodulatory drugs (IMiDs) to provide synergistic anti-myeloma activity after ASCT.To that end, we utilized CRBN transgenic mice to investigate the efficacy of TIGIT blockade in combination with lenalidomide, the standard of care IMiD used in maintenance therapy after clinical ASCT. Briefly, B6 Vk*MYC myeloma-bearing (MM-bearing) mice were lethally irradiated and transplanted with B6 bone marrow (BM) and a suboptimal dose of T cells followed by anti-TIGIT or control Ig (100 mg twice weekly) for 5 weeks with lenalidomide (50 mg/kg daily gavage) or control diluent from D+14 for 3 weeks (Figure 1A). The combination of anti-TIGIT and lenalidomide provided synergistic anti-myeloma efficacy evidenced by prolonged median survival (109 days in combination vs < 60 days in monotherapy/control-treated mice, p<0.01; Figure B). Myeloma M bands were also suppressed in the combination treated mice relative to monotherapy or cIg-treated mice (p<0.01; Figure 1). Analysis of BM CD8 T cells 6 weeks after ASCT demonstrated that combination therapy significantly decreased terminal exhaustion (TOX + TIM3 + CD101 + PD-1 + DNAM-1 ─) with an average of only 20% of CD8 T cells with an exhausted phenotype in the combination group compared to greater than 50% exhausted CD8 T cells in monotherapy or cIg-treated mice (p<0.05; Figure 1C-D). The combination also increased the frequency of central memory and tissue-resident memory subsets (CD49b +CD69 +; p<0.05; Figure 1C-D), and increased IFNγ production from activated (PD-1 +; p<0.05; Figure 1E) cells compared to monotherapy or control Ig-treated mice. Importantly, these phenotypic changes were specific to the BM tumor microenvironment as we observed no effect of combination or monotherapy treatment on CD8 or CD4 T cells in peripheral blood.In sum, these data provide a strong rationale for combining TIGIT inhibition with immunomodulatory drugs to prevent the progression of myeloma. [Display omitted] DisclosuresDriessens: iTeos Therapeutics: Current Employment, Current equity holder in publicly-traded company. Holmberg: Up-To-Date: Patents & Royalties; Bristol Myers Squibb: Research Funding; Janssen: Research Funding; Merck: Research Funding; Millennium-Takeda: Research Funding; Sanofi: Research Funding; Seattle Genetics: Research Funding. Hill: NeoLeukin Therapeutics: Consultancy; Compass Therapeutics: Research Funding; NapaJen Pharma: Consultancy; Generon Corporation: Consultancy; Roche: Research Funding; iTeos Therapeutics: Consultancy, Research Funding; Syndax Pharmaceuticals: Research Funding; Applied Molecular Transport: Research Funding.

Combining Immunocytokine and Ex Vivo Activated NK Cells as a Platform for Enhancing Graft-Versus-Tumor Effects Against GD2+ Murine Neuroblastoma.

Management for high-risk neuroblastoma (NBL) has included autologous hematopoietic stem cell transplant (HSCT) and anti-GD2 immunotherapy, but survival remains around 50%. The aim of this study was to determine if allogeneic HSCT could serve as a platform for inducing a graft-versus-tumor (GVT) effect against NBL with combination immunocytokine and NK cells in a murine model. Lethally irradiated C57BL/6 (B6) x A/J recipients were transplanted with B6 bone marrow on Day +0. On day +10, allogeneic HSCT recipients were challenged with NXS2, a GD2+ NBL. On days +14-16, mice were treated with the anti-GD2 immunocytokine hu14.18-IL2. In select groups, hu14.18-IL2 was combined with infusions of B6 NK cells activated with IL-15/IL-15Rα and CD137L ex vivo. Allogeneic HSCT alone was insufficient to control NXS2 tumor growth, but the addition of hu14.18-IL2 controlled tumor growth and improved survival. Adoptive transfer of ex vivo CD137L/IL-15/IL-15Rα activated NK cells with or without hu14.18-IL2 exacerbated lethality. CD137L/IL-15/IL-15Rα activated NK cells showed enhanced cytotoxicity and produced high levels of TNF-α in vitro, but induced cytokine release syndrome (CRS) in vivo. Infusing Perforin-/- CD137L/IL-15/IL-15Rα activated NK cells had no impact on GVT, whereas TNF-α-/- CD137L/IL-15/IL-15Rα activated NK cells improved GVT by decreasing peripheral effector cell subsets while preserving tumor-infiltrating lymphocytes. Depletion of Ly49H+ NK cells also improved GVT. Using allogeneic HSCT for NBL is a viable platform for immunocytokines and ex vivo activated NK cell infusions, but must be balanced with induction of CRS. Regulation of TNFα or activating NK subsets may be needed to improve GVT effects.

Post-Transplant Cyclophosphamide Contributes to Impaired Graft-Versus-Leukemia Effects and Regulates Graft-Versus-Host Disease in the Murine Stem Cell Transplant Model

IntroductionPost-transplant cyclophosphamide (PTCy) for controlling alloreactivity after allogeneic hematopoietic stem cell transplantation (HSCT) is an effective prophylaxis for acute and chronic graft-versus-host disease (GVHD). HLA-haploidentical HSCT using PTCy is a promising option for patients with hematological malignancy who require allo-HSCT and lack an immediately available HLA-identical related or unrelated donor. In contrast, a relatively high relapse rate was reported in HLA-haploidentical HSCT using PTCy with non-myeloablative conditioning compared to HLA-matched related or unrelated HSCT using conventional GVHD prophylaxis. The effects of GVHD have been shown to correlate partially with those of graft-versus-leukemia (GVL), and therefore, PTCy treatment might contribute to the increase in relapse. However, the rationale for the high relapse rate in HSCT using PTCy compared to conventional HSCT remains poorly understood. To address this issue, we explored the effect of PTCy on survival, leukemia progression and immune reconstruction in a murine transplant model.Material & MethodsTwelve-week old C57BL/6 (B6) mice were irradiated with 5 Gy in one fraction on day 0 and transplanted with 1.0 × 107 spleen cells and 1.0 × 107 bone marrow cells from Balb/c mice. All mice were purchased from Japan SLC Inc. and maintained in a pathogen-free environment. Mice consumed normal chow and autoclaved hyperchlorinated water (pH 4) after the transplantation. Mice were not treated (control) or received cyclophosphamide 100 mg/kg at days 3 and 4 intraperitoneally. A leukemia cell line that was derived from the B6 bone marrow and transformed with the BCR/ABL fusion gene (BA-1) was used for leukemogenic experiments. One million BA-1 cells were intravenously infused into the recipients 25 (±4) days after transplantation with no additive treatment. We continuously examined weight, GVHD score, peripheral blood cell counts, and peripheral blood cell prolife of the recipient mice, and finally investigated spleen and bone marrow cells. The chimeric balances among host-residual H-2Db+ cells and donor graft-derived H-2Dd+ cells in CD3+ T cells were monitored. To evaluate the function of activated T cells in the spleen, CD62L+, CD44+, and PD-1+ marker levels in CD8+ and CD4+ cells were quantitatively monitored at various time points. GVHD scoring was based on weight loss, posture, activity, fur texture, and skin integrity (severity score 0 to 2 for each variable, maximum index 10) as previously reported. Leukemia progression was defined by the presence of 0.05% BA-1 cells in peripheral blood mononuclear cells as detected by GFP expression. All experimental procedures were approved by an institutional review committee of the Osaka City University.ResultsControl B6 recipients developed severe weight loss and GVHD after transplantation and died of GVHD at around day 60, whereas the recipients with PTCy had significantly improved weight, GVHD score, and survival compared to control mice (figure1). These results were comparable to those of previous reports. In the leukemogenic experiment, the control recipients showed significantly longer survival than the ones with PTCy (figure2). Recipients with PTCy had a higher rate of leukemia progression compared to the control at day 30 after BA-1 injection, although there were no significant differences in the chimeric balances among donor graft-derived H-2Dd+ T cells both before and at day 7, 14, 21, 28, and 35 after BA-1 injection. Prior to BA-1 injection, CD62L+ CD44- naïve CD8+/CD4+ T cell number was low, and CD62L- CD44+ effector memory CD8+/CD4+ T cell number was high in the spleen of the control recipients compared to that in PTCy recipients. Moreover, the numbers of CD44+ PD-1+ memory CD8+/CD4+ T cells were also overwhelmingly high in the spleen of control recipients. These results showed that use of PTCy after allo-HSCT promoted T cell tolerance to alloantigens by eliminating antigen-exposed T cells and reduced the probability of rejection in leukemia as a consequence.ConclusionOur data indicated that PTCy treatment after allo-HSCT contributes to impaired GVL effects by reducing the number of antigen-exposed T cells and improves GVHD regulation in a murine stem cell transplant model. [Display omitted] DisclosuresHino:Novartis: Research Funding.

Memory T Cells from Minor Histocompatibility Antigen-Vaccinated Donors Improve Engraftment with Low Intensity Conditioning

While allogeneic hematopoietic stem cell transplantation (alloSCT) is largely employed to treat malignant disorders, it can also cure inherited and acquired nonmalignant disorders. For example, inherited diseases such as sickle cell anemia, thalassemias, and varied immunodeficiencies can be cured with alloSCT. Autoimmune diseases can also be cured via the abilities of both the conditioning regimen and alloimmune T cells to eradicate autoimmune T and B cells. And importantly, tolerance to solid organ grafts can be created via transplantation of hematopoietic cells from the organ donor. However, the utility of alloSCT in these settings is limited by the morbidity and mortality of the procedure, including graft-vs-host disease (GVHD). Some progress has been made in lowering the toxicities in these settings. However, these approaches frequently result in mixed chimerism, which may not be sufficient to treat autoimmune diseases, and may lead to unreliable engraftment overall. Previously we reported that in mouse models of alloSCT memory CD8+ T cells from mice vaccinated against the single minor histocompatibility antigen (miHA) H60, which is mostly expressed on hematopoietic cells, dramatically improves graft-vs-leukemia (GVL) against H60-expressing leukemias, without causing GVHD. In the present work we have tested the utility of memory CD8 cells reactive to H60 (TMH60) in promoting engraftment. Following 6 Gy or 8Gy of total body irradiation (TBI), B6.H60 (H-2b, Ly9.1-, H60+) mice were transplanted with C3H.SW (H-2b, Ly9.1+, H60-) bone marrow (BM), with memory T cells from H60 vaccinated mice (TMH60) containing 3500 tetramer+ cells (TetH60) or with TM from unvaccinated mice as negative control. At TBI doses of 8 Gy and 6 Gy, the addition of TM from H60 vaccinated mice (TMH60), as compared to TM from unvaccinated mice, modestly improved engraftment. We next lowered the dose of TBI further. At 5 Gy, recipients of TMH60 had multilineage engraftment of donor-derived hematopoietic cells in bone marrow (BM), spleen and lymph nodes (LN) nine weeks after transplantation. In contrast, mice with TM failed to engraft. Importantly, mice did not develop clinical GVHD. At a TBI dose of 4 Gy, recipients with TMH60 had poor donor engraftment, while TMH60 recipients treated with an anti-CD40 agonist antibody (FGK45) on day 0 achieved full multilineage engraftment in spleen, LN and BM. In addition we achieved mixed chimerism in some TMH60 and anti-CD40 treated mice conditioned with 3 Gy. We also tested the utility of TM targeting the Kb-restricted SIINFEKL peptide derived from OVA (TM-OVA) in a mouse model wherein OVA expression is restricted to hematopoietic cells. We used B6.OVA mice, which express OVA ubiquitously, as BM donors and created B6.OVA–>B6 BM chimeras (H-2b, Ly9.1-, OVA+). We retransplanted these chimeras with C3H.SW (H-2b, Ly9.1+, OVA-) BM with C3H.SW TM-OVA, containing 3500 tetramer+ cells, or TM from unvaccinated mice following 5 Gy or 4 Gy of TBI. TM-OVA, but not TM promoted full multilineage engraftment in mice conditioned with 5 Gy in blood of mice analyzed two weeks after transplantation. At dose of 4 Gy, TM-OVA induced full donor engraftment, but only with the addition of anti-CD40. This experiment is ongoing. In sum, our data demonstrate that TM reactive against a miHA expressed on hematopoietic cells can dramatically promote donor multilineage engraftment without GVHD, and that the effectiveness of these TM can be augmented by stimulating CD40. Future experiments will test miHA-reactive TM with chemotherapy-based conditioning, in haploidentical models and the utility of blocking PD-1, which we have found greatly augments GVL mediated by miHA-reactive TM. This approach holds the promise of achieving consistent donor engraftment without GVHD, a prerequisite for the widespread use of alloSCT in treatment of autoimmune diseases and for tolerance induction in tandem with organ transplantation. DisclosuresNo relevant conflicts of interest to declare.

Ablation of IRF4 in donor T cells eliminates GVHD while partially preserving GVT in experimental bone marrow transplantation

Abstract Objective We discovered that the transcription factor IRF4 governs T cell function in heart transplantation (Immunity 47:1114). Here, using a bone marrow (BM) transplantation model, we studied the role of IRF4 in regulating T cell function in graft-vs-host disease (GVHD) and graft-vs-tumor (GVT). Methods 9 Gy irradiated Balb/c recipients were i.v. injected: 1) with 5 × 106 T cell-depleted CD45.1+ B6 BM cells; 2) with or without 0.1 × 106 Balb/c A20-luciferase lymphoma cells; and 3) with or without 1 × 106 CD45.2+ wild type (WT), Irf4−/−, or Irf4−/−Foxp3sf/Y (Treg-deficient Irf4−/−) B6 donor T cells. GVHD, GVT, survival, and donor T cell states were determined. Results In the absence of A20 infusion, all recipients without T cell transfer (n=10), or transferred with Irf4−/− (n=10) or Irf4−/−Foxp3sf/Y (n=5) donor T cells survived long-term (&amp;gt;60 days post BM transplantation). Recipients transferred with WT donor T cells had developed lethal GVHD [mean survival time (MST) = 9.5±2 days; n =8]. Among A20 infused groups, recipients without T cell transfer died due to lymphoma progression (MST=22±2.80 days; n =15). Transfer of Irf4−/− donor T cells led to a moderate GVT effect and significantly prolonged survival (MST=33.4 ± 11.22 days; n =15; p&amp;lt;0.001 vs no T cell). Transferred WT donor T cells exerted potent GVT activity but caused lethal GVHD (12.56 ± 6.02 days; n = 18). Mechanistically, Irf4−/− donor T cells developed into effector T cells, but barely infiltrated into GVHD tissues and were prone to apoptosis. Conclusions IRF4 ablation in donor T cells eliminates GVHD in a Treg independent manner while partially preserving GVT. This reveals the possibility to separate GVHD and GVT effects by modulating transcription factors in T cells.

Combination Therapy of Allogeneic HSCT and In Vivo Activated iNKT Cells Prevents Acute Gvhd While Augmenting GVL Effect

Adult acute lymphoblastic leukemia (ALL) is a highly aggressive cancer with poor clinical prognosis and high relapse rate. Whereas allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment for highly differentiated hematologic malignancies, allo-HSCT provides only limited benefits for treating ALL with relapses frequent. Additionally, the development of graft-versus-host-disease (GVHD) remains a major concern for allo-HSCT recipients. Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that express both a semi-invariant T cell receptor and natural killer cell markers. They recognize lipid antigens presented by non-polymorphic CD1d and have been reported to directly target CD1d+ solid tumors. Many B-ALL, including the P210 B-ALL cell line, are also CD1d+. Clinically, increased iNKT cells have simultaneously been correlated with reduced risk of developing acute GVHD and lower rates of malignant relapse. Because iNKT cells comprise only a tiny fraction of total T cells, we established a protocol to insert an iNKT TCR into murine HSC, thereby generating retrogenic mice with 5-10x expansion of iNKT cells in spleen and liver. Although engineered T cell therapy, including CD19-CART therapy, has proven to be effective in inducing remission in many B-ALL patients, when used as a monotherapy, relapse remains a major obstacle. We therefore tested whether a combination of allo-HSCT and iNKT cell therapy could robustly prevent GVHD while preserving graft-versus-leukemia effects (GVL). To test whether the addition of iNKT cells into allo-graft was capable of preventing acute GVHD (aGVHD), lethally irradiated BALB/C were given B6 bone marrow (2.5 x 106) and whole splenocytes (2.5 x 106, 5 x 106, or 10 x 106) from either B6 or retrogenic B6-iNKT mice. Mice that received 5 x 106 B6 splenocytes developed lethal aGVHD and became moribund by Day 10. Half of the mice that received 2.5 x 106 B6 splenocytes develop lethal aGVHD and died by Day 20. In contrast, only half of the mice that received 5 x 106 or 10 x 106 B6-iNKT splenocytes developed lethal aGVHD by Day 20, and all recipients of 2.5 x 106 B6 splenocytes remained alive by Day 20, suggesting the addition of iNKT cells into allograft may reduce the severity of aGVHD. To test whether adding iNKT cells to allograft could enhance GVL without causing GVHD, BALB/C mice with pre-established P210 B-ALL leukemia were given allo-HSCT with or without iNKT cell addition. Initially, our data showed freshly isolated iNKT cells were rapidly cleared following transplantation. We therefore included lethally irradiated P210 loaded with α-Galactosylceramide (α-Galcer) as adjuvant, and were able to see robust expansion of iNKT cells in vitro and in vivo (&gt;20% vs &lt;1% in WT in BM and Liver). Additionally, we found that HSCT recipients that also received iNKT cells were able to fully clear bone marrow residing B-ALL cells by Day8, compared to less than half in control, suggesting an important role of iNKT cells for targeting bone marrow residing B-ALL cells to prevent relapse. Taken together, combination therapy of allogeneic HSCT with infusion of donor-type iNKT cells activated via irradiated α-Galcer-loaded host-type ALL represent a novel approach for preventing GVHD and augmenting GVL effect against ALL. Disclosures No relevant conflicts of interest to declare.

Inhibiting EZH2 function prevents and treats chronic graft vs host disease (cGVHD) by disrupting germinal center formation and plasma cell maturation

Abstract To determine whether targeting EZH2, a histone methyltransferase necessary for germinal center (GC) formation, could reduce murine cGVHD (cGVHD), we first used a clinically relevant murine model of cGVHD, which recapitulates multi-organ cGVHD with bronchiolitis obliterans (BOS). Bone marrow (BM) from EZH2 fl/fl CD19 Cre B6 mice and WT T cells, or WT B6 BM and EZH2 fl/fl CD4 Cre B6 T cells were given to conditioned B10.BR mice. EZH2 expression in BM and T cells was obligatory for cGVHD. To determine whether EZH2 function could be targeted to treat cGVHD, we tested a novel EZH2 small molecule inhibitor, JQ5 with a favorable toxicity and efficacy profile to other inhibitors. Mice with established cGVHD given JQ5 had dramatically improved cGVHD. Similar results were seen in a sclerodermatous cGVHD model. To assess possible causes of this improvement we performed RNA-seq and ATAC-seq data of MACS purified CD19+ B cells obtained from control and JQ5 treated BOS cGVHD mice. Unsupervised gene set enrichment analysis and weighted gene correlation network analysis showed that among gene clusters associated with JQ5 treatment there was an enrichment for genes expressed in naïve B cells compared to plasma cells (GSEA4142) and an impairment of genes associated with late stage GC B cells compared to early follicular B cells (GSEA11961). This suggested that JQ5 impaired GC B cell and plasma cell maturation. Consistent with these data, splenic follicular cells, GC size and number, and lung resident PCs were reduced compared to cGVHD controls. Taken together these results show that EZH2 is necessary for the initiation of cGVHD and show for the first time that EZH2 inhibition using a novel inhibitor JQ5 can reverse established cGVHD in mice with BOS or scleroderma.

Donor T Cells Maintain Myeloma-Immune Equilibrium after Autologous Stem Cell Transplantation and Concurrent Immunotherapy Promotes Cure

Autologous stem cell transplantation (ASCT) remains an important consolidation treatment for multiple myeloma (MM) patients, even in the era of novel agents. The prolongation of plateau-phase induced by ASCT is generally attributed to intensive cytoreduction. However, ASCT generates inflammation and profound lymphodepletion, which may result in hitherto unexpected immunological effects. To investigate potential immunological contributions to myeloma control after ASCT, we developed preclinical models of transplantation for MM using Vk*MYC myeloma that generates bony lytic lesions, a serum M band and marrow plasmacytosis that are hallmarks of clinical disease.Myeloma-bearing B6 recipients underwent myeloablative conditioning and were transplanted with naïve B6 bone marrow (BM) grafts with or without T cells from donors that were myeloma-naïve (SCT) or had low M bands at the time of harvest to mimic ASCT. Surprisingly, we demonstrate the broad induction of T cell-dependent myeloma control with enhanced median survival in recipients of grafts containing T cells compared to T cell depleted (TCD) BM alone (SCT= 91 days and ASCT > 100 days post-transplant vs TCD BM alone= 44 days; p<0.0001). Myeloma was most efficiently controlled when recipients were transplanted with memory T cells (CD44+) from autologous grafts (median survival: ASCT-CD44+ T cells >90 days post-transplant vs. CD44─ T cells = 50 days; p = 0.0006). Importantly, T cells adoptively transferred from recipients surviving > 120 days (MM-primed) protected secondary recipients compared to T cells from naïve donors (median survival: MM-primed > 120 days post-transplant vs 65 days naïve T cells; p = 0.0003). Furthermore, MM-primed CD8 T cells were restricted in TCR repertoire and provided protection in a myeloma clone-specific fashion, indicative of a tumor-specific T cell response.Despite this immune-mediated control of myeloma after SCT, progression still occurred in the majority of recipients. We phenotyped CD8+ T cells from the BM of MM-relapsed, MM-controlled and MM-free (that had never seen myeloma) mice 8 weeks after SCT. Expression of the inhibitory receptors, programmed cell death protein 1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) on BM CD8+ T-cells strongly correlated with myeloma cell number (r = 0.729, p<0.0001 and r = 0.796, p<0.0001 respectively). Additionally, the co-stimulatory/adhesion receptor CD226 (DNAM-1) was markedly downregulated as myeloma progressed (r = - 0.865, p<0.0001), as was interferon-γ secretion (r = - 0.76, p = 0.0022). t-SNE analysis confirmed an irreversible exhaustion signature at myeloma progression, characterized by the absence of DNAM-1 and co-expression of PD-1, TIM-3, TIGIT together with CD101 and CD38. Immune-checkpoint inhibition (CPI) early post-SCT, using antibodies against PD-1 or TIGIT facilitated long-term myeloma control (median survival in both treatment arms > 120 days post-SCT vs. 60 and 68 days respectively; p <0.05). Furthermore, TIGIT blockade limited CD8+ T cell exhaustion, increased CD107a and IFNγ secretion and expanded a memory CD8+ T cell population in the BM. Genetic deletion of either IFNγ or the IFNγ receptor from the donor graft resulted in dramatic myeloma progression after SCT. Consequently, treatment with a CD137 (4-IBB) agonist early after SCT profoundly augmented CD8+IFNγ+GranzymeB+ T-cell expansion in the BM, such that majority of treated animals eliminated myeloma and survived long-term. These data provide insights into an unappreciated mechanism of action of ASCT whereby myeloma immune-equilibrium is established and suggest that combination with immunotherapeutic strategies is a rational approach to generate long term disease control. DisclosuresSmyth:Bristol Myers Squibb: Other: Research agreement; Tizona Therapeutics: Research Funding.

CD8+ Effector T Cell Migration into the Bone Marrow of Transplanted Mice: Influence of Gvhd, Leukemia Cells and Variable Dependence on Chemokine Receptors

The graft-versus-leukemia (GVL) effect, mediated by alloreactive T cells, reduces the risk of leukemia relapse after allogeneic hematopoietic stem cell transplantation. While much work has focused on how T cells kill leukemia cells, less attention has been paid to the process whereby activated alloreactive T cells find leukemic targets. We initially focused on how chemokine receptor signaling impacts effector T cells egress from blood, entry into the marrow space and subsequent scanning of leukemia cells. We further tested how this process is influenced by transplantation and the consequences of alloreactive T cells. We tracked the migration of CD8+ RAG1-/- OT-1 TCR transgenic effectors (generated by in vivo vaccination of B6 mice seeded with naïve OT-1 cells) in recipients. OT-1 cells recognize the SIINFEKL peptide from ovalbumin and are not reactive in our models. OT-1 effectors were treated (or not) with pertussis toxin (PTX) to block chemokine receptor signaling, and labeled with fluorescent dyes so as they could be identified by flow cytometry and two photon intravital microscopy (2PIM). PTX+ and PTX- OT-1 cells were transferred together into the following recipients: unmanipulated B6 mice, irradiated (B6xBALB/c) CB6F1mice transplanted with B6 bone marrow (BM) cell, with or without B6 T cells. In some experiments on day 12 post-transplant, F1 mice also received WT or IFN-γR-/- B6 blast crisis CML cells (BC-CML) generated by transduction of BM with retrovirus encoding bcr-abl and the NUP98/HOXA9 fusion as we have described (J Clin Invest. 2017;127(7):2765-2776). OT-1 effectors were infused on day 14 and 3 hours later the calvarium was analyzed by 2PIM to enumerate PTX treated or untreated OT-1 effectors that were inside or outside the blood vessels. Blood vessels were identified using labeled dextran or Qdot 655.PTX treatment was highly effective as PTX-treated OT-1 were nearly completely excluded from lymph node (LN). PTX treatment reduced OT1 migration into the BM of unmanipulated B6 mice and similarly in F1 recipients without T cells. However, the presence of a GVH response reduced the recruitment of PTX- OT-1 cells into BM. The ratio of OT-1 outside/inside vessels was still higher in the PTX- group; however, this was mostly driven by an increase of PTX+ OT-1 in vessels in GVH mice. These data suggest that GVH may reduce effector T cell recruitment to the BM, though some of this effect could be due to effectors trafficking instead to other locations, perhaps GVHD target tissues.The presence of WT BC-CML leukemia cells in F1 GVH recipients increased recruitment of OT-1 PTX- effectors into the BM, both relative to PTX+ OT-1 and in total numbers compared to experiments wherein no BC-CML cells were infused. We considered the possibility that IFN-g-stimulation of BC-CML cells may have released chemokines that recruited PTX- OT-1. However, IFNγR-/- BC-CML also increased PTX- OT-1 recruitment in GVHD mice to a similar degree. Experiments are ongoing to better understand how BC-CML cells promote PTX-inhibitable effector T cell recruitment. DisclosuresShlomchik:NapaJen: Consultancy.

The Commensal Neisseria musculi Modulates Host Innate Immunity To Promote Oral Colonization.

Neisseria musculi, isolated from the oral cavity of wild-caught mice, does not colonize most inbred mouse strains. N. musculi does weakly (50%) colonize C57BL/6J (B6) mice but readily colonizes CAST/EiJ (CAST) mice. In this study, we examined whether differences in the CAST and B6 host response could elucidate mechanisms governing N. musculi colonization. In vivo stimulation of B6 or CAST splenocytes with wild type (WT) Neisseria or Escherichia coli LPS showed that CAST mice had a blunted inflammatory response, producing significantly lower levels of IL-6 than B6 mice. The use of specific genetic knockouts highlighted a need for an intact innate immune system to prevent colonization. B6–RAG-1−/− mice were colonized at a similar rate as WT B6 mice, whereas B6-MyD88−/− and TLR4−/− mice were readily colonized like CAST (100%) mice. Sequence analysis revealed a unique point mutation in TLR4 in CAST mice. However, crosses to TLR4−/− mice and analysis of recombinant inbred Collaborative Cross mice showed that TLR4 from CAST mice was not sufficient to allow Neisseria colonization. In vitro stimulation of B6 bone marrow–derived macrophages or splenocytes with WT Neisseria yielded low levels of IL-6 compared with LPS stimulation. Surprisingly, UV-inactivated Neisseria induced high levels of IL-6, suggesting suppression of IL-6 production is an active bacterial process. Consistent with a critical role for IL-6 in preventing colonization, mice deficient for the IL-6 receptor were efficiently colonized, indicating host IL-6 production plays a critical role in determining host colonization susceptibility.

Neutrophils from Both Susceptible and Resistant Mice Efficiently Kill Opsonized Listeria monocytogenes.

Inbred mouse strains differ in their susceptibility to infection with the facultative intracellular bacterium Listeria monocytogenes, largely due to delayed or deficient innate immune responses. Previous antibody depletion studies suggested that neutrophils (polymorphonuclear leukocytes [PMN]) were particularly important for clearance in the liver, but the ability of PMN from susceptible and resistant mice to directly kill L. monocytogenes has not been examined. In this study, we showed that PMN infiltrated the livers of BALB/c/By/J (BALB/c) and C57BL/6 (B6) mice in similar numbers and that both cell types readily migrated toward leukotriene B4 in an in vitro chemotaxis assay. However, CFU burdens in the liver were significantly higher in BALB/c mice than in other strains, suggesting that PMN in the BALB/c liver might not be able to clear L. monocytogenes as efficiently as B6 PMN. Unprimed PMN harvested from either BALB/c or B6 bone marrow killed L. monocytogenes directly ex vivo, and pretreatment with autologous serum significantly enhanced killing efficiency for both. L. monocytogenes were internalized within 10 min and rapidly triggered intracellular production of reactive oxygen species in a dose-dependent manner. However, PMN from gp91phox-deficient mice also readily killed L. monocytogenes, which suggested that nonoxidative killing mechanisms may be sufficient for bacterial clearance. Together, these results indicate that there is not an intrinsic defect in the ability of PMN from susceptible BALB/c mice to kill L. monocytogenes and further suggest that if PMN function is impaired in BALB/c mice, it is likely due to locally produced modulating factors present in the liver during infection.

The Nrf2 activator dimethyl fumarate inhibits acute graft-versus-host disease with retention of graft-versus-tumor effects

Abstract Dimethyl fumarate (DMF), an activator of Nrf2, has been shown to have anti-inflammatory and immunomodulatory properties without significant immunosuppression. we therefore hypothesized that DMF could be a new candidate drug for the treatment of aGVHD with retention of GVT effect. Lethally irradiated BALB/c mice transplanted with B6 bone marrow plus splenocytes were administered DMF or vehicle solution by gavage daily from day -3 to day 3. DMF treatment inhibited aGVHD, accompanied by less weight loss and lower clinical score. DMF is capable of significantly inhibiting proliferation of alloreactive T cells in a dose-dependent manner in mixed lymphocyte reaction assay. Furthermore, the percentages of CD69+T cells and IFN-γ expressing T cells, were significantly increased in aGVHD target organs from DMF recipients. In addition, DMF treatment markedly increased the frequencies of Treg cells in target organs. Depletion of CD25+ cells in DMF recipients aggravated aGVHD mortality compared with IgG control recipients. Moreover, Treg polarizing assay suggested that DMF could promote Treg cell differentiation in vitro in a dose-dependent manner. In addition, No protective effects of DMF were observed in mice transplanted with BM and challenged with leukemia cells. However, DMF treatment significantly prolonged the survival in tumor-bearing mice that received an allogeneic BMT with donor splenocytes. we also observed that donor T cells recovered from DMF-treated recipients and controls showed comparable cytolytic activity against leukemia cells. In conclusion, our findings demonstrated that activation of Nrf2 with DMF may be of significant use in preventing aGVHD and preserving GVT responses.

Impairment of myeloid-derived suppressor cell expansion and enhancement of dendritic cell differentiation of MyD88-deficient bone marrow cells in graft-versus-host disease

Abstract Myeloid differentiation 88 (MyD88) is an adaptor molecule in toll-like receptor-associated signaling. To examine the effect of MyD88-deficiency in myeloid cell differentiation under inflammatory condition, we transplanted bone marrow (BM) cells isolated from MyD88-deficient C57BL/6 (B6) mice together with B6 T cells into MHC-matched allogeneic BALB.B strain mice. This transplantation induced more serious graft-versus-host disease in the BALB.B BM recipients, compared with the GVHD induced in the control BALB.B mice which received wild type B6 BM and T cells. The aggravation of GVHD was associated with impaired expansion of CD11b+Gr1+ myeloid derived suppressor cells (MDSCs) from the MyD88-deficient BM cells during the GVHD development. Instead of MDSC expansion, myeloid cells from MyD88-deficient BM cells showed enhanced apoptosis and dendritic cell (DC) differentiation, which resulted in enhancement of activation of allo-reactive B6 T cells. These results provide information helpful for the understanding of the role of MyD88 in myeloid cell differentiation and development of strategy to reduce the GVHD severity.

Single cell analysis of transitional B cells reveals autocrine IFNβ sustains a dynamic type I IFN network in lupus

Abstract Increased selection of transitional B cells reactive with nucleic acid antigens is a prominent feature of systemic lupus erythematosus (SLE). We found that transitional stage 1 (T1) B cells from both human SLE patients and BXD2 lupus mice intrinsically express significantly higher levels of IFNα and IFNβ compared to normal individuals. IFNβ expression in T1 B cells was significantly correlated with the development of mature autoreactive 9G4+ B cells in SLE patients and La+ B cells in BXD2 mice. IFNβ was also specifically required for optimal B cell survival and TLR7 responses. In single T1 B cells isolated from Ifnb−/− vs. WT B6 bone marrow chimeras, upregulation of Ifnα7 was completely abrogated in single Ifnb−/− T1 B cells, while other genes (Ifna1, Ifna4, Cd86, Tlr7) were significantly but incompletely diminished implicating both autocrine and paracrine IFNβ activity. To resolve these signals in an autoimmune environment, single cell gene expression analysis was carried out on sorted BXD2 T1 B cells. Hierarchical clustering revealed three distinct gene expression patterns. Cluster 1 cells were “IFN producers” characterized by high expression of type I IFN genes, but low co-expression of IFN receptor genes, Ifnar1 and Ifnar2. Cluster 2 “IFN responders” exhibited the highest co-expression of both Ifnar1 and Ifnar2 as well as immunomodulatory IFN response genes (Irg1, IL6, Cd69, Cd86). Cluster 3 had low expression of type I IFN and IFNAR genes. Clusters 1 and 2 exhibited the highest Tlr7 expression, while Cluster 3 had the highest expression of Baffr and Tlr9. The heterogeneity of IFN signatures at the T1 stage suggests that varying interferonogenic phenomena can differentially prime T1 B cells for subsequent responses and cell fates.

The IL-27/Wsx-1 Axis Modulates T Cell Responses and Regulates Acute Graft-Versus-Host Disease after Allogeneic Bone Marrow Transplantation

IL-27 is a member of the IL-12 cytokine family that consists of EBV-induced gene 3 (EBI3), and p28. IL-27 is produced by activated antigen-presenting cells such as dendritic cells (DCs) and macrophages, and it signals through a heterodimeric receptor (IL-27R) consisting of the WSX-1 and the gp130. Emerging evidence has shown that IL-27 has both pro- and anti-Inflammatory effects. Recent study demonstrated a protected role IL-27p28 in acute graft-versus-host disease (aGVHD) in parent-to-F1 murine aGVHD model (Marillier et al., Eur. J. Immunol. 2014). However, the precise role of IL-27 in the development of aGVHD remains largely unknown.In this study, we carried out studies in a murine aGVHD model of fully MHC-mismatched myeloablative bone marrow transplantation (C57BL/6 to BALB/c). Lethally irradiated BALB/c mice transplanted with WT B6 bone marrow (BM) plus IL-27-/-(IL-27p28flox/flox-CD11c-cre (Itgax-p28f/f)) splenocytes had significantly accelerated aGVHD mortality comparing with those received WT BM plus splenocytes (P<0.001). However, similar aGVHD mortality was observed between mice transplanted with WT BM plus WT splenocytes and IL-27-/-BM plus WT splenocytes, and IL-27-/-BM plus IL-27-/- splenocytes, suggesting that IL-27 from splenocytes, not BM-derived cells, had protective effect in aGVHD. Additionally, mice were transplanted with IL-27-/- BM and splenocytes, and then treated with rmIL-27 on days 0 and 7 post BMT. rmIL-27 treated mice had longer survival and alleviated the clinical signs of aGVHD compared to PBS-treated mice (P<0.05). We next explored the mechanisms by which IL-27 protects mice against aGVHD. We found the expression of the cell surface IL-27 receptor, WSX-1 and gp130, were increased on both CD4+ and CD8+T cells after allo-stimulation. Moreover, IL-27 inhibited cell proliferation of allo-reactive T cells in mix lymphocytes reactivation (MLR) in vitro. We also found that the percentages of CD69+CD4+, CD69+CD8+T cells, as well as IFN-γ expression by CD4+ and CD8+T, were significantly increased in the spleen, liver and intestinal intraepithelial lymphocytes (IEL) from mice transplanted with WT BM and IL-27-/- splenocytes. Furthermore, we observed increased frequencies and numbers of Treg cells and MDSCs in aGVHD target organs in mice reconstituted with IL-27-/-splenocytes. Together, these results indicated that IL-27 alleviated aGVHD may through suppressing Th1 cell responses and promoting immune suppressing cells, including MDSCs and Treg cells.To further clarify the role of IL-27 in aGVHD, mice were transplanted with WT BM plus IL-27R-/-(B6N.129P2-Il27ratm1Mak/J) splenocytes, the results, however, showed that IL-27R deficiency in donor T cells significantly attenuated aGVHD, which was unpredicted, and was contrary to that of IL-27 deficiency results. Additional studies showed that IL-27R deficiency in T cells inhibited allo-reactive T cells proliferation and IL-2, IFN-γ production in MLR assay. The percentages of CD69+ T cells and IFN-γ+ CD4+ and CD8+T were significantly decreased, while the MDSCs and Treg cells wereincreased in aGVHD target organs from mice transplanted IL-27R-/-splenocytes. These results indicated that lack of IL-27R signaling resulted in downregulation of intrinsic immune responses which led to alleviation of aGVHD.Previous study reported that soluble form of IL-27Ra is highly existed in human serum and may severs as a natural IL-27 antagonist (Dietrich et al., JI. 2014). We hypothesized that sIL-27Ra may function as a decoy receptor to regulate aGVHD through inhibiting IL-27 signaling. To test this hypothesis, WT recipients were given exogenous soluble mouse IL-27 Ra Fc chimera protein known to block the binding of IL-27 to the membrane bound form of IL-27R. WT recipients given IL-27 Ra-Fc to block IL-27R/IL-27 interaction had significantly exacerbated GVHD mortality. The percentages of CD69+ T cells and IFN-γ+ CD4+ and CD8+T were significantly increased in aGVHD target organs from mice received IL-27 Ra fusion protein.In conclusion, this is the first demonstration in the same allogeneic BMT model that IL-27 deficiency augments but IL-27R deficiency alleviates acute GVHD. Our studies provide further evidence for the protective role of rmIL-27, and promoting effect of sIL-27R in the same aGVHD model. This study warrants further investigations of the possible therapeutic applications of IL-27 in acute GVHD. DisclosuresNo relevant conflicts of interest to declare.

Characterizing and Augmenting Peripheral Tolerance in in Utero Hematopoietic Cell Transplantation

Background: In utero hematopoietic cell transplantation (IUHCT) results in long-term, multilineage chimerism without myeloablation/immunosuppression. It has the potential to treat a number of congenital hematologic disorders, including as Sickle cell disease. Central tolerance, through which donor-reactive host T cells and host-reactive donor T cells are deleted, is instrumental to the induction of donor specific tolerance (DST) and the prevention of graft-versus-host-disease (GVHD). Central deletion, however, is incomplete, and donor-reactive host T cells remain following allogeneic IUHCT. Furthermore, central deletion alone cannot explain the absence of GVHD either following IUHCT using non-T cell depleted bone marrow (BM) or in chimeric animals that undergo postnatal donor lymphocyte infusion (DLI) with subsequent conversion to >90% allogeneic donor cell engraftment (Hayashi et al., Blood 2002;100:804-12). Studies in both mice and humans have demonstrated the importance of regulatory T cells, including traditional CD25+FoxP3+ Tregs and CD49b+LAG-3+ Type 1 regulatory T (Tr1) cells, in maintaining tolerance following postnatal allogeneic transplantation. We hypothesized that peripheral regulatory T cells are increased in chimeric animals following IUHCT and that they contribute to the establishment of DST and prevention of GVHD.Methods: 10x106 BM cells from 6-8 week old C57Bl/6 (B6) FoxP3GFP mice (H2Kb, CD45.2+) were injected intravenously (IV) into gestational day 14 Balb/c FoxP3GFP fetal recipients (H2Kd, CD45.2+). Mice were sacrificed at 2, 4, 8, and 24 weeks of age. Splenocytes were analyzed for the expression of CD4, H2Kb, H2Kd, CD49b, LAG-3, CD25, and FoxP3 to determine the percentage of traditional Tregs and Tr1 cells within the CD4+ population as well as for the expression of intracellular IL-10 - a key mediator of regulatory T cell function. A GVHD model was developed in which 10x106 B6 BM cells were injected IV into day of life 0 (P0) naive Balb/c mice. The ability of IUHCT-induced regulatory T cells to suppress GVHD was assessed in two groups: 1) 10x106 B6 BM cells injected into P0 mice that had undergone IUHCT and 2) 10x106 B6 BM cells co-injected with 5x106 CD4+ splenocytes harvested from 2 week old chimeric mice into P0 naive Balb/c mice. To evaluate the role of regulatory T cells following DLI in chimeric mice, 30x106 B6CD45.1 (H2Kb, CD45.1+) splenocytes were injected IV into 4 week old chimeric recipients. Mice were sacrificed at 48 hours and 2 weeks post-DLI, and regulatory T cells were measured as a percentage of CD45.2+CD4+ cells in the spleen.Results: IUHCT was associated with a significant increase in Tr1 cells, predominantly of donor origin, compared to naive controls (Figure 1). There was a corresponding increase in intracellular IL-10 expression in donor CD4+ splenocytes in chimeric animals compared to naive B6 FoxP3GFP controls at 2 weeks of age (MFI 7.07 vs. 3.26, p=0.0017). Elevated levels of regulatory T cells following IUHCT also had a functional effect in the GVHD model (Figure 2). Mice that had undergone IUHCT (group 1) demonstrated a trend toward improved survival compared to naive mice when injected with B6 BM at P0. Naive mice injected at P0 with B6 BM plus CD4+ splenocytes from chimeric mice (group 2) demonstrated significant improvement in survival compared to naive recipients of B6 BM alone (p=0.049). Although we detected no increase in traditional Tregs after IUHCT alone, we noted a 3-fold increase in traditional Tregs of host origin in chimeric animals at 48 hours post-DLI (25.5% vs. 8.3%, p<0.001).Conclusion: The induction of tolerance following IUHCT is important for maintaining successful long-term engraftment and preventing GVHD. We demonstrate an increase in nontraditional regulatory T cells following IUHCT which can minimize GVHD as well as an increase in traditional Tregs following DLI in an established model of complete allogeneic engraftment. These studies suggest an important role for regulatory T cell populations in the induction of tolerance following IUHCT. They are significant in light of a potential obstacle to the clinical translation of IUHCT: the window of central tolerance induction in humans occurs at a gestational age that will pose significant technical challenges. Augmentation of regulatory T cell-dependent peripheral tolerance is one approach by which IUHCT could be delayed to a technically feasible point later in gestation. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Inhibition of HIF Prolyl Hydroxylases Mitigate Gut Graft-Versus-Host Disease

The etiology of graft-versus-host disease (GVHD) is rooted in the alloreactive response of donor T cells present in the hematopoietic graft resulting in the destruction of the patient's tissues, particularly the gastrointestinal tract. Gut GVHD affects up to 50% of patients, is a leading cause of death, and has overlapping features with inflammatory bowel disease (IBD). Increased gut permeability, alterations of the gut microbiome and intestinal stem cell niche damage predispose the gut to the local and systemic effects of GVHD, and is exacerbated by the inability of the gut to adequately regenerate.Severe shifts in metabolism and reduced oxygen (O2) availability in the inflamed gut are major underlying factors in the pathogenesis of IBD. Two related transcription factors, hypoxia-inducible factor-1 (HIF-1) and HIF-2, originally discovered as master regulators of the adaptive response to hypoxia, have been recently shown to be gut protective and promote mucosal healing in response to injury. Using a MHC mismatched B10.BR→B6 bone marrow transplant (BMT) model, we previously found that loss of intestinal epithelial (IE) HIF-1α or HIF-2α worsened survival compared to wild-type mice and exhibited increased GVHD-induced-histopathology. Thus, we hypothesized that HIF-1 and HIF-2 protects and repairs the gut from conditioning and GVHD-related damage.HIF-1 and HIF-2 are heterodimers consisting of an O2-labile HIF-1α or HIF-2α subunit respectively and a constitutively expressed HIF-1β subunit. The recent discovery that iron-dependent prolyl hydroxylase enzymes (PHD1-3) can trigger the O2-dependent proteasomal degradation of the HIF-1α subunit has led to the development of pan-PHD inhibitors (PHDi) that activate HIF-1 and HIF-2. PHDi such as dimethyloxallyl glycine (DMOG) and AKB-4924 have been shown to attenuate colitis and radiation-induced gut toxicity in animal models. We thus sought to determine whether PHDi could also ameliorate gut GVHD in the B10.BR→B6 BMT model. B6 mice were lethally irradiated (10Gy, split dose) and transplanted with 5x106 T-cell depleted bone marrow (BM) cells from B10.BR donors with 2x106 enriched T cells from spleens and lymph nodes (BM+T). B6 mice transplanted with only T cell depleted BM cells served as our non-GVHD control group. B6 mice were treated with AKB-4924 (AKB, 5 mg/kg) or vehicle (β-cyclodextrin) via intraperitoneal delivery, beginning 1 day prior to BMT and for 6 consecutive days post-BMT. Treatment with AKB prevented significant weight loss, compared to vehicle-treated mice, 7 days post-BMT (n=6, p<.002). Histopathologic assessment of the jejunum of AKB treated mice after 7 days revealed fewer apoptotic cells and an increased number of Paneth and goblet cells compared to vehicle-treated mice. Immunohistochemical staining for lysozyme showed that AKB prevented GVHD-induced Paneth cell ablation compared to their vehicle BMT counterpart (2.8 Lyz+ cells/crypt vs 0.84 Lyz+ cells/crypt, n=3, p<.003). Additionally, we evaluated the effects of AKB on T cell alloreactivity. In mixed lymphocyte reaction Elispot assays, AKB and DMOG, reduced the alloreactive interferon (IFN)-γ response in B10.BR anti-B6 (p<0.001) and MHC matched, minor antigen-mismatched B6 anti-BALB.B cultures (p<0.05). Furthermore, AKB reduced the number of gut-infiltrating CD3+ T cells compared to vehicle-treated BMT mice (39 vs 155.1 CD3+ cells/high-power field, n=3, p<.0003). Our results provide the framework to validate the therapeutic potential of PHDi as an intestinal regenerative strategy in mitigating GVHD. DisclosuresPeters:Aerpio Therapeutics: Employment, Equity Ownership.

Activation of Interferon Regulatory Factor 3 (IRF3) is Required in Both T cells and APCs for Optimal Expression of IFN-γ, IL-17, and Granzyme B during T cell responses.

Abstract Recently we saw a link between activation of Interferon Regulatory Factor (IRF)-3 and expression of IFN-γ, as well as Granzyme B (GrB) from T cells during viral infections in C57Bl/6 (B6) and IRF3KO mice. To investigate this further, we stimulated splenic mononuclear cells (SMC) from B6 and IRF3KO mice in vitro with anti-CD3±anti-CD28± poly I:C, a TLR3/IRF3 agonist. We show during responses with anti-CD3/anti-CD28 that IRF3 is activated within 24 h in B6 SMC and expression of IFN-γ, IL-17 and GrB is impaired in IRF3KO SMC. Adding poly I:C with anti-CD3/anti-CD28 accelerated IRF3 activation and significantly increased IFN-γ, but not IL-17 or GrB, from IRF3KO SMC. Interestingly, Foxp3 expression and percentage of Foxp3+ T cells were significantly lower in unstimulated IRF3KO SMC compared with B6 SMC. To determine the contribution of IRF3 activation in T cells vs APCs, B6 and IRF3KO bone marrow dendritic cells (DCs; 3×104) were mixed in various combinations with WT or IRF3KO purified T cells (3×105), as well as anti-CD3±anti-CD28± poly I:C. After 48 h, IFN-γ, IL-17, and GrB expression was greatest in cultures where both B6 DCs and B6 T cells were mixed. IFN-γ, IL-17 and GrB decreased when either IRF3KO DCs or IRF3KO T cells were part of the mixture. Least production of IFN-γ, IL-17, and GrB occurred when DCs and T cells were from IRF3KO mice. These results suggest that IRF3 in both APCs and T cells is required to achieve optimum production of IFN-γ, IL-17, and GrB during T cell responses. Moreover, the data unexpectedly show that IRF3 is activated during T cell immune responses without a TLR3 agonist. The factor(s) responsible for IRF3 activation during APC/T cell responses is unclear but appears to be a soluble factor whose expression is stimulated through CD28.

BCAP (B-cell adaptor for phosphoinositide 3-kinase) plays a role in development of Marginal Zone B cells in mice

Abstract B-cell adaptor for phosphoinositide 3-kinase (BCAP) connects B cell receptor (BCR) signaling to the phosphoinositide 3-kinase (PI3K)-Akt pathway. Upon BCR engagement, tyrosine kinases Syk and Btk phosphorylate BCAP and phosphorylated BCAP provides the binding site for the regulatory subunit (p85) of PI3K, leading to relocation of PI3K to cytoplasmic membrane and PI3K activation. In response to CD19 engagement, BCAP phosphorylation is also required for p85 binding of BCAP prior to PI3K and Akt activation. It has been shown that disruption of the BCAP gene in the chicken DT40 B cell line inhibits BCR-mediated phosphatidylinositol 3,4,5-trisphosphate generation, leading to an impaired Akt response through a significant reduction in the recruitment of PI3K to glycolipid-enriched microdomains (lipid rafts). Furthermore, using BCAP knock out (KO) mice, BCAP has been shown to be essential for normal Follicular B and B1 B cell development and function. Here we report that the development of another peripheral B cell subpopulation, Marginal Zone B (MZB) cells, is impaired in BCAP KO mice as well. The numbers of MZB (B220+CD23−CD24midCD21high) cells and their surface expression level of CD80 are significantly reduced, compared to the littermate or age-matched B6 controls. The serum IgG3 level of the BCAP KO mice is also significantly lower than that of control mice. This result is further supported by an in vitro co-culture system, using bone marrow (BM) cells with the OP9-DL1 cells, in which the development of MZB cells from BCAP KO BM is also reduced, compared to B6 BM. Moreover, addition of exogenous BAFF could not fully restore the development of MZB cells from BCAP KO BM. Our data ascribes a role of BCAP to MZB cell development in mice.

Third Party Cytokine-Induced Killer Cells Protect from Murine Lethal Graft-Versus-Host Disease

Cell adoptive immunotherapy protect from lethal graft-versus-host disease (GVHD) while preserving graft-versus-tumor effects is ideal in allogeneic hematopoietic stem cell transplantation (HSCT) in patients with hematologic malignancies. Moreover, the use of third party cells would open a huge source of availability and feasibility across major histocompatibility barriers. In fact, some studies demonstrated that infusion of third party derived cells such as whole bone marrow cells or iNKT cells could ameliorate lethal GVHD in a murine model.Cytokine-induced killer (CIK) cells are ex vivo-expanded T lymphocytes expressing both natural killer and T-cell markers. We have previously reported that donor-type CIK cells have potential to separate graft-versus-tumor effects from GVHD by eliminating host dendritic cells due to the enhanced killing activity by interferon-gamma. Actually, donor-typed CIKs infusion for refractory hematological malignancies after HSCT has been started in some clinical trails. In the present study, we examine the effect of third party cells against GVHD protection, in particular third party CIK cells for prevention from lethal GVHD in a murine model of allogeneic HSCT.To test this, lethally irradiated host Balb/c (H-2d) mice were given C3H (H-2k) or B6 (H-2b) bone marrow cells and splenocytes to induce lethal GVHD with/without third-party cells (C57/BL6 or C3H) such as CIK cells, whole splenocytes, or bone marrow derived dendritic cells (BMDCs) on day 0, which were cultured from bone marrow cells with GM-CSF. Mice receiving third party CIK cells showed much less GVHD and significant survival benefit compared those with third party splenocytes, or BMDCs as shown in the figure below. Interestingly, when infusion of third party splenocytes was delayed until day 4 after bone marrow transplantation, host mice receiving splenocytes survived much better compared with mice receiving splenocytes on day 0, even though reduced number of third party splenocytes was used. This result indicated that timing of third party cell infusion was quite important, especially when third party splenocytes were used. As expected, the mice given reduced number of third CIK cells also showed excellent survival with much less GVHD. Taken together, our results clearly demonstrated that third party CIK cells have strong potential to prevent lethal GVHD without attention to timing of cell infusion.Next, to further investigate the advantage of third party CIK cells rather than whole splenocytes, we compared the hematological reconstitution between the mice given third party CIK cells on day 4 after BMT and whole splenocytes. Both mice showed much less GVHD with the same level, however the recovery of white blood cell count on day 21 after BMT was significantly better in the mice receiving third party CIK cells.In conclusion, infusion of third party CIK cells has strong potential to prevent lethal GVHD with faster hematological recovery after HSCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.