Human Mixed Lymphocyte Reaction Research Articles

Allostimulation leads to emergence of a human B cell population with increased expression of HLA class I antigen presentation–associated molecules and the immunoglobulin receptor FcRL5

In the extensive literature characterizing lymphocyte contributions to transplant-related pathologies including allograft rejection and graft-versus-host disease, T cell–focused investigation has outpaced investigation of B cells. Most B cell–related reports describe regulatory and antibody-producing functions, with less focus on the potential role of antigen-presenting capacity. Using in vitro human mixed lymphocyte reactions (MLRs) to model allostimulation, we analyzed responder B cells using transcriptional analysis, flow cytometry, and microscopy. We observed emergence of an activated responder B cell subpopulation phenotypically similar to that described in individuals with graft-versus-host disease or allograft rejection. This population had markedly increased expression of FcRL5 (Fc receptor like 5) and molecules associated with human leukocyte antigen class I antigen presentation. Consistent with this phenotype, these cells demonstrated increased internalization of irradiated cell debris and dextran macromolecules. The proportion of this subpopulation within MLR responders also correlated with emergence of activated, cytotoxic CD8+ T cells. B cells of similar profile were quite infrequent in unstimulated blood from healthy individuals but readily identifiable in disaggregated human splenocytes and increased in both cases upon allostimulation. Further characterization of the emergence and function of this subpopulation could potentially contribute to identification of novel biomarkers and targeted therapeutics relevant to curbing transplant-related pathology.

Comparative Inhibitory Effects of Tacrolimus, Cyclosporine, and Rapamycin on Human Anti-Pig Xenogeneic Mixed Lymphocyte Reactions.

Long-term immunosuppressive maintenance therapy is necessary to prevent the rejection of xenografts. However, it is still unclear which oral immunosuppressant is most suitable for pig-to-human xenotransplantation . A xenogeneic mixed lymphocyte reaction (MLR) system was established using peripheral blood mononuclear cells (PBMCs) isolated from wildtype (WT) or GTKO/CMAHKO/β4GalNT2KO (TKO) pigs as stimulator cells and human PBMCs as responder cells. Various concentrations of tacrolimus (Tac), cyclosporine (CsA), or rapamycin (Rapa) were added to the MLR system as interventions. The inhibitory effects of the three immunosuppressants on the proliferation and cytokine production of human T cells were studied and compared. The inhibitory effect of anti-CD154 mAb alone or in combination with Tac/CsA/Rapa on xenoreactive MLR was also investigated. PBMCs from both WT and TKO pigs stimulated significant proliferation of human T cells. Tac had a strong inhibitory effect on human T-cell proliferation stimulated by pig PBMCs. CsA inhibited human T-cell proliferation in a typical dose-dependent manner. When Tac and CsA concentrations reached 5 and 200ng/mL, respectively, the proliferation rates of CD3+/CD4+/CD8+ T cells were reduced almost to a negative level. Even at high concentrations, Rapa had only a moderate inhibitory effect on xenogeneic MLR. The inhibitory effects of these three immunosuppressants on xenogeneic T-cell responses were further confirmed by the detection of CD25 expression and supernatant cytokines (IL-2, IL-6, IFN-γ, TNF-α, IL-4, IL-10, and IL-17). Although anti-CD154 mAb monotherapy showed only moderate inhibitory effects on xenoreactive T-cell proliferation, low-dose anti-CD154 mAb combined with low-dose Tac, CSA, or Rapa could produce significant synergistic inhibitory effects. Tac is more efficient than CsA or Rapa in inhibiting xenogeneic T-cell responses in vitro. If used in combination with anti-CD154 mAb, all the three immunosuppressants can achieve satisfactory synergistic inhibitory effects.

Abstract A077: A novel, tumor-targeted immunocytokine comprising an anti-PD-L1 Affimer® fused to IL-15 exhibits potent anti-tumor activity

Abstract Affimer® biotherapeutics are novel 15kDa scaffold proteins based on the human protease inhibitor Stefin A. Affimer® proteins lack disulfide bonds and post translational modifications including glycosylation. Large, diverse phage display libraries have been created which incorporate peptide loops into the scaffold backbone. We have previously identified high-affinity competitive binders to human programmed death-ligand 1 (PD-L1) using phage display which block interaction with programmed death 1 (PD-1). Lead anti-PD-L1 Affimer® proteins were formatted by fusing to human Fc or as in-line-fusions to a serum albumin binding Affimer® to extend pharmacokinetic half-life. Here we describe novel immunocytokines that consist of half-life extended Affimer® antagonist to PD-L1 fused to human Interleukin 15 (IL-15) receptor alpha sushi domain and human IL-15 (PDL1-IL15 Affimer® fusion proteins). PD-L1 is upregulated on tumor cells, macrophages, and dendritic cells in the tumor microenvironment. PD-L1 engagement with PD-1 on T cells inhibits T cell proliferation, transcriptional activation, and cytokine release, resulting in suppressed anti-tumor response. IL-15 signals through the IL-2 receptor beta chain and the common gamma chain (IL-2Rβ/γ) and induces differentiation and proliferation of CD8+ T cells and Natural Killer (NK) cells; it does not stimulate regulatory T cells (Tregs). Immunostimulatory cytokines have therapeutic potential but are often associated with toxicity when administered systemically. PDL1-IL15 Affimer® fusion proteins have the potential to bring the immunomodulatory cytokine directly to the tumor and reverse the immunosuppressive tumor microenvironment by enhancing T cell activation, while also introducing a checkpoint inhibitor to augment the anti-tumor immune response. We characterized binding and activity of PDL1-IL15 Affimer® fusion proteins in in vitro assays. PDL1-IL15 Affimer® fusion proteins bind both PD-L1 and IL-2Rβ/γ and exhibit PD-1:PD-L1 blocking activity and IL-2Rβ/γ activation. PDL1-IL15 Affimer® fusion proteins reversed T cell exhaustion in a human mixed lymphocyte reaction assay: PDL1-IL15 Affimer® fusion proteins showed greater activity than either the PD-L1 or the IL-15 component alone. Furthermore, PDL1-IL15 Affimer® fusion proteins enhanced immune cell killing of tumor cells in co-culture assays of human peripheral blood mononuclear cells and tumor cells. In summary, data suggests that PDL1-IL15 Affimer® fusion proteins can reverse T cell exhaustion and induce immune cell activation to elicit an anti-tumor effect. Ongoing in vivo work aims to investigate the efficacy and tolerability profile of PDL1-IL15 Affimer® fusion proteins to direct future development of this therapeutic. In addition, the data will support wider utility of the Affimer® platform to develop new ways of targeting the tumor while reducing systemic toxicity. Citation Format: Victoria Juskaite, Deepa Avisetti, David H Jones, Ahmad Sobri, Gregory Billenness, Hanna Buist, Tik Nga Tong, Norhakim Yahya, Fiona McLaughlin. A novel, tumor-targeted immunocytokine comprising an anti-PD-L1 Affimer® fused to IL-15 exhibits potent anti-tumor activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A077.

The immunoregulatory effect of the TREM2-agonist Sulfavant A in human allogeneic mixed lymphocyte reaction.

Sulfavant A (SULF A) is a synthetic derivative of naturally occurring sulfolipids. The molecule triggers TREM2-related maturation of dendritic cells (DCs) and has shown promising adjuvant activity in a cancer vaccine model. the immunomodulatory activity of SULF A is tested in an allogeneic mixed lymphocyte reaction (MLR) assay based on monocyte-derived dendritic cells and naïve T lymphocytes from human donors. Flow cytometry multiparametric analyses and ELISA assays were performed to characterize the immune populations, T cell proliferation, and to quantify key cytokines. Supplementation of 10 µg/mL SULF A to the co-cultures induced DCs to expose the costimulatory molecules ICOSL and OX40L and to reduce release of the pro-inflammatory cytokine IL-12. After 7 days of SULF A treatment, T lymphocytes proliferated more and showed increased IL-4 synthesis along with downregulation of Th1 signals such as IFNγ, T-bet and CXCR3. Consistent with these findings, naïve T cells polarized toward a regulatory phenotype with up-regulation of FOXP3 expression and IL-10 synthesis. Flow cytometry analysis also supported the priming of a CD127-/CD4+/CD25+ subpopulation positive for ICOS, the inhibitory molecule CTLA-4, and the activation marker CD69. These results prove that SULF A can modulate DC-T cell synapse and stimulate lymphocyte proliferation and activation. In the hyperresponsive and uncontrolled context of the allogeneic MLR, the effect is associated to differentiation of regulatory T cell subsets and dampening of inflammatory signals.

Porcine-Stimulated Human Tr1 Cells Showed Enhanced Suppression in Xenoantigen Stimulation Response.

Type 1 regulatory T (Tr1) cells play a fundamental role in maintaining and inducing immune tolerance. Our preliminary study demonstrated that an interleukin- (IL-) 10-mediated pathway is a possible regulatory mechanism underlying the xenoantigen-specific human Treg enhanced suppressive capacity. Here, we developed a feasible protocol for expanding IL-10-induced xenoantigen-specific human Tr1 cells in vitro which would be more efficient in transplantation immunotherapy efficiency. In this study, xenoantigen-specific Tr1 cells are generated from human naive CD4+ T cells expanded for two subsequent xenoantigen-stimulation cycles with recombinant human IL-10. The phenotype and suppressive capacity of xenoantigen-stimulated Tr1 cells are assessed, and the mechanism of their suppression is studied. Tr1 cells can be induced by porcine xenoantigen stimulation combined with IL-10, IL-2, and IL-15, displaying an increased expression of CD49b, CTLA-4, and LAG-3 without expressing Foxp3 which also showed an effector memory Treg phenotype and expressed high levels of CD39. After xenoantigen stimulation, the IL-10 and IL-5 gene expression in Tr1 cells increased, secreting more IL-10, and xenoantigen-stimulated Tr1 cells changed their T cell receptor (TCR) Vβ repertoire, increasing the expression of TCR Vβ2, TCR Vβ9, and TCR Vβ13. In a pig to human mixed lymphocyte reaction (MLR), xenoantigen-stimulated Tr1 cells displayed enhanced suppressive capacity via CD39 in a dose-dependent manner. Moreover, IL-5 could affect the proliferation of xenoantigen-specific Tr1 cells, but not their phenotypes' expression. This study provides a theory and feasible method for immune tolerance induction in clinical xenotransplantation.

Hyaluronan synthesis inhibition impairs antigen presentation and delays transplantation rejection

A coat of pericellular hyaluronan surrounds mature dendritic cells (DC) and contributes to cell-cell interactions. We asked whether 4-methylumbelliferone (4MU), an oral inhibitor of HA synthesis, could inhibit antigen presentation. We find that 4MU treatment reduces pericellular hyaluronan, destabilizes interactions between DC and T-cells, and prevents T-cell proliferation in vitro and in vivo. These effects were observed only when 4MU was added prior to initial antigen presentation but not later, consistent with 4MU-mediated inhibition of de novo antigenic responses. Building on these findings, we find that 4MU delays rejection of allogeneic pancreatic islet transplant and allogeneic cardiac transplants in mice and suppresses allogeneic T-cell activation in human mixed lymphocyte reactions. We conclude that 4MU, an approved drug, may have benefit as an adjunctive agent to delay transplantation rejection.

FS222, a CD137/PD-L1 Tetravalent Bispecific Antibody, Exhibits Low Toxicity and Antitumor Activity in Colorectal Cancer Models.

With the increased prevalence in checkpoint therapy resistance, there remains a significant unmet need for additional therapies for patients with relapsing or refractory cancer. We have developed FS222, a bispecific tetravalent antibody targeting CD137 and PD-L1, to induce T-cell activation to eradicate tumors without the current toxicity and efficacy limitations seen in the clinic. A bispecific antibody (FS222) was developed by engineering CD137 antigen-binding sites into the Fc region of a PD-L1 IgG1 mAb. T-cell activation by FS222 was investigated using multiple in vitro assays. The antitumor efficacy, survival benefit, pharmacodynamics, and liver pharmacology of a murine surrogate molecule were assessed in syngeneic mouse tumor models. Toxicology and the pharmacokinetic/pharmacodynamic profile of FS222 were investigated in a non-human primate dose-range finding study. We demonstrated simultaneous binding of CD137 and PD-L1 and showed potent T-cell activation across CD8+ T-cell activation assays in a PD-L1-dependent manner with a CD137/PD-L1 bispecific antibody, FS222. FS222 also activated T cells in a human primary mixed lymphocyte reaction assay, with greater potency than the monospecific mAb combination. FS222 showed no signs of liver toxicity up to 30 mg/kg in a non-human primate dose-range finding study. A surrogate molecule caused significant tumor growth inhibition and survival benefit, concomitant with CD8+ T-cell activation, in CT26 and MC38 syngeneic mouse tumor models. By targeting CD137 agonism to areas of PD-L1 expression, predominantly found in the tumor microenvironment, FS222 has the potential to leverage a focused, potent, and safe immune response augmenting the PD-(L)1 axis blockade.

Heparin affects the induction of regulatory T cells independent of anti-coagulant activity and suppresses allogeneic immune responses.

Heparin is a widely used anti-coagulant that enhances anti-thrombin (AT) activity. However, heparin also suppresses immune and inflammatory responses in various rodent models and clinical trials, respectively. The mechanism by which heparin suppresses immune responses is unclear. The effect of heparin on regulatory T cells (Tregs ) in allogeneic immune responses was analysed using an acute graft-versus-host disease (aGVHD) mouse model and mixed lymphocyte reactions (MLRs). In-vitro culture systems were utilized to study the effects of heparin on Tregs . Heparin administration reduced mortality rates and increased the proportion of Tregs in the early post-transplantation period of aGVHD mice. In both murine and human MLRs, heparin increased Tregs and inhibited responder T cell proliferation. Heparin promoted functional CD4+ CD25+ forkhead box protein 3 (FoxP3)+ Treg generation from naive CD4+ T cells, increased interleukin (IL)-2 production and enhanced the activation of pre-existing Tregs with IL-2. Heparin-induced Treg increases were not associated with anti-coagulant activity through AT, but required negatively charged sulphation of heparin. Importantly, N-acetyl heparin, a chemically modified heparin without anti-coagulant activity, induced Tregs and decreased mortality in aGVHD mice. Our results indicate that heparin contributes to Treg -mediated immunosuppression through IL-2 production and suggest that heparin derivatives may be useful for immunopathological control by efficient Treg induction.

Autophagy-lysosome inhibitor chloroquine prevents CTLA-4 degradation of T cells and attenuates acute rejection in murine skin and heart transplantation

Background: The immune checkpoint cytotoxic T lymphocyte antigen-4 (CTLA-4), induced upon T cell activation but degraded quickly, has been targeted in the clinical therapy of advanced cancers and autoimmune diseases. However, whether inhibiting CTLA-4 degradation ameliorates transplant rejection remains unknown.Methods: The CTLA-4 expression in activated murine T cells treated with the inhibitors mediating protein degradation was detected by flow cytometry (FCM). CD45.1 mice, which received TEa T cells and underwent heart transplantation, were administrated with the inhibitor. Subsequently, CTLA-4 expression of TEa T cells was analyzed. Murine skin and heart transplantation models were built, then the survival and histopathology of the allografts, and T cell subsets in the spleens of each group were compared.Results: Chloroquine (CQ) was identified as an inhibitor of CTLA-4 degradation, which augmented both surface and total CTLA-4 expression in T cells. It considerably prolonged the skin and heart allograft survival time and reduced the infiltration of inflammatory cells in allografts. Besides decreasing the frequencies of the CD4+ and CD8+ effector T cells, especially IFN-γ producing T cells, CQ also increased the proportion of regulatory T cells in the spleen. The CTLA-4 blockade abrogated the benefits of CQ on the survival of heart allografts. Moreover, CQ enhanced CTLA-4 expression in activated human T cells and reduced the secretion of IFN-γ in human mixed lymphocyte reaction.Conclusion: Targeting CTLA-4 degradation provides a novel means to prevent transplant rejection and induce transplant tolerance.

Abstract 1288: Blocking tumor-associated immune suppression with BAY-218, a novel, selective aryl hydrocarbon receptor (AhR) inhibitor

Abstract Tumor cells co-opt multiple pathways in order to evade attack by infiltrating immune cells. One such mechanism is the upregulation of indole-2,3-dioxygenase (IDO1) and/or tryptophan-2,3-dioxygenase (TDO2), both of which are first-step, rate-limiting enzymes degrading tryptophan to the immunosuppressive metabolites kynurenine (KYN) and kynurenic acid (KA). KYN and KA bind and activate the aryl hydrocarbon receptor (AhR), which is expressed in many cell types and is well known for its immunosuppressive effects. Targeting of the AhR with an inhibitor may therefore provide a novel immunotherapeutic approach for enhancing anti-tumoral immune responses and treating cancer. Here we describe the identification and functional immune characterization of BAY-218, a novel, selective and potent AhR small molecule inhibitor. Mechanistically, BAY-218 inhibited AhR nuclear translocation, dioxin response element (DRE)-luciferase reporter expression and AhR-regulated target gene expression induced by both exogenous and endogenous AhR ligands. In vitro, BAY-218 rescued TNFα production from KA-suppressed LPS-treated primary human monocytes. Furthermore, BAY-218 enhanced T cell cytokine production in a human mixed lymphocyte reaction (MLR) and a mouse antigen-specific bone-marrow-derived dendritic cell (BMDC)-OT-I T cell co-culture. In the MLR, BAY-218 increased anti-PD1 antibody-mediated IL-2 and IFNγ secretion, while an IDO inhibitor did not, indicating that BAY-218 is able to block AhR activation mediated by ligands outside of the IDO-KYN pathway. In vivo, BAY-218 enhanced anti-tumoral immune responses and reduced tumor growth in the syngeneic mouse tumor models CT26 and B16-OVA. FACS analysis of leukocytes infiltrating B16-OVA tumors demonstrated that administration of BAY-218 increased the frequency of tumor-infiltrating CD8+ T cells and NK cells while decreasing GR1+ myeloid cells and CD206+M2 macrophages. Furthermore, BAY-218 enhanced therapeutic efficacy of an anti-PD-L1 antibody in the CT26 model. In summary, AhR inhibition with BAY-218 stimulates pro-inflammatory monocyte and T cell responses in vitro and drives anti-tumor immune responses, resulting in decreased tumor growth, in vivo. Thus, inhibiting AhR represents a novel immunotherapeutic approach for blocking AhR-mediated tumor-associated immunosuppression. Citation Format: Ilona Gutcher, Christina Kober, Lars Roese, Julian Roewe, Norbert Schmees, Florian Prinz, Matyas Gorjanacz, Ulrike Roehn, Benjamin Bader, Horst Irlbacher, Detlef Stoeckigt, Rafael Carretero, Katharina Sahm, Iris Oezen, Hilmar Weinmann, Ingo V. Hartung, Bertolt Kreft, Michael Platten. Blocking tumor-associated immune suppression with BAY-218, a novel, selective aryl hydrocarbon receptor (AhR) inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1288.

PD-L2 generates a signal through the PD-1 co-receptor distinct from that of PD-L1

Abstract Immune checkpoint blockade of the PD-1/PD-L1/PD-L2 signalling axis yields potent anti-tumor responses by disinhibiting T cell effector function. Mouse and human data demonstrating an inhibitory role for PD-L1 has resulted in FDA approval of anti-PD-L1 antibodies; however, PD-L2 remains sparsely studied despite being the higher affinity of the two ligands for PD-1. Recent findings demonstrate that PD-L2 is widely expressed by human malignancies. PD-L1 and PD-L2 have also been shown to induce distinct conformational changes on engaging human PD-1. We utilized a Jurkat T cell based bioluminescent assay of T cell activation and PD-1 inhibition to determine the unique mechanism of action of human PD-L2. In this system, human PD-L2 generates a solely co-inhibitory signal, but at reduced inhibitory potential relative to PD-L1. We find that unlike murine PD-L2, human PD-L2 appears to have a dedicated co-inhibitory function as we find no significant engagement of the human homolog of its murine co-stimulatory partner RGMb. Reverse phase protein array analysis of human PD-L1 and PD-L2 engagement of PD-1 on Jurkat T cells revealed significant differences in downstream T cell signalling generated by each ligand. We also find that human PD-L1 and PD-L2 have disparate impacts on proliferation wherein PD-L2 supports a preferential arrest of T cells in the S-phase of the cell cycle. We observed that combination blockade of PD-L1 and PD-L2 improves on the blockade of PD-L1 alone resulting in increased production of IL-2 and IFNγ in primary human mixed lymphocyte reactions. Our findings address critical gaps in our knowledge about the basic function of PD-L2 and will inform future attempts to manipulate the PD-1 co-inhibitory circuit.

Long Non-Coding RNA Regulation of Allogeneic T-Cells

The mechanisms governing human allogeneic T-cell responses remain incompletely understood which limits the wider clinical application of allogeneic BMT. Long non-coding RNAs (lncRNA) are an emerging class of non-coding RNAs that control gene expression with tissue and context specificity. However, their role in alloimmunity is unknown. To determine whether lncRNAs regulate T cell alloimmunity we performed RNA-seq of donor T-cells from clinical BMT patients that received increasing amounts of allogeneic stimulation. Hierarchical clustering and differential expression analysis showed that increasing allogeneic stimulation drove differential expression of lncRNAs. To validate the sequencing data, we confirmed the differential expression of 6 lncRNAs in an independent patient cohort by qRT-PCR and confirmed the altered expression of 4 of these candidate lncRNAs in a human mixed lymphocyte reaction (MLR). The database FANTOM-CAT showed strong enrichment of Linc00402 (88 fold) and RP11-348F1.3 (18 fold) in human T-cells, and CD4 and CD8 human T-cells demonstrated similar expression of Linc00402 and RP11-348F1.3. Linc00402 is predicted to be conserved, and its expression was detected by qRT-PCR primer tiling in mouse T-cells. The differential expression upon allo-stimulation of the Linc00402 mouse orthologue, GM34199, was confirmed both in vitro with an MLR and in vivo using both major and minor histocompatibility disparate murine models of GVHD. The functional validation of human RP11-348F1.3, human Linc00402, and Linc00402’s murine orthologue, GM34199, was tested by anti-sense oligonucleotide-mediated knockdown, which inhibited human Linc00402 (55% of control, p

RNA-Seq of Human T Cells after BMT Identifies Linc00402 As a Novel Regulator of Human T Cell Alloimmunity

The mechanisms governing human allogeneic T cell responses remain incompletely understood which limits the wider clinical application of allo-BMT. Long non-coding RNAs (lncRNA) are an emerging class of non-coding RNAs that control gene expression with tissue and context specificity. However, their role in alloimmunity is unknown. To determine whether lncRNAs regulate T cell alloimmunity we performed RNA-seq of donor T cells following clinical BMT to identify and characterize differentially-expressed lncRNAs. CD3+ T cells 30 days post myeloablative BMT were isolated in a discovery cohort from 3 different groups, each with increasing amounts of allo-antigen stimulation: 1) autologous donors (Auto) to control for lymphopenia induced proliferation; 2) matched unrelated donors (MUD), which receive minor histocompatibility allo-stimulation and are exposed to immune suppression; and 3) mis-matched unrelated donors (MMUD), which receive both minor and major histocompatibility allo-stimulation and also receive immune suppression. Subjects with GVHD, infection, who received corticosteroids, or who received T cell depleting therapy were excluded. Hierarchical clustering showed distinct grouping of MMUD samples, but an intermingling of the Auto and MUD samples suggesting that increasing allogeneic stimulation drove differential transcript expression. Gene ontology analysis of protein coding genes showed enrichment for pathways operant in activated T cells, thereby providing validity to our approach. The differentially expressed lncRNAs mainly showed increasing expression with increasing allo-stimulation (i.e. higher expression in the MMUD group) (Figure 1). We validated the sequencing data with qRT-PCR for 6 of 8 lncRNAs with sequencing padj < 0.05 relative to the Auto group (see those marked with an * in Figure 1).To further validate the sequencing data, we confirmed the differential expression of 5 lncRNAs in an independent patient cohort (n=8-11 per group) by qRT-PCR (see those underlined in Figure 1). Linc00402 was chosen as the lead candidate (increased 3 fold in the MMUD group, p < 0.01). Its expression along with the 4 other lncRNAs were further confirmed to be regulated by allo-stimulation following in vitro human mixed lymphocyte reactions (MLRs) (Linc00402's expression changed 4.7 fold, p < 0.001). The database FANTOM-CAT showed strong enrichment of Linc00402 (88 fold) in human T cells relative to other cell/tissue types, thereby highlighting its tissue specific expression. This was validated in CD4 and CD8 human T cells which demonstrated similar expression of Linc00402.We next explored for murine orthologues of Linc00402 and the other 4 differentially expressed lncRNAs using Transmap. Expression of 3 of the 4 putative orthologues that were identified by Transmap was detected by qRT-PCR primer tiling (see those in bold in Figure 1). Their differential expression upon allo-stimulation, including the putative Linc00402 orthologue, GM34199, was confirmed in vitro with an MLR (22 fold change in expression of GM34199, p < 0.01) and in vivo using both major and minor histocompatibility disparate murine models of GVHD (B6gBalb/c and B6gC3H.SW, respectively) at 7 and 14 days post BMT in which GM34199's expression changed 2.5 to 13 fold (p < 0.01).The functional validation of human Linc00402 and murine GM34199 was tested by anti-sense oligonucleotide-mediated knockdown, which inhibited both human (55% of control, p < 0.01) and murine (32% of control, p < 0.01) allogeneic T cell proliferation. Further mechanistic studies revealed that Linc00402 is predominantly expressed in the cytoplasm following subcellular fractionation.These results indicate that Linc00402 in humans and its putative murine orthologue are differentially expressed by allogeneic T cells and regulate their function. In light of its tissue and allogeneic context specific expression, Linc00402 could be an attractive target for improving outcomes after allogeneic BMT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract LB-193: Protein phosphatase 2A inhibition,with a novel small molecule inhibitor, LB-100, achieves durable immune-mediated antitumor activity when combined with PD1 blockade in a preclinical model

Abstract LB-100 is a novel, first-in-class, small molecule inhibitor of protein phosphatase 2A (PP2A) recently shown in a Phase I trial to be well-tolerated at doses associated with stabilization of progressive solid tumors (Chung. Clin Cancer Res. 2017). PP2A has been implicated in mediating Akt signaling downstream of CTLA-4 (Parry. MolCell Biol. 2005). An in vivo pooled short hairpin RNA screen identified PP2A as a key regulator in suppressing T-cell proliferation in the tumor microenvironment (Zhou. Nature. 2014) and to be essential for regulatory-T-cell (Treg) function (Apostolidis. Nat Immunol. 2014). We hypothesized that pharmacologic inhibition of PP2A could enhance cancer immunity. We assessed the effect of LB-100 on T-cells in human allogenic mixed lymphocyte reactions, in which CD8+ or CD4+ T cells were co-cultured with monocyte-derived dendritic cells. We found a dose dependent increase in T cell proliferation in CD8+ and CD4+ cells and an increase in IFNγ secretion in CD4+ T cells. We investigated the effect of LB-100 plus anti-PD-1 antibody on CD4+ T cells in the same assay. The combination enhanced proliferation and IFNγ production compared to anti-PD-1 alone. For in vivo syngeneic studies, BALB/c mice were implanted subcutaneously in the right flank with CT26 colon carcinoma cells (5 x10^5 cells). 12 days after implantation, mice with tumors between 30-100 mm3 were randomized into four treatment groups (placebo, LB-100 - 0.16 mg/kg, anti-PD1- 10 mg/kg, and combination). Treatment was given every 2 days up to 28 days. 10 days after treatment, there was a significant decrease in tumor growth in the combination group compared to placebo or anti-PD-1 alone. 7/11 (63.6%) of mice treated with the combination and none in the other treatment groups achieved complete remission (CR). FACS analysis of the tumor infiltrating lymphocytes (TIL) after 10 days of treatment demonstrated enhanced IFNγ production in CD8+ T cells in the combination group compared to either treatment alone. There was also a marked decrease in FoxP3+ Treg cells in LB-100 treated group compared to placebo (3% vs 12% of CD4+ T cells, p&amp;lt;0.05). Furthermore, mice achieving CR were resistant to tumor growth when re-inoculated with CT26 cells. Mice subjected to CD8+ T cell ablation using depleting antibodies, were unable to reject CT26 tumors - 0/8 (0%) despite treatment with combination therapy. This indicates that the anti-tumor effect of LB100 with anti-PD-1 treatment is CD8+ T cell mediated. In conclusion, we have shown in a syngeneic animal model that the PP2A inhibitor, LB-100, has synergistic potential in conjunction with checkpoint blockade supporting investigation of its ability to enhance immunotherapy in the clinic. Citation Format: Winson S. Ho, Herui Wang, John S. Kovach, Rongze Lu, Zhengping Zhuang. Protein phosphatase 2A inhibition,with a novel small molecule inhibitor, LB-100, achieves durable immune-mediated antitumor activity when combined with PD1 blockade in a preclinical model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-193. doi:10.1158/1538-7445.AM2017-LB-193

Indoleamine 2,3-dioxygenase, by degrading L-tryptophan, enhances carnitine palmitoyltransferaseI activity and fatty acid oxidation, and exerts fatty acid-dependent effects in human alloreactive CD4+ T-cells.

Indoleamine 2,3-dioxygenase (IDO) is expressed in antigen-presenting cells and by degrading L-tryptophan along the kynurenine pathway suppresses CD4+ T-cell proliferation, induces apoptosis and promotes differentiation towards a regulatory as opposed to an effector phenotype. Recent findings revealed that the above effects may be mediated through alterations in T-cell metabolism. In this study, the effect of IDO on fatty acid β-oxidation in CD4+ T-cells was evaluated in human mixed lymphocyte reactions(MLRs) using the IDO inhibitor, 1-DL-methyl-tryptophan. Protein analysis of CD4+ T-cells isolated from the MLR showed that L-tryptophan degradation acts by activating the general control non‑derepressible2 kinase and aryl-hydrocarbon receptor in T-cells. In the absence of IDO inhibition, fatty acid oxidation increased along with increased activity of carnitine palmitoyltransferaseI(CPT1), the latter due to the increased expression of CPT1 isoenzymes and alterations in acetyl-CoA carboxylase2, the enzyme that controls CPT1 activity. Increased fatty acid oxidation due to the action of IDO was accompanied by an increased expression of forkhead boxP3 (FoxP3) and a decreased expression of related orphan receptorγt(RORγt), the signature transcription factors of regulatory T-cells and T helper 17cells, respectively. However, in MLR and in the presence of fatty acid in the culture medium, IDO did not inhibit proliferation. Additionally, fatty acid protected the CD4+ T-cells against apoptosis. Thus, IDO, by degrading L-tryptophan, enhances CPT1 activity and fatty acid oxidation, and exerts fatty acid-dependent effects in human alloreactive CD4+ T-cells.

Integrated Pharmacokinetic/Pharmacodynamic Analysis for Determining the Minimal Anticipated Biological Effect Level of a Novel Anti-CD28 Receptor Antagonist BMS-931699.

BMS-931699 (lulizumab pegol), a domain antibody (dAb) conjugated with 40-kDa branched polyethylene glycol, is a human anti-CD28 receptor antagonist under development for the treatment of inflammatory and autoimmune diseases. In the present work, the minimal anticipated biologic effect level (MABEL) was determined for BMS-931699 by integrating all the available preclinical data. The relevance of the in vitro mixed lymphocyte reaction (MLR) assay to a whole blood CD28 receptor occupancy (RO) assessment, as well as the relationship between the CD28 RO and the inhibition of T-cell-dependent antibody response to keyhole limpet hemocyanin in vivo, was demonstrated through an integrated pharmacokinetic/pharmacodynamic analysis using anti-hCD28 dAb-001 (differing from BMS-931699 by two additional amino acids at the N-terminus) and a mouse surrogate. Based on this analysis, the EC10 value (0.32 nM) from the human MLR assay and the human plasma volume (0.04 l/kg) were employed to calculate the MABEL (0.01 mg) of BMS-931699 in humans, with a CD28 RO predicted to be ≤10%. The estimated MABEL dose was threefold higher than the value derived from the binding constant and twofold less than the MABEL converted from animal efficacy studies based on the body surface area. Furthermore, it was 2900-fold lower than the human equivalent dose derived from the no observed adverse effect level in monkeys (15 mg/kg/week for 5 doses, intravenous dosing) with a 10-fold safety factor applied. Therefore, the MABEL dose represented a sound approach to mitigate any potential risk in targeting CD28 and was successfully used as the first-in-human starting dose for BMS-931699.

Effect of Cell Density and HLA-DR Incompatibility on T-Cell Proliferation and Forkhead Box P3 Expression in Human Mixed Lymphocyte Reaction

BackgroundThe proliferation rates of human T cells in vitro are affected by some factors such as initial T-cell number, dose of stimulating cells, and duration of culture. The transcription factor forkhead box P3 (FoxP3) has been used to identify regulatory T cells in humans and is thought to correlate with tolerance to allogeneic organ transplant. Thus, it is important to optimize conditions to expand FoxP3 cell proliferation to improve engraftment of allogeneic organ transplants. MethodsWe studied proliferative responses and FoxP3 expression in divided T cells with the use of flow cytometric analysis of Ki-67 in culture of different concentrations of responding cells (6 × 106, 4 × 106, 2 × 106, 1 × 106, and 0.5 × 106cells/mL), different types of stimulating cells (lymphocytes and low density cells), and different numbers of HLA mismatches. ResultsThe proportion of CD3(+) cells, CD4(+)CD25(+) cells, and CD4(+)CD25(+)FoxP3(+) cells among mononuclear cells were highest at initial cell concentration of 2 × 106 responder cells/mL with lymphocytes as stimulators at day-5 mixed lymphocyte reaction (MLR). They were highest at a concentration of 4 × 106 responder cells/mL with low density cells as stimulators. The recovery (%), proportion of CD3(+) cells, CD4(+)CD25(+) cells, and CD4(+)CD25(+)FoxP3(+) cells with 2 HLA-DR incompatibility were significantly higher than those of 1 HLA-DR incompatibility at day-5 MLR. ConclusionsInitial cell concentration and HLA-DR incompatibility can affect the generation of FoxP3+ T cells in human MLR. These factors could be considered for efficient generation of Tregs for clinical trials in the future.

6-Substituted 9-fluoroquino[3,2-b]benzo[1,4]thiazines display strong antiproliferative and antitumor properties.

6-Substituted 9-fluoroquino[3,2-b]benzo[1,4]thiazines - a new type of tetracyclic azaphenothiazines-were obtained from of 6H-9-fluoroquinobenzothiazine by the introduction of appropriate substituents to the thiazine nitrogen atom (alkyl, aminoalkyl, amidoalkyl, sulfonamidoalkyl and nitrogen half-mustard groups). The compounds displayed differential cytotoxic as well as antiproliferative actions against human peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin A (PHA). In addition, they suppressed lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-α) production by whole blood human cell cultures. Two compounds (4 and 15, with the propargyl and methanesulfonamidopropyl groups) were selected for further experiments because of lack of cytotoxicity and strong antiproliferative actions. Compound 4 showed strong suppressive actions on growth of L1210, SW948, A-431 and CX-1 tumor cell lines which were close to those of cisplatin, the reference drug (e.g. GI50 of 2.28 μg/mL vs. 1.86 μg/mL for L1210 cells). Further, the compound appeared to be equally effective as cyclosporine A (CsA) in the inhibition of human two-way mixed lymphocyte reaction (MLR). The compound did not significantly inhibit interleukin 2 (IL-2)-induced growth of CTLL-2 cell line. In addition, inhibition of prostaglandin (PG) synthesis by indomethacin or block of PG receptors did not interfere with the inhibitory effect of the compound on PHA-induced cell proliferation. Therefore, it is likely that the compound acts by inhibiting cell cycle as proposed for other phenothiazines. Further studies are required for the elucidation of the mechanism of action and therapeutic utility of these compounds in more advanced in vivo models.

Anti‐CD2 producing pig xenografts effect localized depletion of human T cells in a huSCID model

We investigated whether graft produced anti-human CD2, mediated by adenovirus (Adv) transduction of pig neonatal islet cell clusters (pNICC), would protect xenografts in a humanized mouse model from immune attack and whether such immunosuppression would remain local. A mouse anti-human CD2 Ab (CD2hb11) previously generated by us was genetically engineered to produce chimeric and humanized versions. The three forms of CD2hb11 were named dilimomab (mouse), diliximab (chimeric) and dilizumab (humanized). All 3 forms of CD2hb11 Ab were tested for their ability to bind CD3(+) human T cells and to inhibit a human anti-pig xenogeneic mixed lymphocyte reaction (MLR). They were administered systemically in a humanized mouse model in order to test their ability to deplete human CD3(+) T cells and whether they induced a cytokine storm. An adenoviral vector expressing diliximab was generated for transduction of pNICC. Humanized mice were transplanted with either control-transduced pNICC or diliximab-transduced pNICC and human T cells within grafts and spleens were enumerated by flow cytometry. Dilimomab and diliximab inhibited a human anti-pig xenogeneic response but dilizumab did not. All 3 forms of CD2hb11 Ab bound human T cells in vitro though dilimomab and diliximab exhibited 300-fold higher avidity than dilizumab. All 3 anti-CD2 Abs could deplete human CD3(+) T cells in vivo in a humanized mouse model without inducing upregulation of activation markers or significant release of cytokines. Humanized mice transplanted with diliximab-transduced pNICC afforded depletion of CD3(+) T cells at the graft site leaving the peripheral immune system intact. Local production of a single Ab against T cells can reduce graft infiltration at the xenograft site and may reduce the need for conventional, systemic immunosuppression.

Chemical Synthesis and Immunosuppressive Activity of Dipalmitoyl Phosphatidylinositol Hexamannoside

Phosphatidylinositol mannosides (PIMs) isolated from mycobacteria have been identified as an important class of phosphoglycolipids with significant immune-modulating properties. We present here the synthesis of dipalmitoyl phosphatidylinositol hexamannoside (PIM(6)) 1 and the first reported functional biology of a synthetic PIM(6). Key steps in the synthetic protocol included the selective glycosylation of an inositol 2,6-diol with a suitably protected mannosyl donor and construction of the glycan core utilizing a [3 + 4] thio-glycosylation strategy. The target 1 was purified by reverse phase chromatography and characterized by standard spectroscopic methods, HPLC, and chemical modification by deacylation to dPIM(6). The (1)H NMR spectrum of synthetic dPIM(6) obtained from 1 matched that of dPIM(6) obtained from nature. PIM(6) (1) exhibited dendritic cell-dependent suppression of CD8(+) T cell expansion in a human mixed lymphocyte reaction consistent with the well established immunosuppressive activity of whole mycobacteria.