Allogeneic Antigen-presenting Cells Research Articles

Assessment of Corneal Graft Outcomes in a Murine Model of Endothelial Keratoplasty.

Objectives: In this study, we establish a protocol for evaluating the outcomes of endothelial keratoplasty, including graft survival, rejection, or failure. Additionally, we also evaluate the alloimmune response in graft recipients. Methods: We performed EK using C57BL/6 (allogeneic) and BALB/c (syngeneic) as donors and BALB/c mice as recipients. Slit-lamp examination and optical coherence tomography were performed for clinical evaluations for 16 weeks post-procedure. Criteria for the assessment of corneal opacity were established and the animals were graded weekly. Additionally, we assessed corneal endothelial cell density by harvesting the corneas and staining with zonula occludens-1 (ZO-1). Lastly, lymph nodes were collected, and CD4+ T cells were MACS-sorted and co-cultured with syngeneic or allogeneic antigen-presenting cells (APCs) to assess the IFN-γ expression levels by alloreactive Th1 cells (ELISPOT) in response to the direct (donor) or indirect (host) pathways of sensitization. Results: We observed graft failure in four animals, including irreversible corneal opacity, graft detachment, and anterior synechiae in the first four weeks. The remaining animals were graded between 0 and 5 as per the established criteria. The total and graft corneal thickness and endothelial cell density progressively worsened with a higher grade of corneal opacity. The direct allosensitization of Th1 cells was significantly higher in mice with a higher grade of corneal opacity. At 16 weeks follow-up, the grafts remained stable with low opacity scores in syngeneic EK recipients; however, the opacity scores were higher and variable in allogeneic EK recipients. Conclusions: These findings establish a standardized protocol to assess the graft outcomes in a murine model of EK. Furthermore, we delineate the underlying immunological pathway that contributes to the immune-mediated rejection of grafts in this model.

Deletion of AMPK Reduces Oxidative Metabolism in Gvhd-Causing Human T Cells

BACKGROUND Allogeneic hematopoietic stem cell transplantation (AlloHSCT) is a curative therapy for many hematological disorders including primary immunodeficiencies and relapsed/refractory leukemia. However, the therapeutic application of AlloHSCT is limited by acute graft-versus-host-disease (GVHD), where donor T cells recognize and destroy recipient tissues in the skin, liver, and gastrointestinal tract. GVHD occurs following 30-50% of allogeneic transplants and is a leading cause of non-relapse morbidity and mortality. Recent efforts to treat GVHD have improved but attempts to prevent GVHD remain an important area of ongoing investigation. Following transplantation, alloreactive T cells exhibit heightened energy requirements that present a promising area for therapeutic intervention. Previously, we have shown that murine T cells upregulate activity of the cellular energy sensor AMPK-activated protein kinase (AMPK) during GVHD initiation and furthermore, that transfer of AMPK-deficient T cells increased recipient survival while preserving leukemia clearance and immune reconstitution. Whether these findings translate to human cells is unknown. Here, we report successful targeting of AMPK in primary human T cells and demonstrate clear similarities in phenotype between murine and human T cells lacking AMPK. METHODS To delete AMPK, primary human T cells were isolated from healthy donors and electroporated with Cas 9 ribonuclear protein complexes targeting the AMPKa1 locus. Deletion of AMPK was assessed via DNA sequencing (TIDE analysis) and phosphorylation of downstream target Unc-51-like kinase (ULK1) by immunoblot. Oxidative metabolism and spare respiratory capacity (SRC) were determined via extracellular flux analysis (Agilent Seahorse). Mitochondrial transmembrane potential (TMRM) and cell phenotype (mitochondrial density, apoptosis) were evaluated by flow cytometry after cells stimulation. Intracellular cytokine expression was assessed following 72 hours in a mixed lymphocyte reaction (MLR), followed by re-stimulation with PMA/Ionomycin in the presence of Brefeldin A. AMPK-deficient murine T cells from AMPKa1/a2 dKO C57BL/6J mice were transplanted into allogeneic B6D2F1 recipients for 7 days for comparative analysis. RESULTS CRISPR treatment of primary human T cells efficiently deleted AMPK as measured by both DNA sequencing (92.3% insertion/deletion frequency by TIDE analysis) and immunoblot analysis of downstream ULK1 phosphorylation. When re-stimulated in physiological glucose (5.5 mM), AMPK-deficient human T cells significantly decreased mitochondrial respiration (Fig. 1A) as measured by both maximal oxygen consumption (174.6 ± 27.73 vs 277.3 ± 22.88, p<0.0001) and spare respiratory capacity (SRC, 126.9 ± 22.78 vs 207.1 ± 19.68, p<0.0001). This result was strikingly similar to the metabolism of post-transplant murine T cells lacking AMPK, which exhibited a proportional decrease in SRC (-6.499 ± 5.156 vs 29.11 ± 7.064, p<0.0001) and maximal oxygen consumption (46.82 ± 10.11 vs 80.01 ± 6.946, p<0.0001). AMPK-deficient human T cells also experienced decreased cellular viability (43.4% vs 51.8%) as well as limited mitochondrial membrane potential (Fig. 1B); however, no differences in mitochondrial density were observed. Upon co-culture with allogeneic antigen presenting cells, AMPK-deficient human CD4+ T cells produced lower levels of the pro-inflammatory cytokines TNFa (97.7 MFI vs 209 MFI) and IL-2 ( 391 vs 454 MFI). DISCUSSION Here, we demonstrate a method to effectively delete AMPK in human T cells, a modification which decreases oxidative metabolism identical to what is observed in AMPK-deficient murine T cells. Specifically, human T cells lacking AMPK decreased their maximal mitochondrial respiration and spare respiratory capacity, a result we attribute to decreased mitochondrial transmembrane potential without changes in mitochondrial mass. Together with decreased pro-inflammatory cytokine production, we predict these changes will limit the viability and function of alloreactive T cells in vivo. As such, future studies will assess the disease potential of AMPK-deficient human T cells in a modified model of xenogeneic GVHD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Allogeneic Donor T Cells Directly Attack Renal Endothelial and Epithelial Cells in Mice

Acute graft-versus-host disease (aGVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT). The skin, liver, and gastrointestinal (GI) tract are widely considered the main target organs of aGVHD, whereas the kidney is not widely recognized as a target organ. Clinical and preclinical studies suggest that the kidney may be targeted by allogeneic donor T cells, and acute kidney injury (AKI) is a relatively frequent complication of allo-HCT. However, renal biopsy is rarely performed in the early phase of allo-HCT, making it difficult to identify the cause of AKI. Here, we tested the hypothesis that the kidney is a target for aGVHD by determining whether allogeneic donor T cells directly attack renal endothelial or epithelial cells in murine models of aGVHD. To avoid potential confounding nephrotoxic effects, our models did not use calcineurin inhibitor-based aGVHD prophylaxis. We first tested our hypothesis using an MHC-mismatched model of aGVHD. Briefly, BALB/crecipients were lethally irradiated and transplanted on day 0 with 5x106 bone marrow (BM) and 0.5x106 CD90.2+ splenic T cells from either syngeneic BALB/c or allogeneic MHC-mismatched C57BL/6 (B6) donors. On day 14 after HCT, urinary protein and N-acetyl-β-D-glucosaminidase (NAG), a marker of tubular injury, were elevated in allogeneic recipients. Similar results were obtained using an MHC-haploidentical B6 into B6D2F1 model and an MHC-matched multiple minor antigen mismatched B6 into C3H.sw model. Renal histopathology in an MHC-mismatched model (B6 into BALB/c) showed peritubular vasculitis as well as donor-derived H-2Kb positive CD3+ T cells in the glomerulus, peritubular capillaries, interstitium, and perivascular areas of allogeneic recipients. These data suggested that donor T cells directly infiltrated the kidneys. Next, we examined the characteristics of the T cells infiltrating the kidney using flow cytometry. Both donor-derived CD4+ and CD8+ T cells were significantly more abundant in the allogeneic group. These increased T cells demonstrated significantly elevated surface markers of activation (CD69) and exhaustion (PD-1 and Tim-3) in allogeneic animals. In addition, secretion of proinflammatory cytokines (TNF-α and IFN-γ) and cytotoxic proteins (granzyme B) was increased by renal infiltrating donor T cells in allogeneic recipients. To determine whether allogenic T cells directly damaged kidneys, the expression of neutrophil gelatinase-associated lipocalin (NGAL), a marker of acute kidney injury, and Elafin, a marker of cutaneous GVHD, were measured. Both were increased in the kidneys of allogeneic animals suggesting donor T cells directly damage kidneys. To confirm whether allogeneic donor T cells directly attack renal cells, we performed an in vitro cytotoxic T lymphocyte (CTL) killing assay using alloreactive T cells as effector cells and renal cells as targets. BALB/c or B6-derived T cells were stimulated with lethally irradiated syngeneic or allogeneic antigen-presenting cells. These activated T cells were then co-cultured with BALB/c-derived renal endothelial or proximal tubular epithelial cells. Apoptotic (TUNEL-positive) cells were increased in both renal endothelial and proximal tubular epithelial cells cultured with alloreactive T cells. These studies demonstrated that donor T cells directly attack renal endothelial and tubular epithelial cells and mediate kidney-specific aGVHD after allo-HCT. Thus, our data supports the growing body of literature suggesting that the kidney is also an aGVHD target organ. Elucidating kidney-intrinsic mechanisms of aGVHD may lead to improved strategies for reducing renal dysfunction following allo-HCT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Absence of NLRP6 in Donor T Cells Exacerbates Gvhd

The Nlrp6 (NOD-like receptor family pyrin domain containing 6) inflammasome is important for intestinal epithelial cell innate immune responses and for maintaining gut homeostasis by preventing microbial dysbiosis. Contrary to its role in epithelial cell inflammasome-mediated responses, we recently showed that Nlrp6 in gut epithelial cells exacerbates GVHD in a manner independent of the inflammasome or gut microbiota. However, donor allogeneic T cells are also critical for GVHD development, yet, the function of Nlrp6 in allogeneic T cells is unknown. We hypothesized that Nlrp6 deficient donor T cells would ameliorate experimental GVHD.To test our hypothesis, WT-BALB/crecipients were lethally irradiated and transplanted on day 0 with 5x10 6 bone marrow and 1.0x10 6 splenic CD90 +T cells from either syngeneic WT-BALB/c, allogeneic MHC-mismatched WT-B6 or Nlrp6 -/- donors. Contrary to our hypothesis, the survival of allogeneic recipients of Nlrp6 -/- donor T cells was significantly worse than those receiving WT-B6 T cells (p<0.05). Nlrp6 -/- donor T cells also caused greater GVHD mortality and morbidity in an MHC mismatched haploidentical B6 into B6D2F1 model (p<0.05) and an MHC mismatched B10.BR into B6 model. Similar results were obtained using B6 into BALB/c and B6 into B6D2F1 models performed at the University of Michigan, suggesting our results were not unique to local environmental factors. By contrast, GVHD severity and mortality were similar in an MHC matched multiple minor antigen mismatched B6 into C3H.sw model. Because the B6 into C3H.sw model is largely driven by CD8+ T cells whereas the previous models are mediated by both CD4+ and CD8+ T cells, we examined whether Nlrp6 separately regulates CD4+ and CD8+ T cell-mediated GVHD. In order to test this, we transplanted C3H.sw recipients as above except we infused either 1x10 6 CD4+ or CD8+ T cells from B6-WT or Nlrp6 -/- animals. GVHD severity and mortality (P<0.05) were enhanced only when Nlrp6 -/- CD4+ T cells were transplanted. These data suggested that Nlrp6 regulates allogeneic T cell responses in a subset-specific manner.To explore how Nlrp6 regulates intrinsic responses in donor T cell subsets, we tested naïve T cell proliferation in vitro after allogeneic or non-specific TCR stimulation. Consistent with the lack of increased GVHD induced by CD8+ Nlrp6 -/- donor T cells in the B6 into C3H.sw model, Nlrp6 -/- CD4+ but not CD8+ T cells proliferated more than WT-B6 CD4+ or CD8+ T cells, respectively, when stimulated with either anti-CD3/CD28 antibodies or lethally irradiated allogeneic antigen presenting cells in a mixed lymphocyte reaction. In addition, activation-induced apoptosis was decreased in Nlrp6 -/- CD4+ T cells compared to WT T cells. Importantly, Treg suppressive function was not altered in Nlrp6 -/- T cells. Therefore, increased proliferative responses and resistance to activation-induced apoptosis may have contributed to the enhanced GVHD caused by Nlrp6 -/- donor T cells.Increased Th1 and Th17 polarization is associated with worse GVHD. Because only CD4+ Nlrp6 -/- T cells enhanced GVHD, we tested whether Nlrp6 influenced T helper cell differentiation into Th1, Th17, and Th2 subsets. Consistent with our in vivo data, Th1 in vitro differentiation was enhanced in Nlrp6 -/- CD4+ T cells. To determine the molecular signaling events altered by Nlrp6 deficiency, we tested various T cell activation signaling pathways and found that phosphorylation of ZAP-70 was increased in Nlrp6 -/- T cells. These data suggested that Nlrp6 in donor T cells may regulate allo-immune responses via ZAP-70 pathway.GVH and graft-versus-tumor (GVT) responses are intricately linked. Because CD8+ responses were not affected by Nlrp6 deficiency, we hypothesized that GVT responses would be unaltered in Nlrp6 -/- donor T cells. Indeed, Nlrp6 -/- T cells showed equivalent in vivo GVL responses to MLL-AF4 leukemia cells as WT-T cells. Hence Nlrp6in donor T cells is not required for GVT responses.Altogether our data suggested that Nlrp6 negatively-regulates allogeneic donor CD4+ T cell responses, possibly via negative regulation of ZAP-70 signaling, resulting in mitigation of GVHD and maintenance of robust GVT responses. DisclosuresIshizawa: AbbVie: Research Funding; Eisai: Honoraria; Chugai: Honoraria; Ono: Honoraria; Celgene: Honoraria; Takeda: Honoraria; Bayer: Research Funding; Bristol Myers Squibb: Speakers Bureau; Pfizer: Research Funding; Kyowa Kirin: Consultancy; SymBio: Honoraria, Research Funding; Otsuka: Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau; Sanofi: Research Funding; IQVIA: Research Funding.

NKG2D Negatively Regulates CD4 T Cells in Allo-Response in Mice

NKG2D is a C-type lectin-like receptor which mainly expresses on mouse NK, activated CD8 and some γδ T cells. NKG2D is also found to express on human and mouse CD4 T cells, but the role of NKG2D in CD4 T cells is essentially unknown. We asked what subsets of CD4 T cells could express NKG2D; we activated and polarized CD4 T cells into Th0, Th1,Th2, Th17 and Foxp3+ Tregs in vitro and measured NKG2D expression on those subsets. We found that only a proportion of Th1 cells expressed NKG2D, whereas little or no NKG2D was expressed on other subsets of T cells. In a mixed lymphocyte reaction (MLR) assay, NKG2D was induced on a subset of CD4 T cells after stimulation with allogeneic antigen presenting cells (APCs) (Figure 1A). NKG2D+CD4+ T cells expressed higher levels of LAG-3, CD49b, CD80, FasL and PD-L1 (Figure 1B) and secreted more IL-10 and IFN-γ than NKG2D- CD4+ T cells (Figure 1C).Previous reports by others showed that NKG2D on NK and activated CD8 T cells contributing to graft versus host disease (GVHD) pathogenesis have been well-established, but the expression and function of NKG2D on CD4 T cells in GVHD development are unknown.To address these questions, we utilized a CD4-driven, MHC-mismatched aGVHD model of allogeneic bone marrow transplantation (allo-BMT), C57BL/6 to BALB/c. We monitored the development of NKG2D+CD4+ T cells in allogeneic recipients. We observed that NKG2D was continuously induced on CD4 T cells during the whole aGVHD process - gradually increased, peaked on day 14 and then slowly decreased after allo-BMT. By comparing the severity of aGVHD in recipients transferred with WT versus NKG2D-/- CD4 T cells. We observed that recipients transferred with NKG2D-/- CD4 T cells showed more severe GVHD demonstrated by a higher lethality (Figure 1D) and more severe diarrhea compared to WT counterpart. Mechanistically, NKG2D-/- CD4 T cells had a higher capacity of migrating to gut and liver than WT CD4 T cells reflected by the higher expression of α4β7 and CXCR3. Furthermore, NKG2D+CD4+ T cells secreted less proinflammatory cytokine IL-17 and more anti-inflammatory cytokine IL-10. Strikingly, NKG2D expression coincided with T-bet, but excluded RORγt or Foxp3 expression of CD4 T cells. Given NKG2D+CD4+ T cells expressed regulatory markers and secreted IL-10 and IFN-γ; we hypothesized that NKG2D+CD4+ T cells may serve as suppressive cells (e.g. Tr1 cells). To test this hypothesis, we sorted NKG2D+CD4+ and NKG2D-CD4+ T cells from recipients of allogeneic BMT, and performed in vitro suppressive assay. We observed that NKG2D+CD4+ T cells had a higher suppressive activity than NKG2D-CD4+ T cells. (Figure 1E)Taken together, the current work demonstrates that NKG2D may serve as a negative regulator on CD4 T cells, and NKG2D+CD4+ subset can function as a regulatory cell subset in the development of GVHD after allo-BMT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

In vitro and In vivo CD8+ T Cell Suppression Assays.

CD8+CD28- T suppressor cells (Ts) have been documented to promote immune tolerance by suppressing effector T cell responses to alloantigens following transplantation. The suppressive function of T cells has been defined as the inhibitory effect of Ts on the proliferation rate of effector T cells. 3H-thymidine is a classical immunological technique for assaying T cell proliferation but this approach has drawbacks such as the inconvenience of working with radioactive materials. Labeling T cells with CFSE allows relatively easy tracking of generations of proliferated cells. In this report, we utilized antigen presenting cells (APCs) and T cells matched for human leukocyte antigen (HLA) class I or class II to study CD8+CD28- T cell suppression generated in vitro by this novel approach of combining allogeneic APCs and γc cytokines. The expanded CD8+CD28- T cells were isolated (purity 95%) and evaluated for their suppressive capacity in mixed lymphocyte reactions using CD4+ T cells as responders. Here, we present our adapted protocol for assaying the Ts allospecific suppression of CFSE-labeled responder T cells.

Human CD8+CD28\u2212 T suppressor cells expanded by common gamma chain (\u03b3c) cytokines retain steady allospecific suppressive capacity in vivo

BackgroundCD8+CD28− T suppressor (Ts) cells play critical role in transplant tolerance. Our previous study has generated CD8+CD28− Ts cells in vitro which exert robust allospecific suppressive capacity in vitro.ResultsCD8+CD28− Ts cells were expanded by stimulating human CD8+ T cells with allogeneic antigen presenting cells in the presence of the common gamma chain cytokines IL-2, IL-7 and IL-15 in vitro, and were further verified in vitro through day 7 to 11 for their persistency of the allospecific suppressive capacity. When CD8+CD28− Ts cells were adoptively transferred into NOG mice, their capacity to inhibit CD4+ T cell proliferation in allospecific manner remained potent on 11 days after their injection. The mechanisms for expansion of CD8+CD28− Ts cells by the common gamma chain cytokines were investigated. These included promoting CD8+CD28− T cells proliferation, converting CD8+CD28+ T cells to CD8+CD28− T cells and decreasing CD8+CD28− T cell death. Furthermore, the expanded CD8+CD28− Ts cells showed upregulation of the co-inhibitory molecule Tim-3 and down-regulation of the cytotoxic molecule granzyme B.ConclusionsIn summary, these results demonstrated that the in vitro-expanded human CD8+CD28− T cells retained potent allospecific suppressive capacity in vivo and depicted multiple mechanisms for the expansion of Ts cells, which might promote further bench to clinic research.

Abstract B062: Enabling the rapid generation of allogeneic artificial antigen presenting cell (aAPC) Red Cell Therapeutics with a loadable MHC system

Abstract Next generation sequencing technologies, coupled with personal neoantigen identification approaches, have significantly improved the capability to develop patient-specific T-cell-based therapies targeting tumors. Current peptide neoantigen vaccine approaches are promising, but do not adequately stimulate and expand patient T cells to the levels required to achieve robust efficacy. To address these limitations, Rubius Therapeutics has developed allogeneic artificial antigen presenting cells (aAPCs), which express the required signals for complete T cell activation: antigenic peptide-MHC complex, costimulatory ligand and cytokine. By engineering red cells to express immunomodulatory signals 1, 2 and 3, these aAPCs dramatically expand antigen-specific T cells in vivo and promote T cell memory and effector function. To use the aAPC approach with personal neoantigens, Rubius Therapeutics has developed a loadable MHC system that enables the rapid generation of aAPCs. Unloaded MHC class I molecules typically do not express robustly on the cell surface, as innate biophysical instability and internal cellular quality control mechanisms prevent MHC molecules that lack a loaded peptide from displaying on the cell surface. We demonstrate here that the empty MHC class I complex can be stably presented on the red cell surface. This was achieved by fusing wild-type MHC class I [human] HLA-A2 and β2 microglobulin to the glycophorin A transmembrane domain. Of note, disulfide engineering of HLA-A2 did not change expression when compared to wild-type constructs on the red cells. Fluorescently labelled peptides were used to measure the kinetics of peptide loading, which demonstrated that disulfide-engineering dramatically increased binding rates. Further, peptide competition experiments indicated slightly increased affinity for peptide with disulfide engineered HLA-A2. Functional testing revealed that addition of exogenous HPV E7 peptide and co-incubation of loaded cells with Jurkat-Lucia NFAT cells expressing HPV E7-specific TCR cells demonstrated TCR-specific activity. Additional TCR activity assays showed that peptide loaded onto empty wild-type HLA-A2 was stable up to 3 days, while disulfide-engineered HLA-A2 activity was abrogated after 1 day. Finally, this concept was extended to other HLA genes, demonstrating that our approach could achieve expression of a variety of HLA alleles on the red cell surface, including MHC class II alleles in the HLA-DR and HLA-DP gene families. Collectively, these results demonstrate that Rubius’ loadable aAPC system is highly generalizable and can be applied to produce aAPC populations presenting multiple antigenic peptides across a range of functional alleles on the red cell surface. Rubius’ allogeneic aAPC system represents a novel approach to generate effective personal neoantigen-specific therapies in a wide patient population with requisite ease of manufacturability Citation Format: Christopher L Moore, Sneha Pawar, Mellissa Nixon, Timothy J Lyford, Douglas C McLaughlin, Shamael R Dastagir, Abigail Bracha, Lori Melancon, Christopher L Carpenter, Thomas J Wickham, Tiffany F Chen. Enabling the rapid generation of allogeneic artificial antigen presenting cell (aAPC) Red Cell Therapeutics with a loadable MHC system [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B062. doi:10.1158/1535-7163.TARG-19-B062

Abstract 3260: Engineered red-cell therapeutics (RCT) as artificial antigen presenting cells promotein vivoexpansion and anti-tumor activity of antigen specific T cells

Abstract T cell-based therapies have demonstrated efficacy in a small subset of cancers; however, they have the potential to proliferate uncontrollably and manufacturing these therapies at scale has proven difficult. To address this limitation, Rubius Therapeutics has genetically engineered red cells to create allogeneic artificial antigen presenting cells (RCT-aAPCs) that express MHC class I loaded with a tumor specific antigen, together with costimulatory molecules that recapitulate normal APC-T cell interactions. These RCT-aAPC cells are designed to expand and activate tumor-specific T cells already present within the patient, thus eliminating the need to individually manufacture patient-derived T cells. As a proof of principle, red cells were engineered to express mouse MHC class I H-2Kb loaded with OVA 257-264 peptide and murine 4-1BBL. These cells induced in vitroT cell proliferation of OVA antigen-specific OT1 cells, whereas red cells expressing only MHC I or 4-1BBL did not induce proliferation. The RCT-aAPC expanded OT1 cells demonstrated an activated phenotype with increased CD44 expression, secretion of both IFNγ and IL2, as well as antigen-specific tumor killing of EG7.OVA tumor cells. To test in vivo efficacy, a mouse surrogate RCT-aAPC was created using murine red blood cells chemically conjugated with H-2Kb OVA and the m4-1BBL molecule. CellTrace Violet (CTV)-labeled OT1 cells were adoptively transferred into B6 Cd45.1 mice followed by intravenous dosing of the RCT-aAPC several hours later. Significant OT1 proliferation was observed 3-4 days post-dosing as measured by CTV dilution. Administration of a second RCT-aAPC dose at this time drove &amp;gt;200-fold expansion of OT1 cells with a memory-like phenotype in the peripheral blood and secondary lymphoid organs. Using a similar dosing strategy, administration of RCT-aAPC to mice bearing EG7.OVA tumors caused 60% tumor growth inhibition by Day 7 after dosing, which corresponded with the increased expansion of the OT1s. Treatment with RCT-aAPC significantly prolonged survival compared to the control group (p-val = 0.0024). After interacting with RCT-aAPC, antigen-specific T cells, traffic to the lymph nodes and tumor as demonstrated by OT1 presence at these sites. Based on the proof of concept using a murine system, human RCT-aAPCs expressing [human] 4-1BBL and [human] HLA-A2 loaded with an HPV E7 peptide were developed to expand and activate HPV E7-specific T cells. These RCT-aAPC cells activated TCR signaling in primary HPV E7-specific T cells as measured by upregulation of Nur77 expression and in engineered HPV E7-specific TCR Jurkat lines, measured using an NFAT luciferase reporter assay. Further validation of RCT-aAPC is ongoing and will be the focus for future clinical development in patients with HPV-positive cancers. Citation Format: Xuqing Zhang, Shamael R. Dastagir, Naren Subbiah, Mengyao Luo, Vikram Soman, Sneha Pawar, Douglas C. McLaughlin, Nicholas Bayhi, Viral Amin, Torben Straight Nissen, Christopher L. Carpenter, Thomas J. Wickham, Tiffany F. Chen. Engineered red-cell therapeutics (RCT) as artificial antigen presenting cells promotein vivoexpansion and anti-tumor activity of antigen specific T cells [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 3260.

Theoretical modeling reveals that regulatory T cells increase T-cell interaction with antigen-presenting cells for stable immune tolerance

The immune system in tolerance maintains cell diversity without responding to self-antigens. Foxp3-expressing CD25+CD4+ regulatory T cells (Tregs) inhibit T-cell activation through various molecular mechanisms. However, several key questions are still not resolved, including how Tregs control the immune response on the basis of their self-skewed T-cell receptor repertoire and how Tregs avoid impeding relevant immunity against pathogens. Here, we show that Tregs promote the proliferation of conventional T cells in the presence of excessive co-stimulation when murine T cells are stimulated in vitro with allogeneic antigen-presenting cells (APCs). Antigen-specific Tregs increase the number of cells interacting with dendritic cells (DCs) by increasing the number of viable DCs and the expression of adhesion molecules on DCs. Theoretical simulations and mathematical models representing the dynamics of T-APC interaction and T-cell numbers in a lymph node indicate that Tregs reduce the dissociation probability of T cells from APCs and increase the new association. These functions contribute to tolerance by enhancing the interaction of low-affinity T cells with APCs. Supporting the theoretical analyses, we found that reducing the T-cell numbers in mice increases the ratio of specific T cells among CD4+ T cells after immunization and effectively induces autoimmune diabetes in non obese diabetes mice. Thus, as a critical function, antigen-specific Tregs stabilize the immune state, irrespective of it being tolerant or responsive, by augmenting T-APC interaction. We propose a novel regulation model in which stable tolerance with large heterogeneous populations proceeds to a specific immune response through a transient state with few populations.

Modeling Human Immunity In Vitro: Improving Artificial Lymph Node Physiology by Stromal Cells

There has recently been an increasing demand for modeling the human immune system in vitro and the use of such models for testing immune responses against pharmaceutical drugs, cosmetics, and chemicals. The human artificial lymph node (HuALN) model has been developed for testing biopharmaceuticals and vaccines assessing immunomodulation, immunogenicity, and immunotoxicity. The system uses peripheral blood mononuclear cells (PBMCs) of leucapheresis material or whole blood donations of healthy volunteers for long-term three-dimensional (3D) culture in a perfused bioreactor system. A part of the current development is the implementation of lymph node (LN) stromal-like cells differentiated/obtained from mesenchymal stromal cells (MSCs) to improve structural organization and functionality. Development, structural organization, and function of lymphoid tissues and organ system are dependent on the interaction between hematopoietic and nonhematopoietic cells. In this study, we show the identification of a suitable differentiation protocol for LN stromal-like cells and their use for cocultivation in the HuALN model to improve the formation of microorganoids in long-term culture. During cocultivation with allogeneic PBMCs and antigen-presenting dendritic cells, MSC-derived stromal cells form a 3D network after seeding into the hydrogel matrix. The MSC network attracts PBMCs and influences the drug-induced immune responses, which are monitored by cytokine analysis in culture supernatants.

Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor.

Adoptive immunotherapy with regulatory T cells (Tregs) is a promising treatment for allograft rejection and graft-versus-host disease (GVHD). Emerging data indicate that, compared with polyclonal Tregs, disease-relevant antigen-specific Tregs may have numerous advantages, such as a need for fewer cells and reduced risk of nonspecific immune suppression. Current methods to generate alloantigen-specific Tregs rely on expansion with allogeneic antigen-presenting cells, which requires access to donor and recipient cells and multiple MHC mismatches. The successful use of chimeric antigen receptors (CARs) for the generation of antigen-specific effector T cells suggests that a similar approach could be used to generate alloantigen-specific Tregs. Here, we have described the creation of an HLA-A2-specific CAR (A2-CAR) and its application in the generation of alloantigen-specific human Tregs. In vitro, A2-CAR-expressing Tregs maintained their expected phenotype and suppressive function before, during, and after A2-CAR-mediated stimulation. In mouse models, human A2-CAR-expressing Tregs were superior to Tregs expressing an irrelevant CAR at preventing xenogeneic GVHD caused by HLA-A2+ T cells. Together, our results demonstrate that use of CAR technology to generate potent, functional, and stable alloantigen-specific human Tregs markedly enhances their therapeutic potential in transplantation and sets the stage for using this approach for making antigen-specific Tregs for therapy of multiple diseases.

A Novel PD-L1-targeting Antagonistic DNA Aptamer With Antitumor Effects.

The PD-1/PD-L1 axis is a major pathway involved in tumor immune evasion. Here, we report the novel PD-L1 antagonizing DNA aptamer (aptPD-L1) and demonstrate an integrated pipeline that expedites therapeutic aptamer development. Aptamer can exert antibody-mimic functions and is advantageous over antibody for its chemically synthetic nature, low immunogenicity, and efficient tissue penetration. Our results showed that aptPD-L1 blocked the binding between human PD-1 and PD-L1. Experiments using murine models showed that aptPD-L1 promoted in vitro lymphocyte proliferation and suppressed in vivo tumor growth without the induction of observable liver or renal toxicity. Analyses on the aptPD-L1-treated tumors further revealed elevated levels of infiltrating CD4+ and CD8+ T cells, intratumoral IL-2, TNF-α, interferon (IFN)-γ and the C-X-C motif chemokines, CXCL9 and CXCL10. The CD8+ T cells in the aptPD-L1-treated tumors had higher CXCR3 expression level compared to the random-sequence oligonucleotides-treated ones. Besides, the length and density of CD31+ intratumoral microvessels were significantly decreased in the aptPD-L1 treatment group. Collectively, our data suggested that aptPD-L1 helps T cell function restoration and modifies tumor microenvironment. These chemokines might orchestrate together to attract more T cells into the tumor tissues to form the positive amplification loop against tumor growth, indicating the translational potential of aptPD-L1 in cancer immunotherapy.

Immunological benefits by ginseng through reciprocal regulation of Th17 and Treg cells during cyclosporine-induced immunosuppression

BackgroundIt is not clear whether ginseng affects cyclosporine A (CsA)-induced desirable immunosuppressive action. In this study, we evaluated the immunological influence of combined treatment of ginseng with CsA. MethodsUsing CD4+ T cells from mouse spleens stimulated with the T cell receptor (TCR) or allogeneic antigen-presenting cells (APCs), we examined the differentiation of naïve T cells into T helper 1 (Th1), Th2, Th17, and regulatory T cells (Tregs), and their cytokine production during treatment by Korean Red Ginseng extract (KRGE) and/or CsA. The influence of KRGE on the allogeneic T cell response was evaluated by mixed lymphocyte reaction (MLR). We also evaluated whether signal transducer and activator of transcription 3 (STAT3) and STAT5 are implicated in this regulation. ResultsUnder TCR stimulation, KRGE treatment did not affect the population of CD4+interferon gamma (IFNγ)+ and CD4+interleukin (IL)-4+ cells and their cytokine production compared with CsA alone. Under the Th17-polarizing condition, KRGE significantly reduced the number of CD4+IL-17+ cells and CD4+/phosphorylated STAT3 (p-STAT3)+ cells, but increased the number of CD4+CD25+forkhead box P3 (Foxp3)+ cells and CD4+/p-STAT5+ cells compared with CsA alone. In allogeneic APCs-stimulated CD4+ T cells, KRGE significantly decreased total allogeneic T cell proliferation. Consistent with the effects of TCR stimulation, KRGE reduced the number of CD4+IL-17+ cells and increased the number of CD4+CD25+Foxp3+ cells under the Th17-polarizing condition. ConclusionKRGE has immunological benefits through the reciprocal regulation of Th17 and Treg cells during CsA-induced immunosuppression.

Promiscuous T-cell responses to drugs and drug-haptens

Promiscuous T-cell responses to drugs and drug-haptens

Specificity of JAK-Kinase Inhibition Determines Impact on T-Cell Function

Inhibitors of JAK2-kinase (Ruxolitinib, Momelotinib) are already approved or currently investigated in advanced clinical trials for treatment of myeloproliferative neoplasia (MPN). Besides their effect on mutated JAK2-kinase these compounds inhibit wildtype JAK and thereby impair JAK-STAT-signaling, which is an important pathway for proliferation and activation of other cell types such as human T-cells. Accumulating evidence suggests that they may also exert substantial immunosuppressive activity. Very recent reports highlighting hepatitis B reactivation complemented the series of severe infections in ruxolitinib-treated patients among which cryptococcus neoformans pneumonia, toxoplasmosis retinitis, disseminated tuberculosis, and progressive multifocal leukencephalopathy are the most alarming.We hypothesized that JAK-kinase inhibitors may act as immunosuppressant drugs by impairment of T-cell responses through inhibition of T-cell signaling (JAK-STAT pathway) and that specificity of JAK-kinase inhibition may be of major importance for the degree of T-cell inhibition. Therefore we investigated the effects of pharmacological JAK-kinase inhibition on healthy donor (HD-) and MPN patient T-cells. Selective inhibitors of JAK2-kinase (BSK805) and JAK3-kinase (BQM245) as well as clinically relevant inhibitors of JAK1/2-kinases (Ruxolitinib and Momelotinib) were used for pharmacologic inhibition. The SRC-kinase inhibitor Dasatinib served as a positive control for T-cell inhibition. Knockdown of specific JAK-kinases by RNAi was used to control for target specificity.In regard to T-cell receptor (TCR)-mediated signaling we investigated bona fide signaling molecules downstream of the TCR by Western Blotting. Besides SRC-kinases like LCK also ZAP70, PLCG1 and the MAPK/ERK pathway have been described to play a pivotal role in T-cell activation. In our data set, selectivity of JAK-kinase inhibition (JAK2, JAK3 or JAK1/2) influenced TCR-signaling in regard to overall tyrosine phosphorylation but also in regard to downstream effectors such as ERK.As activation and proliferation of primary T-cells is a critical step in immune responses against viral and tumor antigens we aimed to investigate the influence of JAK-kinase inhibition on activation and proliferation of human T-cells. T-cells from healthy donors were stimulated using either PHA 0.5% or CD3/CD28 beads to ensure a more T-cell receptor specific stimulation. CD69 expression was used as a marker for T-cell activation and CFSE staining was applied to assess for T-cell proliferation. Using CD3/CD28 stimulation, CD69 expression was almost abrogated following Dasatinib treatment and proliferation was significantly reduced. Applying relevant doses of specific JAK2 and JAK3 inhibitors to isolated T-cells did neither influence CD69 expression nor T-cell proliferation. These findings are confirmed by RNAi. In contrast, clinically relevant doses of JAK1/2 inhibitors Ruxolitinib and Momelotinib, respectively reduced CD69 expression and T-cell proliferation. Likewise, T-cells derived from MPN patients treated with Ruxolitinib revealed decreased CD69 expression and decreased proliferative capacity upon stimulation, compared to untreated patients or HD-controls.In order to investigate T-cell function, we assessed for allo-reactivity in a mixed lymphocyte culture. Human pan-T-cells were co-cultured with allogeneic antigen presenting cells. T-cell reactivity – as measured by 3H-thymidine incorporation – was significantly impaired by Ruxolitinib and Momelotinib. Specific inhibition of JAK2 or JAK3 kinase, however, did not affect T-cell reactivity. These effects could be confirmed using T-cells derived from Ruxolitinib-treated MPN patients. Investigation of leukemia- and virus-antigen-specific T-cell responses are currently under way to gain deeper insight regarding this clinically relevant scenario.Taken together, specificity of JAK-kinase inhibition influences the inhibitory potential on T-cell function. JAK1 kinase seems to play an important role in T-cell activation, as unspecific inhibitors of JAK1 & JAK2 Kinase inhibit T-cell function while selective inactivation of JAK2 kinase leaves T-cell function almost unaffected. Heterogeneity in T-cell function of Ruxolitinib-treated patients is an important finding that deserves detailed investigation. DisclosuresHeidel:Novartis: Consultancy.

Targeting VLA-4 to Reduce GvHD

▪The therapeutic benefits of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for hematologic malignancies are primarily derived from anti-leukemia effect that is mediated by T cells in donor grafts. Unfortunately, these T cells also mediate graft-versus-host disease (GvHD), the major complication of allo-HSCT.We have recently reported that blockade of alloreactive donor T cell trafficking to the GvHD target organs significantly reduces GvHD in both a MHC fully-mismatched (B6 (H-2b) → Balb/c (H-2d)) (75% vs 0% overall survival) and a minor-mismatched (B6 (H-2b) → B6x129 (H-2b)) allo-HSCT models (100% vs 10% overall survival) compared to WT T cells (Choi et al., Blood 2012). In this study, we examined if inhibition of VLA-4, which is required for transendothelial migration and access to GvHD target organs, could reduce GVHD. VLA-4 consists of two subunits α4 (CD49d) and β1 (CD29). To genetically eliminate VLA-4 from allogeneic donor T cells, we generated Tie-2 cre+ α4-/- mice (B6, H-2b, CD45.2+) in which α4 is knocked out by Tie-2 cre, which is expressed in hematopoietic cells. Splenic pan T cells were isolated from these mice and T cell-depleted bone marrow cells (TCD BM) from congenic B6 mice (CD45.1+). 5x106 TCD BM and 5x105 splenic pan T cells were transplanted into lethally irradiated allogeneic Balb/c recipient mice (H-2d, CD45.2+). We found that VLA-4 deficient T cells significantly reduced GvHD compared to WT T cells (Fig. A). VLA-4 deficient T cells had no significant effect on donor engraftment and achieved a complete donor chimerism. In addition, recipients transplanted with VLA-4 deficient T cells had significantly better histopathology score (Fig. B). Similar results were observed when donor T cells were infused at day 11 after allo-HSCT.While we speculate defective T cell trafficking to GvHD target organs would be the primary reason for the reduced GvHD in the recipients of VLA-4 deficient T cells, we examined other possible mechanisms as well, such as T cell alloreactivity and proliferation. We found that VLA-4 deficient T cells proliferated at the same rate as WT T cells in the presence of anti-CD3/CD28 antibody-coated beads. However, VLA-4 deficient T cells proliferate less in the presence of allogeneic antigen presenting cells (APCs) compared to WT T cells. Interestingly, VLA-4 deficient T cells upregulate CTLA-4 and GZMB significantly more than WT T cells after in vitro activation. These data suggest that not only T cell trafficking to GvHD target organs but also altered T cell phenotype and function might help explain the observed reduced GvHD. Genetic deletion of α4 in T cells will also alter expression of α4b7. Since antibodies to b7 have also been shown to reduce GvHD (Waldman et al., Blood, 2006) our current studies will focus on determining which of these pathways (α4b1 vs. α4b7) is most important in mitigating GvHD by using genetic knockout models, antibodies to b1 and b7 and small molecule inhibitors of α4b1 vs. α4b7.In conclusion, our data suggests that VLA-4 represents a promising therapeutic target for future efforts to mitigate GvHD after allo-HSCT. In addition, this strategy can be exploited in other diseases and settings in addition to allo-HSCT, such as solid organ transplantation, chronic inflammatory diseases and autoimmune diseases. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Alloreactive regulatory Tcells generated with retinoic acid prevent skin allograft rejection.

CD4(+) CD25(+) Foxp3(+) regulatory T (Treg) cells mediate immunological self-tolerance and suppress immune responses. Retinoic acid (RA), a natural metabolite of vitamin A, has been reported to enhance the differentiation of Treg cells in the presence of TGF-β. In this study, we show that the co-culture of naive Tcells from C57BL/6 mice with allogeneic antigen-presenting cells (APCs) from BALB/c mice in the presence of TGF-β, RA, and IL-2 resulted in a striking enrichment of Foxp3(+) T cells. These RA in vitro-induced regulatory T (RA-iTreg) cells did not secrete Th1-, Th2-, or Th17-related cytokines, showed a nonbiased homing potential, and expressed several cell surface molecules related to Treg-cell suppressive potential. Accordingly, these RA-iTreg cells suppressed T-cell proliferation and inhibited cytokine production by Tcells in in vitro assays. Moreover, following adoptive transfer, RA-iTreg cells maintained Foxp3 expression and their suppressive capacity. Finally, RA-iTreg cells showed alloantigen-specific immunosuppressive capacity in a skin allograft model in immunodeficient mice. Altogether, these data indicate that functional and stable allogeneic-specific Treg cells may be generated using TGF-β, RA, and IL-2. Thus, RA-iTreg cells may have a potential use in the development of more effective cellular therapies in clinical transplantation.

Antibody-functionalized peptidic membranes for neutralization of allogeneic skin antigen-presenting cells

We report herein application of an in situ material strategy to attenuate allograft T cell responses in a skin transplant mouse model. Functionalized peptidic membranes were used to impede trafficking of donor antigen-presenting cells (dAPCs) from skin allografts in recipient mice. Membranes formed by self-assembling peptides (SAPs) presenting antibodies were found to remain underneath grafted skins for up to 6days. At the host–graft interface, dAPCs were targeted by using a monoclonal antibody that binds to a class II major histocompatibility complex (MHC) molecule (I-Ad) expressed exclusively by donor cells. Using a novel cell labeling near-infrared nanoemulsion, we found more dAPCs remained in allografts treated with membranes loaded with anti-I-Ad antibodies than without. In vitro, dAPCs released from skin explants were found adsorbed preferentially on anti-I-Ad antibody-loaded membranes. Recipient T cells from these mice produced lower concentrations of interferon-gamma cultured ex vivo with donor cells. Taken together, the data indicate that the strategy has the potential to alter the natural course of rejection immune mechanisms in allogeneic transplant models.

Human Hepatic Stellate Cells Inhibit T-Cell Response Through B7-H1 Pathway

The liver is an immunologic privileged organ; liver allografts are accepted across major histocompatibility complex barriers in many species. However, hepatocyte transplants are acutely rejected, suggesting a role for liver nonparenchymal cells in regulating the immunoresponse. We have shown potent immunoregulatory activity of hepatic stellate cells (HSCs) in mice. The aim of this study was to examine the immunoregulatory activity of human HSCs. HSCs were isolated from normal human livers for analyses of their impact on T-cell response. HSCs expressed low HLA-DR and costimulatory molecules CD40 and CD80 but constitutively expressed high levels of CD54. Interferon-γ stimulated HSCs to express B7-H1 in a dose-dependent manner and produce the suppressive cytokines interleukin-6, interleukin-10, and transforming growth factor-β but did not affect expression of HLA-DR, CD40, and CD80. Human HSCs did not stimulate allogeneic T-cell proliferative response, indicating that they are not professional antigen-presenting cells. HSCs markedly inhibited T-cell response elicited by either allogeneic antigen-presenting cells or CD3/CD28 beads, which was associated with increases in activated CD4 and CD8 T-cell apoptosis. Addition of anti-B7-H1 blocking antibody significantly reversed the inhibitory effect. Human HSCs demonstrate potent immunoregulatory activity via B7-H1-mediated induction of apoptosis in activated T cells. Understanding of the involved mechanisms may lead to development of novel therapeutic approaches for treatment of liver diseases.