Professional APCs Research Articles

Functional Heterogeneity of Umbilical Cord Blood Monocyte-Derived Dendritic Cells.

Human umbilical cord blood (UCB) represents a unique resource for hematopoietic stem cell transplantation for children and patients lacking suitable donors. UCB harbors a diverse set of leukocytes such as professional APCs, including monocytes, that could act as a novel source for cellular therapies. However, the immunological properties of UCB monocytes and monocyte-derived dendritic cells (MoDCs) are not fully characterized. In this study, we characterized the phenotype and functions of UCB-MoDCs to gauge their potential for future applications. UCB exhibited higher frequencies of platelets and lymphocytes as well as lower frequencies of neutrophils in comparison with adult whole blood. Leukocyte subset evaluation revealed significantly lower frequencies of granulocytes, NK cells, and CD14+CD16- monocytes. Surface marker evaluation revealed significantly lower rates of costimulatory molecules CD80 and CD83 while chemokine receptors CCR7 and CXCR4, as well as markers for Ag presentation, were similarly expressed. UCB-MoDCs were sensitive to TLR1-9 stimulation and presented quantitative differences in the release of proinflammatory cytokines. UCB-MoDCs presented functional CCR7-, CXCR4-, and CCR5-associated migratory behavior as well as adequate receptor- and micropinocytosis-mediated Ag uptake. When cocultured with allogeneic T lymphocytes, UCB-MoDCs induced weak CD4+ T lymphocyte proliferation, CD71 expression, and release of IFN-γ and IL-2. Taken together, UCB-MoDCs present potentially advantageous properties for future medical applications.

Abstract 3983: Antigen-presenting cancer-associated fibroblasts provide an alternate mechanism for CD4 T cell activation in murine pancreatic ductal adenocarcinoma

Abstract Despite the advent of immune checkpoint inhibitors (ICI) that have markedly changed the treatment landscape for select tumors, pancreatic ductal adenocarcinoma (PDAC) remains unresponsive to such an approach. Features of the PDAC tumor microenvironment that contribute to treatment resistance, including the influence of cancer-associated fibroblasts (CAF) on immune and tumor cells, remain poorly understood. Antigen-presenting CAFs (apCAFs) express MHC-II and activate CD4 T cells in genetically engineered PDAC mouse models, but are rarely found in human PDAC. In a human PDAC atlas consisting of six published single-cell RNA-sequencing datasets, we identified 370 apCAFs out of 8736 total CAFs, the majority of which were from one dataset. Understanding apCAF regulation can reveal how to promote their differentiation and enhance T cell activating capacity in PDAC. Methods: Two KPC clones that differ in sensitivity to ICIs, sKPC (sensitive) and rKPC (resistant), derived from the autochthonous KPC murine model of PDAC, were implanted orthotopically into the pancreas or subcutaneously into the flank of syngeneic C57BL/6 mice. Tumors were harvested at 14-28 days post implantation for quantification of T cells, dendritic cells, and CAFs. To evaluate function, apCAFs were sorted and CD11c+ and CD4+ T cells were isolated from tumors. Naïve CD4+ T cells were also isolated from OT-II mice. apCAFs and CD11c+ cells were pulsed with OVA and co-cultured with OT-II CD4+ T cells, or co-cultured with sKPC tumor infiltrating CD4+ T cells in the presence of endogenous peptides. Results: Total CAFs are equivalent between tumors, but apCAFs are significantly more abundant in sKPC tumors. Furthermore, the apCAF to dendritic cell ratio within these tumors ranged from 5:1 to 50:1. CD11c+ dendritic cells sorted from sKPC tumors and pulsed with OVA peptide robustly activate naïve OT-II CD4+ T cells, as expected from professional APCs; sorted apCAFs also activate naïve OT-II CD4+ T cells, although to a lesser degree. When apCAFs or CD11c+ dendritic cells are cultured with tumor-infiltrating CD4+ T cells, however, they upregulate CD4 activation markers to a similar extent. Conclusions: apCAFs are more abundant in tumors derived from ICI-sensitive KPC clones and greatly outnumber dendritic cells. Functionally, apCAFs from sKPC tumors stimulate tumor-derived CD4 T cells to the same degree as CD11c+ dendritic cells, implicating a role in activation of the immune response in ICI-sensitive tumors. Although a small proportion of the tumor microenvironment, the apCAF subset may be enhanced in number and function by CAF-targeted modulators to affect the antitumor response; transcriptomics analyses are currently underway to identify regulators of apCAFs. Such a strategy could provide a solution to the currently unmet need for new and more effective treatments for PDAC patients. Citation Format: Saumya Maru, Lalitya Andaloori, Kathryn Howe, Kabeer Munjal, James Leatherman, Edward J. Pearce, Elizabeth M. Jaffee. Antigen-presenting cancer-associated fibroblasts provide an alternate mechanism for CD4 T cell activation in murine pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3983.

Abstract 3915: BMS-986442 (AGEN1777), a novel TIGIT/CD96 bispecific antibody, demonstrates superior monotherapy and combination activity versus conventional anti-TIGIT antibodies in preclinical models

Abstract TIGIT immune checkpoint blockade can enhance anti-tumor immunity, however conventional anti-TIGIT antibodies have shown suboptimal activity in the clinic. Here, we present the first preclinical disclosure of BMS-986442 (AGEN1777), a novel and fully human Fc-enhanced bispecific antibody against TIGIT and CD96 co-receptor, that demonstrates superior pharmacological activity compared to conventional TIGIT monospecific antibodies (mAbs). Gene expression of TIGIT, CD96, and the co-stimulatory counter-receptor CD226 in tumor samples from patients with cancer was identified using bulk and single cell RNA-sequencing. To target this interaction, Fc-engineered knob-in-hole bispecific antibodies against TIGIT and CD96 were developed and affinity optimized to achieve potent and complete blockade of TIGIT and CD96 ligand interactions. BMS-986442 and corresponding monospecific anti-TIGIT and anti-CD96 monoclonal antibodies were evaluated with or without anti-PD-1 in a variety of human T and NK cell stimulation assays. Mouse surrogate TIGIT/CD96 bispecific antibodies were developed and evaluated as monotherapy or together with anti-PD-1 in colorectal CT26 and breast orthotopic 4T1 tumor bearing mice. In CT26 and 4T1 tumor-bearing mouse models, the Fc-enhanced TIGIT/CD96 bispecific antibody demonstrated superior tumor control compared to either anti-TIGIT, anti-CD96, or the combination of anti-TIGIT and anti-CD96 mAbs. Tumor control was dependent on co-engaging activating Fcγ receptors, as an Fc-silent bispecific antibody lacked anti-tumor activity. In the tumor microenvironment, efficacy was associated with increased frequency of CD226+ effector and TCF-1+ GrzB- memory precursor CD8 T cells, increased frequency and activation of cytotoxic NK cells, and increased activation of professional APCs, including B cells, MHC Class II monocytes, and dendritic cells as determined by expression of CD40, CD83, and CD86. In primary human in vitro assays, BMS-986442 potently enhanced T cell responsiveness and activation compared with conventional anti-TIGIT mAbs alone and in combination with anti-PD-1 (nivolumab). Lastly, BMS-986442 promoted superior NK cell activation in tumor co-culture assays. In conclusion, BMS-986442 demonstrated a differentiated FcγR-dependent mechanism-of-action to enhance innate and adaptive immune responses compared with anti-TIGIT or anti-CD96 mAbs. BMS-986442 is currently advancing in phase I clinical studies in patients with advanced solid cancers (NCT05543629). Citation Format: Rebecca Ward, Nicola Ramsay, Spencer Campbell, Hema Patel, Mark Bushell, Benjamin M. Morin, Beth Wensley, David A. Savitsky, Pilar Garcia-Broncano, Bishnu Joshi, Emmanuel Briend, Olga Ignatovich, Zahra Jawad, Nils-Petter Rudqvist, Nicholas Wilson, Dhan Chand. BMS-986442 (AGEN1777), a novel TIGIT/CD96 bispecific antibody, demonstrates superior monotherapy and combination activity versus conventional anti-TIGIT antibodies in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3915.

IMMU-31. B-CELL THERAPY (BVAX) PROMOTES CD8 T CELLS' PERSISTENCE AND FUNCTION IN THE GLIOBLASTOMA MICROENVIRONMENT BY MAINTAINING THEIR STEM-LIKE FEATURES

Abstract The last decade witnessed tremendous improvement in immunotherapy to treat cancers. Its application in GBM has been particularly challenging due to the immunosuppressive tumor microenvironment, which greatly challenges T-cell infiltration and function. We developed a B-cell therapy with potent antigen-presenting functions that promote CD8+ tumor tropism and persistence in the tumor microenvironment. The present study investigates how BVax activates CD8+ T cells differently from other professional APCs like dendritic cells (DC) that could explain the persistence of CD8+ T cells in the tumor microenvironment. Upon in vitro activation by coculturing with BVax, most CD8+ T cells proliferated into TCF+ PD1- intermediate state, with CD44+ CD62L+ memory phenotype. Less than 10% of CD8+ were in the terminally-proliferation state. In contrast, DCs preferentially expanded TCF- PD1+ terminally-proliferating CD8+ T cells that were exhibiting CD44+ CD62L- effector phenotype. In vivo, activation of CD8+ T cells by co-injecting BVax or DC with CD8+ T cells to CT2A-bearing mice indicated that BVax promoted the proliferation of tumor-reactive CD8 T cells while maintaining the low PD1+ expression. Re-exposing the ex vivo-activated CD8+ T cells to tumor by adoptive transferring them to CT2A tumor-bearing mice revealed that BVax elicited immunological memory and potentiated GzmB+ production of CD8+ T cells. This study suggests that BVax and DC have distinctive interactions with CD8+ T cells, with the former poise CD8+ T cells at a more stem-like state, allowing them to differentiate upon tumor encounter.

Sec22b and Stx4 Depletion Has No Major Effect on Cross-Presentation of PLGA Microsphere-Encapsulated Antigen and a Synthetic Long Peptide InVitro.

The induction of CTL responses by vaccines is important to combat infectious diseases and cancer. Biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres and synthetic long peptides are efficiently internalized by professional APCs and prime CTL responses after cross-presentation of Ags on MHC class I molecules. Specifically, they mainly use the cytosolic pathway of cross-presentation that requires endosomal escape, proteasomal processing, and subsequent MHC class I loading of Ags in the endoplasmic reticulum (ER) and/or the endosome. The vesicle SNARE protein Sec22b has been described as important for this pathway by mediating vesical trafficking for the delivery of ER-derived proteins to the endosome. As this function has also been challenged, we investigated the role of Sec22b in cross-presentation of the PLGA microsphere-encapsulated model Ag OVA and a related synthetic long peptide. Using CRISPR/Cas9-mediated genome editing, we generated Sec22b knockouts in two murine C57BL/6-derived APC lines and found no evidence for an essential role of Sec22b. Although pending experimental evidence, the target SNARE protein syntaxin 4 (Stx4) has been suggested to promote cross-presentation by interacting with Sec22b for the fusion of ER-derived vesicles with the endosome. In the current study, we show that, similar to Sec22b, Stx4 knockout in murine APCs had very limited effects on cross-presentation under the conditions tested. This study contributes to characterizing cross-presentation of two promising Ag delivery systems and adds to the discussion about the role of Sec22b/Stx4 in related pathways. Our data point toward SNARE protein redundancy in the cytosolic pathway of cross-presentation.

Characterization of the immunosuppressant activity of the novel biogenic amine 3HKA and its analog derivatives

Abstract Tryptophan (Trp) is an essential amino acid which in mammalian cells 90% is processed through the kynurenine pathway. Kynurenine metabolites are among the most potent regulators of immune responses. Herein we report the characterization of a novel immunosuppressant biogenic amine, 3-hydroxy-L-kynurenamine (3HKA), generated through an uncharacterized lateral pathway of Trp catabolism, involving its decarboxylation by the aromatic amino acid decarboxylase. 3HKA is generated by both professional (Dendritic Cells, DC) and non-professional (Lymphatic Endothelial Cells) APC. Quantitative phosphoproteomic analysis of human-monocytes (CD14 +) derived DCs, identified key signaling transduction receptors, IFNR1, and signaling molecules from the JAK/STAT and cAMP mediated PKA pathways, regulated by 3HKA. Cell-based assays coupled with ELISA platforms showed that 3-HKA inhibited the IFN-g-mediated STAT1/NF-kB pathway in human DCs, and consequent decreased the release of pro-inflammatory chemokines and cytokines IL-1, TNFa, IL-6, and IL12p70. In vivo, 3-HKA reduced the intestinal inflammation in a model of dextran sodium sulfate-induced colitis. Furthermore, flow cytometry analysis revealed that 3-HKA increased the number of T-cells expressing 2, 3 or 4 exhaustion markers (PD-1, CTLA4, LAG3 or TIM3), exclusively in CD8 +CD4 −cells. We used, in silico approaches for ligand and pharmacophore-based drug discovery and developed 30 novel 3-HKA analogues with ADMET predicted drug-like properties. Structure-activity relationships studies, leaded to identify compounds with improved anti-inflammatory profiles. The novel anti-inflammatory 3-HKA analogues showed promising activity for the treatment of autoimmune diseases

Skewed fate and hematopoiesis of CD34+ HSPCs in umbilical cord blood amid the COVID-19 pandemic.

Umbilical cord blood (UCB) is an irreplaceable source for hematopoietic stem progenitor cells (HSPCs). However, the effects of SARS-CoV-2 infection and COVID-19 vaccination on UCB phenotype, specifically the HSPCs therein, are currently unknown. We thus evaluated any effects of SARS-CoV-2 infection and/or COVID-19 vaccination from the mother on the fate and functionalities of HSPCs in the UCB. The numbers and frequencies of HSPCs in the UCB decreased significantly in donors with previous SARS-CoV-2 infection and more so with COVID-19 vaccination via the induction of apoptosis, likely mediated by IFN-γ-dependent pathways. Two independent hematopoiesis assays, a colony forming unit assay and a mouse humanization assay, revealed skewed hematopoiesis of HSPCs obtained from donors delivered from mothers with SARS-CoV-2 infection history. These results indicate that SARS-CoV-2 infection and COVID-19 vaccination impair the functionalities and survivability of HSPCs in the UCB, which would make unprecedented concerns on the future of HSPC-based therapies.

Migrating Type 2 Dendritic Cells Prime Mucosal Th17 Cells Specific to Small Intestinal Commensal Bacteria.

Dendritic cells (DCs) are professional APCs equipped with MHC-restricted Ags, costimulations, and cytokines that effectively prime and differentiate naive T cells into distinct functional subsets. The immune signals that DCs carry reflect the route of Ag uptake and the innate stimuli they received. In the mucosal tissues, owing to the great variety of foreign Ags and inflammatory cues, DCs are predominantly activated and migratory. In the small intestine, CD4 Th17 cells are abundant and have been shown to be regulated by DCs and macrophages. Using a mouse commensal bacteria experimental model, we identified that the early priming step of commensal-driven Th17 cells is controlled by bona fide Zbtb46-expressing DCs. CCR7-dependent migration of type 2 DCs (DC2s) from the small intestine to the mesenteric lymph nodes (MLNs) is essential for the activation of naive CD4 T cells. The migratory DC2 population in the MLNs is almost exclusively Esam+ cells. Single-cell RNA sequencing highlighted the abundance of costimulatory markers (CD40 and OX40) and chemokines (Ccl22 and Cxcl16) on MLN migratory DCs. Further resolution of MLN migratory DC2s revealed that the Th17-polarizing cytokine IL-6 colocalizes with DC2s expressing CD40, Ccl17, and Ccl22. Thus, early Th17 cell differentiation is initiated by a small subset of migratory DC2s in the gut-draining lymph nodes.

MARCH1 Controls an Exhaustion-like Program of Effector CD4+ T Cells Promoting Allergic Airway Inflammation.

Persistent antigenic signaling leads to T cell exhaustion, a dysfunctional state arising in many chronic infections and cancers. Little is known concerning mechanisms limiting exhaustion in immune-stimulatory diseases such as asthma. We report that membrane-associated RING-CH1 (MARCH1), the ubiquitin ligase that mediates surface turnover of MHC class II (MHCII) and CD86 in professional APCs, plays an essential role in restraining an exhaustion-like program of effector CD4+ T cells in a mouse model of asthma. Mice lacking MARCH1 or the ubiquitin acceptor sites of MHCII and CD86 exhibited increased MHCII and CD86 surface expression on lung APCs, and this increase promoted enhanced expression of immune-inhibitory receptors by effector CD4+ T cells and inhibited their proliferation. Remarkably, ablation of MARCH1 in mice with established asthma reduced airway infiltration of eosinophils and Th2 cells. Thus, MARCH1 controls an exhaustion-like program of effector CD4+ T cells during allergic airway inflammation and may serve as a therapeutic target for asthma.

Gift bags from the sentinel cells of the immune system: The diverse role of dendritic cell-derived extracellular vesicles.

Dendritic cells (DCs) are professional APCs of the immune system that continuously sample their environment and function to stimulate an adaptive immune response by initiating Ag-specific immunity or tolerance. Extracellular vesicles (EVs), small membrane-bound structures, are released from DCs and have been discovered to harbor functional peptide-MHC complexes, T cell costimulatory molecules, and other molecules essential for Ag presentation, immune cell regulation, and stimulating immune responses. As such, DC-derived EVs are being explored as potential immunotherapeutic agents. DC-derived EVs have also been implicated to function as a trafficking mechanism of infectious particles aiding viral propagation. This review will explore the unique features that enable DC-derived EVs to regulate immune responses and interact with recipient cells, their roles within Ag-presentation and disease settings, as well as speculating on a potential immunological role of apoptotic DC-derived EVs.

Activation of Transcription Factor 4 in Dendritic Cells Controls Th1/Th17 Responses and Autoimmune Neuroinflammation.

Dendritic cells (DCs) are professional APCs that play a crucial role in initiating robust immune responses against invading pathogens while inducing regulatory responses to the body's tissues and commensal microorganisms. A breakdown of DC-mediated immunological tolerance leads to chronic inflammation and autoimmune disorders. However, cell-intrinsic molecular regulators that are critical for programming DCs to a regulatory state rather than to an inflammatory state are not known. In this study, we show that the activation of the TCF4 transcription factor in DCs is critical for controlling the magnitude of inflammatory responses and limiting neuroinflammation. DC-specific deletion of TCF4 in mice increased Th1/Th17 responses and exacerbated experimental autoimmune encephalomyelitis pathology. Mechanistically, loss of TCF4 in DCs led to heightened activation of p38 MAPK and increased levels of proinflammatory cytokines IL-6, IL-23, IL-1β, TNF-α, and IL-12p40. Consistent with these findings, pharmacological blocking of p38 MAPK activation delayed experimental autoimmune encephalomyelitis onset and diminished CNS pathology in TCF4ΔDC mice. Thus, manipulation of the TCF4 pathway in DCs could provide novel opportunities for regulating chronic inflammation and represents a potential therapeutic approach to control autoimmune neuroinflammation.

Polyglutamine-Related Aggregates Can Serve as a Potent Antigen Source for Cross-Presentation by Dendritic Cells.

Protective MHC class I-dependent immune responses require an overlap between repertoires of proteins directly presented on target cells and cross-presented by professional APC, specifically dendritic cells. How stable proteins that rely on defective ribosomal proteins for direct presentation are captured for cell-to-cell transfer remains enigmatic. In this study, we address this issue using a combination of in vitro (C57BL/6-derived mouse cell lines) and in vivo (C57BL/6 mouse strains) approaches involving stable and unstable versions of OVA model Ags displaying defective ribosomal protein-dependent and -independent Ag presentation, respectively. Apoptosis, but not necrosis, of donor cells was found associated with robust global protein aggregate formation and captured stable proteins permissive for cross-presentation. Potency of aggregates to serve as Ag source was directly demonstrated using polyglutamine-equipped model substrates. Collectively, our data implicate global protein aggregation in apoptotic cells as a mechanism that ensures the overlap between MHC class I epitopes presented directly or cross-presented by APC and demonstrate the unusual ability of dendritic cells to process stable protein aggregates.

Disease-Specific Expression of Conjunctiva Associated Lymphoid Tissue (CALT) in Mouse Models of Dry Eye Disease and Ocular Allergy.

Conjunctiva-associated tissue (CALT) is assumed to play a crucial role in the immune system of the ocular surface. Its function in several ocular surface diseases (OSD) is still not fully understood. This study investigates the function of CALT in mouse models of dry-eye disease and ocular allergy. Since antigen-presentation is the central similarity in the pathologies, this study focuses on antigen-presentation in CALT Morphology and the expression of CALT, which was investigated in mice after induction of dry-eye, ocular allergy, topical antigen-stimulation, and after local depletion of phagocytic cells. Antigen uptake was investigated after the application of fluorescent ovalbumin (OVA). OSD influences the appearance and morphology of CALT in a disease-dependent manner. Ocular allergy leads to an increase and dry-eye disease to a decrease in number and size of CALT. The development of CALT is dependent on the presence of APCs. Professional APCs are present in CALT, and soluble antigen is transported into the follicle. CALT appearance is disease-specific and indicative of differing functions. Although the specific involvement of CALT in OSD needs further study, the existence of functional APCS and antigen-uptake supports the hypothesis that CALT is an immunological key player at the ocular surface.

Extracellular Release of Antigen by Dendritic Cell Regurgitation Promotes B Cell Activation through NF-κB/cRel.

Dendritic cells (DCs) are professional APCs, which sample Ags in the periphery and migrate to the lymph node where they activate T cells. DCs can also present native Ag to B cells through interactions observed both in vitro and in vivo. However, the mechanisms of Ag transfer and B cell activation by DCs remain incompletely understood. In this study, we report that murine DCs are an important cell transporter of Ag from the periphery to the lymph node B cell zone and also potent inducers of B cell activation both in vivo and in vitro. Importantly, we highlight a novel extracellular mechanism of B cell activation by DCs. In this study, we demonstrate that Ag released upon DC regurgitation is sufficient to efficiently induce early B cell activation, which is BCR driven and mechanistically dependent on the nuclear accumulation of the transcription factor NF-κB/cRel. Thus, our study provides new mechanistic insights into Ag delivery and B cell activation modalities by DCs and a promising approach for targeting NF-κB/cRel pathway to modulate the DC-elicited B cell responses.

Lactoferrin modulates BMDCs to be tolerogenic leading to enhancement of Foxp3+iTreg differentiation as well as suppression of T cell proliferation

Abstract DCs are professional APCs for initiating both immune response and immune tolerance. Tolerogenic DCs are generally known as regulatory DCs that suppress immune responses through regulation of T cells such as induction of T cell anergy/apoptosis and Treg. Since we have demonstrated that lactoferrin (LF) induces functional Tregs and has a protective effect against IBD, we explored the tolerogenic effect of LF on mouse bone marrow-derived DC (BMDC) and its underlying mechanisms. LF enabled BMDCs to suppress the proliferation of allogenic CD4+ T cells, and elevated levels of Arg-1, IDO-1, and CD39/73 (known as key T cell suppressor molecules). Consistently, the suppressive activity of LF-treated DCs (LF-DCs) was disappeared by inhibitors of Arg-1 and CD39. Finally, LF-DCs enhanced TGFβ1-induced OVA-specific Foxp3+ T cell population. Collectively, these results suggest that lactoferrin effectively causes DCs to be tolerogenic by both direct suppression of T cell proliferation and indirect enhancement of Treg differentiation.

Inflammation-induced tissue damage mimicking GvHD in human skin models as test-platform for immunotherapeutics.

With cellular products being on the front run there is a rising demand for non-animal-based test platforms to predict, study and treat undesired immunity. Here, we generated human organotypic skin models from human biopsies isolating and expanding keratinocytes, fibroblasts and microvascular endothelial cells finally allowing to seed these components on a collagen matrix or a biological vascularized scaffold matrix in a bioreactor. Afterwards, we were able to induce inflammation-based tissue damage by pre-stimulated mismatched allogeneic lymphocytes and/or inflammatory cytokine containing supernatants histomorphologically mimicking severe graft versus host disease (GvHD) of the skin. The effects could be prevented by the addition of immunosuppressants to the models. Consequently, these models would harbor a promising potential to serve as a test platform for the prediction, prevention and treatment of GvHD. This would also allow functional studies of immune effectors and suppressors including but not limited to allodepleted lymphocytes, gamma-delta T cells, regulatory T cells and mesenchymal stromal cells which would otherwise be limited to animal models. Thus, the current test platform developed with the limitation given that no professional APC are in place could highly reduce animal testing for investigation of novel immune therapies.

Ex vivo assessment of in vivo DC-targeted antibodies in pre-clinical models.

APCs play a key role at initiating adaptive immune responses by presenting antigens to lymphocytes and DCs are professional APCs. It is critical to understand the differential antigen capture and presentation ability of different DC subsets, which is important for DC-targeted immunotherapy. In this section, we give a brief introduction to different antigen presentation pathways and introduce the key concept of cross-presentation, the major antigen presentation pathway used for anti-viral and anti-tumoral immune responses. CD205, a DC restricted receptor, is highly expressed on certain DCs subsets. We find CD205-mediated antigen uptake to be a useful model for studying antigen uptake and defects. These methods provide an introduction to CD205-mediated pre-clinical delivery of antigens to cross-presenting DCs, which can be adapted to the study of targeting to multiple receptors and other C-type lectins. This is a promising strategy to detect the antigen capture capacity and to study the key players orchestrating tolerance and immunity ex vivo.

Analysis of MHC Class I Processing Pathways That Generate a Response to Vaccinia Virus Late Proteins.

Use of recombinant viral vectors encoding nonnative Ags is an attractive mechanism for the generation of protective Ab, CD4+ T cell (TCD4+), and CD8+ T cell (TCD8+) responses in vivo following immunization. However, the life cycle and tropism of the viral vector, and its interactions with various components of the immune system, must be fully understood to maximize the efficacy of any vaccination strategies. Ab and TCD4+ responses typically target native Ags driven by late promoters in vaccinia virus (VACV)-based vectors. However, it has been demonstrated that model Ags driven by late promoters in recombinant VACV vectors do not stimulate TCD8+ responses, whereas identical Ags driven by early promoters stimulate strong responses. Conversely, TCD8+ can be generated against some natural late VACV Ags. We explored this dichotomy by investigating the Ag presentation pathways responsible for presentation of natural late VACV Ags in mice. We found that all of the late VACV Ags we examined could be cross-primed (i.e., presented by uninfected professional APC), as well as directly presented by infected dendritic cell populations. However, one Ag was only presented by professional APC populations and was not the target of a protective TCD8+ response. Therefore, there is no generalized blockade in Ag presentation of late VACV Ags, and expression of nonnative Ags driven by a late promoter allows production of large quantities of Ag that may allow simultaneous targeting of both TCD4+ and Ab responses, as well as TCD8+ responses, in the future.

Peri-Transplant Steroids Enhance GVL and Attenuate GVHD By Redistributing Alloreactive Donor T Cells from the Gut into the Bone Marrow

Leukemia relapse represents a failure of graft-versus-leukemia (GVL) and remains the major limitation of allogeneic stem cell/bone marrow transplantation (BMT). Graft-versus-host disease (GVHD) within the gastrointestinal (GI) tract is the principal determinant of transplant-related mortality and is initiated by a network of alloantigen presentation by professional and non-professional APC that prime donor T cells in the GI tract and related lymphoid structures. Since GVL and lethal GVHD are mediated by donor T cells at spatially distinct sites; bone marrow (BM) and the GI tract respectively, we sought tractable approaches to spatially separate alloreactive responses at these two locations. The administration of high dose steroids in the peri-transplant period is permissive of T cell replete HLA-haploidentical BMT and significant GVL effects (Ogawa H, et al. BBMT. 2006). We utilized murine haploidentical BMT models (B6D2F1 → B6C3F1, B6 → B6D2F1) with recipient background MLL/AF9 primary acute myeloid leukemia (AML), with or without dexamethasone (Dex) administration (5 mg/kg/day i.p., days -1 to +5). Dex-treatment improved transplant survival (from 25% to 68% at day 100, P=0.0012) with significant reductions in GVHD histopathology specifically in the colon (histopathology scores 8.7±1.0 vs 4.6±0.8, P< 0.05), despite excellent leukemia control. To understand this paradox, we analyzed the kinetics of donor T cell expansion after BMT. In the mesenteric lymph node (mLN), Dex treatment significantly suppressed the expansion of both CD4 and CD8 T cells (3.3±0.3 x 105 vs 1.4±0.3 x 105, P< 0.001 and 4.2±0.4 x 105 vs 2.1±0.4 x 105, P< 0.01 respectively) and the activation of CD4 T cells (CD25 MFI: 2021±146 vs 1056±102, P< 0.01). In contrast, donor effector/memory CD44+ CD8 T cells were expanded in the BM of Dex treated recipients (1.9±0.3 x 105 vs 3.1±0.4 x 105, P< 0.05) that demonstrated high per cell cytolytic activity against leukemia (specific lysis: 65±2.4 % vs 62±2.6 % in untreated vs Dex-treated, P> 0.05). Surprisingly, there was no difference in proliferation (cell tracking dye dilution: 63±5.5 % vs 57±5.5 % in untreated vs Dex-treated, P> 0.05) or apoptosis (caspase-3: 6.6±0.4 % vs 6.1±0.6 %, caspase-8: 20±1.6 % vs 17±3.3 % in untreated vs Dex-treated, respectively, P> 0.05) of CD4 T cells in the mLN between the two groups. We undertook experiments with luciferase expressing T cells and noted that Dex-treatment preferentially inhibited T cell accumulation in the GI tract, but not marrow after BMT. Thus, it appeared that Dex treatment preferentially re-distributed donor T cells from the GI tract to the bone marrow. We next determined if Dex exerted effects via direct signaling to the donor T cell. We thus transplanted glucocorticoid receptor (GR)-deficient or intact T cells (GRfl/fl lck-Cre mice). Dex-treatment reduced donor CD4 T cell expansion in the mLN independent of their expression of the GR (untreated vs Dex-treated: 2.8±0.6 x 105 vs 1.2±0.3 x 105, lckCREGRfl/fl and 2.4±0.3 x 105 vs 1.4±0.4 x 105, GRfl/fl littermates, P< 0.05 both groups). Thus steroid effects were mediated indirectly, putatively via effects on recipient alloantigen presentation. There was a marked reduction in recipient dendritic cells (DC) and macrophages expressing the Ea peptide within MHC class II in the GI tract of Dex-treated recipients (terminal Ileum YAe+ DC number 896±93 vs 356±40, P< 0.01, YAe+ macrophage number 1035±136 vs 355±97, P< 0.01). In conjunction with this, expression of the gut homing integrin a4b7 expression was reduced in CD4 T cells from Dex treated recipient mLN (25±1.6 % vs 17±1.7 %, P< 0.01), while the marrow homing integrin VLA-4 (a4b1) was increased (a4: 62±2.2 % vs 75±1.6 %, P< 0.001, b1: 52±2.5 % vs 61±1.6 %, P< 0.05) in donor CD8 T cells from Dex treated recipient BM. Finally, Dex treatment enhanced GVL against a second primary AML (BCR/ABL-NUP98/HOXA9) relative to untreated recipients and those receiving post-transplant cyclophosphamide (PT-Cy) (relapse rate: 0% vs 40% vs 100% at day 35 in Dex vs untreated vs PT-Cy, PT-Cy vs Dex-treated, P< 0.0001; untreated vs Dex-treated, P=0.029). These data suggest a potential therapeutic strategy to modulate antigen presentation in the GI tract and consequent integrin imprinting that minimizes GVHD lethality whilst enhancing GVL within BM. Disclosures No relevant conflicts of interest to declare.

Effects of Live Attenuated Vaccine and Wild Type Strains of Edwardsiella ictaluri on Phagocytosis, Bacterial Killing, and Survival of Catfish B Cells.

Edwardsiella ictaluri, a Gram-negative facultative intracellular pathogen, is the causative agent of enteric septicemia of catfish (ESC). The innate functions of B cells have been demonstrated in several teleost fish, including zebrafish, rainbow trout, and channel catfish. Recently, our group has developed several protective E. ictaluri live attenuated vaccines (LAVs). However, the innate role of catfish B cells to phagocytose and destroy E. ictaluri wild-type (WT) and live attenuated vaccine (LAV) strains has not been evaluated. In this study, we assessed the efficacy of E. ictaluri WT and two LAVs on phagocytosis, microbial killing, and survival of catfish anterior kidney (AK) B cells. Initially, we documented active uptake of E. ictaluri WT and two LAVs in B cells by flow cytometry and light microscopy. Then, we observed the E. ictaluri strains-induced phagosome and/or phagolysosome formation in the cytoplasm of catfish magnetically sorted IgM+ B cells. Furthermore, we demonstrated that AK B cells were able to destroy the internalized E. ictaluri WT and LAV strains efficiently. Finally, we documented early and late apoptotic/necrotic manifestations induced by E. ictaluri in catfish AK B cells. In conclusion, our results suggest that both LAVs and WT strain initiate similar innate immune responses such as active phagocytic uptake, induced bactericidal activity as well as promote early and late apoptotic changes in catfish B cells. Our data suggest that phagocytic and microbicidal B cells may serve as professional APCs in initiation of protective adaptive immune responses against ESC in channel catfish.