HOD Mice Research Articles

On the derived models of self-iterable universes

We show that if the universe is self-iterable and κ \kappa is an inaccessible limit of Woodin cardinal then A D R + “ Θ \mathsf {AD}_{\mathbb {R}}+“\Theta is regular” holds in the derived model at κ \kappa . The proof is fine-structure free, and only assumes basic knowledge of iteration trees and iteration strategies. Our proof can be viewed as the fine-structure free version of the well-known fact that A D R + “ Θ \mathsf {AD}_{\mathbb {R}}+“\Theta is regular” is true in the derived models of hod mice that have inaccessible limit of Woodin cardinals (see for example Sargsyan [Mem. Amer. Math. Soc. 236 (2015), p. viii+172]). However, the proof uses a different set of ideas and is more general.

Tolerization of CD4+ Recent Thymic Emigrants Is Required to Prevent RBC-Specific Autoimmunity

BackgroundLoss of humoral tolerance to red blood cell (RBC)-specific antigens may lead to the development of pathogenic autoantibodies and result in autoimmune hemolytic anemia, a severe and sometimes fatal disease. Recently, it has been demonstrated that although RBC-specific autoreactive B cells escape central tolerance (and can secrete autoantibodies), stringent T cell tolerance is a stopgap to autoimmunity; however, T cell tolerance mechanisms utilized against RBC antigens is largely unknown.Thymic central tolerance is a process by which newly-formed autoreactive T cells undergo negative selection by deletion, anergy, or diversion into a regulatory T cell (Treg). This process ensures that newly-minted T cells are self-tolerant and functional. Elimination of >95% of thymocytes occurs during central tolerance. However, while rigorous, central tolerance sometimes fails and autoreactive T cells escape to the periphery. The youngest peripheral T cells, recent thymic emigrants (RTEs), are functionally distinct from mature T cells and are more susceptible to peripheral tolerance mechanisms in response to autoantigens. Herein, we assess central and peripheral T tolerance to RBC-specific autoantigens.MethodsHOD transgenic mice express an RBC-specific fusion protein consisting of hen egg lysozyme (HEL), ovalbumin (OVA), and human blood group, Duffy (D). To assess tolerance, HOD mice were bred with OTII mice, CD4+ T cell receptor (TCR) transgenic mice with specificity for OVA (contained within HOD). HODxOTII F1s were immunized with 100ug of OVA/CFA. To assess central tolerance, thymi from 8-12 week old HODxOTII F1 mice were harvested and stained with antibodies to identify OTIIs and Tregs. For RTE analysis, HOD mice were bred with OTII.Rag2p-GFP (referred to as HODxRTE mice), OTII T cells that express GFP during TCR rearrangement. GFP+ OTII RTEs were stained with antibodies against CD73, FR4, and PD1. To assess function, GFP+ OTII RTEs were sorted and 3x105 RTEs were infused into naïve B6 mice. Recipient mice received a 100uL packed HOD RBC transfusion and RTE proliferation was assessed at 3 days by Ki67 staining.ResultsHOD+OTII+ mice had profound tolerance to the HOD RBC autoantigen; immunization with OVA/CFA elicited strong antibody responses in HOD-OTII+ littermates, but not autoreactive HOD+OTII+ mice. To determine whether OTIIs underwent central tolerance, thymi were analyzed. No significant differences in total numbers of thymocytes or CD4+ OTIIs was observed in autoreactive HOD+OTII+ mice, compared to control HOD-OTII+. Additional phenotyping for co-expression of CD25 and FoxP3 (a phenotype consistent with Tregs) revealed no difference, suggesting autoreactive OTII T cells were not diverted into a regulatory program. Thus, negative selection of RBC autoreactive CD4+ T cells does not occur in the thymus.Given that RTEs are subject to tolerization in response to peripherally expressed autoantigens, HODxRTE F1s were used to assess whether RBC-specific autoreactive OTIIs are tolerized as RTEs. RTEs were divided into GFPhi (< 1 week in the periphery), GFPint (2-3 weeks in the periphery), and GFPneg (mature T cells). In HOD+RTE+ mice, GFPhi RTEs had significantly higher levels of PD1, a marker of exhaustion, compared to controls. As RTEs matured, PD1 expression slightly decreased, but remained high compared to controls. Additionally, as autoreactive OTII RTEs matured, a significant number OTIIs expressed CD73hiFR4hi, a phenotype consistent with anergy. On average, 5% of GFPneg OTIIs from HOD-RTE+ mice expressed CD73hiFR4hi, compared to over 60% in autoreactive HOD+RTE+ mice. To assess function, sorted RTEs from HODxRTE mice were adoptively transferred into naïve mice and assessed for proliferation post HOD RBC transfusion. All RTEs sorted from HOD-RTE+ mice proliferated (>95% of OTIIs were Ki67+). However, RTEs derived from HOD+RTE+ mice had diminished proliferative capacity upon maturation (Ki67+ RTEs: GFPhi 65%, GFPint 35%, GFPneg 15%).ConclusionsThese data demonstrate that RBC-specific autoreactive CD4+ T cells are neither deleted nor diverted into a Tregs during thymic central tolerance, rather peripheral tolerance mechanisms occurring during RTE maturation is required. Together, these data provide insight into RBC-specific T cell tolerance mechanisms, pinpoint when they occur, and demonstrate that if RTE maturation does not occur, RBC autoimmunity may ensue. DisclosuresNo relevant conflicts of interest to declare.

Covering with Chang models over derived models

We present a covering conjecture that we expect to be true below superstrong cardinals. We then show that the conjecture is true in hod mice. This work is a continuation of the work that started in [4], and the main conjecture of the current paper is a revision of the UB-Covering Conjecture of [4].

Autoantigen presentation by splenic dendritic cells is required for RBC-specific autoimmunity.

Failure of humoral tolerance to red blood cell (RBC) antigens may lead to autoimmune hemolytic anemia (AIHA), a severe and sometimes fatal disease. Previous studies have shown that although tolerance is robust in HOD mice, autoantibodies are generated upon adoptive transfer of OTII CD4+ T cells, which are specific for an epitope contained within the HOD antigen. These data imply that antigen-presenting cells (APCs) are presenting RBC-derived autoantigen(s) and are capable of driving T-cell activation. Given that multiple APCs participate in erythrophagocytosis, we used a transgenic approach to determine which cellular subsets were required for autoantigen presentation and subsequent autoreactive T-cell activation. HOD mice, which express an RBC-specific antigen consisting of hen egg lysozyme, ovalbumin, and human blood group molecule Duffy, were bred with IAbfl/fl and Cre-expressing transgenic animals to generate mice that lack I-Ab expression on particular cell subsets. OTII CD4+ T cell proliferation was assessed in vivo in HOD+ I-Abfl/fl xCre+ mice and in vitro upon coculture with sorted APCs. Analysis of HOD+ I-Abfl/fl xCre+ mice demonstrated that splenic conventional dendritic cells (DCs), but not macrophages or monocytes, were required for autoantigen presentation to OTII CD4+ T cells. Subsequent in vitro coculture experiments revealed that both CD8+ and CD8- DC subsets participate in erythrophagocytosis, present RBC-derived autoantigen and stimulate autoreactive T-cell proliferation. These data suggest that if erythrocyte T-cell tolerance fails, DCs are capable of initiating autoimmune responses. As such, targeting DCs may be a fruitful strategy for AIHA therapies.

Transfusion with Stored Antigen-Negative Blood Impairs T Cell Cross Priming to Red Cell Alloantigen in a Subsequent Transfusion

BACKGROUND: Donor red blood cell (RBC) alloantigens are efficiently cross-presented by recipient antigen presenting cells (APCs), thereby priming recipient naive CD8 T cells; however, this process and its downstream immunological consequences are poorly understood. RBC antigen cross presentation has been linked to cytotoxic T cell responses driving bone marrow graft rejection, but in other systems it causes tolerance. The conventional dendritic cell (cDC) subset, cDC1, is required for cross-presentation of RBC antigen. Macrophages are insufficient for cross-presentation in the absence of cDC1s, but do promote RBC antigen cross-presentation by cDCs in vitro. After prolonged storage, a portion of transfused RBCs are rapidly cleared by the spleen. In murine models, transfused stored RBCs have multiple effects on splenic APCs, including architectural distortion, dendritic cell activation, and macrophage depletion via ferroptosis due to increased erythrophagocytosis. Thus, the antigen presentation machinery controlling alloimmunization to RBC antigens may be influenced by the storage duration of previously transfused RBCs.AIMS: To better understand how recent transfusions may influence the immunogenicity of future transfusions, we used a mouse model to determine whether prior transfusion of stored, antigen-matched RBCs influences CD8 T cell cross priming to antigen mismatched RBCs in a subsequent transfusion.METHODS: In an in vivo cross-priming assay, OVA-expressing RBCs from transgenic HOD mice (with RBC surface expression of hen egg lysozyme, ovalbumin (OVA), and Duffy antigens) were the transfused antigen source and naïve OT-1 CD8 T cells, which express an MHC class I restricted OVA-specific TCR transgene, were the responders. Magnetically-selected CD8+ OT-1 cells, labeled with CellTrace Far Red (CTFR), were adoptively transferred into MHC identical (H-2Kb) UBC-GFP C57BL6/J recipients. After 16 hours, these mice were transfused with fresh or stored (12 days in CPDA-1), leukoreduced UBC-GFP RBCs or PBS. Five hours after the initial transfusion, recipients were transfused again with fresh leukoreduced HOD RBCs. To determine the effect of stored RBC transfusion on T cell priming to a non-RBC associated antigen, groups of mice carrying adoptively transferred OT-1 T cells were transfused with fresh or stored RBCs and then challenged 5 hours later with soluble OVA protein (10 µg, IV). After 4 days, splenic OT-1 T cells were analyzed for proliferation (by CTFR dye dilution) and activation (by CD44 and CD122 expression).RESULTS: Proliferation and activation of OT-1 T cells in response to challenge with soluble OVA protein were significantly increased in mice that had been previously transfused with stored RBCs compared to those transfused with fresh RBCs (Fig. 1A). In contrast, OT-1 T cell activation in response to HOD RBC transfusion was decreased in mice that had previously received a stored RBC transfusion, as compared to those receiving fresh RBCs or PBS (Fig. 1B).CONCLUSION: The increased T cell priming after soluble OVA challenge in the mice previously transfused with stored vs. fresh RBCs may be related to the increased expression of co-stimulatory molecules on DCs after stored RBC transfusion. However, T cell priming to the RBC-associated antigen requires additional factors, which are disrupted by previous transfusion of stored, but not fresh, RBCs. Splenic red pulp macrophages potentiate OT-1 T cell cross priming by dendritic cells in vitro and become severely depleted after stored RBC transfusion in vivo. Thus, macrophage help may be needed for T cell cross priming to an RBC antigen in vivo, and stored RBC transfusions may disrupt macrophage function, thereby affecting the immunogenicity of subsequent RBC alloantigen exposure.Figure 1. Effect of fresh and stored RBC transfusion on priming after antigenic challenge with soluble OVA or HOD RBCs.Representative contour plots show proliferation-induced dye dilution and CD44 expression on OT-1 cells in the spleen 4 days after transfusion of fresh (left) or stored (right) RBCs followed 5 hours later with IV injection of soluble OVA protein (A) or HOD RBCs (B). Dot plots at the right of representative contour plots show compiled data from two independent experiments quantifying the percentage of activated and proliferated cells (CTFR diluted, CD44hi) in the OT-1 subgate (mean ± SEM, n = 4-9). *P < 0.05, **P < 0.01, unpaired t test. DisclosuresNo relevant conflicts of interest to declare.

Dendritic Cell Cross Presentation of RBC Antigens in-Vivo Is Not Affected By RBC Storage Duration and Requires Red Pulp Macrophage "Help" in-Vitro

BACKGROUND: Chronic transfusion is associated with an increased risk of bone marrow graft rejection. In prior studies with mouse models, minor histocompatibility antigens in red blood cell (RBC) and platelet products were presented in the context of recipient MHC class I to prime recipient cytotoxic T cells; these represent cross presentation and cross priming, respectively. Once primed in the recipient, these T cells may attack allogeneic bone marrow grafts. Thus, understanding the mechanism of transfusion-induced T cell cross priming may lead to new methods to reduce bone marrow graft rejection in chronically transfused patients. Cross presentation is performed by a specialized subset of antigen presenting cells (APCs) - CD11b lo, XCR1+ dendritic cells (XCR DCs). Transfusion of RBCs after prolonged refrigerated storage induces erythrophagocytosis and pro-inflammatory gene expression in the spleen. Fluorescent tracking of transfused RBCs showed that splenic red pulp macrophages (RPMs) ingest the majority of damaged RBCs, but RPMs are weak APCs. Splenic dendritic cells, including XCR DCs, also display increased uptake of stored vs. fresh RBCs. These data suggested that refrigerated storage may increase cross presentation of RBC antigens, thereby enhancing T cell cross priming.AIMS: To compare T cell cross priming after transfusion of fresh and refrigerator-stored RBCs, a mouse model was used with transgenic OVA-carrying RBCs as the antigen source and transgenic naive MHC class I-restricted (H-2Kb) OVA-specific T cells (OT-1) as responders. In parallel, an in-vitro system was established to determine the cellular elements required for cross presentation of RBC antigens.METHODS: Purified CD8 T cells from OT-1 mice were loaded with the cell proliferation tracking dye Cell Trace Far Red (CTFR) and adoptively transferred (4 x 10e6 per mouse) into cohorts of 8-12 week old C57BL/6 GFP+ mice (n=3 per group). The next day mice were transfused with 400 uL of fresh (<24 hours old), or stored RBCs (14 days old) from transgenic HOD mice (RBCs express surface HEL, OVA, and Duffy antigens). After 96 hours, flow cytometry was used to assess OT-1 cell proliferation by dye dilution and the expression of activation markers CD44, CD122 and CD62L. Control transfusions with non-HOD GFP+ RBCs confirmed the antigen specificity of the response, and transfusions of HOD blood that had undergone RBC lysis ruled out direct antigen presentation by MHC identical white cells in the transfusate. For the in-vitro cross presentation assay, 1x10e5 CTFR-loaded OT-1 cells were plated in U-bottom 96 well plates with all possible combinations of fresh or stored HOD RBCs (50 x 10e6 per well), bone marrow Flt3L-derived C57BL/6 murine dendritic cells (Flt3L-DCs, 5 X 10e4 per well), and magnetically selected BALB/c (H-2Kd) RPMs (5 x 10e4 per well). MHC mismatch rules out antigen presentation by BALB/c RPMs (H-2Kd) to OT-I T cells (H-2Kb).RESULTS: Transfusion of both fresh and stored HOD RBCs induced vigorous proliferation and activation of OT-1 cells. After 4 days, no differences were seen in the proliferation and activation profiles of OT-1 cells in mice receiving fresh (78±4% CD44hi, CTFR diluted) vs. stored (79±5%) HOD RBCs. The in-vitro cross presentation assay showed weak to absent OVA-specific OT-1 proliferation with co-cultures including fresh or stored HOD RBCs alone, and fresh or stored HOD RBCs plus Flt3L-DCs. In contrast, cultures including RPMs and Flt3L-DCs showed enhanced OVA-specific OT-1 proliferation with fresh (29±10% CTFR diluted) and stored (25±2%) HOD RBCs.CONCLUSION: Cross priming by fresh RBCs was stronger than expected based on prior studies showing minimal RBC uptake by XCR DCs after fresh GFP + RBC transfusion. Cross priming in this system may be more sensitive due to the high frequency of antigen specific T cells and, thus, is saturated by the small antigen load delivered by fresh transfusion. It is also possible that direct uptake of RBCs by DCs harms the cross-presentation machinery and offsets the effect of increased RBC antigen delivery. Optimal cross-presentation of RBC antigen may require cooperation with cells functionally specialized for metabolizing RBC by-products. Indeed, the in-vitro study shows RPMs promote DC-mediated cross presentation of antigens on fresh and stored RBCs. Thus, RPMs may share ingested RBC antigen with adjacent DCs and increase DC activation by secreting inflammatory cytokines.. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Peripheral Tolerance Regulates RBC-specific CD4+ T Cells in a Novel Mouse

Abstract T cell tolerance mechanisms towards tissue-restricted antigens are well described and include deletion, anergy, or regulatory cells; however tolerance mechanisms against RBC-restricted antigens are poorly understood. Herein, we utilized the HOD mouse to investigate T cell tolerance to RBC-specific antigens. The HOD mouse expresses a fusion protein consisting of hen egg lysosyme (HEL), ovalbumin (OVA), and human blood group, Duffy driven by an RBC-specific promoter. The HOD mouse was crossed to TCR transgenic OTII mouse to generate HOD mice with autoreactive CD4+ T cells (OTII+HOD+). Young OTII+HOD+ mice do not have detectable autoantibodies, suggesting immune tolerance. Analysis of thymii 8 week-old mice revealed similar numbers of OTII CD4+ SP thymocytes between OTII+HOD+ and OTII+HOD−, suggesting that central tolerance plays little role in RBC specific CD4+ T cells. However, in the periphery, several tolerance mechanisms were identified in OTII+HOD+ mice. Autoreactive splenic CD4+ T cells in OTII+HOD+ mice underwent deletion, expressed higher levels of inhibitory markers PD-1, LAG3, CD5, and had higher numbers of CD25+Foxp3+ regulatory T cells, when compared to OTII+HOD−. Furthermore, CD4+ T cells from OTII+HOD+ had significantly reduced proliferation in response to cognate antigen, suggesting anergy or functional inactivation. Analysis of antigen presenting cells in OTII+HOD+ revealed higher frequencies of myeloid DC, known to express high levels of MHC II and propensity to activate CD4+ T cells. Finally, a break in tolerance was observed in 30% of OTII+HOD+ at 6 months, as indicated by anti-HOD antibodies. Future studies will focus on identifying defective tolerance mechanism that contributed to autoantibody genesis.

DOCK8-Deficient Mice Reveal a Crucial Role for Dendritic Cell Activity in the Initiation of the Allogeneic Response to Transfused Red Blood Cells

The generation of antibodies against transfused red blood cells (RBCs) can pose a serious health risk, especially in chronically transfused patients requiring life-long transfusion support; yet our understanding of what immune signals or cells dictate when someone will become alloimmunized is lacking. The relative role of dendritic cells, B cells and macrophages in the induction of RBC alloimmunization remain unclear. Given the now well established role of innate immune signals in regulating adaptive immunity, understanding if and how innate immunity is triggered during transfusion may allow development of therapies to prevent alloimmunization in chronically transfused subjects such as those with myelodysplasia or hemoglobinopathies. We have established a murine model system in which we can evaluate both the role of particular innate immune stimuli as well as particular cells of the immune system in regulating the allogeneic response to transfused RBCs. A particularly useful transgenic "HOD mouse" has been engineered, which encodes a triple fusion protein and provides a unique tool to directly assess both RBC-specific T and B cell responses. This RBC-specific antigen contains the model protein antigen hen egg lysozyme (HEL) fused to chicken ovalbumin (OVA) fused to the human Duffyb blood group antigen (HEL-OVA-Duffy) as an integral membrane protein under control of the beta globin promoter. Transfusion of genetically targeted mice lacking various innate immune cells or receptors allows us to screen for important immune pathways regulating the response to allogeneic RBCs.Using these models, we recently discovered that mice lacking the GEF (guanine nucleotide exchange factor) DOCK8 fail to develop alloimmunity to transfused RBCs. Dendritic cells in these knockout mice fail to migrate to T cells due to lack of coordinated actin rearrangement governed by this GEF. Both B cell and T cell activation in the spleen to the transgenic transfused RBCs is abrogated. Inclusion of OVA in the alloantigen of the HOD mice allows us to readily study naïve CD4+ T cell activation following transfusion by using the OTII T cell receptor (TCR) transgenic mice in which essentially all T cells express one antigen receptor specific for a peptide of OVA. By tracking rounds of cell division we found that adoptively transferred OTII undergo more than 5-8 rounds of division in the spleen three days following transfusion of HOD RBCs in WT recipients. In contrast, no OTII proliferation was observed in DOCK8-deficient mice following OTII adoptive transfer and HOD RBC transfusion, suggesting that T cells are failing to receive activation signals by splenic antigen presenting cells. Our preliminary data now suggest that DOCK8-deficient dendritic cells are able to process and present RBC-derived antigens, but do not migrate to T cell zones in the spleen to prime naïve RBC-specific T cells. The need for dendritic cell migration within the spleen in the induction of alloimmunity to transfused RBCs has not been addressed; these mice allow us for the first time to answer these fundamental immunologic questions during transfusion. Future work will aim to determine how dendritic cell movement within the spleen is regulated during transfusion and the specific role of splenic dendritic cell subsets in CD4+ T cell priming to allogeneic RBCs. DisclosuresNo relevant conflicts of interest to declare.

IgG-Mediated Immune Suppression to Erythrocytes by Polyclonal Antibodies Can Occur in the Absence of Activating or Inhibitory Fcγ Receptors in a Full Mouse Model.

Polyclonal anti-D has been used to prevent RhD-negative mothers from becoming immunized against RhD positive fetal erythrocytes, and this mechanism has been referred as Ab or IgG-mediated immune suppression (AMIS). Although anti-D has been highly successful, the inhibitory mechanisms remain poorly understood. Two major theories behind AMIS involve the binding of IgG to activating or inhibitory FcγR, which can induce either erythrocyte clearance or immune inhibition, respectively. In this work, we explored the absolute role of activating and inhibitory FcγR in the AMIS mechanism using the HOD mouse model of RBC immunization. HOD mice contain a RBC-specific recombinant protein composed of hen egg lysozyme (HEL), OVA and human transmembrane Duffy Ag, and erythrocytes from HOD mice can stimulate an immune response to HEL. To assess the contribution of activating and inhibitory FcγR to AMIS, C57BL/6 versus FcRγ-chain(-/-) or FcγRIIB(-/-) mice were used as recipients of HOD-RBC alone or together with anti-HEL Abs (i.e., AMIS) and the resulting immune response to HEL evaluated. We show that anti-HEL polyclonal Abs induce the same degree of AMIS effect in mice lacking these IgG binding receptors as compared with wild-type mice. In agreement with this, F(ab')2 fragments of the AMIS Ab also significantly reduced the Ab response to the HOD cells. In conclusion, successful inhibition of in vivo Ab responses to HOD-RBC by polyclonal IgG can occur independently of activating or inhibitory FcγR involvement. These results may have implications for the understanding of RhD prophylaxis.

Hod mice and the Mouse Set Conjecture

Introduction Hod mice Comparison theory of Hod mice Hod mice revisited Analysis of HOD Hod pair constructions A proof of the mouse set conjecture Appendix A. Descriptive set theory primer Index Bibliography

Regulatory T Cells Are Not Required for Prevention of RBC-Specific Autoantibody Generation

Introduction: Loss of humoral tolerance to red blood cell (RBC) antigens may lead to the generation of pathogenic autoantibodies and result in autoimmune hemolytic anemia (AIHA), a severe and potentially fatal disease. Failure of tolerance to RBC antigens occurs with considerable frequency (1-3 cases/1,000 adults) and prevalence of AIHA is as high as 30% in persons with compromised B and/or T cell tolerance mechanisms. However, RBC-specific tolerance mechanisms are poorly understood. To elucidate the immune tolerances to RBC autoantigens, we utilized HOD mice. The HOD mouse expresses an RBC-specific transgene consisting of hen egg lysozyme (HEL), ovalbumin (OVA), and Duffy. Using the HOD model, we previously demonstrated B cell tolerance to RBC-specific HOD antigen is incomplete; however, T cell tolerance is stringent. HOD mice have similar detectable frequencies of HOD-specific CD4+ T cells compared to B6 mice. Although present, autoreactive HOD-specific CD4+ T cells are non-functional. Circumventing T cell tolerance by adoptive transfer, HOD mice make high titer anti-HOD autoantibodies in vivo. Thus, despite the presence of autoreactive B cells, no HOD-reactive antibodies are detectable unless CD4+ T cells are given, indicating T cell tolerance is a stopgap to autoimmunity. Methods: Leukocytes from C57BL/6 (B6) and HOD mice were harvested and OVA-specific CD4+ T cell responses were assessed by tetramer-pulldown assays with pooled tetramers I-Ab-OVA 329-337/326-334. Isolated cells were stained for surface and intracellular markers and analyzed via flow cytometry. For in vivo analysis, mice were treated with 300ug anti-CD25 (clone PC-61) depleting antibody or isotype control; a subset of antibody-treated mice was immunized with OVA/CFA. Antibodies bound to HOD RBCs were determined by direct antibody test. Anti-HOD antibodies were quantified by indirect immunofluorescence using HOD RBCs as targets. Results: Tetramer pull-down assays revealed similar numbers of OVA-reactive CD4+ T cells from HOD and B6 mice (mean 56 and 40, respectively, p = 0.3). However, cell surface and intracellular marker staining demonstrated that HOD mice had higher numbers of OVA-tetramer reactive CD4+ T cells that express regulatory markers CD25 and FoxP3, and exhaustion marker PD1 as compared to control B6 mice. Inhibitory CTLA4 expression was not detectable on OVA-reactive CD4+ T cells from HOD or B6 mice. To test whether regulatory T cells were required for RBC-specific immune tolerance, HOD and B6 mice were treated with CD25 depleting antibody or isotype control antibody. Anti-CD25 antibody treated mice had a significant reduction of CD25+ cells 4 days post treatment (p &lt; 0.001, 2 independent experiments). Similarly, there was a significant reduction in FoxP3+CD25+CD4+ T cells (Tregs) in anti-CD25 treated mice (p &lt; 0.001), compared to isotype. Mice received weekly injections of anti-CD25 or isotype antibody to maintain depletion for one month. A subset of mice received an OVA/CFA immunization. Sustained CD25+ depletion did not result in anti-HOD autoantibody generation. Further, there was no change in the endogenous frequency of OVA-reactive CD4+ T cells between HOD and B6 mice, regardless of antibody treatment. Similarly, HOD mice treated with depletion (or isotype) antibody and immunized with OVA/CFA did not make detectable anti-HOD autoantibodies. Consistent with lack of detectable autoantibodies, no expansion of OVA-tetramer reactive CD4+ T cells was observed in HOD mice. In contrast, B6 mice (treated with anti-CD25 or isotype antibody) had a detectable expansion of OVA-specific CD4+ T cells as a result of immunization. Conclusions: The data demonstrate a phenotypic difference between the OVA-reactive CD4+ T cells from HOD and B6 mice, with an increase in number of Tregs detectable in HOD mice. Administration of anti-CD25 antibody significantly reduced the number of overall CD25+ cells and Tregs. Prolonged depletion of these cellular subsets did not elicit autoantibodies in HOD mice. Further, immunization of CD25 depleted mice with a strong immune stimulus (OVA/CFA, known to expand OVA-reactive T cells in B6 mice), did not induce anti-HOD autoantibodies nor did it expand OVA-specific autoreactive CD4+ T cells in HOD mice. Together, these data demonstrate that CD25+ cells are not required for the maintenance of RBC-specific T cell tolerance and suggest a role for other regulatory mechanisms. Disclosures No relevant conflicts of interest to declare.

NLRP10-Deficient Mice Reveal a Crucial Role for Dendritic Cell Activity in the Initiation of the Allogeneic Response to Transfused Red Blood Cells

The generation of antibodies against transfused red blood cells (RBCs) can pose a serious health risk, especially in chronically transfused patients requiring life-long transfusion support; yet our understanding of what immune signals or cells dictate when someone will become alloimmunized is lacking. Every non-autologous red cell unit has multiple antigens foreign to the transfused recipient; some people respond to these foreign antigens with an adaptive immune response and some do not. Given the now well established role of innate immune signals in regulating adaptive immunity, understanding if and how innate immunity is triggered during transfusion may allow development of therapies to prevent alloimmunization in chronically transfused subjects such as those with myelodysplasia or hemoglobinopathies. We have established a murine model system in which we can evaluate both the role of particular innate immune stimuli as well as particular cells of the immune system in regulating the allogeneic response to transfused red blood cells. A particularly useful transgenic “HOD mouse” has been engineered, which encodes a triple fusion protein and provides a unique tool to directly assess both RBC-specific T and B cell responses. This RBC-specific antigen contains the model protein antigen hen egg lysozyme (HEL) fused to chicken ovalbumin (OVA) fused to the human Duffybblood group antigen (HEL-OVA-Duffy) as an integral membrane protein under control of the beta globin promoter. Transfusion of genetically targeted mice lacking various innate immune receptors allows us to screen for important immune pathways regulating the response to allogeneic RBCs.Using these models, we recently discovered that mice lacking the NOD-like receptor NLRP10 fail to develop alloimmunity to transfused red blood cells. Surprisingly, the early innate immune cytokine response, including IL-6, IL-1beta and TNF-alpha, was unaffected in mice lacking NLRP10. Yet both B cell and T cell activation in the spleen to the transgenic transfused RBCs was abrogated. Inclusion of OVA in the alloantigen of the HOD mice allows us to readily study naïve CD4+ T cell activation following transfusion by using the OTII T cell receptor (TCR) transgenic mice in which essentially all T cells express one antigen receptor specific for a peptide of OVA. By tracking rounds of cell division we found that adoptively transferred OTII undergo more than 5-8 rounds of division in the spleen three days following transfusion of HOD RBCs in WT recipients. In contrast, no OTII proliferation was observed in NLRP10-deficient mice following OTII adoptive transfer and HOD RBC transfusion, suggesting that T cells are failing to receive activation signals by splenic antigen presenting cells. We have previously demonstrated that NLRP10-deficient dendritic cells fail to migrate from peripheral tissues such as the skin to draining lymph nodes. Our preliminary data now suggest that NLRP10-deficient dendritic cells are able to process and present RBC-derived antigens, but do not migrate to T cell zones in the spleen to prime naïve RBC-specific T cells. The relative role of dendritic cells, B cells and macrophages in the induction of erythrocyte alloimmunization remain unclear. Further, the need for DC migration within the spleen in the induction of alloimmunity to transfused RBCs has not been addressed. These mice allow us for the first time to answer these fundamental immunologic questions during transfusion. Future work will aim to determine how dendritic cell movement within the spleen is regulated during transfusion in NLRP10-deficient mice and the specific role of splenic dendritic cells in CD4+ T cell priming to allogeneic RBCs. DisclosuresNo relevant conflicts of interest to declare.

Cellular Protection Against Antibodies Occurs Through a Complement-Independent Pathway

Abstract 3290 Background:Although mechanisms exist whereby T cells differentiate self from non-self, occasionally tolerance breaks down and T cells target self-antigens. Under these circumstances, healthy cells possess unique receptors that serve to inhibit potential cell-mediated destruction. However, unlike cell-mediated tissue injury, antibody-secreting cells elicit their effects at remote sites, precluding direct feedback from target tissue to secreting cells. As a result, target cells appear to have evolved mechanisms of inhibiting antibody-mediated destruction. Indeed, patients with reduced levels of complement inhibitory factors experience severe hemolysis following antibody engagement. However, regulatory components responsible for protection against complement-independent antibody effecter functions remain poorly understood. RBCs provide a unique tool in the examination of cellular fate following antibody engagement as RBCs fail to synthesize new antigen and also fail to proliferate. As a result, we utilized a model of antibody-RBC binding to determine whether RBCs possess complement-independent mechanisms of cellular resistance to antibody-induced hemolysis. Methods:B6, C3 KO or FcγR KO mice were passively immunized with an anti-Fy3 IgG2a monoclonal antibody. Fluorescent dye labeled RBCs that transgenically express a chimeric antigen including the Fy3 epitope from human Duffy (HOD mice) were then transfused into immunized or nonimmunized control mice. Over a timecourse, peripheral blood was obtained and transfused RBCs were visualized by flow cytometry, followed by examination of clearance, surface C3, bound IgG and Fy3 antigen. To discriminate old from new RBCs following transfusion, RBCs were harvested from mice 35 days post-biotinylation, transfused into immunized or nonimmunized mice and examined for differential clearance by strepavidin staining and flow cytometry. To further evaluate RBC resistance to antibody-induced clearance, HOD RBCs were harvested 2 days following transfusion and re-transfused into immunized or nonimmunized mice followed by examination for clearance, surface C3, bound IgG and Fy3 antigen. Results:Immunized mice rapidly cleared over half of HOD RBCs within 2 hours; however, a significant number of resistant cells remained in circulation despite the persistence of antibody levels capable of clearing additional RBCs following subsequent transfusion. No clearance was observed in FcγR KO mice but normal clearance and resistance was observed in C3 KO mice. Moreover, no complement degradation products were detected following transfusion into wt mice. RBCs 35 days and older displayed comparable antibody-induced clearance as younger RBCs, suggesting that resistance did not reflect preferential loss of older RBCs. Furthermore, HOD RBCs remained resistant to clearance following re-transfusion into recently immunized recipients. Examination of HOD antigen levels demonstrated a rapid decrease in HOD antigen that correlated with the development of resistance. Similar to clearance, HOD antigen levels decreased in C3 KO but not FcγR KO. Conclusion:These results suggest that in addition to known complement-dependent mechanisms of cellular resistance to antibody-induced hemolysis, a novel pathway exists whereby Fcγ receptors not only participate in clearance, but also appear to facilitate specific loss of antibody reactive antigen, which may be responsible for a population of RBCs resistant to further clearance. Taken together, these results indicate that RBCs possess multiple mechanisms for cellular protection against antibodies that may be relevant in autoimmune hemolytic anemia and other antibody-mediated processes. Disclosures:No relevant conflicts of interest to declare.

Central T Cell Tolerance and Peripheral B Cell Tolerance for An RBC Autoantigen Are Incomplete in Healthy Mice; Implications for AIHA Pathogenesis

Abstract 693 Introduction:Breakdown of humoral tolerance to red blood cell (RBC) antigens can result in autoimmune hemolytic anemia (AIHA), a severe and potentially fatal disease. The pathogenesis of AIHA is poorly understood. To investigate the baseline biology of tolerance to self-antigens expressed on RBCs, we utilized a murine transgenic mouse with RBC-specific expression of a model antigen consisting of a triple fusion protein of hen egg lysozyme (HEL), ovalbumin (Ova), and human blood group molecule Duffy; HEL-OVA-Duffy (HOD mouse). Methods:Wild-type C57BL/6 (B6) mice or HOD mice (on a B6 background) were immunized with HEL/CFA or OVA/CFA to test immune responses to antigens contained within HOD. Some animals were immunized with peptides as opposed to whole protein. Anti-HOD antibodies were quantified by indirect immunofluorescence using HOD RBCs as targets. Anti-HEL IgG was quantified by ELISA and anti-HEL secreting B cells were enumerated by ELISPOT. CD4+ T cell responses were assessed by tetramer staining and tetramer pull-down assays using I-Ab-OVA-329-337/326-334. T cell tolerance was specifically broken by adoptive transfer of OT-II CD4+ T cells into HOD mice (OT-II T cells recognize OVA323-339 presented by I-Ab). Effects of HOD antigen expression on B cell development were evaluated by crossing the HOD mouse with an anti-HEL BCR knockin mouse (SwHEL mouse) that is capable of normal class switching. Results:Immunization of B6 mice with OVA/CFA induced high titer antibodies reactive with HOD RBCs; in contrast, no anti-HOD was detected in HOD mice immunized with OVA/CFA. Similarly, no anti-HEL was detected in HOD mice immunized with HEL/CFA, whereas wild-type B6 mice had high anti-HEL titers (p<0.05). These data demonstrate overall humoral tolerance to the HOD antigen. Using pull-down assays, OVA-tetramer reactive T cells were detected in both B6 and HOD mice, with similar endogenous frequencies (mean numbers are 40 and 53 T cells, respectively; at least 6 mice analyzed), suggesting that central tolerance did not eliminate HOD reactive T cells. However, upon immunization with OVA peptide, B6 but not HOD mice had a detectable expansion of OVA-tetramer reactive CD4+ T cells, indicating that peripheral tolerance was preventing HOD autoreactive CD4+ T cells from participating in an immune response. To assess B cell tolerance to the HOD antigen, T cell tolerance was circumvented through adoptive transfer or OTII splenocytes (specific for the OVA323-339 peptide) into HOD mice. Anti-HEL autoantibodies were detected in HOD mice but not control B6 mice (p<0.001). Antibody production correlated with a 10–20 fold increase of anti-HEL antibody secreting cells, as determined by ELISPOT. Autoantibody production in HOD mice was not due to passenger B cells from the OTII donor, an artifact of excess CD4+ T cell number, or bystander activation as no autoantibodies were observed upon adoptive transfer with OTIIs on a Rag knockout background, irrelevant CD4+ T cells from SMARTA mice, or activated CD4+ T cells from TCR75 mice. To test the effects of HOD antigen expression on development of autoreactive B cells, HOD mice were crossed with SwHEL BCR transgenic mice (that express anti-HEL) and the F1 mice were analyzed. HEL-reactive B cells were visualized using multimeric HEL conjugated to allophycocyanin. In HOD-SwHEL+ mice, approximately 46±14% of immature bone marrow B cells were reactive with HEL, compared to 15±12% in HOD+SwHEL+ mice (p=0.043, 3 independent experiments, 5 mice total). Conclusions:These data demonstrate that tolerance to an RBC specific antigen is complete in the CD4+ T cell, but not the B cell compartment. CD4+ T cell tolerance appears to be more an effect of peripheral tolerance than central deletion, as OVA-tetramer reactive CD4+ T cells were visible in HOD mice but did not activate upon immunization with their cognate antigen. In contrast, while the HODxSwHEL F1 mice demonstrate that some B cell tolerance to HOD occurs, the induction of autoantibodies by introducing CD4+ autoreactive T cells (OT-II) demonstrates that B cell tolerance to the HOD antigen is incomplete in HOD mice. Together, these data suggest that a breakdown in T cell tolerance is all that is required for the pathogenesis of AIHA. As the T cell tolerance appears not to be deletional, it is predicted that environmental factors leading to a breakdown in peripheral tolerance of CD4+ T cells would be sufficient to induce AIHA. Disclosures:Zimring:Immucor Inc,: Research Funding.

Mini-Workshop: Feinstrukturtheorie und Innere Modelle

This workshop presented recent advances in fine structure and inner model theory. There were extended tutorials on hod mice and the Mouse Set Conjecture, suitable extender sequences and their fine structure, and the construction of true K below a Woodin cardinal in ZFC. The remaining talks involved precipitous ideals, stationary set reflection, failure of SCH in ZF, nonthreadable square sequences, reverse mathematics, forcing axioms, covering properties of canonical inner models, and “set theoretic geology.”

Activation and Effector Phenotype of CD8+ T Cells Crossprimed by a Minor Histocompatibility Antigen Expressed on Red Blood Cells

Background: Transfusion immunology research has traditionally focused on humoral immunity to RBC antigens, with study of CD4+ T cell and B cell responses. However, minor histocompatibility antigens (mHAs) on transfused RBCs in theory can also be cross-presented by recipient APCs to CD8+ T cells. This has clinical relevance in the context of transfusion patients who undergo transplantation, as several lines of evidence suggest that immunization to mHAs can lead to rejection of bone marrow and solid organ transplants. To test the hypothesis that mHAs on RBCs are capable of inducing immune responses through cross priming of CD8+ T cells, we created a novel transgenic mouse (HOD mouse), which expresses Ovalbumin (OVA) on RBCs but neither on leukocytes nor platelets.Methods: 1×107 OT-I splenocytes (TCR transgenic for the OVA derived peptide SIINFEKL presented by H-2Kb) were adoptively transferred into B6.Thy1.1 mice. 100μl of packed HOD RBCs from whole or leukoreduced blood were then transfused intravenously. Control mice were transfused with FVB whole blood (HOD mice are on a FVB background). Three to 4 days post transfusion the OT-I T cells were visualized in the spleen by staining with anti-CD8 antibody and the Kb/SIINFEKL tetramer. Proliferation was evaluated by CFSE dilution. The activation phenotype was studied by staining with antibodies against CD44 and CD62L. Expression of effector molecules was studied by staining with antibodies against CD107a, Granzyme B, IFNγ, and IL-2.Results: Compared to background analysis of OT-I T cells in control mice that received FVB blood, OT-I T cells in HOD recipients had a maximal expansion 24 fold higher in mice transfused with HOD leukoreduced blood (p=.0001), and 26 fold in mice transfused with HOD whole blood (p=.0672). Compared to naïve OT-I T cells, cross-primed OT-I T cells were CD44high, CD62Llow, CD107ahigh, suggesting activation and degranulation. Expression of IFNγ was also increased. However, cross-primed OT-I T cells were Granzyme Blow and did not have increased IL-2 expression. Lack of increased Granzyme B and IL-2 was not an artifact of the assay not working, as strong expression of both Granzyme B and IL-2 were detected in OT-I T cells stimulated with OVA in vitro.Discussion: Overall these results demonstrate that RBC antigens can be cross-primed into the MHC class I pathway by recipient APCs. However, activation of CD8+ T cells specific for the RBC antigen demonstrates differentiation into an incomplete effector phenotype. The effect is unlikely due to contaminating donor leukocytes in the transfused unit since OVA is not expressed by WBCs. Moreover, we did not see any differences in proliferation or activation phenotype between the mice transfused with whole blood and leukoreduced blood. Activation was not due to direct presentation by residual cells, as the HOD donor mice are on an FVB (H-2q) background and cannot directly present mHAs to the H-2b restricted OT-I T cells. The reason for an incomplete effector profile is unclear, but may be because transfused RBCs are a sterile introduction of foreign antigen. These results also suggest better leukoreduction is not a viable solution to the problem of transfusion-induced immunization to mHAs, as the antigen is coming from the RBCs themselves. Matching for relevant mHAs or use of immunomodulatory treatments may be required.

A Novel Model of Autoimmunity to Self Red Blood Cell Antigens

Background: Autoimmune hemolytic anemia (AIHA) represents a serious and potentially life-threatening illness. However, the mechanisms by which red blood cell (RBC) autoreactive B cells escape tolerance and subsequently receive the signals required to differentiate into plasma cells are unknown. It has been reported that B1 B cells, a special B cell lineage that arises from gut lymphatics, appear to be resistant to self-tolerance to RBC antigens. However, the existing murine models do not allow experimental juxtaposition of animals with and without the autoantigen. Moreover, in existing models the mice develop AIHA with systemic activation of complement, crosslinking of Fc receptors, phagocytosis of RBCs, and generation of large quantities of hemoglobin breakdown products. Although these events are relevant to the pathology of AIHA, they obscure the underlying immunology of the system. To circumvent these problems, we have designed a novel model of autoimmunity to self RBC antigens that allows the analysis of B cell tolerance in the absence of ongoing hemolytic pathology.Materials/Methods: We generated the HOD mouse, which has RBC specific expression of the model humoral antigen hen egg lysozyme (HEL), linked to the cell membrane by a human blood group antigen (Duffy). HOD mice were crossed with B cell receptor transgenic IgHEL mice that express IgM specific for the HEL antigen. Heterozygotes were used for breeding to generate progeny that include HOD positive × IgHEL positive (“double positives”) and HOD negative × IgHEL positive (“controls”). Anti-HEL expressing B cells were identified by flow cytometry in spleen, bone marrow, and lymph nodes by the anti-HEL transgenic allotype (IgMa) and CD19 expression. B cell maturation was monitored by B220 expression. B1 B cells in the peritoneal cavity were identified utilizing CD11b (anti-MAC1). Serum anti-HEL IgM was determined by flow cytometric cross matching and HEL-specific ELISA (IgG is not secreted by anti-HEL BCR transgenic mice as they do not class switch).Results: Double positive mice had significantly fewer B cells expressing anti-HEL (CD19+, IgMa+) than did control mice (7 fold fewer in spleen, 4 fold fewer in bone marrow, and essentially none in lymph nodes, as measured by % of total lymphocytes). These differences were observed predominantly in mature (B220 high) but not immature (B220 low) B cells. In contrast, while there was a dramatic decrease of conventional B cells in the peritoneum of double positive mice, B1 B cells persisted. These observations were not an artifact of expression of the HOD transgene, as no differences were seen in control HOD positive × IgHEL negative mice. Analysis of serum demonstrated that anti-HEL IgM levels in double positive mice were approximately 50% greater than that of control mice.Conclusions: Utilizing our novel model of B cell tolerance to RBC autoantigens, we report a decrease of self reactive B cells in the spleen, bone marrow, and lymph nodes. This difference is likely generated during or after B cell maturation, as the decrease is restricted to B220 high mature B cells. However, autoreactive B1 B cells escape deletion in the double positive mice and persist in the peritoneum. Although the absolute number of self reactive B cells is significantly lower in the double positive as compared to control mice, autoantibody levels (anti-HEL IgM) are higher. Thus, the small percentage of autoreactive B cells that escape tolerance likely produce large amounts of autoantibody. Unlike models previously described by other investigators, the mice that have lost anti-RBC tolerance described herein show no signs of illness and have a hematocrit similar to that of control mice. Thus, our model provides a unique opportunity to perform well controlled studies on the mechanisms of tolerance to self-RBC antigens in the absence of systemic immune activation that occurs during clinical AIHA. Ongoing studies are investigating the role of B1 B cells on autoimmunity in this model system, as well as potential therapies to eliminate such cells.