Transgenic CD8 Research Articles

Lung Parenchyma–Derived IL-6 Induces Alloantigen Specific Th17 Differentiation Within The Lung and Idiopathic Pneumonia Syndrome After Allogeneic Stem Cell Transplantation

Idiopathic Pneumonia Syndrome (IPS) is a fulminant and largely untreatable form of GVHD of the lung following allogeneic stem cell transplantation (SCT). It characteristically develops in the third week after SCT in patients receiving prior total body irradiation (TBI). We have previously shown that IFNγ signaling within lung parenchyma is critical for preventing disease (Burman AC et al, Blood 2007, 110:1064). Using IL-17A-fate reporter grafts (where YFP is expressed if a T cell has ever made IL-17) we now show that Th17 cells preferentially accumulate in the lung (but not other sites) after experimental allo-SCT in recipients in which IFNγ is neutralized (lung Th17 cells: 6.6x104 ± 0.2 vs. 0.8x104 ± 0.01, anti-IFNγ vs. control treated, P = 0.002). To understand whether this accumulation of Th17 cells in the lung and IPS in general was an alloreactive (i.e. GVHD) dependent phenomena, we transplanted luciferase expressing B6 TEa TCR transgenic CD4+ T cells specific for alloantigen (I-Ed) expressed in B6D2F1 recipients in the presence or absence of IFNγ neutralization. Remarkably, allospecific donor TEa T cells again accumulated in the lung in the absence of IFNγ (BLI: 2.2x105 ± 0.6 vs. 0.54x105 ± 0.08 ph/sec/cm2/sr, P = 0.008), but not other sites, excluding responses to other antigens (e.g. pathogen-derived) as a cause for IPS. Critically, we show that donor T cell-derived IFNγ was vital for inhibiting IL-6, TGFβ and IL-21 mRNA expression in lung parenchymal cells after SCT. Protein analysis confirmed that IL-6 was markedly elevated in lung tissue lysate 5 days after allo-SCT in IFNγR–/– recipients compared to WT (128 ± 25 pg/ml vs. 20 ± 5 pg/ml, P = 0.002). In contrast, IL-6 was elevated to equivalent levels in sera of both WT and IFNγR–/– SCT recipients. Studies using IL-6–/– recipients demonstrated that IL-6 was critical for Th17 differentiation within the lung and subsequent IPS. In the absence of IFNγ, IL-6 promoted Th17 cells to preferentially expand in the lungs (14.6x104 ± 0.03 vs. 3.8x104 ± 0.01, WT vs. IL-6–/–, P = 0.04), produce IL-17A (3734 ± 736 pg/ml vs. 521 ± 142 pg/ml, WT vs. IL-6–/–, P = 0.003) and induce IPS (lung pathology scores: 7.3 ± 0.9 vs. 5.0 ± 0.3, WT vs. IL-6–/–; P = 0.02). Consistent with Th17 differentiation as the final pathological pathway, systemic blockade of IL-17A or the transplantation of grafts lacking the IL-17 receptor also profoundly attenuated disease (lung pathology scores: 3.9 ± 0.5 vs. 6.7 ± 0.6, anti-IL-17A vs. control treated, P = 0.002; and 4.5 ± 0.4 vs. 8.1 ± 1.0, IL-17RA–/–- vs. WT, P= 0.003, respectively). Thus IFNγ is the critical cytokine for suppressing an environment conducive to Th17 differentiation within the lung and IPS after allogeneic SCT.To study the clinical relevance of these findings we examined IL-6 dysregulation in a cohort of patients receiving allogeneic peripheral blood stem cell transplantation at our institution. We confirm that plasma levels of IL-6 are also significantly elevated in SCT recipients within the first 2 weeks of transplantation (30.6 ± 8.8 pg/ml vs. 8.3 ± 3.5 pg/ml, day +7 vs. day -7, P = 0.008), particularly in patients receiving Cy/TBI (12 Gy) compared to reduced intensity Flu/Mel (120mg/m2) conditioning (74.4 ± 38.4 pg/ml vs. 23.4 ± 7.1 pg/ml, P = 0.04). Furthermore, standard calcineurin and methotrexate–based immune suppression completely prevented systemic IFNγ generation after SCT, thus inducing an IL-6–high, IFNγ–deplete environment highly conducive to the development of IPS. These data confirm IL-6 and IL-17 as logical therapeutic targets to prevent IPS after clinical SCT utilizing TBI-based conditioning. Disclosures:No relevant conflicts of interest to declare.

The RUNX1-Regulated Cebpa +37 Kb Enhancer Directs Human CD4 Transgene Expression To Long-Term Hematopoietic Stem Cells and Preferentially To Myeloid Compared With Lymphoid Or Erythroid Progenitors

We found that adult mice lacking RUNX1 have increased monopoiesis relative to granulopoiesis correlated with reduce Cebpa RNA expression in myeloid progenitors and that RUNX1 binds and activates Cebpa reporters via two sites in the Cebpa promoter and four conserved sites in a 450 bp +37 kb enhancer (Guo et al, Blood, 2012). This enhancer interacts with p300 and contains the H3K4me1 enhancer mark in myeloid cells. Previously reported ChIP-Seq data had demonstrated interaction of RUNX1 with the +37 kb enhancer, along with GATA2, SCL, Gfi-1b, MEIS1, Fli-1, ERG, PU.1, Lyl1, and LMO2. We have now generated transgenic C57BL/6 mice in which the Cebpa +37 kb enhancer positioned upstream of its 845 bp promoter (-720 to +125) directs expression of cytoplasmically truncated human CD4 (hCD4). Two founders with germ line transmission have been identified – both have detectable hCD4 RNA in marrow, at approximately 10-fold increased levels in one line compared to the other, with hCD4 expression detectable by FACS only in the higher expressing line. Analysis of 4-7 mice reveals that hCD4 expression is detected only in a small subset of mature hematopoietic cells, present in 6% of Mac-1+Gr-1+ neutrophils, 31% of Mac-1+Gr-1- monocytes, 4% of CD3+ T cells, 3% of CD19+ or 7% of B220+ B-cells, and 5% of Ter119+ erythroid cells. 6.5+/-1.9% of total marrow mononuclear cells and 13+/-4% of Lin- marrow cells express hCD4. At the progenitor level, hCD4 was evident in 56+/-7% of CMP, 70+/-5% of GMP, 36+/-9% of CLP, and only 1.5+/-1.0% of MEP. There was>100-fold enrichment of myeloid CFU but a 2-fold decrease in B-lymphoid colonies comparing hCD4+ versus hCD4- fractions of total marrow. Staining LSK cells for CD34 and FcγR demonstrated that 91+/-10% of multi-potent progenitors (MPP), 61+/-8% of ST-HSC, and 17+/-8% of LT-HSC express hCD4. Similarly, staining for the SLAM markers demonstrated that 19+/-9% of LSK-CD48-CD150+ LT-HSC express hCD4. Mean fluorescence index of hCD4 staining was about 2-fold higher in LSK, MPP, ST-HSC, CMP, and GMP compared with CLP and LT-HSC and about 3-fold higher than mature blood myeloid cells. 2E6 hCD4+ or hCD4- CD45.2 marrow cells were transplanted into lethally irradiated CD45.1 syngeneic recipients. 5/5 mice receiving hCD4- cells died at 2 weeks, reflecting lack of even progenitor engraftment, whereas 5/5 mice receiving hCD4+ cells were alive at 20 weeks, reflecting expression of the transgene in functional LT-HSC, with secondary transplanted mice viable at 4 week to date. The marrow cells of surviving mice were almost exclusively CD45.2, with hCD4+ expression in Lin- or Lin-c-kit+ marrow cells similar to that of donor mice. Although our second founder does not allow hCD4 detection by FACS, analysis of hCD4 RNA in marrow stem and progenitor subsets for this line is in progress, as is competitive transplantation of hCD4- versus hCD4+ marrow cells from the higher-expressing line. Murine E13.5 fetal liver hematopoietic stem and progenitor cells express hCD4 in a pattern similar to adult marrow. In zebrafish, the murine Cebpa +37 kb enhancer directs ZsGreen expression specifically to the caudal hematopoietic territory (CHT), analogous to the murine fetal liver, at 72 hour post-fertilization. Murine Cebpa +37 kb enhancer:ZsGreen transgene expression, lacking the Cebpa promoter, overlaps in the CHT with that of murine Runx1 +23 kb enhancer:mCherry in compound transgenic fish. The Cebpa and Runx1 enhancers direct similar tdTomato expression from a Sleeping Beauty vector in the K562 cell line, and these vectors are being compared for expression in hESC derived hematopoietic cells. In summary, the Cebpa gene contains an enhancer active in both LT-HSC and myeloid progenitors. Further characterization of its regulation may provide insights into the development of these hematopoietic stem and progenitor subsets and into how Cebpa expression is impaired in myelodysplastic syndromes or in acute myeloid leukemia. In addition, the Cebpa enhancer may prove useful in efforts to optimize LT-HSC formation from hESC. Disclosures:Zon:FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Stocks, Stocks Other; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Excitotoxic neuronal cell death during an oligodendrocyte-directed CD8+ T cell attack in the CNS gray matter

BackgroundNeural-antigen reactive cytotoxic CD8+ T cells contribute to neuronal dysfunction and degeneration in a variety of inflammatory CNS disorders. Facing excess numbers of target cells, CNS-invading CD8+ T cells cause neuronal cell death either via confined release of cytotoxic effector molecules towards neurons, or via spillover of cytotoxic effector molecules from 'leaky’ immunological synapses and non-confined release by CD8+ T cells themselves during serial and simultaneous killing of oligodendrocytes or astrocytes.MethodsWild-type and T cell receptor transgenic CD8+ T cells were stimulated in vitro, their activation status was assessed by flow cytometry, and supernatant glutamate levels were determined using an enzymatic assay. Expression regulation of molecules involved in vesicular glutamate release was examined by quantitative real-time PCR, and mechanisms of non-vesicular glutamate release were studied by pharmacological blocking experiments. The impact of CD8+ T cell-mediated glutamate liberation on neuronal viability was studied in acute brain slice preparations.ResultsFollowing T cell receptor stimulation, CD8+ T cells acquire the molecular repertoire for vesicular glutamate release: (i) they upregulate expression of glutaminase required to generate glutamate via deamination of glutamine and (ii) they upregulate expression of vesicular proton-ATPase and vesicular glutamate transporters required for filling of vesicles with glutamate. Subsequently, CD8+ T cells release glutamate in a strictly stimulus-dependent manner. Upon repetitive T cell receptor stimulation, CD25high CD8+ T effector cells exhibit higher estimated single cell glutamate release rates than CD25low CD8+ T memory cells. Moreover, glutamate liberation by oligodendrocyte-reactive CD25high CD8+ T effector cells is capable of eliciting collateral excitotoxic cell death of neurons (despite glutamate re-uptake by glia cells and neurons) in intact CNS gray matter.ConclusionGlutamate release may represent a crucial effector pathway of neural-antigen reactive CD8+ T cells, contributing to excitotoxicity in CNS inflammation.

CD301b+ Dermal Dendritic Cells Drive T Helper 2 Cell-Mediated Immunity

Unlike other types of T helper (Th) responses, whether the development of Th2 cells requires instruction from particular subset of dendritic cells (DCs) remains unclear. By using an in vivo depletion approach, we have shown that DCs expressing CD301b were required for the generation of Th2 cells after subcutaneous immunization with ovalbumin (OVA) along with papain or alum. CD301b⁺ DCs are distinct from epidermal or CD207⁺ dermal DCs (DDCs) and were responsible for transporting antigen injected subcutaneously with Th2-type adjuvants. Transient depletion of CD301b⁺ DCs resulted in less effective accumulation and decreased expression of CD69 by polyclonal CD4⁺ T cells in the lymph node. Moreover, despite intact cell division and interferon-γ production, CD301b⁺ DC depletion led to blunted interleukin-4 production by OVA-specific OT-II transgenic CD4⁺ T cells and significantly impaired Th2 cell development upon infection with Nippostrongylus brasiliensis. These results reveal CD301b⁺ DDCs as the key mediators of Th2 immunity.

Differential migration of passenger leukocytes and rapid deletion of naive alloreactive CD8 T cells after mouse liver transplantation

Donor passenger leukocytes (PLs) from transplanted livers migrate to recipient lymphoid tissues, where they are thought to induce the deletion of donor-specific T cells and tolerance. Difficulties in tracking alloreactive T cells and PLs in rats and in performing this complex surgery in mice have limited progress in identifying the contribution of PL subsets and sites and the kinetics of T cell deletion. Here we developed a mouse liver transplant model in which PLs, recipient cells, and a reporter population of transgenic CD8 T cells specific for the graft could be easily distinguished and quantified in allografts and recipient organs by flow cytometry. All PL subsets circulated rapidly via the blood as soon as 1.5 hours after transplantation. By 24 hours, PLs were distributed differently in the lymph nodes and spleen, whereas donor natural killer and natural killer T cells remained in the liver and blood. Reporter T cells were activated in both liver and lymphoid tissues, but their numbers dramatically decreased within the first 48 hours. These results provide the first unequivocal demonstration of the differential recirculation of liver PL subsets after transplantation, and show that alloreactive CD8 T cells are deleted more rapidly than initially reported. This model will be useful for dissecting early events leading to the spontaneous acceptance of liver transplants.

Influenza A Virus Infection Impairs Mycobacteria-Specific T Cell Responses and Mycobacterial Clearance in the Lung during Pulmonary Coinfection

Individuals infected with mycobacteria are likely to experience episodes of concurrent infections with unrelated respiratory pathogens, including the seasonal or pandemic circulating influenza A virus strains. We analyzed the impact of influenza A virus and mycobacterial respiratory coinfection on the development of CD8 T cell responses to each pathogen. Coinfected mice exhibited reduced frequency and numbers of CD8 T cells specific to Mycobacterium bovis bacille Calmette-Guérin (BCG) in the lungs, and the IFN-γ CD8 T cell response to BCG-encoded OVA was decreased in the lungs of coinfected mice, when compared with mice infected with BCG alone. Moreover, after 2 wk of infection, mice coinfected with both pathogens showed a significant increase in the number of mycobacteria present in the lung compared with mice infected with BCG only. Following adoptive transfer into coinfected mice, transgenic CD8 T cells specific for OVA(257-264) failed to proliferate as extensively in the mediastinal lymph nodes as in mice infected only with BCG-OVA. Also noted was a reduction in the proliferation of BCG-specific CD4 transgenic T cells in mice coinfected with influenza compared with mice infected with BCG alone. Furthermore, phenotypic analysis of CD11c(+) dendritic cells from mediastinal lymph nodes of the infected mice showed that coinfection was associated with decreased surface expression of MHC class II and class I. Thus, concurrent pulmonary infection with influenza A virus is associated with decreased MHC expression on dendritic cells, reduced activation of BCG-specific CD4 and CD8 T cells, and impaired clearance of mycobacteria.

Immunosuppressive Property within the Streptococcus pneumoniae Cell Wall That Inhibits Generation of T Follicular Helper, Germinal Center, and Plasma Cell Response to a Coimmunized Heterologous Protein

We previously demonstrated that intact, inactivated Streptococcus pneumoniae (unencapsulated strain R36A) inhibits IgG responses to a number of coimmunized soluble antigens (Ags). In this study, we investigated the mechanism of this inhibition and whether other extracellular bacteria exhibited similar effects. No inhibition was observed if R36A was given 24 h before or after immunization with soluble chicken ovalbumin (cOVA), indicating that R36A acts transiently during the initiation of the immune response. Using transgenic cOVA-specific CD4(+) T cells, we observed that R36A had no significant effect on T-cell activation (24 h) or generation of regulatory T cells (day 7) and only a modest effect on T-cell proliferation (48 to 96 h) in response to cOVA. However, R36A mediated a significant reduction in the formation of Ag-specific splenic germinal center T follicular helper (GC Tfh) and GC B cells and antibody-secreting cells in the spleen and bone marrow in response to cOVA or cOVA conjugated to 4-hydroxy-3-nitrophenylacetyl hapten (NP-cOVA). Of note, the inhibitory effect of intact R36A on the IgG anti-cOVA response could be reproduced using R36A-derived cell walls. In contrast to R36A, neither inactivated, unencapsulated, intact Neisseria meningitidis nor Streptococcus agalactiae inhibited the OVA-specific IgG response. These results suggest a novel immunosuppressive property within the cell wall of Streptococcus pneumoniae.

Nematode-Derived Proteins Suppress Proliferation and Cytokine Production of Antigen-Specific T Cells via Induction of Cell Death

In order to establish long-lasting infections in their mammalian host, filarial nematodes have developed sophisticated strategies to dampen their host’s immune response. Proteins that are actively secreted by the parasites have been shown to induce the expansion of regulatory T cells and to directly interfere with effector T cell function. Here, we analyze the suppressive capacity of Onchocerca volvulus -derived excreted/secreted proteins. Addition of two recombinant O . volvulus proteins, abundant larval transcript-2 (OvALT-2) and novel larval transcript-1 (OvNLT-1) to cell cultures of T cell receptor transgenic CD4+ and CD8+ T cells suppressed antigen-specific stimulation in vitro. Ovalbumin-specific CD4+ DO11.10 and OT-II T cells that had been stimulated with their cognate antigen in the presence of OvALT-2 or OvNLT-1 displayed reduced DNA synthesis quantified by 3H-thymidine incorporation and reduced cell division quantified by CFSE dilution. Furthermore, the IL-2 and IFN-γ response of ovalbumin-specific CD8+ OT-I T cells was suppressed by OvALT-2 and OvNLT-1. In contrast, another recombinant O . volvulus protein, microfilariae surface-associated antigen (Ov103), did not modulate T cell activation, thus serving as internal control for non-ESP-mediated artifacts. Suppressive capacity of the identified ESP was associated with induction of apoptosis in T cells demonstrated by increased exposure of phosphatidylserine on the plasma membrane. Of note, the digestion of recombinant proteins with proteinase K did not abolish the suppression of antigen-specific proliferation although the suppressive capacity of the identified excreted/secreted products was not mediated by low molecular weight contaminants in the undigested preparations. In summary, we identified two suppressive excreted/secreted products from O . volvulus , which interfere with the function of antigen-specific T cells in vitro.

Dendritic cell activation, phagocytosis and CD69 expression on cognate T cells are suppressed by n‐3 long‐chain polyunsaturated fatty acids

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are bioactive n-3 long-chain polyunsaturated fatty acids (LCPUFAs) in fish oil that exert immunosuppressive effects. A significant amount of literature shows that n-3 LCPUFAs suppress dendritic cell (DC) function in vitro; however, few studies have determined if the effects are emulated at the animal level. In this study, we first focused on the functional consequences of 5% (weight/weight) fish oil on splenic CD11c(+) DCs. Administration of n-3 LCPUFAs, modelling human pharmacological intake (2% of total kcal from EPA,1·3% from DHA), to C57BL/6 mice for 3 weeks reduced DC surface expression of CD80 by 14% and tumour necrosis factor-α secretion by 29% upon lipopolysaccharide stimulation relative to a control diet. The n-3 LCPUFAs also significantly decreased CD11c(+) surface expression and phagocytosis by 12% compared with the control diet. Antigen presentation studies revealed a 22% decrease in CD69 surface expression on transgenic CD4(+) T lymphocytes activated by DCs from mice fed fish oil. We then determined if the functional changes were mechanistically associated with changes in lipid microdomain clustering or plasma membrane microviscosity with n-3 LCPUFAs, as reported for B and T lymphocytes. Fish oil administration to mice did not influence cholera-toxin induced lipid microdomain clustering or microviscosity, even though EPA and DHA levels were significantly elevated relative to the control diet. Overall, our data show that n-3 LCPUFAs exert immunosuppressive effects on DCs, validating in vitro studies. The results also show that DC microdomain clustering and microviscosity were not changed by the n-3 LCPUFA intervention used in this study.

Introduction of Exogenous T-cell Receptors Into Human Hematopoietic Progenitors Results in Exclusion of Endogenous T-cell Receptor Expression

Current tumor immunotherapy approaches include the genetic modification of peripheral T cells to express tumor antigen-specific T-cell receptors (TCRs). The approach, tested in melanoma, has led to some limited success of tumor regression in patients. Yet, the introduction of exogenous TCRs into mature T cells entails an underlying risk; the generation of autoreactive clones due to potential TCR mispairing, and the lack of effective negative selection, as these peripheral cells do not undergo thymic selection following introduction of the exogenous TCR. We have successfully generated MART-1-specific CD8 T cells from genetically modified human hematopoietic stem cells (hHSC) in a humanized mouse model. The advantages of this approach include a long-term source of antigen specific T cells and proper T-cell selection due to thymopoiesis following expression of the TCR. In this report, we examine the molecular processes occurring on endogenous TCR expression and demonstrate that this approach results in exclusive cell surface expression of the newly introduced TCR, and the exclusion of endogenous TCR cell surface expression. This suggests that this stem cell based approach can provide a potentially safer approach for anticancer immunotherapy due to the involvement of thymic selection.

The RNA-binding protein HuR coordinately regulates GATA-3 and Th2 cytokine gene expression in dose dependent manner (P6262)

Abstract Posttranscriptional control by RNA binding proteins (RBPs) is poorly understood. Th2 mediated diseases such as asthma, are driven by GATA-3, IL-4 and IL-13. The RBP, HuR, regulates IL-4 and IL-13 by increasing mRNA stability. We identified GATA-3, IL-4 and IL-13 as HuR targets and hypothesized HuR may be coordinately regulating Th2 differentiation by controlling mRNA stability and translatability. We made a HuR CD4+T transgenic mouse, and a HuR conditional knock out mouse (HuRfl/fl) to ablate HuR in T cells. HuR over-expression in transgenic CD4+ T cells stabilized GATA-3, IL-4 and IL-13 mRNAs, and increased mRNA, protein and Th2 polarization. HuR knock-down resulted in decreases in GATA-3, IL-4 and IL-13 expression. Findings were confirmed in human naïve and memory T cells. We defined HuR binding sites in GATA-3. Th2 cells with low HuR levels (26%) from HuRfl/+ mice, had decreases in IL-4, IL-13 and GATA-3 mRNA but not protein. Surprisingly, CD4+ T cells from HuRfl/fl mice (93% knockdown) showed increased IL-2, IL-4, IL-13 mRNA and protein. IL-4 transcription increased but mRNA stability remained unchanged. IL-2 and IL-13 transcription were unaltered but there were increases in mRNA stability. Polysomal gradient analysis revealed similar translation. We analyzed gene expression in KO vs. control CD4+ T cells to determine HuR targets. Ova challenge model of asthma corroborated ex vivo results. These data suggest HuR levels regulate Th2 cytokines and Th2 differentiation.

The effects of Trypanosoma cruzi infection and vaccination on priming T. cruzi-specific CD4+ T cells. (P3346)

Abstract Trypanosoma cruzi is a protozoan parasite and the causative agent of Chagas disease. The T. cruzi protein trans-sialidase (TS) is currently under investigation as a vaccine candidate. There are defects in CD4+ T cell responses after T. cruzi infection as compared to TS vaccination. We sought to compare T cell receptor transgenic (Tg) TS-specific CD4+ T cell responses induced by infection versus vaccination through RNA microarrays. Tg TSaa57-74-specific CD4+ T cells were purified from naïve transgenic mice and activated in vitro by peptide-pulsed DCs or T. cruzi-infected and peptide-pulsed DCs, as well as in vivo by TS-DNA vaccination or T. cruzi infection. At various time points post activation, RNA was extracted from purified Tg CD4+ T cells and analyzed by microarray. In vitro, infection of DCs resulted in an increase in the expression of the signaling molecule Map3k10, the cytokine Il21, and factors involved in cell death/survival in CD4+ T cells. Following in vivo activation, proliferation was similar among Tg T cells stimulated with TS vaccination and T. cruzi infection. The microarray will reveal genes differentially expressed in response to T. cruzi infection with further analysis ongoing. These in vitro and in vivo studies using our transgenic T. cruzi-specific CD4+ T cells suggest that T. cruzi infection and TS vaccination differentially activate CD4+ T cells which may explain the defects in CD4+ T cells observed following T. cruzi infection.

Autoreactive T cell activation is sufficient for breaking B cell tolerance in the setting of functional antigen-specific dominant tolerance (P4543)

Abstract Systemic lupus erythematosus is a multisystem autoimmune disease characterized by anti-nuclear autoantibody (ANA) production by activated autoreactive B cells and the persistence and activation of autoreactive T cells. Animal models of lupus provide evidence that T and B cells interact to drive autoimmunity. Whether a break in tolerance in the T cell or B cell compartment could lead to a break in tolerance in the other compartment is unclear. Several studies have addressed this issue using transgenic autoreactive B cells, but whether significant elaboration of autoreactive B cells in the normal repertoire can be driven by activated T cells has not been shown. To address this question, we developed a mouse model in which naïve or activated transgenic autoreactive CD4+ T cells are transferred into hosts expressing their cognate pseudo-autoantigen (autoAg) on B cells and other antigen presenting cells (APCs). Interestingly, autoAg-specific Th1 or Th2 cells, but not naïve cells, drive ANA production by endogenous autoreactive B cells. In bone marrow chimeras, autoAg expression by radioresistant APCs was crucial for sufficient suppression of B cell activation by activated T cells. Additonally, we found that radioresistant APCs most effectively activate autoAg-specific splenic Tregs. We have shown that T cell activation is an important checkpoint in the prevention of ANA production by autoreactive B cells and that antigen-specific Tregs most effectively control ANA production.

Host conditioning approach and choice of target epitope determine the success of adoptive T cell immunotherapy against oncogene-induced established tumors (P2068)

Abstract Host conditioning regimens can dramatically enhance adoptive T cell-based therapies for some cancers. We investigated the unique and combined contributions of whole body irradiation (WBI) and agonist anti-CD40 conditioning to successful adoptive immunotherapy in a mouse model of autochthonous cancer. Line Rip1-Tag4 mice develop pancreatic islet cell tumors due to expression of the SV40 large T antigen oncogene from the rat insulin promoter. Using transfer of T cell receptor transgenic CD8+ T cells specific for two unique determinants in T antigen (designated TCR-I or TCR-IV), we demonstrate that successful control of established tumors in these mice is both regimen- and epitope-dependent. WBI conditioning alone minimally enhanced T cell accumulation and persistence, while anti-CD40 alone transiently enhanced the subdominant TCR-I but not the immunodominant TCR-IV response. Combining anti-CD40 with WBI conditioning enhanced the magnitude and duration of both T cell responses, but only TCR-I cells accumulated and persisted at high levels in the pancreas. Mice that received combined conditioning plus TCR-I transfer had an increased life span, and showed histological signs of anti-tumor immunity. Lack of therapeutic impact by TCR-IV T cells was associated with development of an unusual differentiation phenotype following antigen recognition in vivo. The results indicate that target selection and conditioning regimen dramatically influence the efficacy of adoptive T cell therapy.

The role of germinal centers in T cell receptor revision (P1421)

Abstract Vβ5 transgenic CD4 T cells are tolerized to the endogenous superantigen Mtv-8 by either deletion or TCR revision. Revision occurs when a peripheral CD4 T cell downregulates Vβ5 and undergoes RAG-mediated recombination and surface expression of an endogenous TCR. Given that revision occurs in germinal centers, and that follicular helper T cells, or Tfh, in Vβ5 Tg mice increase in an Mtv-8-dependent manner, we are studying whether revising cells have a Tfh phenotype and whether germinal center interactions are required for revision. Revising cells have a transcription factor and surface phenotype resembling that of Tfh, with low Blimp-1 and elevated Bcl-6, CXCR5, PD-1, IL-21 receptor, and OX-40 expression. The frequency of post-revision T cells increases in mice heterozygous for Blimp-1, a transcriptional repressor of Tfh formation, but revision intermediates are unaffected. The adaptor molecule SAP is required for prolonged B and T cell interactions in the germinal center, and SAP null mice have reduced numbers of post-revision T cells, but no reduction in revision intermediates. These results suggest that revising T cells share some traits with Tfh and that Tfh formation and prolonged B-T cell interactions are required for the completion, but not initiation, of TCR revision. Ongoing studies will analyze revision in the absence of Bcl-6, a transcription factor required for Tfh formation, and TNFR1, a receptor required for the formation of germinal center structures.

The contribution of individual TCR signaling components to thymic selection (P4540)

Abstract In the thymus, T cell progenitors undergo an extensive selection program that shapes the repertoire of T cell receptors to ensure that mature T cells released into the periphery are tolerant to peptides derived from self proteins but able to recognize foreign peptide antigens. The engagement of the T cell receptor on a developing thymocyte triggers the formation of a proximal signaling complex at the plasma membrane and initiates a calcium signal, which together activate multiple downstream signaling cascades crucial for the thymocyte’s survival and maturation. One of the major signaling pathways initiated is the Ras/MAP kinase cascade, which plays an essential role during the positive selection of thymocytes. As thymic positive selection does not occur efficiently in 2-dimensional co-cultures, we are using a thymic slice preparation that provides the 3-dimensional stromal architecture of the thymus. We aim to study the signaling properties of MHC class I-restricted T cell receptor transgenic CD4+CD8+ thymocytes during positive selection either by flow cytometry or by two-photon laser scanning microscopy. We are using pharmacological inhibitors to understand the contribution of individual T cell receptor signaling components to positive selection. We hope to dissect both their longer-term effect on the thymocytes’ development, as well as their interconnection and potential compensatory mechanisms.

Late cognate antigen interactions are required for CD4+ T cell survival during influenza infection. (P6146)

Abstract At the termination of an influenza infection, the majority of antigen-specific effector CD4 T cells undergo Bim-driven apoptosis leaving a small, heterogeneous population of memory cells capable of protecting against subsequent infection. The kinetics of the CD4 response is known to be influenced by the levels of inflammation and antigen, but the contribution of these to contraction and memory formation is unclear. To analyze these we isolated OT-II transgenic CD4 T cells at various days following PR8-OVAII infection and transferred them to either uninfected, PR8-infected (inflammation), or PR8-OVAII-infected (inflammation plus antigen) hosts. In hosts lacking cognate antigen, effectors underwent minimal cytokine-driven proliferation and began to contract as early as two days post transfer, while those in PR8-OVAII-infected hosts proliferated 3-fold and underwent apoptosis after a delay of 2-3 days, leading to greater recovery. To dissect how CD4 effectors nearing the time of contraction interpret external signals, we isolated donor OT-II CD4 effectors on days 6-8 following PR8-OVAII infection and cultured them with or without TCR stimulation. TCR signaling resulted in increased cell recovery, enhanced proliferation, and reduced ratios of Bim:Bcl2 promoting effector survival. Blocking IL-2 diminished the recovery of TCR-stimulated effectors, suggesting that the beneficial impact of late TCR signaling is dependent on autocrine IL-2 signaling.

Germinal center B cells but not TFH require T cell-derived IL21 in a model of autoimmune arthritis (P4068)

Abstract IL21 is a pluripotent cytokine known to promote germinal center B cell differentiation and is thought be an autocrine factor for TFH differentiation and maintenance. The effects of autocrine IL21 on TFH have been addressed in several mouse models, but its requirement is still disputed. We investigated the importance of IL21 as a T cell autocrine factor in a model of autoimmune arthritis. Adoptive transfer of KRN transgenic CD4+ T cells into appropriate hosts drives germinal center formation and autoantibody production against glucose-6-phosphate isomerase, leading to inflammation and joint destruction. By comparing transfer of T cells deficient in IL21 to transfer of T cells lacking IL21R we were able to distinguish whether T cells themselves require IL21 to induce disease or whether it is required exclusively by other cell types. We found that T cells deficient in IL21 did not induce germinal center formation or autoantibody production, but had normal TFH differentiation. Transfer of T cells lacking IL21R was similar to wild-type transfer in antibody titer, GC B cell frequency, and TFH frequency both early and late in disease. To test whether IL21R signaling was required by autoreactive B cells to induce disease, KRN T cells and IL21R sufficient or deficient B cells were transferred into T and B cell deficient hosts. Mice that received IL21R deficient B cells did not develop arthritis, demonstrating that B cells require IL21 from TFH for a proper germinal center response.

Abstract 3960: Whole body irradiation extends the duration of antitumor immunity toward established prostate tumors by adoptively transferred CD8+ T cells in TRAMP mice.

Abstract Adoptive T cell transfer following whole body irradiation (WBI) can induce dramatic tumor regression in a subset of metastatic melanoma patients. We previously found that this approach also eliminates established autochthonous SV40 T-antigen induced murine brain tumors. However, the broader utility of this immunotherapy approach has not been systematically evaluated in solid tumors of different tissue origin. Here, we assessed the efficacy of applying WBI with adoptive T cell transfer to an SV40 T-antigen driven murine model of prostate cancer (TRAMP) previously shown to rapidly tolerize tumor-specific CD8+ T cells. TRAMP mice bearing established prostate tumors were treated with 475cGy WBI followed by adoptive transfer of T-antigen-specific T cell receptor transgenic CD8+ T cells. Accumulation, phenotype and function of the transferred T cells were monitored by flow cytometry, while therapeutic impact on mouse prostate tumors was assessed by histological scoring. Transferred T cells proliferated in both irradiated and non-irradiated TRAMP mice, but accumulated to significantly higher levels and persisted longer in the secondary lymphoid organs and prostate of irradiated mice. WBI also significantly increased the proportion of T cells that produced the effector molecule granzyme B. Prostate-infiltrating T cells in both treatment groups exhibited an effector differentiated phenotype, although neither developed a significant population of IFNγ-producing cells. Gross observations revealed that the urogenital tract in irradiated TRAMP mice was significantly smaller than that of non-irradiated mice two weeks after transfer, corresponding with a modest but statistically significant drop in pathological score. By three weeks after transfer, when prostate-infiltrating T cells had dramatically contracted in irradiated TRAMP mice, cumulative histological scores were generally equivalent although only mice in the non-irradiated treatment group had progressed to adenocarcinoma. Our results suggest that the addition of irradiation extends the persistence of functional tumor-specific T cells in the highly tolerogenic microenvironment of the TRAMP prostate. T cell accumulation was associated with a short-term delay in progression of established tumors. Citation Format: Lindsay Ward-Kavanagh, Timothy Cooper, Todd D. Schell. Whole body irradiation extends the duration of antitumor immunity toward established prostate tumors by adoptively transferred CD8+ T cells in TRAMP mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3960. doi:10.1158/1538-7445.AM2013-3960

CD47 reduces activation‐induced CD4+ T cell death in MOG peptide‐dependent Experimental Autoimmune Encephalomyelitis

CD47 is involved in immune cell recruitment and adaptive immune responses. We have recently reported CD47 plays an important role in CD4 T cell recruitment to sites of inflammation in vivo and in vitro. Here we have examined the role of CD47 in a murine experimental autoimmune encephalomyelitis (EAE) model. Surprisingly, immunization of CD47−/− mice with myelin oligodendrocyte glycoprotein (MOG)35–55 peptide does not induce EAE or CNS inflammation. In contrast, transfer of activated, MOG‐specific TCR transgenic WT CD4 T cells caused disease in WT and CD47−/− mice, suggesting a defect in the immune cell activation. Accordingly, eight days post MOG immunization, the number of CD4+ T cells in spleens and draining lymph nodes was significantly reduced in CD47−/− vs WT mice (43 and 30% reduction, respectively). A MOG‐dependent proliferation assay using CD4 T cells and dendritic cells revealed the defect lay in CD4 T cells from CD47−/− mice (84% reduction). Unexpectedly, we detected greater CD4 T cell activation by MOG immunization in CD47−/− vs WT mice, however, CD47−/− CD4 T cells showed much greater apoptosis in vivo (Annexin‐V and TUNEL staining) and in vitro (2.3 fold increase in Annexin‐V/7‐AAD staining). Our results suggest that CD47 contributes to CD4 T cell recruitment and resistance to self‐peptide activation‐induced cell death in this EAE model. This work was supported by NIH P01HL03628 and AHA 11POST7730055.