RAG-2 KO Mice Research Articles

Consequences of Aging on Memory CD4 T-Cell Responses

Previous studies have shown an accumulation of memory CD4+ T cells (TMs) that was associated with aging. Although aging has been linked to a shift from naïve to a memory-dominant population, very little is known about the consequences of those changes on CD4 driven alloimmune responses. We studied age-dependent immune responses to cardiac allografts from DBA/2 donor mice into old and young B6 recipients during a primary immune response and following sensitization. Cardiac transplant survival data reflected age-specific modifications of CD4+ TMs during an initial alloantigen encounter and following sensitization. Primary allografts were rejected in a significantly slower fashion in older recipients (6.8 ± 0.7 vs. 10.0 ± 1.3d, p< 0.001). In sharp contrast, cardiac allografts transplanted following an initial sensitization with donor-specific skin grafts at -4 wks were rejected at a comparable and accelerated tempo in both, old and young recipients (4.9 ± 0.1 vs. 5.6 ± 0.6 d, p=ns). Next, wE delineated the impact of aging on CD4+ TMs before and following donor-specific sensitization. We found that the frequency of CD4+ TMs increased significantly with age in naive mice. However, old naive CD4+ TMs expressed less IFN-γ, IL-2 and IL-17 (IFN-γ, IL-17, p< 0.01; IL-2; p< 0.05 compared to young CD4+ TMs). Of note, following sensitization, old CD4+ TMs demonstrated increased IL-17 and IFN-γ but decreased IL-2 levels (IFN-γ, p< 0.05; IL-17, p< 0.01; IL-2, p< 0.05). Moreover, peripheral memory like CD4+CD25+CD44hiFoxp3+ Tregs which have the capacity to secret IL-17 had significantly increased following donor-specific sensitization in old recipients (p< 0.01). Next, we performed adoptive transfer experiments of old and young CD4+TMs from sensitized animals into RAG-1 KO mice testing their functional capacity in vivo. We were able to confirm an age-independent capacity of effector memory CD4+ T cells following sensitization as skin transplants were effectively and rapidly rejected by both young and old primed effector memory CD4+T cells. In vitro, we observed a similar colonial expansion of old and young sensitized CD4+ memory T cells by 3H incorporation (p=ns). Old memory T-cells are functional compromised. However, effector capacities appeared intact and were age-independent following sensitization. The functional capacities of memory CD4+ T cells during primary and secondary immune responses are of critical clinical significance when transplanting older recipients.

O014 Critical role of Th17 pro-inflammatory cytokines to delay skin wound healing

Introduction The presence of pathogens in skin wound is known to delay wound healing. We previously shown that the proinflammatory cytokines Th17 (IL-22 and IL-17A and F) in combination with TNF, IL-1 β and oncostatin M (OSM, IL-6 family) synergistically induce in vitro and in vivo the production of antibacterial peptides and chemokines by human and mouse keratinocytes (Guilloteau et al., J Immunol. 2010), promote proliferation and inhibit their differentiation. Methods In order to investigate the presence and the role of these cytokines during wound healing, we developed a C57BL/6 mouse model of cutaneous wound healing, with or without inoculation of wounds by the bacteria S. aureus and P. aeruginosa , able to infect skin. Results Wound excision on mice induces the early expression of IL-1 β , TNF and OSM, but the expression of IL-22 and IL-17A/ F is not detected so far. Bacteria wound inoculation not only increases the expression of IL-1 β , TNF and of OSM, but induce a strong expression of IL-22 and IL-17A and F. A same expression pattern is observed in infected human skin wounds. Injection of these five cytokines in the wound edges induce a delayed wound healing similar to that induced by the bacterial mixture. The same experiments in septic conditions but in RAG2-KO mice (deficient in mature lymphocytes) show an accelerated wound healing kinetic, comparable to that of uninfected wild type mice. RAG2-KO skin lesions expressed IL-1 β and OSM as wild type mice do, whereas expression of IL-17 and IL-22 is weak or no detectable. It shows that the expression of these cytokines is dependent on the presence of T cells. Finally, supplementation of infected wound edges of RAG2-KO mice by a IL-22, IL-17A and IL-17F mixture delays wound healing at the same level as that of infected wild type mice. Conclusion These results demonstrate the synergistic and specific effects of IL-22 and IL-17 in the wound healing delay process, regardless of the presence of bacteria intra se .

Intrinsic unresponsiveness of Mertk−/− B cells to chronic graft-versus-host disease is associated with unmodulated CD1d expression

Activation and migration of marginal zone B (MZB) cells into follicular (FO) regions of the spleen has been proposed as one of the mechanisms that regulate the development of autoreactive B cells. The mer receptor tyrosine kinase (Mertk) mediates apoptotic cell clearance and regulates activation and cytokine secretion. In the well-studied class II chronic GVH model of bm12 cells into B6 hosts, we observed that Mertk deficient B6 mice did not generate autoantibodies in response to this allogeneic stimulus. We posited that Mertk is important in MHC-II-mediated B cell signaling. In the present study, we show that B cells from Mertk−/− mice but not WT B6 mice exhibited decreased calcium mobilization and tyrosine phosphorylation when stimulated by MHC-II cross-linking. The finding that Mertk was important for class II signaling in B cells was further supported by the preponderance of a-allotype autoantibodies in cGVH in RAG-KO mice reconstituted with a mixture of bone marrow from Mertk−/− mice (b-allotype) and C20 mice (a-allotype). MZB cells from Mertk−/− mice were unable to down regulate surface CD1d expression and subsequent inclusion in the MZ, associated with significantly lower germinal center responses compared to MZB cells from WT. Moreover, Mertk−/− mice treated with an anti-CD1d down regulating antibody responded significantly to bm12 cells, while no response was observed in Mertk−/− mice treated with control antibodies. Taken together, these findings extend the role of Mertk to include CD1d down regulation on MZB cells, a potential mechanism limiting B cell activation in cGVH.

Wiskott–Aldrich Syndrome Protein Deficiency in Innate Immune Cells Leads to Mucosal Immune Dysregulation and Colitis in Mice

Immunodeficiency and autoimmune sequelae, including colitis, develop in patients and mice deficient in Wiskott-Aldrich syndrome protein (WASP), a hematopoietic cell-specific intracellular signaling molecule that regulates the actin cytoskeleton. Development of colitis in WASP-deficient mice requires lymphocytes; transfer of T cells is sufficient to induce colitis in immunodeficient mice. We investigated the interactions between innate and adaptive immune cells in mucosal regulation during development of T cell-mediated colitis in mice with WASP-deficient cells of the innate immune system. Naïve and/or regulatory CD4(+) T cells were transferred from 129 SvEv mice into RAG-2-deficient (RAG-2 KO) mice or mice lacking WASP and RAG-2 (WRDKO). Animals were observed for the development of colitis; effector and regulatory functions of innate immune and T cells were analyzed with in vivo and in vitro assays. Transfer of unfractionated CD4(+) T cells induced severe colitis in WRDKO, but not RAG-2 KO, mice. Naïve wild-type T cells had higher levels of effector activity and regulatory T cells had reduced suppressive function when transferred into WRDKO mice compared with RAG-2 KO mice. Regulatory T-cell proliferation, generation, and maintenance of FoxP3 expression were reduced in WRDKO recipients and associated with reduced numbers of CD103(+) tolerogenic dendritic cells and levels of interleukin-10. Administration of interleukin-10 prevented induction of colitis following transfer of T cells into WRDKO mice. Defective interactions between WASP-deficient innate immune cells and normal T cells disrupt mucosal regulation, potentially by altering the functions of tolerogenic dendritic cells, production of interleukin-10, and homeostasis of regulatory T cells.

323. Expression of Broadly Neutralizing Antibodies (NAbs) Against HIV in RAG KO Mice

323. Expression of Broadly Neutralizing Antibodies (NAbs) Against HIV in RAG KO Mice

Interaction of TNF and TNFR2 stabilizes the phenotype and function of CD4+FoxP3+ regulatory T cells in the inflammatory environment (179.16)

Abstract Our previous results showed that TNF-TNFR2 signaling promotes activation and expansion of Tregs. Thus we hypothesize that TNF-TNFR2 signaling may play a critical role in maintaining phenotypic and functional stability of Tregs during an inflammatory response. We were able to confirm that, in colitis model induced by transfer of naïve CD4 cells into Rag KO mice, the disease could be inhibited by co-transfer of WT Tregs, but not by co-transfer of TNFR2-deficient Tregs. Furthermore, in the colon lamina propria of the colitis model, the majority of WT Tregs maintained FoxP3 expression. In contrast, increased number of TNFR2-deficient Tregs lost FoxP3 expression. In vitro, FoxP3 expression by highly purified Tregs was reduced to almost undetectable levels after potent TCR-stimulation, which was partially abrogated by TNF. This effect of TNF was blocked by anti-TNFR2 Ab, but not by anti-TNFR1 Ab. In addition, TNF was not able to increase FoxP3 expression by TNFR2-deficient Tregs undergoing TCR-stimulation. A full mouse genome array study revealed that, as compared with TNFR2- Tregs, TNFR2+ Tregs from normal mice expressed 4-fold lower levels of a gene encoding SATB1, in line with a recent study show that the repression of this genome organizer play a crucial role in maintenance of Treg phenotype and function. Thus, our data clearly show that the TNF-TNFR2 signaling is critical for the phenotypic and functional stability of Treg in the inflammatory environment.

Colitis in mice with WASP deficiency in innate immune cells is associated with impairment in IL-10 production (71.10)

Abstract Colitis occurs in mice and some patients deficient in the Wiskott-Aldrich Syndrome protein (WASP), a hematopoietic-specific molecule that regulates the actin cytoskeleton. This colitis is lymphocyte-dependent, but severe disease is observed in chimeric mice with WT T cells and WASP KO innate immune cells, generated by transfer of WT CD4+ T cells into WASP/RAG DKO mice. Given the immunomodulatory function of IL-10, coupled with our findings that mixed bone marrow chimeras are free of colitis, we investigated whether alterations in IL-10 play a role in our colitis model. We found reduced IL-10 transcript levels in mesenteric lymph nodes of chimeric mice. To tease out the cellular source of decreased IL-10 production, WASP/RAG DKO DCs were stimulated in vitro and observed to produce less IL-10. We then transferred IL-10GFP CD4+ cells and found a reduction in the IL-10-expressing proportion of T cells in WASP/RAG DKO compared to RAG KO mice. Lastly, exogenous administration of IL-10-Ig completely abrogated disease in chimeric mice. In summary, besides impaired Treg homeostasis and function, severe colitis in our chimeric mice is associated with reduced IL-10 production in MLNs and in vitro stimulated DCs. Taken together with prevention of disease with IL-10 treatment, colitis in this model may result from decreased IL-10 production by WASP KO innate immune cells.

Role of innate signaling pathways in the regulation of antiviral IgG2c responses (108.9)

Abstract Long-term antibody responses provide protection from re-infection and recrudescence of persistent viruses. IgG2a/c is a predominant immunoglobulin isotype in most virus infections. Using a polyomavirus (PyV) mouse model, our lab has shown that MyD88 KO mice generate low levels of virus-specific IgG after the acute phase of infection, have reduced numbers of long-lived plasma cells and make very low levels of IgG2c, but normal levels of IgG1. Investigating the pathways upstream and downstream of MyD88, here we show that in response to PyV infection (i) Unc93B1 mutant mice, which are triple deficient in TLR3, 7 and 9 have diminished IgG2c, but normal IgG1 responses; (ii) TLR9 KO and TLR7 KO mice do not have greatly altered IgG isotype responses as observed in MyD88 KO mice or the Unc93B1 mutant mice, suggesting that these receptors play a redundant role; and (iii) mice lacking IRF5, a transcriptional regulator downstream of MyD88, have greatly diminished IgG2c and normal IgG1 responses, similar to MyD88 KO mice. The isotype switching defect observed in IRF5 KO mice is B cell intrinsic, as transfer of IRF5 KO B cells and WT T cells into RAG KO mice results in diminished antiviral IgG2c secretion compared to mice reconstituted with B6 B and T cells. Therefore, our studies suggest that TLR7/9-MyD88-IRF5 pathways in B cells play an essential role in the generation of IgG2a/c responses to virus infection.

CD40L expression by CD8+ T cells is essential for successful rejection of SV40 T antigen expressing tumor cells in the absence of CD4+ T cells (162.38)

Abstract Cytotoxicity is considered to be the main effector function of CD8+ T cells in the immune system. However, we demonstrate here that CD40L, one of the central effector molecules of CD4+ helper T cells, is expressed by a substantial part of human and murine CD8+ T cells after activation. Human central as well as effector memory CD8+ T cells comprise up to 50% CD40L+ cells after polyclonal activation. These CD40L+ CD8+ T cells display a non-cytotoxic phenotype and resemble functionally various distinct CD4+ T helper cell subtypes with respect to expression of IL2 and IFNγ or IL4 as well as with respect to their potential to induce activation of B cells and maturation of DCs in vitro. To investigate the functional relevance of CD40L expression by CD8+ T cells we analyzed a murine anti-tumor response. During the whole course of the response 50% of the tumor-specific CD8+ T cells expressed CD40L. While application of CD40L competent wt CD8+ T cells to Rag1ko mice challenged with the tumor cells prevented the establishment of solid tumors, injection of CD40Lko CD8+ T cells resulted in a non-controlled tumor progression similar to non-treated tumors. Our results disclose an essential functional relevance of CD40L expressed by CD8+ T cells. Particularly in situations where CD4+ T-cell help is diminished, MHC-II antigen presentation is reduced or limited danger signals are present, CD40L+ CD8+ T cells may exert essential helper responsibilities for a competent cellular immune response.

Bone Marrow Cells in Murine Colitis: Multi-Signal Analysis Confirms Pericryptal Myofibroblast Engraftment without Epithelial Involvement

BackgroundThe contribution of bone marrow-derived cells to epithelial tissues in the inflamed gut remains controversial. Recent reports have suggested that cell fusion between bone marrow-derived cells and the intestinal epithelium takes place in inflammatory conditions.MethodsIn attempts to confirm this, we have undertaken gender mis-matched bone marrow (BM) transplants from male Swiss Webster (SWR) mice to B and T cell-deficient female Rag2 KO mice which, 4 weeks later, were given 5% dextran sodium sulphate in drinking water to induce acute colitis. A further BM-treated group of animals with a graft versus host-like condition was also studied. We developed a new method to combine up to three brightfield or fluorescent lectin- or immuno-histochemical signals with fluorescent in situ hybridisation for the Y and X chromosomes to enable us unequivocally to identify BM-derived male cells which presented as different cell types in the gastrointestinal tract.Principal FindingsIn rolled preparations of whole intestines we scanned around 1.5 million crypts at many tissue levels. In no instance did we see a Y chromosome-positive cell in the epithelial compartment, which was not also CD45-positive. We saw no evidence of cell fusion, based on combined X and Y chromosome analysis. Levels of CD45-positive stromal and lymphoid cells and pericryptal myfibroblasts (positive for α-smooth muscle actin) increased with time up to a plateau, which resembled the level seen in untreated control grafted animals. We saw very few Y chromosome-positive endothelial cells in intestinal stromal vessels.ConclusionsWe conclude that whole BM transplantation does not result in intestinal epithelial engraftment in this model. Our new methods can usefully assist in multi-signal analyses of cell phenotypes following BM transplant and in models of chimaerism and regenerative medicine.

Immune cell-mediated neuroprotection is independent of estrogen action through estrogen receptor-alpha

It has been well documented that both estrogen and immune cells (CD4+ T cells) mediate neuroprotection in the mouse facial nerve axotomy model. Estrogen has been shown to play an important role in regulating the immune response. However, it is unclear whether immune cell-mediated neuroprotection is dependent on estrogen signaling. In this study, using FACS staining, we confirmed that the majority of CD4+ T cells express high levels of estrogen receptor-alpha (ERα), suggesting that CD4+ T cell-mediated neuroprotection may be modulated by estrogen signaling. We previously found that immunodeficient Rag-2KO mice showed a significant increase in axotomy-induced facial motoneuron death compared to immunocompetent wild-type mice. Therefore, we investigated axotomy-induced facial motoneuron loss in immunodeficient Rag-2KO mice that received 17β-estradiol treatment or adoptive transfer of immune cells from mice lacking functional ERα. Our results indicate that while estradiol treatment failed to rescue facial motoneurons from axotomy-induced cell death in Rag-2KO mice, immune cells lacking ERα successfully restored facial motoneuron survival in Rag-2 KO mice to a wild-type level. Collectively, we concluded that CD4+ T cell-mediated neuroprotection is independent of estrogen action through ERα.

Gender is a determinant of the severity of experimental colitis

METHODS: Native marrow(NM): Colitis was induced in 18-20 week old C57BL/6J wild type (WT) and AT1aRKO mice with 2.5% DSS for 5 days. Bone marrow chimera (BMC): Bone marrow (BM) derived myeloid cells (devoid of EC) from 8 week old WT and AT1aRKO mice, were transferred into 10-12 week old RAG1-KO mice, and DSS colitis was induced eight weeks later. Groups were matched with non-DSS treated mice. Outcomes were disease severity, istopathology, and mucosal mRNA expression of pro-inflammatory ytokines using RT-PCR.

Homeostatic control of conjunctival mucosal goblet cells by NKT-derived IL-13

AbstractAlthough the effects of the interleukin 13 (IL-13) on goblet cell (GC) hyperplasia have been studied in the gut and respiratory tracts, its effect on regulating conjunctival GC has not been explored. The purpose of this study was to determine the major IL-13-producing cell type and the role of IL-13 in GC homeostasis in normal murine conjunctiva. Using isolating techniques, we identified natural killer (NK)/natural killer T (NKT) cells as the main producers of IL-13. We also observed that IL-13 knockout (KO) and signal transducer and activator of transcription 6 knockout (STAT6KO) mice had a lower number of periodic acid Schiff (PAS)+GCs. We observed that desiccating stress (DS) decreases NK population, GCs, and IL-13, whereas it increases interferon-γ (IFN-γ) mRNA in conjunctiva. Cyclosporine A treatment during DS maintained the number of NK/NKT cells in the conjunctiva, increased IL-13 mRNA in NK+ cells, and decreased IFN-γ and IL-17A mRNA transcripts in NK+ and NK− populations. C57BL/6 mice chronically depleted of NK/NKT cells, as well as NKT cell-deficient RAG1KO and CD1dKO mice, had fewer filled GCs than their wild-type counterparts. NK depletion in CD1dKO mice had no further effect on the number of PAS+ cells. Taken together, these findings indicate that NKT cells are major sources of IL-13 in the conjunctival mucosa that regulates GC homeostasis.

Enhanced Induction of Regulatory Macrophages Upon Azathioprine/Infliximab Combination Treatment In Vitro

G A A b st ra ct s to determine whether Treg defects resulted from either increased apoptosis or decreased maintenance of Foxp3 expression in Tregs. Methods/Results: WASP-deficient DCs led to decreased In Vitro generation of adaptive Tregs. More importantly, upon transfer of Foxp3cells into recipient mice, there was a marked reduction in the generation of Foxp3+ inducible Tregs in WASP/RAG DKO mice compared to RAG KO controls. The defects in WT Treg function in chimeric mice could not be explained by alterations in the maintenance or apoptosis of Foxp3+ donor cells in WASP/RAG DKO mice compared to RAG KO mice. Employing an In Vitro suppression assay, WT Tregs functioned normally when MLN DCs isolated from either WT or WASP-deficient mice were used as APCs. In contrast, there was aberrant Treg function when DCs were isolated from WASP/RAG DKO mice. Conclusions: WASP deficiency in innate immune cells leads to defective Treg generation and function In Vivo. Similarly, there was reduced generation of WT Tregs In Vitro in the presence of WASPdeficient DCs. However, Treg function was compromised only when DCs were harvested from WASP/RAG DKO mice but not WASP-deficient mice, suggesting an intrinsic aberrancy in WASP-deficient DCs unmasked in a lymphopenic setting. In summary, colitis in chimeric mice may result from DC-driven alterations in Treg number and function.

Abstract 1794: A novel combination therapeutic approach for targeting murine cancers with IL-13 receptor alpha 2 DNA vaccine and an immunotoxin

Abstract Iinterleukin-13 receptor α2 (IL-13Rα2), a cancer testis antigen, is frequently over-expressed on human solid tumors and can be effectively targeted by a recombinant fusion immunotoxin composed of IL-13 and a mutated form of Pseudomonas exotoxin (IL13-PE). We have previously demonstrated that cDNA vaccine to IL-13Rα2 or IL13-PE alone can cause regression of established murine cancer. Here, we demonstrate that combination therapy with DNA vaccine and IL13-PE can cause a remarkable antitumor effect in advanced tumor metastasis models induced by 4T1 breast carcinoma and MCA304 sarcoma. Murine metastatic tumor models were developed by i.v. injection of either 1×105 4T1 or 1×106 MCA304 cells. Mice with established 4T1 metastasis received twice daily i.p. injections of IL13-PE (50 ug/kg/injection) on five consecutive days beginning day 7 followed by i.m. DNA vaccination (100 μg/day) on day 18, 23, 28 and 33. The combination therapy significantly (p&amp;lt;0.01) prolonged overall survival (56 days) of animals compared to control (32 days), IL-13PE alone (43 days) or DNA vaccine alone (40 days). Similar results were observed in MCA304 sarcoma tumor model. Mice treated with combination therapy showed higher CTL activity and IFN-γ release from splenocytes in vitro compared to the controls in both tumor models. Immunofluorescence analyses exhibited infiltration of CD4+ and CD8+ T cells in lung tissues of immunized mice. The role of CD4+ and CD8+ T cells in tumor immunity was further confirmed by similar treatment of tumor-bearing RAG2 KO mice (deficient in T and B cells), which showed no response to therapy. Interestingly, tumor bearing mice treated with combination therapy recruited much lower number of myeloid-derived suppressor cells (MDSC) in lungs than PBS treated controls. These results indicate that combination therapy with IL13-PE followed by DNA vaccine may be a new therapeutic approach of treating human cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1794. doi:10.1158/1538-7445.AM2011-1794

Absence of the Type I IL-4 Receptor reduces IFNγ production and increases the severity of airway inflammation in an ovalbumin induced model of asthma (55.8)

Abstract The TH2 cytokines, IL-4 and IL-13, play critical roles in inducing allergic lung inflammation and also drive alternative activation of macrophages (AAM). Although both cytokines share receptor subunits and signaling proteins, there is evidence that IL-4 and IL-13 have differential roles in asthma pathogenesis. While important in TH2 differentiation, the contribution of IL-4 signaling via the Type I receptor, in modulating airway inflammation remains unclear. Here, we adoptively transferred wild-type ova primed CD4+ T cells into C57BL/6, Rag2KO, or gamma c (γc) KO mice. γcKO mice developed increased peribronchial and perivascular inflammation in the lungs upon ova challenge, compared to WT and Rag2KO mice. Moreover, significantly higher numbers of eosinophils were present in the bronchoalveolar lavage (BAL) and lung tissue in γcKO mice. Characteristic AAM proteins FIZZ1 and YM1 were expressed in lung epithelial cells in both mouse strains, but greater intensity of YM1 expression was observed in γcKOs. The number of FIZZ1+ or YM1+ airways was also significantly higher in these mice. It is known that γcKO mice lack NK cells; Type I R signaling in NK cells induces IFNγ production. Indeed, we found reduced secretion of IFNγ in the BAL in γcKO mice as compared to Rag2KO mice. These results suggest that the Type I R acts to suppress allergic lung inflammation through IFNγ production and, in the presence of TH2 effectors, the Type II R can mediate allergic responses in its absence.

Absence of the Type I IL-4 receptor increases the severity of airway inflammation in a murine model of asthma (141.6)

Abstract The TH2 cytokines, IL-4 and IL-13, play critical roles in inducing allergic lung inflammation, eosinophilia, mucus hypersecretion and also drive alternative activation of macrophages (AAM). Although both cytokines share receptor subunits and signaling machinery, it is becoming apparent that IL-4 and IL-13 have differential roles in asthma pathogenesis. While clearly important in TH2 differentiation, the contribution of IL-4 signaling via the Type I receptor in allergic responses remains unclear. Here, we adoptively transferred wild-type ova primed CD4+ T cells into C57BL/6, Rag2KO, or gamma c (γc) KO mice. We found that γc KO mice developed increased inflammation around the airways and blood vessels in the lungs upon ova challenge when compared to WT and Rag2KO mice. Moreover, significantly higher numbers of eosinophils were present in the bronchoalveolar lavage (BAL) and lung tissue in γc KO mice. Immunohistochemical studies showed that all mouse strains expressed characteristic AAM proteins FIZZ1 and YM1 in lung epithelial cells, while macrophages expressed only YM1. Interestingly, γc KO mice had high levels of FIZZ1 protein in their BAL at baseline, which dropped sharply upon ova treatment. These results suggest that the Type I R acts to suppress allergic lung inflammation and, in the presence of TH2 effectors, the Type II R can mediate allergic responses in its absence. Further experiments are required to fully elucidate the role of the Type I IL-4R and γc in asthma.

Vitamin D receptor deficiency alters the ability of CD8 T cells to induce inflammatory bowel disease. (135.14)

Abstract Vitamin D receptor (VDR) KO mice are more susceptible to several different experimental models of inflammatory bowel disease (IBD) including the Rag KO transfer model when naïve VDR KO CD4 T cells are used to induce disease. CD8 T cells from WT mice do not induce IBD when transferred to Rag KO mice. Conversely, VDR KO CD8 T cells induce IBD in Rag KO recipients. There is no difference in the expression of CD62L, CD44, CCR9 and α4β7 or secretion of IFN-γ and TNF-α between WT and VDR KO CD8 T cells. Disease induced by VDR KO CD8 T cells is mild and the recipients do not lose weight or show outward signs of disease. Instead histological evidence of inflammation and increased numbers of CD8 T cells in the IEL characterize the disease. While WT CD8 T cells fail to induce IBD when transferred to Rag KO mice, they do induce a fulminating form of IBD when transferred to Rag/VDR double KO (DKO) mice. Therefore, the host expression of the VDR alters CD8 T cell development and or function. Experiments to probe the cause of CD8 T cell induced pathology in DKO mice are underway.

Loss of FLVCR, a Heme Export Protein, Blocks Thymic T Cell Development at the CD4+CD8+ Double-Positive Stage.

Abstract 913Cats viremic with the feline leukemia virus C (FeLV-C) develop profound anemia with an early block in erythroid development. In infected cells, the FeLV-C envelope protein sequesters its receptor, feline leukemia virus subgroup Creceptor (FLVCR), in the endosomal compartment and prevents FLVCR trafficking to the cell surface. FLVCR is a transmembrane protein and major facilitator superfamily member, which we previously showed exports heme from cells [Cell(2004)118:57]. FLVCR-null mice died in utero with failed definitive erythropoiesis [Science(2008)319:825]. Conditionally-deleted mice (Flvcrflox/flox;Mx-cre) were then generated, and neonatal or adult deletion caused progressive anemia similar to that seen in cats infected with FeLV-C. In addition to its role as an oxygen carrier in hemoglobin, heme has many other important and diverse roles, including regulation of several transcription and translation factors, microRNA processing, and N-end rule ubiquitination.Early reports noted that cats viremic with FeLV-C had thymic aplasia and lymphopenia. However, it was unclear whether these findings were due to a thymus-intrinsic defect, or a secondary effect of severe anemia and resultant stress. In order to study the role of FLVCR in lymphocyte development, we transplanted limiting numbers (2 × 106) of FLVCR-deleted (FLVCR KO) or control (FLVCR +/+ or +/flox;Mx-cre littermate) bone marrow cells into sub-lethally irradiated (500 cGy) Rag 1-deficient (Rag1 KO) mice. Rag 1KO mice cannot recombine T cell receptor (TCR) or immunoglobulin genes and have no T or B cells. The use of sub-lethal irradiation and transplantation of low numbers of FLVCR KO bone marrow cells allowed endogenous Rag1KO hematopoietic cells to contribute to the erythroid lineage, preventing anemia. However, any T or B cells that developed had to arise from the transplanted FLVCR KO bone marrow.As expected, the FLVCR KO-transplanted Rag1 KO mice were not anemic. However, very few T cells were found in the peripheral blood, spleen, or lymph nodes of FLVCR KO-transplanted mice compared to controls (>90% reduction). In contrast, B cell numbers were preserved. Thus, the previously observed thymic aplasia and lymphopenia seen in FeLV-C viremic cats was not due to severe anemia or stress, but to a direct effect of FLVCR loss on T cell development. Since thymic epithelial cells and the majority of myeloid cells were derived from the Rag1 KO host, the block in T cell development was caused by a cell-intrinsic defect in developing FLVCR KO thymocytes. The requirement for FLVCR was at a developmental stage downstream of the common lymphoid progenitor because B cells developed normally.We then analyzed thymic development in Rag 1 KO mice transplanted with FLVCR KO or control bone marrow. While there were similar numbers of CD4-CD8- double-negative thymocytes (DN) and a slight diminution in the number of CD4+CD8+ double-positive thymocytes (DP), there was a dramatic decrease in the number of CD4+ and CD8+ single positive thymocytes (SP) in FLVCR KO-transplanted mice compared to controls (82% reduction). Analysis of surface TCR expression as well as other maturation markers on developing thymocytes showed that TCR beta rearrangement occurred in FLVCR KO-transplanted mice. However, there was progressive loss of FLVCR KO-derived thymocytes at each subsequent stage. Thus, FLVCR is required for T cell development beyond the DP stage. Following TCR beta rearrangement and surface expression, thymocytes proliferate extensively, rearrange TCR alpha genes, and then undergo positive and negative selection. Our findings suggest that excessive heme caused by FLVCR loss impairs T cell development during these stages, and are intriguing because thus far, heme has not been shown to play a role in T cell development. Whether direct heme toxicity impairs thymocyte proliferation and/or induces apoptosis, or whether excessive heme derails the T cell developmental transcriptional/translational programs, remains to be discovered. Disclosures:No relevant conflicts of interest to declare.

Functional recovery and facial motoneuron survival are influenced by immunodeficiency in crush-axotomized mice

Facial nerve axotomy is a well-described injury paradigm for peripheral nerve regeneration and facial motoneuron (FMN) survival. We have previously shown that CD4 + T helper (Th) 1 and 2 effector subsets develop in the draining cervical lymph node, and that the IL-4/STAT-6 pathway of Th2 development is critical for FMN survival after transection axotomy. In addition, delayed behavioral recovery time in immunodeficient mice may be due to the absence of T and B cells. This study utilized a crush axotomy paradigm to evaluate FMN survival and functional recovery in WT, STAT-6 KO (impaired Th2 response), T-Bet KO (impaired Th1 response), and RAG-2 KO (lacking mature T and B cells) mice to elucidate the contributions of specific CD4 + T cell subsets in motoneuron survival and recovery mechanisms. STAT-6 KO and RAG-2 KO mice exhibited decreased FMN survival after crush axotomy compared to WT, supporting a critical role for the Th2 effector cell in motoneuron survival before target reconnection. Long term FMN survival was sustained through 10 wpo after crush axotomy in both WT and RAG-2 KO mice, indicating that target derived neurotrophic support maintains FMN survival after target reconnection. In addition, RAG-2 KO mice exhibited delayed functional recovery compared to WT mice. Both STAT-6 and T-Bet KO mice exhibited partially delayed functional recovery compared to WT, though not to the extent of RAG-2 KO mice. Collectively, our findings indicate that both pro- and anti-inflammatory CD4 + T cell responses contribute to optimal functional recovery from axotomy-induced facial paralysis, while FMN survival is supported by the anti-inflammatory Th2 response alone.