TCR Tg Mice Research Articles

Deciphering the functional role of commensal-induced T-DC clustering along sensory nerves in the lung

Abstract The airway mucosa is a primary site of the immune response to inhaled antigens. However, how immune homeostasis is maintained in this organ during continuous exposure to airborne particles and microbial antigens is not known. Previous studies have shown that the spatial organization of immune components in relationship to each other and to non-hematopoietic components in tissues is critical to their proper function, as is communication with commensal microbes. To interrogate such relationships in the lung, especially the possible connections between immune cells and the densely innervated pulmonary airway, 3D information is required. However, current methods for 3D imaging of optically cleared tissue do not provide the marker depth afforded by high-content 2D imaging. To overcome this constraint, we combined a fast hydrophilic tissue clearing technique, Ce3D, with the recently developed IBEX technique, and created Ce3D-IBEX. To date, up to 20 markers have been visualized in a single sample. Using Ce3D-IBEX, we identified a previously uncharacterized clustering of T cells and dendritic cells in proximity to the pulmonary sensory nerves in naïve mice. Agonistic denervation of the sensory nerves with resiniferatoxin reduced the number of clusters, and the cluster formation was disrupted in germ-free mice as well as OT-I and OT-II TCR-Tg mice. These findings indicate that the clustering requires sensory nerve-derived signals and TCR-mediated recognition of bacterial antigens. Future studies will be aimed at elucidating the functional role of the clusters in homeostasis and disease conditions and determining the molecular basis for neuro-immune communication that regulates cluster formation and activity. All studies were done with approval from the ICUC of the NIH/NIAID. This work was supported by the Intramural Research Program of NIAID, NIH.

Influence of the Th1 Cytokine Environment on CCL5 Production from Langerhans Cells.

In the chronic skin lesions of atopic dermatitis (AD), T helper type 1 (Th1) cells appear in addition to Th2 cells, but the role played by Th1 cells in skin inflammation during the chronic phase remains unknown. Here we examined CCL5 production from Langerhans cells (LCs) in the Th1 cytokine environment. LCs were generated from mouse bone marrow cells, then stimulated with anti-CD40 antibody and the Th1 cytokine, interferon (IFN)-γ. Their CCL5 production was then measured. In addition, the LCs were incubated with naïve CD4+ T cells from a DO 11.10 TCR Tg mouse in the presence of ovalbumin peptide for 5 d, and their IFN-γ, interleukin-4 and CCL5 production was then measured. When LCs were stimulated with the anti-CD40 antibody in the presence of IFN-γ, significant levels of CCL5 production were confirmed. Furthermore, when LCs presented antigen to Th cells in the Th1 cytokine environment, significant levels of CCL5 production were induced. This CCL5 production was associated with IFN-γ activity and CD40L expression by Th cells in the culture. Our present data suggest that LCs augment CCL5 production by responding to IFN-γ while presenting antigen to Th cells, and that this augmentation of CCL5 production would likely contribute to infiltration of eosinophils and other Th1 cells into skin lesions, followed by expansion of chronic inflammation in the skin.

Helios expression in CD4+Foxp3− T cells

Abstract The transcription factor Helios is expressed in the majority of Foxp3+ Treg cells, where it is necessary for the maintenance of self-tolerance. Mice with a Foxp3-specific deficiency in Helios develop systemic immune activation with increased T follicular helper (Tfh) and T helper 1 (Th1) effector responses. Notably, mice with a CD4-specific deletion of Helios do not exhibit autoimmunity, pointing to an additional role for Helios in CD4+ T cells. In fact, Helios is expressed in a minor population of conventional CD4+ T cells and its expression is upregulated upon activation of CD4+ T cells, yet the role of Helios in these cells remains unknown. We generated TCR Tg mice with a CD4-specific Helios deletion and have shown that Helios deficient naive CD4+ T cells are fully able to differentiate into Th1, Th2 and Tfh effector cells in vivo. Helios deficient CD4+T cells show no defect in expansion or the production of cytokines. However, Helios deficiency increased the propensity of naive T cells to differentiate into antigen-specific pTregs under inflammatory as well as tolerizing conditions and attenuated the induction of a memory response. To further characterize CD4+Foxp3−Helios+ cells, Next-generation sequencing (NGS) analysis was performed. This analysis has yielded evidence for a heterogenous population that is comprised of Tfh, Tmem, iNKT and a population of cells that have Treg-like features. In vitro activation assays and qPCR have validated differentially expressed cytokines between Helios+ and Helios− CD4+ T cell populations, in agreement with the NGS data. Together, these findings suggest that Helios may play a role in stabilizing effector function.

Role of CD4+ T Cells in Allergic Airway Diseases: Learning from Murine Models.

The essential contribution of CD4+ T cells in allergic airway diseases has been demonstrated, especially by using various murine models of antigen-induced airway inflammation. In addition to antigen-immunized mouse models employing mast cell-deficient mice and CD4+ T cell-depleting procedure, antigen-specific CD4+ T cell transfer models have revealed the possible development of allergic inflammation solely dependent on CD4+ T cells. Regardless of the classical Th1/Th2 theory, various helper T cell subsets have the potential to induce different types of allergic inflammation. T cell receptor (TCR)-transgenic (Tg) mice have been used for investigating T cell-mediated immune responses. Besides, we have recently generated cloned mice from antigen-specific CD4+ T cells through somatic cell nuclear transfer. In contrast to TCR-Tg mice that express artificially introduced TCR, the cloned mice express endogenously regulated antigen-specific TCR. Upon antigen exposure, the mite antigen-reactive T cell-cloned mice displayed strong airway inflammation accompanied by bronchial hyperresponsiveness in a short time period. Antigen-specific CD4+ T cell-cloned mice are expected to be useful for investigating the detailed role of CD4+ T cells in various allergic diseases and for evaluating novel anti-allergic drugs.

Abstract 2144: Programmed cell death 2 forms coinhibitory microclusters that directly attenuate T cell receptor signaling by recruiting the phosphatase SHP2

Abstract Background: Anti-PD-1 antibodies have made tremendous therapeutic effects on advanced or recurrent non-small cell lung cancer. However, the expression level of PD-L1 in tumor tissue which is the biomarker in the clinical setting is not necessarily correlated with their efficacy. In recent reports, some kinds of tumors express PD-L2 which is another ligand of PD-1. It is possible that the binding between PD-L2 and PD-1 is contributing to this mechanism. Because we intended to analyze the microstructural basis of immunological synapse, we used an imaging system combined with a planar lipid-bilayer incorporating GPI-anchored major histocompatibility complexes (MHCs) and the other ligands all those are required for T cell activation. We then found the "Microcluster", which is a clustering of receptors and their downstream signaling molecules, functioning as a signalosome for T cell antigen recognition and activation. Here, we imaged the behavior of the PD-1-PD-L2 signalosome recruiting a phosphatase and illustrated the machineries how PD-L2 suppress T cell activation in competition with PD-L1 toward PD-1 binding. Methods: We purified GPI-anchored murine MHC, adhesion molecule and PD-L2 (mPD-L2) and reconstituted them into lipid-bilayer on a coverslip. Densities of these ligands were finely adjusted as same as those on typical antigen presenting cells (APCs) or tumor cells. Primary CD4+ T cell isolated from moss cytochrome C-specific TCR-Tg mice in Rag2−/- Pdcd1−/− background or T cell hybridoma expressing the same TCR (2D12) were retrovirally transduced with GFP- or HaloTag-tagged proteins (e.g. PD-1 and phosphatases SHP1/2) and imaged these proteins on the lipid-bilayers by confocal microscopy. Results: We showed PD-1 is translocated to TCRs together forming microclusters and then accumulates at the central region of the immunological synapse in the presence of PD-L2. We also confirmed the rapid (20 seconds after TCR stimulation) and transient recruitment of SHP2, not SHP1, to PD-1 microclusters. PD-1-PD-L2 binding ware biochemically and biologically demonstrated to dephosphorylate the TCR downstream signaling molecules and introduce the reduction of IL-2 production, respectively, correlating with PD-1-PD-L2 microcluster formation. In the presence of both PD-L1 and PD-L2 on a lipid-bilayer, PD-L2 accumulated toward PD-1 microclusters much more stronger than PD-L1 did, suggesting that PD-L2 possesses higher affinity against PD-1 than PD-L1 if they were compared as an each single molecule. We further confirmed more effective suppression of IL-2 production by PD-1-PD-L2 binding. Conclusions: Our results indicate that the function of PD-L1 and PD-L2 is almost the same in the point of suppressing TCR signaling by the recruitment of SHP2 to PD-1 signalosomes during the initiation of T cell activation. Contrary, PD-L2 more potently occupies PD-1 microclusters than PD-L1. We are now evaluating the effects of anti-PD-1/L1/L2 antibody for TCR signaling. Citation Format: Tomohiro Takehara, Ei Wakamatsu, Hiroaki Machiyama, Kenzo Soejima, Koichi Fukunaga, Tadashi Yokosuka. Programmed cell death 2 forms coinhibitory microclusters that directly attenuate T cell receptor signaling by recruiting the phosphatase SHP2 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2144.

Fli-1 Regulates Multiple T-Cell Subsets during Inflammatory Responses and Experimental Graft-Versus-Host Disease

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is a procedure undertaken to cure hematological malignancies, especially leukemia. Graft-Versus-Host Disease (GVHD) is a serious condition that often appears following allo-HCT. Friend Leukemia Virus Integration 1 (Fli-1) is a transcription factor highly expressed in cancers including erythroleukemia and acute myeloid leukemia while also implicated in pathogenesis of systemic lupus. We have interrogated the role of Fli-1 in T-cell responses by generating a novel T-cell specific conditional disruption of fli-1 where essential exons 3 and 4 of the gene are floxed and excised in the presence of CD4-Cre recombinase. Models of acute GVHD (aGVHD) and chronic GVHD (cGVHD) were tested utilizing hematopoietic cells from mice with a heterozygous (fli-1-/+ or Het) or homozygous (fli-1-/-or Null) disruption of the fli-1 gene, or from wild-type (fli-1+/+ or WT) littermate controls. At baseline, T cells among each of these three mouse strains showed no significant difference in CD44/CD62L expression or CD4+FoxP3+ (nTreg) frequencies. In the cGVHD model, BALB/c (H2Kd) recipients were infused with allogeneic B6 (H2Kb) genotype-respective bone marrow and splenocytes in order to induce cGVHD. Recipients that received fli-1-/+CD4Cre+ marrow and splenocytes demonstrated improved survival and mild cGVHD, whereas those that received fli-1-/-CD4Cre+ or WT donor cells developed serve cGVHD (Fig. 1 a-f). Cellular studies from lymphoid organs of cGVHD allo-HCT recipients revealed that disruption of fli-1 was associated with decreased frequencies of donor CD4+ cells expressing IL-17A, IFN-γ, T follicular-like (TFH-like) cell markers, and CD8+ cells expressing PD-1. In aGVHD settings, donor fli-1-/+CD4Cre+ T cells had a decreased ability to induce aGVHD compared to WT donor T cells and fli-1-/- donor T cells (Fig. 2 a-b). When investigating cellular mechanisms in aGVHD settings, we found that fli-1-/+CD4Cre+ T cells produced significantly lower IFN-γ early after T-cell activation in vivo compared to WT and fli-1-/-CD4Cre+ T cells (Fig. 2 c). To then investigate the role of Fli-1 in T cells beyond GVHD, we utilized a colitis model by transferring naïve CD4+ T cells (CD44-CD25-) into Rag2-/- syngeneic recipients. While fli-1+/+ T cells induced severe colitis as expected, fli-1-/- and fli--/+ T cells caused moderate and mild colitis, respectively. These results were consistent with those observed in GVHD models. To elucidate underlying mechanisms, we tested the effects of Fli-1 on antigen-specific T-cells using a MHC-II restricted TCR transgenic (Tg) mouse strain specific for HY-peptide. CD4+CD25- T cells from fli-1+/+, fli-1-/+, or fli-1-/- CD4Cre+ TCR-Tg mice were polarized in vitro with endogenous antigen presenting cells from spleen in presence of HY-peptide under iTreg- or Th17-polarizing conditions. Both fli-1-/+ and fli-1-/- TCR-tg T cells exhibited significant increases in iTreg (CD4+FoxP3+) frequencies and surface expression of iTreg functional markers (CD25, CD39, CD73, NRP-1), while also having significantly decreased frequencies of IL-17A producing T cells compared to WT-TCR-tg T cells. To further explore the molecular mechanisms, we retrieved fli-1+/+, fli-1-/+, or fli-1-/- donor T cells from recipient spleens after allo-HCT and did RNAseq analysis on these cells. RNAseq data reveals significant differences in mRNA expression within acute inflammatory response and positive regulation of the immune response enrichment pathways between fli-1-/+ T cells and littermate fli-1+/+ T cells, and to a lesser extent in fli-1-/- T cells (Fig. 2 d-f). Thus, reducing Fli-1 transcriptional activity represents a potential therapeutic concept toward ameliorating GVHD after allo-HCT, while simultaneously targeting cancers such as leukemia which typically overexpress Fli-1. Disclosures No relevant conflicts of interest to declare.

GVHD Is Sustained By T Cell Maintenance within Target Tissues: Insights from Clonal Tracking and Parabiosis

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplantation (alloSCT). In GVHD, donor T cells recognize recipient tissues as non-self and mount a broad attack that results in multi-organ damage. While there has been some success in diminishing the incidence of GVHD, less progress has been made in treating established or steroid-refractory disease without severe global immunosuppression. This is in part due to a lack of understanding of the underlining mechanisms that sustain GVHD despite chronic T cell antigen exposure.To address this, we developed a mouse GVHD model that allows us to track the progeny of single alloreactive T cell clones. We used this model to test hypotheses on GVHD maintenance: 1) GVHD is driven by the continuous output and trafficking of alloreactive T cells from secondary lymphoid tissues (SLT) into GVHD target organs; and 2) once tissues are seeded with alloreactive T cells from SLT, GVHD is maintained locally within affected tissues. We reasoned that if GVHD is maintained by the continuous SLT output of T cells, the progeny of single alloreactive clones in a target tissue would come into equilibrium with those in SLT and other target tissues. Alternatively, if there is a degree of local tissue GVHD maintenance, our model predicts that clonal progeny should be unequally distributed and not in equilibrium with SLT.We first tested these possibilities using a GVHD model wherein BALB/c RAG2-/- TCR transgenic (Tg) CD4 T cells (TS1) that recognize the S1 peptide derived from influenza hemagglutinin (HA) induce GVHD in BALB/c RAG2-/- mice that ubiquitously express HA (HA104 mice). We generated TS1 TCR Tg mice on 9 congenic backgrounds based on the expression of CD45.1/2, Thy1.1/2 and GFP. We transferred 500 naïve TS1 cells of 1 clonotype (to induce GVHD) along with single naïve TS1 cells from the remaining 8 clonotypes and BALB/c RAG2-/- bone marrow (BM) into lethally irradiated HA104 mice. We recovered a total of 432 single-cell derived TS1 clones (72% of input clones) from tissues of 79 mice, analyzed 7-35 days after transfer. We enumerated the TS1 clonal composition of each tissue (expressed as the % of all TS1 of that tissue) and found disparate clonal distribution across tissues within individual mice. For example, in a representative mouse analyzed at day 33 (Figure 1), the fractions of a single TS1 clone were relatively high in the colon (1.4%) and small intestine intraepithelial lymphocyte (IEL) (4.6%) when compared to the spleen (0.07%), BM (0.02%) and liver (0.03%). These data support that TS1 clones are not equally distributed among tissues and are not in equilibrium with SLT, suggesting that GVHD is at least in part maintained locally. We also analyzed TS1 clonal frequency distribution in a second model. BALB/c RAG2-/- BM and polyclonal T cells were transplanted into F1 (BALB/c HA104xB10.D2) recipients along with 8 single distinct TS1 cells. In this system, GVHD is induced by polyclonal BALB/c cells and TS1 cells are trackers of reactivity to HA. Preliminary experiments also indicate unequal distributions of TS1 clones across tissues in individual mice.In a second approach to test whether GVHD is maintained locally, irradiated HA104 mice were reconstituted with either 500 Thy1.1 or 500 Thy1.2 TS1 cells and CD45.1 or CD45.2 BALB/c RAG2-/- BM. One partner also received congenic TS1 single cells. We performed parabiosis of mice from one group to the other 21-28 days later. We analyzed 4 pairs 4 weeks post-joining, looking first at the blood to establish a baseline for TS1 crossover from the parabiotic partner. In blood, 18.9% ±1.9 of all TS1 were derived from the partner. Importantly, relative to equilibration in blood, there were far fewer partner-derived TS1 cells in all other tissues (Figure 2). Only a few single cell-derived TS1 clones were detected in the partner mouse at very low frequencies, even when they were dominant in tissues of the corresponding partner. Together, these data indicate that once GVHD is established, local maintenance dominates over new TS1 entry. Consistent with this, TS1 cells incorporate BrdU in vivo even at late time points. We are combining proliferation and clonality data at multiple timepoints to develop a mathematical model of GVHD establishment and maintenance. We are also extending our observations in a polyclonal GVHD model wherein the progeny from single alloreactive CD8 cells can be enumerated. DisclosuresShlomchik:NapaJen: Consultancy.

Thymic Development of Beta-Cell Specific Regulatory T Cells in Neonatal NOD Mice

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that involves the destruction of insulin producing β cells found within the pancreatic islets of Langerhans. Recent attention to neonatal thymocyte development has revealed a window during which central tolerance is established in an Autoimmune Regulator (Aire) dependent manner. However, expression of Chromogranin A (ChgA) within the thymus has not been detected; therefore, tolerance to ChgA may be due in part to peripheral dendritic cells (DCs) presenting ChgA to developing thymocytes. The relative contribution of thymically and peripherally derived antigens in the generation of neonatal β cell antigen specific CD4+ regulatory T cells (Tregs) is not well understood. For example, post-translational modification (PTM) of ChgA by fusion with an insulin peptide increases epitope immunogenicity by forming neo-antigens. Pathogenic T cells that are specific for neo-antigens uniquely expressed in the pancreas may escape thymic selection. Therefore, we utilized two experimental designs to test the role of peripheral antigen exposure on neonatal development of β cell specific Tregs. Firstly, we adoptively transferred dendritic cells pulsed with ChgA peptides into BDC2.5 TCR Tg mice and WT NOD mice to study the role of migratory DCs on neonatal development of ChgA Tregs. Secondly, we targeted thymic Langerin+ dendritic cells with anti-Langerin linked to ChgA peptides. In both experimental approaches, we found a robust increase in the ratio and number of ChgA specific Tregs in the presence of the hybrid insulin/chromogranin A peptide (2.5HIP) while also observing an increase in negative selection. The expansion of thymic Tregs was observed in both adult and neonatal mice. Together our data suggests neonatal exposure to PTM peptides enhances thymic development of β cell specific Tregs and may alter T1D pathogenesis. Disclosure Y. Lin: None. T. Lee: None. M. Bettini: None. M. Bettini: None.

Thymic development of regulatory T cells by dendritic cells presenting modified peripheral beta cell antigens.

Abstract Type I Diabetes (T1D) is a T cell-mediated autoimmune disease that destroys insulin producing β cells found within the pancreatic islets of Langerhans. Insulin and Chromogranin A (ChgA) are the only currently known β cell antigens (βAg) necessary to initiate autoimmune diabetes in the non-obese diabetic (NOD) mice; however, expression of ChgA within medullary thymic epithelial cells (mTECs) has not yet been detected. Therefore, tolerance to ChgA may be due in part to peripheral dendritic cells (DCs) presenting ChgA to developing thymocytes and naïve T cells. For example, post-translational modification (PTM) of ChgA by fusion with an insulin peptide increases epitope immunogenicity by forming the neo-antigen, insulin-ChgA fusion peptide (2.5HIP). Pathogenic T cells that are specific for 2.5HIP, which is uniquely expressed in the pancreas, may escape thymic selection due to immunologic ignorance. The relative contribution of thymic and peripheral derived Ag in the selection of βAg specific CD4+ T cells and regulatory T cells (Tregs) remains unclear. To test the role of peripheral Ag exposure on thymic development of βAg specific Tregs, we generated a 50:50 mixed bone marrow chimera using NOD WT (CD45.2 × CD45.1) and BDC2.5 TCR Tg (CD45.1) bone marrow to study the role of peripheral DCs (CD45.2) on developing ChgA T cells. Next, we targeted thymic Langerin+ DCs with anti-Langerin linked to ChgA peptides in BDC2.5 TCR Tg mice. In both experiments, we found robust increase in the ratio and number of ChgA specific Tregs in the presence of the 2.5HIP, in adult and neonatal mice. The results suggest thymic exposure to PTM peptides by DCs enhances β cell specific Treg development and may alter T1D pathogenesis.

Release from Th1-type immune tolerance in spleen and enhanced production of IL-5 in Peyer’s patch by cholera toxin B induce the glomerular deposition of IgA

We examined the pathogenesis of glomerular damage in Th2 type-dependent GATA-3 transgenic (GATA-3 Tg) mice with IgA nephropathy (IgAN). GATA-3 Tg mice were immunized orally using OVA plus cholera toxin B (CTB), and measurement of the serum IgA antibody level and histopathological examination were performed. Marked increases in the serum levels of OVA-specific IgA antibody, IgA and IgG, C3 deposits analogous to those seen in IgAN, and expansion of the matrix in association with mesangial cell proliferation were observed. Furthermore, glomerular IgA deposits were co-localized with mannan-binding lectin (MBL) deposits, which might actually have been abnormal IgA deposits. In GATA-3/TCR-Tg mice that had been orally sensitized with CTB plus OVA and were re-stimulated with OVA in vitro, cultured Peyer’s patch cells showed the enhanced production of IL-5 and supernatants from cultures of spleen cells showed a reduction of TGF-β production with a simultaneous increase in IL-2 production and the recovery of IFN-γ formation. The amount of TGF-β produced by the spleen cells was found to be correlated with the amount of IFN-γ and IL-IL-2 produced by the cells. Also, the percentage of regulatory T cells (Treg) in the spleens of mice sensitized with OVA plus CTB was lower than that in mice orally sensitized with OVA alone. These results suggest that the increased production of IL-5 from Peyer’s patch cells (PPc) and the restored Th1-type immune response might cause the production of abnormal IgA and might induce the deposition of IgA in glomeruli.

Functional Heterogeneity in CD4(+) T Cell Responses Against a Bacterial Pathogen.

To investigate how CD4+ T cells function against a bacterial pathogen, we generated a Listeria monocytogenes-specific CD4+ T cell model. In this system, two TCRtg mouse lines, LLO56 and LLO118, recognize the same immunodominant epitope (LLO190-205) of L. monocytogenes and have identical in vitro responses. However, in vivo LLO56 and LLO118 display vastly different responses during both primary and secondary infection. LLO118 dominates in the primary response and in providing CD8 T cell help. LLO56 predominates in the secondary response. We have also shown that both specific [T cell receptor (TCR)-mediated] and non-specific stimuli (bypassing the TCR) elicit distinct responses from the two transgenics, leading us to conclude that the strength of self-pMHC signaling during development tightly dictates the cell’s future response in the periphery. Herein, we review our findings in this transfer system, focusing on the contribution of the immunomodulatory molecule CD5 and the importance of self-interaction in peripheral maintenance of the cell. We also discuss the manner in which individual TCR affinities to foreign and self-pMHC contribute to the outcome of an immune response; our assertion is that there exists a spectrum of possible T cell responses to recognition of cognate antigen during infection, adding immense diversity to the immune system’s response to pathogens.

Lack of TNFR2 expression by CD4+ T cells limits IL-2 production and promotes Th17 differentiation (BA11P.138)

Abstract Tumor necrosis factor (TNF) plays dualistic pro-inflammatory and immune suppressive roles that lead to unpredictable outcomes of TNF blockade in autoimmune disease. Recent evidence has identified contrasting co-stimulatory effects of TNF on effector T cells and regulatory T cells in controlling autoimmunity. The objective of this study was to determine whether CD4+ T cell autonomous TNFR1 and TNFR2 differentially regulate IL-2 production. We utilize B10.A 5C.C7 TCR Tg Rag2-/- IL-2-GFP reporter mice that are deficient for TNFR1 and/or TNFR2 and memTNFΔ1-12 FoxP3-GFP reporter mice that encode an uncleavable tmTNF to demonstrate that TNFR2, but not TNFR1, and membrane, but not soluble, TNF promote Il2 promoter activity and decrease Il2 mRNA decay to increase IL-2 production in response to CD4+ T cell stimulation. We further demonstrate differential DNA binding of NF-κB in TNFR1- and TNFR2-deficient CD4+ T cells following activation. Adoptive transfer of naive CD4+ T cells from TNFR2-deficient IL-2-GFP heterozygous (IL-2+/-), but not IL-2-GFP homozygous (IL-2-/-), 5C.C7 Rag2-/- TCR Tg mice into B10.A Rag2-/- hosts and subsequent immunization with MCC88-103 + LPS yielded greater IL-17 production upon CD4+ T cell re-challenge in comparison to transfer of TNFR2-sufficient 5C.C7 TCR CD4+ T cells. Taken together, these results suggest that selective blockade of TNFR2 on CD4+ T cells is sufficient to inhibit IL-2 production and promote TH17 differentiation.

Commensal flagellated A4 bacteria inhibit intestinal Th2 responses through induction of innate cell TGFβ production (MUC9P.742)

Abstract Interaction between host and microbiota maintains intestinal immune homeostasis. While commensal bacteria promotes Th1, Th17, and Tregs cells in lamina propria (LP) in the steady condition, it suppresses mucosal Th2 cells. Some specific commensal organisms have defined effects on mucosal immune function, e.g., SFB preferably promotes intestinal Th17 while certain Clostridium spp induce Tregs. However, it is still unclear whether there are specific commensal organisms down-regulating Th2 responses, and the mechanism involved. In this report, we showed that there were more LP Th2 cells in germ-free mice compared to the mice under SPF conditions. A4 bacteria, a commensal isolated from mouse cecum and produces immunodominant microbiota antigen, CBir1 flagellin, presented in intestinal lumen. In CBir1 TCR Tg mice, Th1, Th17 and Treg but not Th2 cells were present in the intestinal LP. When stimulated with CBir1 flagellin, naïve CBir1 Tg T cells differentiated into Th1, Th17 and Tregs but resisted Th2 differentiation. Aaddition of A4 bacteria, as well as pretreatment of APC with A4 bacteria, inhibited Th2 differentiation of OTII T cells stimulated with OVA and of wild-type T cells stimulated with anti-CD3 under Th2 polarizing conditions. A4 bacteria stimulated DC production of TGFβ, blockade of TGFβ abrogated A4 inhibition of Th2 cell development. Collectively, our data reveal that A4 bacteria inhibit intestinal Th2 responses through induction of innate cell production of TGFβ.

Retinal antigen-specific regulatory T cells protect against spontaneous and induced autoimmunity and require local dendritic cells.

BackgroundWe previously reported that the peripheral regulatory T cells (pTregs) generated ‘on-demand’ in the retina were crucial to retinal immune privilege, and in vitro analysis of retinal dendritic cells (DC) showed they possessed antigen presenting cell (APC) activity that promoted development of the Tregs and effector T cells (Teffs). Here, we expanded these findings by examining whether locally generated, locally acting pTregs were protective against spontaneous autoimmunity and autoimmunity mediated by interphotoreceptor retinoid-binding protein (IRBP). We also examined the APC capacity of retinal DC in vivo.MethodsTransgenic (Tg) mice expressing diphtheria toxin receptor (DTR) and/or green fluorescent protein (GFP) under control of the endogenous FoxP3 promoter (GFP only in FG mice, GFP and DTR in FDG mice) or the CD11c promoter (GFP and DTR in CDG mice) were used in conjunction with Tg mice expressing beta-galactosidase (βgal) as retinal neo-self antigen and βgal-specific TCR Tg mice (BG2). Retinal T cell responses were assayed by flow cytometry and retinal autoimmune disease assessed by histological examination.ResultsLocal depletion of the Tregs enhanced actively induced experimental autoimmune uveoretinitis to the highly expressed retinal self-antigen IRBP in FDG mice and spontaneous autoimmunity in βgal-FDG-BG2 mice, but not in mice lacking autoreactive T cells or their target antigen in the retina. The presence of retinal βgal downregulated the generation of antigen-specific Teffs and pTregs within the retina in response to local βgal challenge. Retinal DC depletion prevented generation of Tregs and Teffs within retina after βgal injection. Microglia remaining after DC depletion did not make up for loss of DC-dependent antigen presentation.ConclusionsOur results suggest that local retinal Tregs protect against spontaneous organ-specific autoimmunity and that T cell responses within the retina require the presence of local DC.

Intermediate expression of CCRL1 reveals novel subpopulations of medullary thymic epithelial cells that emerge in the postnatal thymus.

Cortical and medullary thymic epithelial cells (cTECs and mTECs, respectively) provide inductive microenvironments for T-cell development and selection. The differentiation pathway of cTEC/mTEC lineages downstream of common bipotent progenitors at discrete stages of development remains unresolved. Using IL-7/CCRL1 dual reporter mice that identify specialized TEC subsets, we show that the stepwise acquisition of chemokine (C-C motif) receptor-like 1 (CCRL1) is a late determinant of cTEC differentiation. Although cTECs expressing high CCRL1 levels (CCRL1(hi) ) develop normally in immunocompetent and Rag2(-/-) thymi, their differentiation is partially blocked in Rag2(-/-) Il2rg(-/-) counterparts. These results unravel a novel checkpoint in cTEC maturation that is regulated by the cross-talk between TECs and immature thymocytes. Additionally, we identify new Ulex europaeus agglutinin 1 (UEA)(+) mTEC subtypes expressing intermediate CCRL1 levels (CCRL1(int) ) that conspicuously emerge in the postnatal thymus and differentially express Tnfrsf11a, Ccl21, and Aire. While rare in fetal and in Rag2(-/-) thymi, CCRL1(int) mTECs are restored in Rag2(-/-) Marilyn TCR-Tg mice, indicating that the appearance of postnatal-restricted mTECs is closely linked with T-cell selection. Our findings suggest that alternative temporally restricted routes of new mTEC differentiation contribute to the establishment of the medullary niche in the postnatal thymus.

CD8+ TCR transgenic strains expressing public versus private TCR targeting the respiratory syncytial virus K(d)M2(82-90) epitope demonstrate similar functional profiles.

Our previous work has characterized the functional and clonotypic features of two respiratory syncytial virus (RSV) epitope-specific T cell responses in mice. Following single-cell sequencing, we selected T cell receptor sequences to represent both a public and a private clone specific for the dominant KdM282–90 epitope for the generation of T cell receptor transgenic (TCR Tg) mice. We evaluated cells from these TCR Tg strains for three major functions of CD8+ T cells: proliferation, cytokine production and cytolytic activity. In vitro comparisons of the functional characteristics of T cells from the newly-generated mice demonstrated many similarities in their responsiveness to cognate antigen stimulation. Cells from both TRBV13-1 (private) and TRBV13-2 (public) TCR Tg mice had similar affinity, and proliferated similarly in vitro in response to cognate antigen stimulation. When transferred to BALB/c mice, cells from both strains demonstrated extensive proliferation in mediastinal lymph nodes following RSV infection, with TRBV13-2 demonstrating better in vivo proliferation. Both strains similarly expressed cytokines and chemokines following stimulation, and had similar Granzyme B and perforin expression, however cells expressing TRBV13-1 demonstrated better cytolytic activity than TRBV13-2 cells. These new, well-characterized mouse strains provide new opportunities to study molecular mechanisms that control the phenotype and function of CD8+ T cell responses.

Analysis of CNS tissue-specific Tregs and dendritic cells in T cell responses and autoimmunity (MUC7P.757)

Abstract We assessed locally generated, locally acting peripheral regulatory T cells (pTregs) in retinal immune homeostasis and ability of retinal dendritic cells (DC) to support T cell activation/expansion within retina. We used transgenic (Tg) mice expressing beta-galactosidase (bgal mice) as retinal neo-self antigen, TCR Tg mice (BG2) specific for bgal, and mice with labeled, depletable Tregs or DC, based on expression of diphtheria toxin (DTx) receptor and/or GFP controlled by FoxP3 or CD11c promoter (FDG or CDG mice). Treg and DC depletion from retina was done by DTx injection into the eye. T cells and DC were assayed by FACS of retina or blood. Retinal autoimmunity was assessed by histological analysis. After bgal (FDG/bgal mice) or IRBP (FDG mice) immunization, Treg depleted retinas showed increased incidence and severity of autoimmunity. We found local depletion of Tregs from retina sufficient to induce spontaneous autoimmunity in FDG/bgal/BG2 mice but not in single or double Tg mice. Dose of DTx used to eliminate retinal Tregs did not induce retinal autoimmunity if given systemically. DC depletion from the retina prevented Treg and Teff generation within retina after bgal injection. Retinal microglia remaining after DC depletion did not make up for the loss of DC-dependent antigen presentation. We conclude local Tregs within the retina protect against spontaneous organ-specific autoimmunity and local DC must be present within the retina for antigen presentation to T cells.

MHC class II–dependent B cell APC function is required for induction of CNS autoimmunity independent of myelin-specific antibodies

Whether B cells serve as antigen-presenting cells (APCs) for activation of pathogenic T cells in the multiple sclerosis model experimental autoimmune encephalomyelitis (EAE) is unclear. To evaluate their role as APCs, we engineered mice selectively deficient in MHC II on B cells (B-MHC II(-/-)), and to distinguish this function from antibody production, we created transgenic (Tg) mice that express the myelin oligodendrocyte glycoprotein (MOG)-specific B cell receptor (BCR; IgH(MOG-mem)) but cannot secrete antibodies. B-MHC II(-/-) mice were resistant to EAE induced by recombinant human MOG (rhMOG), a T cell- and B cell-dependent autoantigen, and exhibited diminished Th1 and Th17 responses, suggesting a role for B cell APC function. In comparison, selective B cell IL-6 deficiency reduced EAE susceptibility and Th17 responses alone. Administration of MOG-specific antibodies only partially restored EAE susceptibility in B-MHC II(-/-) mice. In the absence of antibodies, IgH(MOG-mem) mice, but not mice expressing a BCR of irrelevant specificity, were fully susceptible to acute rhMOG-induced EAE, also demonstrating the importance of BCR specificity. Spontaneous opticospinal EAE and meningeal follicle-like structures were observed in IgH(MOG-mem) mice crossed with MOG-specific TCR Tg mice. Thus, B cells provide a critical cellular function in pathogenesis of central nervous system autoimmunity independent of their humoral involvement, findings which may be relevant to B cell-targeted therapies.

The T Cell Receptor As An Oncogene: Thymic Expression Of Self-Reactive T Cell Receptors Targeting Survivin Induces T-Cell Lymphoblastic Leukmia

Genetic engineering techniques can now efficiently express T cell receptors recognizing tumor antigens on mature T cells and antitumor effects have been observed with this approach, although persistence of such mature T cells is often limiting. To enhance the potential for long-term persistence, some investigators are considering using genetic engineering to express T cell receptors targeting tumor antigen in developing T cells. To test this approach, we generated three founders of TCR transgenic (Tg) mice with specificity for the H-2b restricted immunodominant epitope of murine survivin, a molecule overexpressed by most cancers. The gene was expressed under the human CD2 promoter, which induces expression of the transgene in DN2 thymocytes and has been used successfully in the generation of multiple previous TCR transgenics. In survivin TCR Tg mice, survivin reactive T cells were predominantly CD8+ and mediated specific immune reactivity toward survivin peptides. Some antitumor reactivity was observed, but it was not potent, and the survivin reactive transgenic T cells were unable to mediate objective tumor regression of survivin bearing tumors in vivo. Surprisingly however, spontaneous T cell acute lymphoblastic leukemia (T-ALL) occurred in progeny of all three founders, beginning at 4-6 months of age and by two years of age, all survivin TCR Tg+ mice succumb to T-cell ALL. Both survivin TCR+Rag+/- and survivin TCR+Rag-/- mice develop leukemia at the same rate. The leukemic cells are CD3+, survivin TCR+, and CD8+ or CD4–/CD8–, and are malignant as evidenced by continous growth in vitro without growth factors and the induction of leukemia following inoculation into C57BL/6 immunocompetent recipients. Alpha gene rearrangement analysis in tumor tissues demonstrated oligoclonality, consistent with a model wherein leukemic transformation is not isolated to a rare clone, but occurs with significant frequency in developing thymocytes. Insertional mutagenesis was judged unlikely since leukemia occurred in three separate founders, and was further ruled out by insertion site analysis of one founder, which demonstrated no evidence for integration of the transgene within an oncogenic locus. We thus postulated that the survivin reactive TCR itself was oncogenic, perhaps as a result of signaling via survivin peptides expressed within the thymus. In support of this, we demonstrate that survivin is expressed in thymic tissue, and prior to the development of leukemia, the survivin-specific TCRs signal, as evidenced by NFAT nuclear translocation, and enhanced BrdU incorporation compared to non-transgenic controls. Furthermore, transgenic mice that lack beta2microglobulin, and thus are unable to present survivin peptides, have a significantly diminished rate of leukemia development (see figure). Interestingly, at least one NOTCH1 mutation was also found in all leukemias, with mutations in the PEST domain being most common (8/8), but 5' deletions (19/25) and mutations in the heterodimerization domain also observed. We also observed T cell acute lymphoblastic leukemia with NOTCH mutations in TCRTg+ mice with specificity to other tumor-associated antigens including WT1 and gp100, albeit with lower penetrance. We conclude that genetic engineering aimed at endowing T lymphoid progenitors with the capacity to recognize tumor associated antigens expressed in the thymus could pose a risk for neoplasia, independent of insertional mutagenesis. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Positive selection of self‐antigen‐specific CD8+ T cells by hematopoietic cells

In contrast to thymic epithelial cells, which induce the positive selection of conventional CD8(+) T cells, hematopoietic cells (HCs) select innate CD8(+) T cells whose Ag specificity is not fully understood. Here we show that CD8(+) T cells expressing an H-Y Ag-specific Tg TCR were able to develop in mice in which only HCs expressed MHC class I, when HCs also expressed the H-Y Ag. These HC-selected self-specific CD8(+) T cells resemble innate CD8(+) T cells in WT mice in terms of the expression of memory markers and effector functions, but are phenotypically distinct from the thymus-independent CD8(+) T-cell population. The peripheral maintenance of H-Y-specific CD8(+) T cells required presentation of the self-Ag and IL-15 on HCs. HC-selected CD8(+) T cells in mice lacking the Tg TCR also showed these features. Furthermore, by using MHC class I tetramers with a male Ag peptide, we found that self-Ag-specific CD8(+) T cells in TCR non-Tg mice could develop via HC-induced positive selection, supporting results obtained from H-Y TCR Tg mice. These findings indicate the presence of self-specific CD8(+) T cells that are positively selected by HCs in the peripheral T-cell repertoire.