Cell Receptor Transgenic Mice Research Articles

Establishment and characterization of a novel murine model for pollen allergy.

Although there have been many studies revealing the mechanism and establishing the therapeutical method for allergic rhinitis, no suitable animal models for allergic rhinitis, especially for pollen allergy, are currently available. We therefore aimed in this study to develop a murine model producing IgE in response to an inhaled antigen without using any adjuvants. Ovalbumin (OVA)-specific T cell receptor transgenic mice (DO11.10) inhaled an OVA solution for one h, twice a week, for six weeks. The resulting increase of OVA-specific IgE in the serum was observed depending on the times of inhalation. Spleen cells from mice that had inhaled the antigen produced more IL-4 and less IFN-γ than those from the control mice in vitro. These results indicate that inhaled antigen enhanced the Th2-type responses and induced IgE production in a T cell-mediated manner. Our findings would contribute to studies on prevention and treatment of pollen allergy.

Bortezomib Inhibits Osteoclastogenesis and Bone Resorption Through Modulation of CHIT1 and YKL40 Expression

Background: VSIG4 acts as a co-inhibitory ligand to negatively regulate T cell proliferation and cytokine production by arresting cell cycle at G0/G1 phase and upregulating tolerance-inducing p27KIP-1. VSIG4 expression is restricted to macrophages. VSIG4 fusion protein also inhibits T cell production of Th1, Th2, and Th17 cytokines. Thus, we hypothesized that endogenous VSIG4 impairs helper T cell functions and then inhibits the subsequent antibody response. Materials and Methods: Mice were immunized subcutaneously with 100 g ovalumin (OVA) in a 1:1 emulsion with phosphate-buffered saline (PBS) and CFA in 100 l PBS. OVAspecific IgM or IgG subclasses were determined using culture supernatants or sera from immunized mice with OVA-coated plates and horseradish peroxidase-conjugated anti-mouse IgG1, -IgG2a, -IgG2b, and -IgG3. To study the VSIG4 role in antibody class switching via regulation of helper T cells, BALB/c mice were immunized intraperitoneally with 500 g of 2,4,6-Trinitrophenyl hapten (TNP)Keyhole Limpet Hemocyanin (KLH) for 14 days. Isolated B cells from TNP-KLH-immunized mice and purified CD4+ T cells from BALB/c wild-type or VSIG4 KO mice immunized s.c. with OVA/CFA were cocultured in the presence of TNPOVA (50 ng/ml) for 5 days. Culture supernatants were collected, and ELISA was performed on a TNP-CGG (100 g/ml)-coated plate to measure anti-TNP IgM and IgG titers. Results: Isotype switching of OVA-specific antibody subclasses to IgG1, IgG2a, IgG2b, and IgG3 was enhanced in OVA-immunized VSIG4 KO mice. 2,4,6-TNPe KLH-primed B cells cocultured with OVAprimed CD4+ T cells from OVA-immunized VSIG4 KO mice in the presence of TNP-OVA showed enhanced isotype switching to IgG subclasses compared to those cocultured with cells isolated from OVA-immunized wild-type (WT) mice. Furthermore, the levels of CD40L expression, the frequency of memory CD4+ T cells, and the production of isotype switching-inducing cytokines increased significantly in OVA-primed CD4+ T cells from VSIG4 knockout (KO) mice. T cells from OVA-specific T cell receptor transgenic mice produced more IFNwhen cocultured with macrophages from VSIG4 KO mice compared to WT mice. Conclusion: Our results demonstrate that macrophage-associated VSIG4 plays a negative role in helper T cell-dependent isotype switching by inhibiting helper T cell activation and differentiation, and suppressing the isotype switching-inducing cytokine production in antigen-primed CD4+ helper T cells.

Interleukin-21-Producing CD4+ T Cells Promote Type 2 Immunity to House Dust Mites

Asthma is a T helper 2 (Th2)-cell-mediated disease; however, recent findings implicate Th17 and innate lymphoid cells also in regulating airway inflammation. Herein, we have demonstrated profound interleukin-21 (IL-21) production after house dust mite (HDM)-driven asthma by using T cell receptor (TCR) transgenic mice reactive to Dermatophagoides pteronyssinus 1 and an IL-21GFP reporter mouse. IL-21-producing cells in the mediastinal lymph node (mLN) bore characteristics of T follicular helper (Tfh) cells, whereas IL-21(+) cells in the lung did not express CXCR5 (a chemokine receptor expressed by Tfh cells) and were distinct from effector Th2 or Th17 cells. Il21r(-/-) mice developed reduced type 2 responses and the IL-21 receptor (IL-21R) enhanced Th2 cell function in a cell-intrinsic manner. Finally, administration of recombinant IL-21 and IL-25 synergistically promoted airway eosinophilia primarily via effects on CD4(+) lymphocytes. This highlights an important Th2-cell-amplifying function of IL-21-producing CD4(+) T cells in allergic airway inflammation.

Severe asthma, airway re-modelling and induced bronchial associated lymphoid tissue produced using novel dust mite specific CD4 T cell receptor transgenic mice (HYP2P.327)

Abstract Patients with severe asthma are very difficult to manage are present a major medical challenge. Development of clinical asthma requires many years of patient exposure to allergens. Mouse asthma models require weeks and are normally transitory and do not lead to allergen-induced dyspnea. We have followed an alternative approach to conventional immunization through the transfer highly polarized CD4 T cell receptor transgenic T cells specific for the dust mite allergen Blo t 5 followed by thrice weekly intranasal recombinant challenge with recombinant allergen to induce persistent high levels of eosinophilia (4 million/mouse lavage) and serum IgE (40,000 ng/ml) and specific IgG1 (30,000 ng/ml). Intranasal challenge of these mice with 25ug of allergen is well tolerated, but a fourfold increase to 100ug induces respiratory symptoms within 15 minutes. These mice show evidence of mucus plugging, increased collagen deposition, mucus hypersecretion and the formation of bronchial associated lymphoid structures (iBALT). Neutralization of IL-4 or IL-13 with monoclonal antibody reduced the response and a combination of these two was most effective. Many drugs reduce respiratory inflammation in mice yet fail to benefit asthmatic patients. We hope that the model described here may help identify new/better drugs for the treatment of patients with severe asthma.

Endogenous VSIG4 negatively regulates the helper T cell-mediated antibody response

VSIG4 acts as a co-inhibitory ligand to negatively regulate T cell proliferation and cytokine production, and its expression is restricted to macrophages. We hypothesized that endogenous VSIG4 impairs helper T cell functions and then inhibits the subsequent antibody response. Isotype switching of ovalbumin (OVA)-specific antibody subclasses to IgG1, IgG2a, IgG2b, and IgG3 was enhanced in OVA-immunized VSIG4 knockout (KO) mice. 2,4,6-Trinitrophenyl hapten (TNP) – Keyhole Limpet Hemocyanin (KLH)-primed B cells cocultured with OVA-primed CD4+ T cells from OVA-immunized VSIG4 KO mice in the presence of TNP-OVA showed enhanced isotype switching to IgG subclasses compared to those cocultured with cells isolated from OVA-immunized wild-type (WT) mice. Furthermore, the levels of CD40L expression, the frequency of memory CD4+ T cells, and the production of isotype switching-inducing cytokines increased significantly in OVA-primed CD4+ T cells from VSIG4 KO mice. T cells from OVA-specific T cell receptor (TCR) transgenic mice produced more IFN-γ when cocultured with macrophages from VSIG4 KO mice compared to WT mice. Thus, our results demonstrate that macrophage-associated VSIG4 plays a negative role in helper T cell-dependent isotype switching by inhibiting helper T cell activation and differentiation, and suppressing the isotype switching-inducing cytokine production in antigen-primed CD4+ helper T cells.

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

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

OX40 agonist immunotherapy expands tumor reactive CD8 T cells with a unique T cell receptor repertoire and synergizes with PDL-1 blockade to promote tumor regression

Previously, the field of tumor immunology has relied on T cell receptor (TCR) transgenic mice and model tumor antigens to study the T cell:tumor cell interaction. However, these models have limitations. Namely, they do not reflect the natural affinity, repertoire, or precursor frequency of tumor antigen-specific T cells. Using a novel TCR affinity tool, the Nur77GFP mouse, we have characterized the signal strength and phenotype of naturally occurring tumor antigen-specific CD8 T cells. Our data suggest that the co-expression of Nur77GFP and PD-1 identifies a population of polyclonal CD8+ tumor infiltrating lymphocytes (TIL) that are tumor antigen-specific. This population produces more IFN-γ in situ and upon re-stimulation with syngeneic but not allogeneic tumor cells. In addition, treatment of tumor bearing mice with an OX40 agonist mAb doubled the frequency of tumor reactive T cells receiving strong TCR signals as compared to the control (~two-fold increase in CD8+Nur77GFPhi p=.0027). PD-1, PDL-1, or CTLA4 blockade also had a modest influence on the frequency of CD8+Nur77GFPhi TIL but this was not as significant as anti-OX40 mAb. To further understand the anti-tumor immune response, CD8+Nur77GFPhi and Nur77GFPlo TIL were sorted and the TCRb CDR3 region was sequenced. We observed clonal expansions of unique Vb sequences exclusively found in the TIL population after anti-OX40 immunotherapy. Unexpectedly, the CD8+ Nur77GFPlo population sorted from mice treated with the anti-OX40 mAb contained very similar Vb sequences and together these represented more than 30% of the TCR repertoire. This suggests that both weak and strong tumor antigens drive TIL expansion after anti-OX40 mAb immunotherapy. Importantly, these repertoire changes were not observed with the control antibody in TIL nor in the spleens of OX40 agonist treated mice. OX40 agonist therapy also increased the frequency of PD-1+ TIL suggesting that targeting this inhibitory molecule in combination with OX40 might improve tumor rejection. In fact, when established tumors (~50mm2) were treated with a combination of anti-OX40 and anti-PDL-1, significant tumor regression and disease free survival was observed in multiple tumor models (C57Bl/6:MCA205 50% survival p < 0.0001, Balb/c:CT26 30% survival p = 0.0105). Tumor rejection corresponded to an increase in the circulation of CD44hi, proliferating, CD8 T cells that was observed only in the combination therapy group as compared to either single agent alone. Therefore, we propose that one mechanism of OX40 agonist immunotherapy is via the priming and expansion of low and high affinity tumor antigen specific CD8 T cells.

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.

CD8+ T cells from a novel T cell receptor transgenic mouse induce liver-stage immunity that can be boosted by blood-stage infection in rodent malaria.

To follow the fate of CD8+ T cells responsive to Plasmodium berghei ANKA (PbA) infection, we generated an MHC I-restricted TCR transgenic mouse line against this pathogen. T cells from this line, termed PbT-I T cells, were able to respond to blood-stage infection by PbA and two other rodent malaria species, P. yoelii XNL and P. chabaudi AS. These PbT-I T cells were also able to respond to sporozoites and to protect mice from liver-stage infection. Examination of the requirements for priming after intravenous administration of irradiated sporozoites, an effective vaccination approach, showed that the spleen rather than the liver was the main site of priming and that responses depended on CD8α+ dendritic cells. Importantly, sequential exposure to irradiated sporozoites followed two days later by blood-stage infection led to augmented PbT-I T cell expansion. These findings indicate that PbT-I T cells are a highly versatile tool for studying multiple stages and species of rodent malaria and suggest that cross-stage reactive CD8+ T cells may be utilized in liver-stage vaccine design to enable boosting by blood-stage infections.

Identification of the genomic insertion site of Pmel-1 TCR α and β transgenes by next-generation sequencing.

The pmel-1 T cell receptor transgenic mouse has been extensively employed as an ideal model system to study the mechanisms of tumor immunology, CD8+ T cell differentiation, autoimmunity and adoptive immunotherapy. The ‘zygosity’ of the transgene affects the transgene expression levels and may compromise optimal breeding scheme design. However, the integration sites for the pmel-1 mouse have remained uncharacterized. This is also true for many other commonly used transgenic mice created before the modern era of rapid and inexpensive next-generation sequencing. Here, we show that whole genome sequencing can be used to determine the exact pmel-1 genomic integration site, even with relatively ‘shallow’ (8X) coverage. The results were used to develop a validated polymerase chain reaction-based genotyping assay. For the first time, we provide a quick and convenient polymerase chain reaction method to determine the dosage of pmel-1 transgene for this freely and publically available mouse resource. We also demonstrate that next-generation sequencing provides a feasible approach for mapping foreign DNA integration sites, even when information of the original vector sequences is only partially known.

Interferon-gamma (IFN-γ)-mediated retinal ganglion cell death in human tyrosinase T cell receptor transgenic mouse.

We have recently demonstrated the characterization of human tyrosinase TCR bearing h3T-A2 transgenic mouse model, which exhibits spontaneous autoimmune vitiligo and retinal dysfunction. The purpose of current study was to determine the role of T cells and IFN-γ in retina dysfunction and retinal ganglion cell (RGC) death using this model. RGC function was measured by pattern electroretinograms (ERGs) in response to contrast reversal of patterned visual stimuli. RGCs were visualized by fluorogold retrograde-labeling. Expression of CD3, IFN-γ, GFAP, and caspases was measured by immunohistochemistry and Western blotting. All functional and structural changes were measured in 12-month-old h3T-A2 mice and compared with age-matched HLA-A2 wild-type mice. Both pattern-ERGs (42%, p = 0.03) and RGC numbers (37%, p = 0.0001) were reduced in h3T-A2 mice when compared with wild-type mice. The level of CD3 expression was increased in h3T-A2 mice (h3T-A2: 174±27% vs. HLA-A2: 100%; p = 0.04). The levels of effector cytokine IFN-γ were also increased significantly in h3T-A2 mice (h3T-A2: 189±11% vs. HLA-A2: 100%; p = 0.023). Both CD3 and IFN-γ immunostaining were increased in nerve fiber (NF) and RGC layers of h3T-A2 mice. In addition, we have seen a robust increase in GFAP staining in h3T-A2 mice (mainly localized to NF layer), which was substantially reduced in IFN-γ (-/-) knockout h3T-A2 mice. We also have seen an up-regulation of caspase-3 and -9 in h3T-A2 mice. Based on our data we conclude that h3T-A2 transgenic mice exhibit visual defects that are mostly associated with the inner retinal layers and RGC function. This novel h3T-A2 transgenic mouse model provides opportunity to understand RGC pathology and test neuroprotective strategies to rescue RGCs.

Inflammation Enhances IL-2 Driven Differentiation of Cytolytic CD4 T Cells

Cytolytic CD4 T cells (CD4 CTL) have been identified in vivo in response to viral infections; however, the factors necessary for driving the cytolytic phenotype have not been fully elucidated. Our previously published work suggests IL-2 may be the master regulator of perforin-mediated cytotoxicity in CD4 effectors. To further dissect the role of IL-2 in CD4 CTL generation, T cell receptor transgenic mice deficient in the ability to produce IL-2 or the high affinity IL-2 receptor (IL-2Rα, CD25) were used. Increasing concentrations of IL-2 were necessary to drive perforin (Prf) expression and maximal cytotoxicity. Granzyme B (GrB) expression and killing correlated with STAT5 activation and CD25 expression in vitro, suggesting that signaling through the high affinity IL-2R is critical for full cytotoxicity. IL-2 signaling was also necessary in vivo for inducing the Th1 phenotype and IFN-γ expression in CD4 T cells during influenza A (IAV) infection. In addition, GrB expression, as measured by mean fluorescent intensity, was decreased in CD25 deficient cells; however, the frequency of CD4 cells expressing GrB was unchanged. Similarly, analysis of cytolytic markers such as CD107a/b and Eomesodermin indicate high IL-2Rα expression is not necessary to drive the CD4 CTL phenotype during IAV infection. Thus, inflammatory signals induced by viral infection may overcome the need for strong IL-2 signals in driving cytotoxicity in CD4 cells.

Lesional-targeting of neuroprotection to the inflammatory penumbra in experimental multiple sclerosis

Progressive multiple sclerosis is associated with metabolic failure of the axon and excitotoxicity that leads to chronic neurodegeneration. Global sodium-channel blockade causes side effects that can limit its use for neuroprotection in multiple sclerosis. Through selective targeting of drugs to lesions we aimed to improve the potential therapeutic window for treatment. This was assessed in the relapsing-progressive experimental autoimmune encephalomyelitis ABH mouse model of multiple sclerosis using conventional sodium channel blockers and a novel central nervous system-excluded sodium channel blocker (CFM6104) that was synthesized with properties that selectively target the inflammatory penumbra in experimental autoimmune encephalomyelitis lesions. Carbamazepine and oxcarbazepine were not immunosuppressive in lymphocyte-driven autoimmunity, but slowed the accumulation of disability in experimental autoimmune encephalomyelitis when administered during periods of the inflammatory penumbra after active lesion formation, and was shown to limit the development of neurodegeneration during optic neuritis in myelin-specific T cell receptor transgenic mice. CFM6104 was shown to be a state-selective, sodium channel blocker and a fluorescent p-glycoprotein substrate that was traceable. This compound was >90% excluded from the central nervous system in normal mice, but entered the central nervous system during the inflammatory phase in experimental autoimmune encephalomyelitis mice. This occurs after the focal and selective downregulation of endothelial p-glycoprotein at the blood-brain barrier that occurs in both experimental autoimmune encephalomyelitis and multiple sclerosis lesions. CFM6104 significantly slowed down the accumulation of disability and nerve loss in experimental autoimmune encephalomyelitis. Therapeutic-targeting of drugs to lesions may reduce the potential side effect profile of neuroprotective agents that can influence neurotransmission. This class of agents inhibit microglial activity and neural sodium loading, which are both thought to contribute to progressive neurodegeneration in multiple sclerosis and possibly other neurodegenerative diseases.

Effect of Orally Administered Lactobacillus brevis HY7401 in a Food Allergy Mouse Model

We had found that orally administered Lactobacillus species were effective immune modulators in ovalbumin (OVA)-sensitized mice. To validate these findings, we investigated the effects of orally administered Lactobacillus brevis HY7401 in OVA-T cell receptor transgenic mice. This strain showed a tendency to induce Th1 cytokines and inhibit Th2 cytokines. All assayed isotypes of OVA-specific antibody were effectively reduced. Systemic anaphylaxis was also relatively reduced with the probiotic administration. These results reveal that L. brevis HY7401 might be useful to promote anti-allergic processes through oral administration.

Pathogenic CD4+ T cells recognizing an unstable peptide of insulin are directly recruited into islets bypassing local lymph nodes

In the nonobese diabetic mouse, a predominant component of the autoreactive CD4(+) T cell repertoire is directed against the B:9-23 segment of the insulin B chain. Previous studies established that the majority of insulin-reactive T cells specifically recognize a weak peptide-MHC binding register within the B:9-23 segment, that to the 12-20 register. These T cells are uniquely stimulated when the B:9-23 peptide, but not the insulin protein, is offered to antigen presenting cells (APCs). Here, we report on a T cell receptor (TCR) transgenic mouse (8F10) that offers important new insights into the biology of these unconventional T cells. Many of the 8F10 CD4(+) T cells escaped negative selection and were highly pathogenic. The T cells were directly recruited into islets of Langerhans, where they established contact with resident intra-islet APCs. Immunogenic insulin had to be presented in order for the T cells to localize and cause disease. These T cells bypassed an initial priming stage in the pancreatic lymph node thought to precede islet T cell entry. 8F10 T cells induced the production of antiinsulin antibodies and islets contained immunoglobulin (IgG) deposited on β cells and along the vessel walls.

Maternal CD4+ and CD8+ T Cell Tolerance Towards a Fetal Minor Histocompatibility Antigen in T Cell Receptor Transgenic Mice1

Tolerance of the maternal immune system in pregnancy is important for successful pregnancy because the semiallogeneic fetus may be subject to antifetal responses. We examined maternal tolerance to the fetus using a murine system in which a model paternally inherited antigen, ovalbumin (OVA), is expressed exclusively in the fetus and placenta. By employing T cell receptor (TCR) transgenic mice specific for major histocompatibility complex class I- or class II-restricted epitopes of OVA (OT-I and OT-II) as mothers, we investigated the fate of fetus-specific CD8⁺ and CD4⁺ T cells, respectively, during gestation. Both OVA-specific CD8⁺ and CD4⁺ T cells displayed an activated phenotype in the peripheral lymphoid tissues of OVA-bred OT-I and OT-II mice, consistent with their encounter of fetal antigen. Whereas a small percentage of OVA-specific CD4⁺ T cells were deleted in the periphery and thymus of OVA-bred OT-II mice, with evidence of TCR downregulation in the remaining T cells, deletion and TCR downregulation were not observed in OVA-bred OT-I mice. Both CD4⁺ and CD8⁺ T cells upregulated inducible costimulator expression in response to the fetal antigen, but only CD4⁺ T cells consistently upregulated the inhibitory receptors programmed cell death 1 and cytotoxic T lymphocyte antigen-4. More regulatory T cells (Tregs) were present in pregnant OVA-bred than in WT-bred OT-II mice, revealing that Tregs expanded specifically in response to the fetal antigen. These data indicate that several mechanisms tolerize fetal antigen-specific maternal CD4⁺ T cells, whereas tolerance of fetal antigen-specific CD8⁺ T cells is less effective. The importance of these mechanisms is underscored by the finding that fetal loss occurs in OVA-bred OT-I but not OT-II mice.

Impaired specific CD8 T cell response with aging is not due to decreased expression of CD90 on TCR transgenic T cells.

BackgroundCD90 (Thy-1) is a small glycoprotein that is particularly abundant on the surface of mouse thymocytes and peripheral T cells, and is often used as a marker in adoptive transfer experiments to distinguish donor and recipient T cells with different CD90 subtypes. We have performed adoptive transfer experiments with T cell receptor transgenic (TCR Tg) mice to study the impaired CD8 T cell response with aging.FindingsAfter stimulation with a CD8 T cell epitope, HA518-524, the response of TCR Tg CD8 T cells from aged mice was decreased compared to the response of TCR Tg T cells from young mice. CD90 expression was also substantially decreased on the TCR Tg CD8 T cells of aged mice. However, the responses of CD90hi and CD90low CD8 T cells of the aged mice were similar in both early activation and proliferation, demonstrating that the impaired Tg T cell response with aging is not associated with the altered CD90 expression on CD8 T cells.ConclusionsThe impaired Tg CD8 T cell response in aged mice is not due to age-associated changes in CD90 expression on Tg CD8 T cells.

Th1 not Th17 cells drive spontaneous MS-like disease despite a functional regulatory T cell response

Multiple sclerosis is considered a disease of complex autoimmune etiology, yet there remains a lack of consensus as to specific immune effector mechanisms. Recent analyses of experimental autoimmune encephalomyelitis, the common mouse model of multiple sclerosis, have investigated the relative contribution of Th1 and Th17 CD4 T cell subsets to initial autoimmune central nervous system (CNS) damage. However, inherent in these studies are biases influenced by the adjuvant and toxin needed to break self-tolerance. We investigated spontaneous CNS disease in a clinically relevant, humanized, T cell receptor transgenic mouse model. Mice develop spontaneous, ascending paralysis, allowing unbiased characterization of T cell immunity in an HLA-DR15-restricted T cell repertoire. Analysis of naturally progressing disease shows that IFNγ(+) cells dominate disease initiation with IL-17(+) cells apparent in affected tissue only once disease is established. Tregs accumulate in the CNS but are ultimately ineffective at halting disease progression. However, ablation of Tregs causes profound acceleration of disease, with uncontrolled infiltration of lymphocytes into the CNS. This synchronous, severe disease allows characterization of the responses that are deregulated in exacerbated disease: the correlation is with increased CNS CD4 and CD8 IFNγ responses. Recovery of the ablated Treg population halts ongoing disease progression and Tregs extracted from the central nervous system at peak disease are functionally competent to regulate myelin specific T cell responses. Thus, in a clinically relevant mouse model of MS, initial disease is IFNγ driven and the enhanced central nervous system responses unleashed through Treg ablation comprise IFNγ cytokine production by CD4 and CD8 cells, but not IL-17 responses.

CD8+T Cells Specific for a Malaria Cytoplasmic Antigen Form Clusters around Infected Hepatocytes and Are Protective at the Liver Stage of Infection

Following Anopheles mosquito-mediated introduction into a human host, Plasmodium parasites infect hepatocytes and undergo intensive replication. Accumulating evidence indicates that CD8(+) T cells induced by immunization with attenuated Plasmodium sporozoites can confer sterile immunity at the liver stage of infection; however, the mechanisms underlying this protection are not clearly understood. To address this, we generated recombinant Plasmodium berghei ANKA expressing a fusion protein of an ovalbumin epitope and green fluorescent protein in the cytoplasm of the parasite. We have shown that the ovalbumin epitope is presented by infected liver cells in a manner dependent on a transporter associated with antigen processing and becomes a target of specific CD8(+) T cells from the T cell receptor transgenic mouse line OT-I, leading to protection at the liver stage of Plasmodium infection. We visualized the interaction between OT-I cells and infected hepatocytes by intravital imaging using two-photon microscopy. OT-I cells formed clusters around infected hepatocytes, leading to the elimination of the intrahepatic parasites and subsequent formation of large clusters of OT-I cells in the liver. Gamma interferon expressed in CD8(+) T cells was dispensable for this protective response. Additionally, we found that polyclonal ovalbumin-specific memory CD8(+) T cells induced by de novo immunization were able to confer sterile protection, although the threshold frequency of the protection was relatively high. These studies revealed a novel mechanism of specific CD8(+) T cell-mediated protective immunity and demonstrated that proteins expressed in the cytoplasm of Plasmodium parasites can become targets of specific CD8(+) T cells during liver-stage infection.

Tob1 plays a critical role in the activation of encephalitogenic T cells in CNS autoimmunity

Reliable biomarkers corresponding to disease progression or therapeutic responsiveness in multiple sclerosis (MS) have not been yet identified. We previously reported that low expression of the antiproliferative gene TOB1 in CD4⁺ T cells of individuals presenting with an initial central nervous system (CNS) demyelinating event (a clinically isolated syndrome), correlated with high risk for progression to MS. We report that experimental autoimmune encephalomyelitis (EAE) in Tob1⁻/ ⁻ mice was associated with augmented CNS inflammation, increased infiltrating CD4⁺ and CD8⁺ T cell counts, and increased myelin-reactive Th1 and Th17 cells, with reduced numbers of regulatory T cells. Reconstitution of Rag1⁻/ ⁻mice with Tob1⁻/⁻ CD4⁺ T cells recapitulated the aggressive EAE phenotype observed in Tob1⁻/⁻ mice. Furthermore, severe spontaneous EAE was observed when Tob1⁻/⁻ mice were crossed to myelin oligodendrocyte glycoprotein–specific T cell receptor transgenic (2D2) mice. Collectively, our results reveal a critical role for Tob1 in adaptive T cell immune responses that drive development of EAE, thus providing support for the development of Tob1 as a biomarker for demyelinating disease activity.