OVA-specific TCR-transgenic Mice Research Articles

Platelet-Targeted Gene Transfer Induces Immune Tolerance in a Primed Model

Our previous studies have demonstrated that platelet-specific gene transfer under control of the αIIb promoter (2b) is effective in inducing immune tolerance not only to the coagulation factor VIII and IX, but also to the non-coagulant protein ovalbumin (OVA) in a model without pre-existing immunity (the non-primed model). In the present study, we explored whether platelet-specific gene transfer could induce immune tolerance in the primed model. To address this question, we first used C57BL6 wild type (WT) mouse models. Both donors and recipients were immunized with OVA to induce anti-OVA antibody development. OVA expression was introduced by 2bOVA lentivirus (LV) transduction of hematopoietic stem cells (HSCs) from WT/CD45.2 donors followed by transplantation into OVA-primed WT/CD45.1 recipients preconditioned with a sub-lethal 6.6Gy total body irradiation (TBI). 2bGFP was used as a control vector in parallel. Animals were analyzed after at least 4 weeks of bone marrow (BM) reconstitution. The viability of OVA genetically modified HSCs was confirmed by PCR in all 2bOVA-transduced recipients. After full bone marrow constitution, which takes about 12 weeks, the engraftment in the 2bOVA group (87.4±4.4%, n=5) was similar to that in the 2bGFP group (87.8±3.9%, n=5) as determined by flow cytometry. The OVA protein level was 6.7±2.8 ng/108platelets (n=5) in 2bOVA transduced recipients. Anti-OVA antibody titers declined with time in both groups. Six months after transplantation, recipients were rechallenged with OVA. All of the anti-OVA titers in 2bGFP-transduced recipients (n=5) increased dramatically, while only one in the 2bOVA group slightly increased. Normalized anti-OVA antibody titers in the 2bOVA group (90.8±152.7) were significantly lower than those in the 2bGFP group (1278±2101) after OVA rechallenge, demonstrating that platelet-specific OVA gene delivery to HSCs can suppress anti-OVA immune responses in OVA-primed WT mice.To explore how immune suppression is established after platelet-specific gene transfer in the OVA-primed model, we transduced HSCs from OVA-specific TCR transgenic mice (OTII/CD45.2, in which greater than 97% CD4 T+ cells are OVA-specific) with 2bOVALV and transplanted into OVA-primed WT/CD45.1 recipients preconditioned with 6.6Gy TBI. Untransduced transplanted mice were used as a control. After BM reconstitution, the OVA protein level was 32.3±6.3 ng/108 platelets (n=5) in the 2bOVA-transduced group. Anti-OVA antibody titers in both groups declined with time. When all the recipients were rechallenged with OVA, 2 of 3 anti-OVA titers in the untransduced group increased, while none of the titers in the 2bOVA group increased. Normalized anti-OVA antibody titers in the 2bOVA group (4.1±4.9) were significantly lower than those in the un-transduced group (152.3±214.5), demonstrating that platelet-specific OVA gene delivery to HSCs can also suppress the anti-OVA immune response in the OVA-primed WT mice that received 2bOVA-transduced HSCs from OTII mice (the OTII/WT model). After BM reconstitution, the engraftments in the 2 groups were similar (81.4±2.6%, and 80.9±2.5%, respectively), but donor-derived CD45.2+CD4+ (OTII/CD4) T cells in the 2bOVA (0.2±0.1%) group were consistently significantly lower than in the untransduced group (4.2±1.1%) in peripheral blood during the entire study course. Also, donor-derived CD45.2+CD4+ T cells in both spleen and lymph nodes were significantly lower in the 2bOVA group compared to the untransduced group. Interestingly, there were no differences in either percentage or total cell number of CD45.2+CD4+ T cells in the thymus between the 2 groups, indicating that central tolerance may not play a role in the immune tolerance induced after platelet-targeted gene therapy. Meanwhile, the frequency and total number of endogenous CD4+ T cells were similar in the 2 groups. Of note, the percentages of donor-derived regulatory T (Treg) cells in the 2bOVA group were significantly higher than those in the untransduced group in peripheral blood, spleen, and lymph nodes, while there were no significant differences in the total numbers of donor-derived Treg cells between the 2 groups.Taken together, our studies demonstrate that platelet-specific gene therapy can induce immune tolerance in a primed system through two distinct pathways, peripheral antigen-specific CD4+ T cell clone deletion and regulatory T cell induction. DisclosuresNo relevant conflicts of interest to declare.

Route of Antigen Presentation Can Determine the Selection of Foxp3-Dependent or Foxp3-Independent Dominant Immune Tolerance.

It has been shown that dominant tolerance, namely in transplantation, requires Foxp3+ regulatory T cells. Although most tolerance-inducing regimens rely on regulatory T cells, we found that induction of tolerance to proteins in aluminum hydroxide can be achieved in Foxp3-deficient mice using nondepleting anti-CD4 Abs. This type of tolerance is Ag specific, and tolerant mice retain immune competence to respond to unrelated Ags. We demonstrated with chicken OVA-specific TCR-transgenic mice that the same tolerizing protocol (CD4 blockade) and the same target Ag (OVA) achieves Foxp3-dependent transplantation tolerance to OVA-expressing skin grafts, but Foxp3-independent tolerance when the Ag is provided as OVA-aluminum hydroxide. In the latter case, we found that tolerance induction triggered recessive mechanisms leading to elimination of effector cells and, simultaneously, a dominant mechanism associated with the emergence of an anergic and regulatory CTLA-4+IL-2lowFoxp3- T cell population, where the tolerance state is IL-10 dependent. Such Foxp3-independent mechanisms can improve the efficacy of tolerance-inducing protocols.

The Requirement of CD8+ T Cells To Initiate and Augment Acute Cardiac Inflammatory Response to High Blood Pressure

Macrophage infiltration and activation in myocardium are hallmarks of acute cardiac inflammatory response to high blood pressure. However, the underlying mechanisms remain elusive. In this article, we report that CD8(+) T cells are required for cardiac recruitment and activation of macrophages. First, mice with CD8 gene-targeted (CD8 knockout) or CD8(+) T cells depleted by Ab showed significantly reduced cardiac inflammatory response to the elevation of blood pressure after angiotensin II (Ang II) infusion, whereas CD8 knockout mice reconstituted with CD8(+) T cells restored the sensitivity to Ang II. More importantly, CD8(+) T cells were required for macrophage infiltration in myocardium and subsequent activation to express proinflammatory cytokines and chemokines. Furthermore, macrophage activation required direct contact with activated CD8(+) T cells, but with TCR dispensable. TCR-independent activation of macrophages was further confirmed in MHC class I-restricted OVA-specific TCR transgenic mice, which showed a CD8(+) T cell activation and cardiac proinflammatory response to Ang II similar to that of wild-type mice. Finally, only myocardium-infiltrated, but not peripheral, CD8(+) T cells were specifically activated by Ang II, possibly by the cardiac IFN-γ that drove IFN-γR(+) CD8(+) T cell infiltration and activation. Thus, this work identified a TCR-independent innate nature of CD8(+) T cells that was critical in initiating the sterile immune response to acute elevation of blood pressure.

Resting B cells as Inducers of Antigen-Specific T Regulatory Cells (83.22)

Abstract We have previously shown that resting B cells are able to expand T regulatory cells through TGF-beta3 whereas activated B cells induce increased apoptosis of T regulatory cells. Thus, we hypothesize that resting B cells can be used to induce antigen-specific T regulatory cells. To test our hypothesis, we isolate resting B cells from mice and load the cells with Ova, and then adoptively transfer the B cells to Ova-specific TCR transgenic mice. We determine numbers of OVA-specific T effector cells and T regulatory cells in the adoptively transferred mice and in control mice that receive untreated resting B cells alone by flow cytometry (Foxp3). We further determine if these T regulatory cells mediate immunosuppressive activities by using proliferation assay in vitro and by in vivo prevention of immune response against challenge with OVA + adjuvant. This study will provide basis to develop approaches to induce antigen-specific T regulatory cells to treat inflammatory diseases such as allergy and autoimmune diseases.

Dendritic cell-derived TNF-α is responsible for development of IL-10-producing CD4 + T cells

Immature dendritic cells (DCs) appear to be involved in peripheral immune tolerance via induction of IL-10-producing CD4 + T cells. We examined the role of TNF-α in generation of the IL-10-producing CD4 + T cells by immature DCs. Immature bone marrow-derived DCs from wild type (WT) or TNF-α −/− mice were cocultured with CD4 + T cells from OVA specific TCR transgenic mice (OT-II) in the presence of OVA 323–339 peptide. The WT DCs efficiently induced the antigen-specific IL-10-producing CD4 + T cells, while the ability of the TNF-α −/− DCs to induce these CD4 + T cells was considerably depressed. Addition of exogenous TNF-α recovered the impaired ability of the TNF-α −/− DCs to induce IL-10-producing T cells. However, no difference in this ability was observed between TNF-α −/− and WT DCs after their maturation by LPS. Thus, TNF-α appears to be critical for the generation of IL-10-producing CD4 + T cells during the antigen presentation by immature DCs.

TH17 Cells Mediate Steroid-Resistant Airway Inflammation and Airway Hyperresponsiveness in Mice

Steroid-resistant asthma comprises an important source of morbidity in patient populations. T(H)17 cells represent a distinct population of CD4(+) Th cells that mediate neutrophilic inflammation and are characterized by the production of IL-17, IL-22, and IL-6. To investigate the function of T(H)17 cells in the context of Ag-induced airway inflammation, we polarized naive CD4(+) T cells from DO11.10 OVA-specific TCR-transgenic mice to a T(H)2 or T(H)17 phenotype by culturing in conditioned medium. In addition, we also tested the steroid responsiveness of T(H)2 and T(H)17 cells. In vitro, T(H)17 cytokine responses were not sensitive to dexamethasone (DEX) treatment despite immunocytochemistry confirming glucocorticoid receptor translocation to the nucleus following treatment. Transfer of T(H)2 cells to mice challenged with OVA protein resulted in lymphocyte and eosinophil emigration into the lung that was markedly reduced by DEX treatment, whereas T(H)17 transfer resulted in increased CXC chemokine secretion and neutrophil influx that was not attenuated by DEX. Transfer of T(H)17 or T(H)2 cells was sufficient to induce airway hyperresponsiveness (AHR) to methacholine. Interestingly, AHR was not attenuated by DEX in the T(H)17 group. These data demonstrate that polarized Ag-specific T cells result in specific lung pathologies. Both T(H)2 and T(H)17 cells are able to induce AHR, whereas T(H)17 cell-mediated airway inflammation and AHR are steroid resistant, indicating a potential role for T(H)17 cells in steroid-resistant asthma.

Induction of central T cell tolerance: Recombinant antibodies deliver peptides for deletion of antigen-specific CD4+8+ thymocytes

In order to prevent or ameliorate autoimmune disease, it would be desirable to induce central tolerance to peripheral self-antigens. We have investigated whether recombinant antibodies (Ab) that deliver T cell epitopes to antigen-presenting cells (APC) in the thymus can be used to induce thymocyte deletion. Troybodies are recombinant Ab with V regions specific for APC surface molecules that have T cell epitopes genetically introduced in their C domains. When MHC class II-specific Troybodies with the lambda2(315)T cell epitope were injected into lambda2(315)-specific TCR transgenic mice, a profound deletion of (CD4+)8+ thymocytes was observed. MHC class II-specific Troybodies were 10-100-fold more efficient than non-targeting peptide Ab, and 500-fold more efficient than synthetic peptide at inducing deletion. Similar findings were observed when MHC class II-specific Troybodies with the OVA(323-339) T cell epitope were injected into OVA-specific TCR transgenic mice. Although deletion was transient after a single injection, newborn mice repeatedly injected with MHC class II-specific Troybodies for 4 weeks, had reduced antigen-specific T cells in peripheral lymphoid tissues and reduced T cell responses. These experiments suggest that Troybodies constructed to target specifically thymic APC could be useful tools for induction and maintenance of central T cell tolerance in autoimmune diseases.

OX40-mediated memory T cell generation is TNF receptor-associated factor 2 dependent.

Tumor necrosis factor receptor-associated factor 2 (TRAF2), an adapter protein that associates with the cytoplasmic tail of OX40, may play a critical role in OX40-mediated signal transduction. To investigate the in vivo role of TRAF2 in OX40-mediated generation of Ag-specific memory T cells, we bred OVA-specific TCR transgenic mice to TRAF2 dominant-negative (TRAF2 DN) mice. Following Ag stimulation and OX40 engagement of TRAF2 DN T cells in vivo, the number of long-lived OVA-specific T cells and effector T cell function was dramatically reduced when compared with wild-type T cells. We also demonstrate that CTLA-4 is down-regulated following OX40 engagement in vivo and the OX40-specific TRAF2 DN defect was partially overcome by CTLA-4 blockade in vivo. The data provide evidence that TRAF2 is linked to OX40-mediated memory T cell expansion and survival, and point to the down-regulation of CTLA-4 as a possible control element to enhance early T cell expansion through OX40 signaling.

"Bystander polarization" of CD4+ T cells: activation with high-dose IL-2 renders naive T cells responsive to IL-12 and/or IL-18 in the absence of TCR ligation.

Responsiveness of CD4+ T cells to the IFN-gamma-inducing cytokines IL-12 and IL-18 is generally thought to be acquired only after stimulation via the TCR. We report herein that stimulation of naive CD4+ T cells with high-dose IL-2 (1000 U/ml) renders these cells responsive to IL-12 and/or IL-18 without a requirement for TCR ligation. Naive CD4+CD62L+ Tcells from normal C57BL/6 mice or from DO11.10/Rag2(-/- )OVA-specific TCR-transgenic mice secreted substantial amounts of IFN-gamma when stimulated concurrently with high-dose IL-2 plus IL-12 or IL-18. mRNA encoding both chains of the IL-12 and the IL-18 receptors was expressed by CD4+ T cells after stimulation with high-dose IL-2. Furthermore, anti-CD3-induced IL-12/IL-18 responsiveness was fully abrogated in the presence of cyclosporin A whereas IL-2-induced IL-12/IL-18 responsiveness was not, reminiscent of the previously reported IL-12+IL-18 innate pathway of T cell activation. Lastly, after stimulation with IL-2+IL-12, naive CD4+ T cells from DO11.10/Rag2(-/- )mice exhibited polarization towards a Th1 phenotype (high IFN-gamma but no IL-4) during secondary stimulation with immobilized anti-CD3. We have coined the term "bystander polarization" to describe this phenomenon and we speculate that bystander polarization of naive CD4+ T cells may occur in vivo during strong antigen-specific immune responses.

Requirement for the lymphocyte semaphorin, CD100, in the induction of antigen-specific T cells and the maturation of dendritic cells.

CD100 belongs to the semaphorin family, several members of which are known to act as repulsive axonal guidance factors during neuronal development. We have previously demonstrated that CD100 plays a crucial role in humoral immunity. In this study, we show that CD100 is also important for cellular immunity through the maturation of dendritic cells (DCs). CD100(-/-) mice fail to develop experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein peptide, because myelin oligodendrocyte glycoprotein-specific T cells are not generated in the absence of CD100. In vitro studies with T cells from OVA-specific TCR-transgenic mice demonstrate that Ag-specific T cells lacking CD100 fail to differentiate into cells producing either IL-4 or IFN-gamma in the presence of APCs and OVA peptide. In addition, DCs from CD100(-/-) mice display poor allostimulatory capabilities and defects in costimulatory molecule expression and IL-12 production. The addition of exogenous soluble rCD100 restores normal functions in CD100(-/-) DCs and further enhances functions of normal DCs. Furthermore, treatment of Ag-pulsed DCs with both soluble CD100 and anti-CD40 before immunization significantly enhances their immunogenicity. This treatment elicits improved T cell priming in vivo, enhancing both primary and memory T cell responses. Collectively, these results demonstrate that CD100, which enhances the maturation of DCs, is essential in the activation and differentiation of Ag-specific T cells.

Naive CD4+ T cells exhibit distinct expression patterns of cytokines and cell surface molecules on their primary responses to varying doses of antigen.

The amount of an Ag used for stimulation affects the type and magnitude of T cell responses. In this study we have investigated the primary response of naive CD4(+) T cells derived from OVA-specific TCR-transgenic mice (OVA23-3) upon stimulation with varying doses of the antigenic peptide, OVA(323-339). IL-4 expression was maximal with 50 nM Ag and decreased significantly with increasing doses. In contrast, IFN-gamma expression, which was also detected at 50 nM Ag, increased with increasing doses. The expression patterns of mRNA for the Th2-specific transcription factors GATA-3 and c-Maf were parallel to that of IL-4. These expression profiles were not altered by the addition of anti-IL-4 plus anti-IL-12 mAbs, suggesting that cytokine receptor signaling is not essential. Naive CD4(+) T cells stimulated with 5 nM Ag elicited IgM secretion from cocultured B cells, whereas those stimulated with 50 nM Ag or more elicited apoptosis of B cells. This may be because at lower doses of Ag (5 nM), naive CD4(+) T cells express CD40 ligand and OX40, whereas at higher doses (50 nM), they express Fas ligand. Clearly, the expression of each type of molecule depends on the Ag dose, and different molecules had different expression patterns. Thus, in the primary response, naive CD4(+) T cells can exhibit different functions depending on the dose of Ag.

Involvement of IL-4-producing Vbeta8.2+ CD4+ CD62L- CD45RB- T cells in non-MHC gene-controlled predisposition toward skewing into T helper type-2 immunity in BALB/c mice.

It was found that freshly isolated BALB/c CD4+ T cells produced high levels of IL-4 and IL-10 in response to immobilized anti-CD3 mAb, while C57BL/6 CD4+ T cells produced low amounts of IL-4 and IL-10. The high IL-4-producing ability of BALB/c mice was demonstrated to be genetically dominant and it was controlled by non-MHC gene (or genes). The cells responsible for IL-4 production in BALB/c mice were defined as TCRVbeta8.2+ CD4+ CD62L- CD45RB- memory-type T cells, which were distinct from NK1.1+ CD4+ NKT cells. Although these memory-type T cells were also detected in C57BL/6 mouse spleen at the same frequency, they showed a functionally different property from BALB/c CD4+ CD62L- CD45RB- T cells in terms of IL-4 production. The fact that germfree BALB/c mouse spleen cells also produced high levels of IL-4 suggested that the IL-4 producer in BALB/c mice might be developed under the influence of unknown factors other than environmental Ags. The CD4+ CD62L- CD45RB- T cells obtained from BALB/c mice accelerated the development of IL-4-producing memory-type CD4+ T cells from CD4+ CD62L+ CD45RB+ naive T cells prepared from OVA-specific TCR-transgenic mice. Therefore, IL-4-producing CD4+ CD62L- CD45RB- T cells might play an important role in the preferential induction of Th2-dominant immunity in BALB/c mouse strain.