Randal K. Gregg

NKT Cell Activation Mediates Neutrophil IFN-γ Production and Renal Ischemia-Reperfusion Injury

Previous work has shown that ischemia-reperfusion (IR) injury (IRI) is dependent on CD4+ T cells from naive mice acting within 24 h. We hypothesize that NKT cells are key participants in the early innate response in IRI. Kidneys from C57BL/6 mice were subjected to IRI (0.5, 1, 3, and 24 h of reperfusion). After 30 min of reperfusion, we observed a significant increase in CD4+ cells (145% of control) from single-cell kidney suspensions as measured by flow cytometry. A significant fraction of CD4+ T cells expressed the activation marker, CD69+, and adhesion molecule, LFA-1high. Three hours after reperfusion, kidney IFN-γ-producing cells were comprised largely of GR-1+CD11b+ neutrophils, but also contained CD1d-restricted NKT cells. Kidney IRI in mice administered Abs to block CD1d, or deplete NKT cells or in mice deficient of NKT cells (Jα18−/−), was markedly attenuated. These effects were associated with a significant decrease in renal infiltration and, in activation of NKT cells, and a decrease in IFN-γ-producing neutrophils. The results support the essential role of NKT cells and neutrophils in the innate immune response of renal IRI by mediating neutrophil infiltration and production of IFN-γ.

On the Role of Dendritic Cells in Peripheral T Cell Tolerance and Modulation of Autoimmunity

Recently, it has become clear that dendritic cells (DCs) are essential for the priming of T cell responses. However, their role in the maintenance of peripheral T cell tolerance remains largely undefined. Herein, an antigen-presenting cell (APC) transfer system was devised and applied to experimental allergic encephalomyelitis (EAE), to evaluate the contribution that DCs play in peripheral T cell tolerance. The CD8α−CD4+ subset, a minor population among splenic DCs, was found to mediate both tolerance and bystander suppression against diverse T cell specificities. Aggregated (agg) Ig-myelin oligodendrocyte glycoprotein (MOG), an Ig chimera carrying the MOG 35–55 peptide, binds and cross-links FcγR on APC leading to efficient peptide presentation and interleukin (IL)-10 production. Furthermore, administration of agg Ig-MOG into diseased mice induces relief from clinical EAE involving multiple epitopes. Such recovery could not occur in FcγR-deficient mice where both uptake of Ig-MOG and IL-10 production are compromised. However, reconstitution of these mice with DC populations incorporating the CD8α−CD4+ subset restored Ig-MOG–mediated reversal of EAE. Transfer of CD8α+ or even CD8α−CD4− DCs had no effect on the disease. These findings strongly implicate DCs in peripheral tolerance and emphasize their functional potency, as a small population of DCs was able to support effective suppression of autoimmunity.

Specific T Regulatory Cells Display Broad Suppressive Functions against Experimental Allergic Encephalomyelitis upon Activation with Cognate Antigen

To date, very few Ag-based regimens have been defined that could expand T regulatory (Treg) cells to reverse autoimmunity. Additional understanding of Treg function with respect to specificity and broad suppression should help overcome these limitations. Ig-proteolipid protein (PLP)1, an Ig carrying a PLP1 peptide corresponding to amino acid residues 139-151 of PLP, displayed potent tolerogenic functions and proved effective against experimental allergic encephalomyelitis (EAE). In this study, we took advantage of the Ig-PLP1 system and the PLP1-specific TCR transgenic 5B6 mouse to define a regimen that could expand Ag-specific Treg cells in vivo and tested for effectiveness against autoimmunity involving diverse T cell specificities. The findings indicate that in vivo exposure to aggregated Ig-PLP1 drives PLP1-specific 5B6 TCR transgenic cells to evolve as Treg cells expressing CD25, CTLA-4, and Foxp3 and producing IL-10. These Treg cells were able to suppress PLP1 peptide-induced EAE in both SJL/J and F1 (SJL/J × C57BL/6) mice. However, despite being effective against disease induced with a CNS homogenate, the Treg cells were unable to counter EAE induced by a myelin basic protein or a myelin oligodendrocyte glycoprotein peptide. Nevertheless, activation with Ag before transfer into the host mice supports suppression of both myelin oligodendrocyte glycoprotein- and myelin basic protein peptide-induced EAE. Thus, it is suggested that activation of Treg cells by the cognate autoantigen is necessary for operation of broad suppressive functions.

Neonatal Exposure to Antigen Primes the Immune System to Develop Responses in Various Lymphoid Organs and Promotes Bystander Regulation of Diverse T Cell Specificities

Neonatal exposure to Ag has always been considered suppressive for immunity. Recent investigations, however, indicated that the neonatal immune system could be guided to develop immunity. For instance, delivery of a proteolipid protein (PLP) peptide on Ig boosts the neonatal immune system to develop responses upon challenge with the PLP peptide later. Accordingly, mice given Ig-PLP at birth and challenged with the PLP peptide as adults developed proliferative T cells in the lymph node that produced IL-4 instead of the usual Th1 cytokines. However, the spleen was unresponsive unless IL-12 was provided. Herein, we wished to determine whether such a neonatal response is intrinsic to the PLP peptide or could develop with an unrelated myelin peptide as well as whether the T cell deviation is able to confer resistance to autoimmunity involving diverse T cell specificities. Accordingly, the amino acid sequence 87–99 of myelin basic protein was expressed on the same Ig backbone, and the resulting Ig-myelin basic protein chimera was tested for induction of neonatal immunity and protection against experimental allergic encephalomyelitis. Surprisingly, the results indicated that immunity developed in the lymph node and spleen, with deviation of T cells occurring in both organs. More striking, the splenic T cells produced IL-10 in addition to IL-4, providing an environment that facilitated bystander deviation of responses to unrelated epitopes and promoted protection against experimental allergic encephalomyelitis involving diverse T cell specificities. Thus, neonatal exposure to Ag can prime responses in various organs and sustain regulatory functions effective against diverse autoreactive T cells.

Neonatal Exposure to Antigen Induces a Defective CD40 Ligand Expression that Undermines Both IL-12 Production by APC and IL-2 Receptor Up-Regulation on Splenic T Cells and Perpetuates IFN-γ-Dependent T Cell Anergy

T cell deletion and/or inactivation were considered the leading mechanisms for neonatal tolerance. However, recent investigations have indicated that immunity develops at the neonatal stage but evolves to guide later T cell responses to display defective and/or biased effector functions. Although neonatal-induced T cell modulation provides a useful approach to suppress autoimmunity, the mechanism underlying the biased function of the T cells remains unclear. In prior studies, we found that exposure of newborn mice to Ig-PLP1, a chimera expressing the encephalitogenic proteolipid protein (PLP) sequence 139–151, induced deviated Th2 lymph node cells producing IL-4 instead of IL-2 and anergic splenic T cells that failed to proliferate or produce IFN-γ yet secreted significant amounts of IL-2. However, if assisted with IFN-γ or IL-12, these anergic splenic T cells regained full responsiveness. The consequence of such biased/defective T cells responses was protection of the mice against experimental allergic encephalomyelitis. In this study, investigations were performed to delineate the mechanism underlying the novel form of IFN-γ-dependent splenic anergy. Our findings indicate that CD40 ligand expression on these splenic T cells is defective, leading to noneffective cooperation between T lymphocytes and APCs and a lack of IL-12 production. More striking, this cellular system revealed a requirement for IL-2R expression for CD40 ligand-initiated, IL-12-driven progression of T cells into IFN-γ production.

Neonatal Immunity Develops in a Transgenic TCR Transfer Model and Reveals a Requirement for Elevated Cell Input to Achieve Organ-Specific Responses

In recent years, it has become clear that neonatal exposure to Ag induces rather than ablates T cell immunity. Moreover, rechallenge with the Ag at adult age can trigger secondary responses that are distinct in the lymph node vs the spleen. The question addressed in this report is whether organ-specific secondary responses occur as a result of the diversity of the T cell repertoire or could they arise with homogeneous TCR-transgenic T cells. To test this premise, we used the OVA-specific DO11.10 TCR-transgenic T cells and established a neonatal T cell transfer system suitable for these investigations. In this system, neonatal T cells transferred from 1-day-old DO11.10/SCID mice into newborn (1-day-old) BALB/c mice migrate to the host’s spleen and maintain stable frequency. The newborn BALB/c hosts were then given Ig-OVA, an Ig molecule carrying the OVA peptide, and challenged with the OVA peptide in CFA at the age of 7 wk; then their secondary responses were analyzed. The findings show that the lymph node T cells were deviated and produced IL-4 instead of IFN-γ and the splenic T cells, although unable to proliferate or produce IFN-γ, secreted a significant level of IL-2. Supply of exogenous IL-12 during Ag stimulation restores both proliferation and IFN-γ production by the splenic T cells. This restorable form of splenic unresponsiveness referred to as IFN-γ-dependent anergy required a transfer of a high number of neonatal DO11.10/SCID T cells to develop. Thus, the frequency of neonatal T cell precursors rather than repertoire diversity exerts control on the development of organ-specific neonatal immunity.

Neonatal tolerant immunity for vaccination against autoimmunity.

Autoimmunity arises when the immune system no longer tolerates self and precipitates lymphocyte reactivity against our own antigens. Although the developing T cell repertoire is constantly purging, self-recognition events do exist when such tight control is evaded and autoreactive lymphocytes escape the thymus (the sites of T cell development) and migrate to the periphery. Upon activation these autoreactive cells may exert aggressive behavior toward ones own tissues and organs leading to autoimmune disease. Multiple sclerosis, Rheumatoid arthritis, and type I diabetes are autoimmune diseases mediated by autoreactive T cells. A logical approach to prevent such autoimmunity would be to reprogram those lymphocytes to tolerate the self antigen. Injection of antigen at the neonatal stage promotes a state of tolerance such that successive encounter with antigen does not precipitate aggressive reactions. The mechanism underlying neonatal tolerance involves priming of T cells whose effector functions do not cause inflammatory reactions upon recognition of antigen but rather induce protective immunity. This form of tolerant immunity provides an attractive strategy for vaccination against autoimmunity. Herein, it is shown that neonatal exposure to a self-peptide-immunoglobulin chimera drives a tolerant immunity toward the self-peptide and protects against the autoimmune disease, experimental allergic encephalomyelitis.