Anthony Foster

The Role of OX40 Ligand Interactions in the Development of the Th2 Response to the Gastrointestinal Nematode Parasite Heligmosomoides polygyrus

In these studies, we examined the effects of OX40 ligand (OX40L) deficiency on the development of Th2 cells during the Th2 immune response to the intestinal nematode parasite Heligmosomoides polygyrus. Elevations in IL-4 production and total and Ag-specific serum IgE levels were partially inhibited during both the primary and memory immune responses to H. polygyrus in OX40L−/− mice. The host-protective memory response was compromised in OX40L−/− mice, as decreased worm expulsion and increased egg production were observed compared with H. polygyrus-inoculated OX40L+/+ mice. To further examine the nature of the IL-4 defect during priming, adoptively transferred DO11.10 T cells were analyzed in the context of the H. polygyrus response. Although Ag-specific T cell IL-4 production was reduced in the OX40L−/− mice following immunization with OVA peptide plus H. polygyrus, Ag-specific T cell expansion, cell cycle progression, CXCR5 expression, and migration were comparable between OX40L+/+ and OX40L−/− mice inoculated with OVA and H. polygyrus. These studies suggest an important role for OX40/OX40L interactions in specifically promoting IL-4 production, as well as associated IgE elevations, in Th2 responses to H. polygyrus. However, OX40L interactions were not required for serum IgG1 elevations, increases in germinal center formation, and Ag-specific Th2 cell expansion and migration to the B cell zone.

IL-2 and Autocrine IL-4 Drive the In Vivo Development of Antigen-Specific Th2 T Cells Elicited by Nematode Parasites

The intestinal nematode parasite, Nippostrongylus brasiliensis, triggers potent type 2 immunity. Using OVA peptide as a model Ag, we have examined the adjuvant effects of this parasite on the in vivo development of Ag-specific Th2 cells from naive DO11.10 T cells. Our findings show that Th2 cells can develop from transferred naive OVA-specific DO11.10 T cells in recipient IL-4−/− mice inoculated with N. brasiliensis plus OVA. However, autocrine IL-4 is required for in situ Th2 cell differentiation since transferred IL-4Rα-deficient DO11.10 T cells showed greatly reduced Th2 cell development in inoculated IL-4−/− recipient mice. Surprisingly, we also found that IL-2 blockade promoted B7-dependent T cell cycling, but inhibited the development of OVA-specific Th2 cells. Furthermore, the effects of IL-2 occurred independently of CD25+ T regulatory cells. These studies establish a previously unrecognized requirement for autocrine IL-4 and IL-2 in Th2 responses elicited by nematode parasites.

Nippostrongylus brasiliensis can induce B7-independent antigen-specific development of IL-4-producing T cells from naive CD4 T cells in vivo.

Th2 immune responses to a number of infectious pathogens are dependent on B7-1/B7-2 costimulatory molecule interactions. We have now examined the Th2 immune response to Nippostrongylus brasiliensis (Nb) in B7-1/B7-2−/− mice and show that Th2 effector cells develop that can mediate worm expulsion and produce substantial Th2 cytokines comparable with wild-type infected mice; however, in marked contrast, B cell Ag-specific Ab production is abrogated after B7 blockade. To examine the mechanism of T cell activation, OVA-specific DO11.10 T cells were transferred to recipient mice, which were then immunized with a combination of Nb plus OVA or either alone. Only the combination of Nb plus OVA triggered T cell differentiation to OVA-specific Th2 cells, suggesting that Nb acts as an adjuvant to stimulate Ag-specific naive T cells to differentiate to effector Th2 cells. Furthermore, using the DO11.10 TCR-transgenic T cell adoptive transfer model, we show that blocking B7-1/B7-2 interactions does not impair nonparasite Ag-specific DO11.10 Th2 cell differentiation; however, DO11.10 T cell cycle progression and migration to the B cell zone are inhibited.

Memory Th2 Effector Cells Can Develop in the Absence of B7-1/B7-2, CD28 Interactions, and Effector Th Cells After Priming with an Intestinal Nematode Parasite

B7-1/B7-2 interactions are required for many Th2-cell mediated primary immune responses including the response that follows infection with the intestinal nematode parasite, Heligmosomoides polygyrus. However, few studies have examined the role of B7-1/B7-2/CD28 interactions in the development of a Th2 memory immune response. We examined the development of the memory Th2 response to H. polygyrus in BALB/c mice deficient in both B7-1 and B7-2 (B7-1/B7-2−/−) and in BALB/c mice deficient in CD28 (CD28−/−). Following primary inoculation with H. polygyrus, adult worms in the gut were cleared with an anti-helminthic drug and mice were subsequently challenge-inoculated with H. polygyrus larvae. The memory Th2 response is readily distinguished by its inhibitory effect on adult worm maturation, resulting in marked reductions in adult worm egg production that are not observed during the primary immune response. Following H. polygyrus challenge inoculation, comparable decreases in egg production and similar increases in mesenteric lymph node cell IL-4 production were observed in B7-1/B7-2−/− and B7-1/B7-2+/+ mice. However, elevations in total serum IgG1 and IgE were reduced, while increases in serum Ag-specific IgG1 and IgE and germinal center formation were blocked in H. polygyrus-challenged B7-1/B7-2−/− mice. In contrast, in H. polygyrus-challenged CD28−/− mice, marked elevations in Ag-specific IgG1 and IgE and increased germinal center formation were observed. The results of these studies demonstrate that effector Th2 memory cells that produce IL-4 and mediate host defense can develop when B7-1/B7-2 interactions, and associated effector Th2 cell development, are blocked during priming. However, humoral immunity is impaired and differentially affected in B7-1/B7-2−/− mice and CD28−/− mice following H. polygyrus challenge.

Donor CD8 T cell activation is critical for greater renal disease severity in female chronic graft-vs-host mice and is associated with increased splenic ICOShi host CD4 T cells and IL-21 expression

Lupus-like renal disease in DBA/2-into-F1 (DBA --> F1) mice is driven by donor CD4 T cells and is more severe in females. Donor CD8 T cells have no known role. As expected, we observed that females receiving unfractionated DBA splenocytes (CD8 intact --> F1) exhibited greater clinical and histological severities of renal disease at 13 weeks compared to males. Surprisingly, sex-based differences in renal disease severity were lost in CD8 depleted --> F1 mice due to an improvement in females and a worsening in males. CD8 intact --> F1 female mice exhibited significantly greater donor and host effector (CD44(hi), CD62L(lo)) CD4 T cells and ICOS(hi) CD4 T follicular helper cells than males. CD8 depleted --> F1 female mice exhibited a reduction in the absolute numbers of host, but not donor CD4 Tfh cells and lost the significant increase in host CD4 effector cells vs. males. Greater female IL-21 expression, a product of Tfh cells, was seen in CD8 intact --> F1 and although reduced was still greater than male CD8 depleted --> F1 mice. Thus, donor CD8 T cells have a critical role in mediating sex-based differences in lupus renal disease severity possibly through greater host ICOS(hi) CD4 T cell involvement.

Donor CD8 T Cells and IFN-γ Are Critical for Sex-Based Differences in Donor CD4 T Cell Engraftment and Lupus-Like Phenotype in Short-Term Chronic Graft-Versus-Host Disease Mice

The transfer of unfractionated DBA/2J (DBA) splenocytes into B6D2F1 (DBA→F1) mice results in greater donor CD4 T cell engraftment in females at day 14 that persists long-term and mediates greater female lupus-like renal disease. Although donor CD8 T cells have no demonstrated role in lupus pathogenesis in this model, we recently observed that depletion of donor CD8 T cells prior to transfer eliminates sex-based differences in renal disease long-term. In this study, we demonstrate that greater day 14 female donor CD4 engraftment is also critically dependent on donor CD8 T cells. Male DBA→F1 mice exhibit stronger CD8-dependent day 8–10 graft-versus-host (GVH) and counter-regulatory host-versus-graft (HVG) responses, followed by stronger homeostatic contraction (days 10–12). The weaker day 10–12 GVH and HVG in females are followed by persistent donor T cell activation and increasing proliferation, expansion, and cytokine production from days 12 to 14. Lastly, greater female day 14 donor T cell engraftment, activation, and cytokine production were lost with in vivo IFN-γ neutralization from days 6 to 14. We conclude the following: 1) donor CD8 T cells enhance day 10 proliferation of donor CD4 T cells in both sexes; and 2) a weaker GVH/HVG in females allows prolonged survival of donor CD4 and CD8 T cells, allowing persistent activation. These results support the novel conclusion that sex-based differences in suboptimal donor CD8 CTL activation are critical for shaping sex-based differences in donor CD4 T cell engraftment at 2 wk and lupus-like disease long-term.

The parent-into-F1 murine model in the study of lupus-like autoimmunity and CD8 cytotoxic T lymphocyte function.

The transfer of homozygous C57Bl/6 (B6) or DBA/2 (DBA) parental strain T cells into normal B6D2F1 mice in the parent-into-F1 (p → F1) model results in a graft-vs.-host disease (GVHD) that takes one of the following two forms: (a) acute GVHD seen with B6 → F1 mice and mediated by donor CD8 cytotoxic T cells that eliminate host lymphocytes and (b) a chronic lupus-like GVHD seen with DBA → F1 mice and mediated by donor CD4 T cell cognate help to autoreactive B cells resulting in autoantibody production and renal disease similar to human lupus. Importantly, these two phenotypes can be distinguished by flow cytometry as early as 2 weeks after donor cell transfer. The p → F1 model can be used to screen for agents that alter lupus development. Additionally, the model is useful for preclinical screening of biologic agents with immunomodulatory potential. Agents that selectively inhibit CD8 T cell function will convert acute GVHD to chronic GVHD in B6 → F1 mice. Conversely, agents that promote CD8 CTL function will convert chronic GVHD to acute GVHD in DBA → F1 mice. Agents that completely suppress T cell function will block both phenotypes. The model is also useful for examining the effects of T cell mutations by transferring mutant T cells into wild-type hosts and assessing the effects on disease phenotype. Differences observed from wild-type T cells → F1 can be directly ascribed to alterations in mutant T cell function. Because of the early 2-week phenotype development, the p → F1 model is well suited to screening of potential immunomodulatory therapeutic compounds and the assessment of T cell mutations on in vivo function.

Administration of FTY720 during Tourniquet-Induced Limb Ischemia Reperfusion Injury Attenuates Systemic Inflammation

Acute ischemia-reperfusion injury (IRI) of the extremities leads to local and systemic inflammatory changes which can hinder limb function and can be life threatening. This study examined whether the administration of the T-cell sequestration agent, FTY720, following hind limb tourniquet-induced skeletal muscle IRI in a rat model would attenuate systemic inflammation and multiple end organ injury. Sprague-Dawley rats were subjected to 1 hr of ischemia via application of a rubber band tourniquet. Animals were randomized to receive an intravenous bolus of either vehicle control or FTY720 15 min after band placement. Rats (n = 10/time point) were euthanized at 6, 24, and 72 hr post-IRI. Peripheral blood as well as lung, liver, kidney, and ischemic muscle tissue was analyzed and compared between groups. FTY720 treatment markedly decreased the number of peripheral blood T cells (p < 0.05) resulting in a decreased systemic inflammatory response and lower serum creatinine levels and had a modest but significant effect in decreasing the transcription of injury-associated target genes in multiple end organs. These findings suggest that early intervention with FTY720 may benefit the treatment of IRI of the limb. Further preclinical studies are necessary to characterize the short-term and long-term beneficial effects of FTY720 following tourniquet-induced IRI.

Induction of Skin Allograft Transplantation Tolerance in Mice Using Human Adipose Derived Stromal Cells

Murine models of allograft transplantation are valuable for understanding the immunological mechanisms of allograft acceptance and rejection, the evaluation of immunosuppressive drugs and strategies, and the restoration of functional defects. Herein, we describe methods to create a skin murine allograft surgical model and how to administer adipose-derived stromal cells (ASC) with limited numbers of donor bone marrow to create stable multilineage donor cell chimerism and indefinite immunological tolerance.

Using Stem Cells to Promote Wound Healing: An Emerging Solution for a Clinical Problem

Wound healing is a multi-faceted and tightly regulated process by which tissues and cells both proximal and distal to the site of injury collaborate to bring about effective closure and a restoration of normal tissue function. The roles of various cellular populations in the healing process often vary with the location and nature of the injury. Defects that result in ineffective or impaired wound closure/healing can have significant and deleterious effects on both the short and long-term health of the individual. Chronic slow healing or non-healing wounds represent a significant cause of morbidity, mortality and costs. Treatment and management options for these types of difficult-to-heal wounds are limited; however there is a growing interest in the use of stem cell based treatment strategies. In addition to giving rise to specialized healthy new tissue, the intrinsic trophic and paracrine actions, anti-inflammatory, and immunomodulatory properties of these cells have been shown to facilitate the recruitment and mobilization of endogenous progenitor cells, to promote tissue regeneration and to attenuate dysregulated cellular and humoral immune-mediated responses within various inflammatory niches. In recent years, both pre-clinical animal models as well as clinical studies have shown promising results for the development of new and effective treatment strategies using a variety of non-embryonic or adult tissue-derived stem/progenitor cell populations. In this chapter we review the in vivo role of stem cells and the stem cell niche during the wound healing process, so as to provide a context for potential exogenous stem cell therapeutic applications, as well as explore some of the recent evidence that supports improved healing and tissue regeneration.