Microbiota-Specific Foxp3+ Regulatory T Cells Could Control Pathological T Helper Responses.

Upon engaging cognate peptide MHC-II complexes (pMHC-IIs), naive CD4+ T cells differentiate and acquire several T helper (Th) fates, guided by a dynamic cytokine milieu following antigenic challenge. This physiological Th fate choice process is often erroneously conflated with a maladaptive pathological process historically termed Th polarization. Here we propose why these two processes are distinct and separable. We posit that, though innate signaling alone is sufficient for Th fate choice in naive CD4+ T cells, Th polarization instead strictly originates from pre-existing cross-reactive memory CD4+ T cells. We further posit that Th polarization is normally prevented by thymus-derived cross-reactive antigen-specific regulatory T cells (Tregs) and inevitably manifests as immunopathology when the Treg repertoire and the microbiota that maintains it are selectively depleted. Bifurcating Th fate choice and polarization delineate Th effector pathways more accurately and tangibly improve the scope of targeted therapies for allergies, autoimmune diseases, and effective vaccines.

Could cross-reactivity rescue Foxp3+ regulatory T cell precursors from thymic deletion?

Thymocytes that bind with high affinity to peptides displayed by MHC class II (pMHC-II) are deleted while low-affinity binders differentiate into naive CD4+ T cells. However, Foxp3+ regulatory T cells (Tregs) seem to defy this binary choice as their precursors require high-affinity interaction with pMHC-II for maturation in the thymus. Here, we rely on the antigen-specific interpretive framework, SPIRAL (Specific ImmunoRegulatory Algorithm), to propose that Tregs escape thymic deletion by forming dyads with IL-2-producing T cells via antigen cross-reactivity. This interpretation reconciles contradictions related to Treg ontogeny in the thymus and their role in modulating antigen-specific immune responses.

Concurrent cross-reactivity of microbiota-derived epitopes to both self and pathogens may underlie the "Hygiene hypothesis".

The current iteration of the "Hygiene hypothesis" proposes precipitous decline in exposure to conserved microbial products and metabolites in individuals in developed countries undermines innate self-nonself "training" of immune system leading to allergy and autoimmunity. However, lack of innate "training" alone fails to account for the antigen-driven nature of these immunopathologies. Here, we advance an alternative, antigen-specific interpretive framework, SPIRAL (Specific ImmunoRegulatory Algorithm) that predicts "loss" of commensal microbiota-derived epitopes cross-reactive to both self and pathogens, rather than conserved microbial moieties or metabolites, underlies the "Hygiene hypothesis." By mechanistically delineating how loss of selective microbiota in predisposed individuals could lead to corresponding "holes" in the epitope-specific Foxp3+ regulatory T cell repertoire and subsequent selective immunopathologies, SPIRAL represents a novel interpretation of cross-reactivity that could enable targeted discovery of microbiota species and their associated Treg epitopes "missing" in the diseases "Hygiene hypothesis" implicates, and provides a roadmap for a novel unified interpretation of self-nonself discrimination and T helper phenotype selection.

Dendritic cells and the immunity/tolerance decision.

The role of dentritic cells in initiating the immune response has been well established. Recent studies point to an important role for DCs in the induction of peripheral tolerance as well. It was proposed that the role of DC in the immunity/tolerance decision could be associated simply with DC maturation states. However, it has been observed that immature DC do not process endocytosed antigens well to form MHC+peptide complexes and therefore self-specific T cells would not be able to recognize their ligands on immature DCs. Then how might immature DCs induce tolerance to self-antigens? Below it is discussed a new mechanism which might control whether the DCs behavior will be tolerogenic or immunogenic. The hypothesis proposed that DCs should have two maturation programs operating in absence/presence of Danger signals leading to mature-tolerogenic and mature-immunogenic phenotypes, respectively.

The novel mechanism of peripheral tolerance.

The circulating pool of lymphocytes contains self- as well as non-self reactive T cells. The mature DCs present as non-self as well as self-epitopes to nai;ve T cells. To avoid autoimmunity the organism has to keep the mature DCs afar from self-specific T cells. I had proposed that the different anatomical distribution of the immature (tolerogenic) and mature (immunogenic) DCs in the peripheral lymphoid tissues may contribute to this mechanism. I had proposed that T cells to reach the mature DC should pass through the layer of immature DCs, which constitutively phagocytose and transport apoptotic cells to the regional LN and present only 'self' epitopes. Thus, self-specific T cells will be trapped by the immature DC layer and be deleted or become anergic. The layer of immature DC will be crossed only by those T cells that are not self-specific.

Chronic infection and the origin of adaptive immune system.

It has been speculated that the rise of the adaptive immune system in jawed vertebrates some 400 million years ago gave them a superior protection to detect and defend against pathogens that became more elusive and/or virulent to the host that had only innate immune system. First, this line of thought implies that adaptive immune system was a new, more sophisticated layer of host defense that operated independently of the innate immune system. Second, the natural consequence of this scenario would be that pathogens would have exercised so strong an evolutionary pressure that eventually no host could have afforded not to have an adaptive immune system. Neither of these arguments is supported by the facts. First, new experimental evidence has firmly established that operation of adaptive immune system is critically dependent on the ability of the innate immune system to detect invader-pathogens and second, the absolute majority of animal kingdom survives just fine with only an innate immune system. Thus, these data raise the dilemma: If innate immune system was sufficient to detect and protect against pathogens, why then did adaptive immune system develop in the first place? In contrast to the innate immune system, the adaptive immune system has one important advantage, precision. By precision I mean the ability of the defense system to detect and remove the target, for example, infected cells, without causing unwanted bystander damage of surrounding tissue. While the target precision per se is not important for short-term immune response, it becomes a critical factor when the immune response is long-lasting, as during chronic infection. In this paper I would like to propose new, "toxic index" hypothesis where I argue that the need to reduce the collateral damage to the tissue during chronic infection(s) was the evolutionary pressure that led to the development of the adaptive immune system.

Brief antigenic stimulation generates effector CD8 T cells with low cytotoxic activity and high IL-2 production.

It is currently believed that a brief antigenic stimulation is sufficient to induce CD8 T cells to complete their differentiation program, become effector T cells, and subsequently generate memory. Because this concept was derived from studies in which only a single effector function was analyzed (either IFN-gamma production or target cell lysis), we wondered whether monitoring for multiple effector functions might reveal novel characteristics of effector CD8 T cells elicited by brief or prolonged Ag exposure. Using an in vitro system to generate effector T cells and an in vivo adoptive transfer model to track donor CD8 T cells, we found that the differentiation programs acquired by CD8 T cells after brief or prolonged antigenic stimulation were different. Although the frequencies of IFN-gamma and TNF-alpha producers were comparable for both effector CD8 T cell populations, there were major differences in cytotoxic potential and IL-2 production. Whereas prolonged (>24 h) Ag exposure stimulated effector CD8 T cells with high cytotoxic activity and low IL-2 production, brief (<24 h) stimulation generated effector CD8 T cells with low cytotoxic activity and high IL-2 production. The latter effector T cells rapidly converted into central memory-like CD8 T cells, exhibited long-term survival in adoptively transferred hosts, and gave robust recall responses upon Ag challenge. These data suggest that not all functions of effector CD8 T cells are equally inherited after brief or prolonged antigenic stimulation.

The JAM Test and its daughter P-JAM: simple tests of DNA fragmentation to measure cell death and stasis.

Cytotoxic T lymphocytes, and other death-inducing agents, have at least two different ways of killing their targets: drilling holes in the target cell membrane, or triggering the targets to commit suicide. The JAM Test is a method that measures the DNA fragmentation that accompanies cell suicide. We label target cells with radioactive DNA-precursor nucleotides and harvest them onto fiberglass filters, which trap large pieces of DNA but pass smaller fragments of apoptotic cells. As a general measure of apoptosis, the JAM Test described here is faster (can be completed in 4 h [or less if labeling is done the night before]), more quantitative, easier, more sensitive, more flexible and cheaper than most other current assays of apoptosis. The P-JAM, also discussed, additionally allows for assessment of death in cells that don't fragment their DNA, and allows for assays of agents that induce cell stasis rather than death.