Type 1 Diabetes: Cellular, Molecular & Clinical Immunology

Theoretical Essay E - Can Getting Excited Over Nothing Provoke Autoimmunity?
Don Bellgrau

In this article I will present a hypothesis regarding the role of a diabetogenic gene in promoting diabetes. Our concepts are formulated around experimental observations from our group regarding a novel T cell activation state found among a majority of peripheral T cells in the diabetes-prone Biobreeding rat. We hypothesize that the novel activation is promoted via an indirect effect wherein a diabetogenic gene creates a unique stimulus in the periphery, space.
The Biobreeding (BB) rat has provided an excellent animal model to study the causes and cures of type I diabetes (reviewed in this publication). Diabetogenicity is controlled by at least four disease susceptibility genes in the rat (1), the most prominent in this species and in all diabetes-prone animals maps to the Class II region of the Major Histocompatibility Complex (MHC). The BB rat was derived from an outbred colony of Wistar rats (2). Selective breeding lead to the development of two sublines, one diabetes-prone (DP) and one diabetes-resistant (DR). The BB-DP and BB-DR sublines appear to differ in only one disease susceptibility locus (3), mapping to a syntenic region of rat chromosome four (4-6). This locus is commonly referred to as lyp for the phenotype that it produces, peripheral T cell lymphopenia (7). Congenic BB-DR rats bred for homozygosity at lyp demonstrate essentially 100% penetrance of the disease while non-lymphopenic BB-DR animals do not develop diabetes spontaneously (4); with the proviso that the animals are maintained under specific pathogen free conditions. For example, infection with Kilham’s rat virus can cause BB-DR animals to become diabetic (8).
Lymphopenia is dramatic in the diabetes-prone BB rat (BB-DP). The peripheral T cell pool is diminished by as much as 80%. While both CD4+ and CD8+ T cells are affected the reduction is most dramatic among CD8+ T cells (9). Lymphopenic animals bear a peripheral T cell pool that is virtually devoid of CD8+ T cells and the few remaining T cells of this phenotype express reduced levels of this co-receptor (10). CD4 co-receptor and T cell receptor expression levels appear normal, however. The T cells from the lymphopenic animal also express low or undetectable levels (henceforth referred to as RT6-) of the RT6 rat differentiation antigen (11). Reconstitution of BB-DP rats with RT6+ T cells from non-lymphopenic BB-DR donors protects the lymphopenic BB-DP recipient from disease (12).
These observations indicated that the diabetogenicity of the lyp gene could be related to its effect on targeting RT6+ regulatory T cells. In the rat, Thy1 is expressed on immature T cells and is coordinately downregulated with the expression, post thymically of RT6. The frequency of Thy1+ T cells is markedly increased in BB-DP rat. However, the BB-DP RT6 gene is intact as the BB allelic product is expressed in a co-dominant fashion on T cells from BB-DP/Wistar F1 animals (13). Therefore the Thy1+ RT6- phenotype that dominates the periphery of the BB-DP rat has been explained as due to a lyp gene mediated block in T cell development. RT6+ regulatory T cells never mature and the periphery of the lymphopenic animals is populated by RT6- immature T cells not regulated by RT6+ mature T cells. This immature, regulatory T cell deficient environment is favorable for the development of diabetes.
Therefore, contained within the RT6- T cells are at least some with autoreactive potential. Why do RT6- T cells with autoreactive potential exist? It is known that the deletion of high affinity anti-self reactive T cells occurs in the thymus during the negative selection process but that peripheral tolerance mechanisms also contribute to maintaining a T cell repertoire that is devoid of overt autoreactivity. Therefore lyp could be exerting its effect by interfering with either or both thymic and peripheral tolerance mechanisms. Peripheral tolerance mechanisms appear abnormal in the BB-DP. We defined one abnormality as an inability of T cells from the BB-DP to be tolerized following superantigen exposure in vivo (14). The thymic infrastructure of the BB-DP rat also has been reported to be abnormal (15) and our laboratory reported a thymic dysfunction that mapped to thymic APC (16).
Thymic and peripheral tolerance induction each involve the paradigm commonly described as the two signal model for T cell activation. A basic tenet of the model is that T cells are signaled not only by an interaction through the antigen specific T cell receptor (TCR) but also through various costimulatory interactions, provided most commonly by costimulatory molecules expressed on antigen presenting cells (APC) (17). It is widely held that signal one (TCR) without signal two (costimulation) can tolerize peripheral T cells either by anergizing them or causing them to be deleted. Thus, in the periphery signal one plus two is generally considered as an activating impetus while signal one alone is a tolerizing signal (18). In the thymus, signal one and two appear to function differently compared to the periphery. Evidence indicates that signal one plus two when provided by thymic APC to immature T cells (CD4+CD8+ cortical thymocytes (19) and a subset of “single positive” more mature, medullary thymocytes (20) initiates deletion or negative selection. The two signal model for negative selection in the thymus helps explain earlier observations proposing that negative selection appeared to be associated with the medulla rather than the cortical region of the thymus (21). While it has been concluded that negative selection can occur in both cortical and medullary regions the perceived dominance of negative selection in the latter may simply reflect an increased frequency of thymic APC in this region.
If thymic negative selection and therefore tolerance is a two signal process in the thymus, while tolerance induction in the periphery is a one signal process, during T cell maturation it becomes possible that the same T cell can respond differently to identical signals. We hypothesized that defects in the way in which T cells respond to these two signals may permit them to escape tolerizing signals, thus promoting the survival of autoreactive T cells. To examine this we developed a two signal activation model in vitro to test T cells at various stages in development. A goal was to determine if T cells from lymphopenic animals responded similarly or not compared to T cells from non-lymphopenic animals. Our approach was to purify T cells such that endogenous sources of costimulation were absent. This could be accomplished by vigorous depletion of antigen presenting cells, resulting in a concomitant enrichment for T cells. Using the periphery as a source of lymphocytes we observed that this purified T cell population from normal rats underwent apoptosis if the TCR was engaged without provision of an exogenous source of costimulation. This was the predicted result based on published data using mice as the T cell source. Costimulation rescued the rat T cells from apoptosis and lead to progression into cell cycle followed by clonal expansion, also as predicted from studies performed with T cells from mice. However, when T cells from lymphopenic BB-DP or lymphopenic Fischer rats were purified and provided with costimulation the T cells were not rescued from apoptosis and did not clonally expand. Instead the stimulus lead to their death by apoptosis (22). The provision of signal one alone was sufficient to push the T cells into cell cycle (23). Such progression into cell cycle did not occur if the T cells were from non-lymphopenic donors. We ascribed the term promiscuous activation to this costimulation-independent progression into cell cycle of T cells from lymphopenic donors.
Costimulation-independent cell cycle progression could be attributed to antigen experienced T cells i.e. T cells that had undergone activation previously. However, T cells from lymphopenic animals did not express a cell surface phenotype consistent with activation. Activation markers such as OX40 and IL-2 receptor were absent from the cell surface of the vast majority of T cells from lymphopenic donors, unless TCR stimulation was provided. The QCA antigen, a resting T cell marker, was also expressed (24;25). Therefore the costimulation-independent progression into cell cycle i.e. promiscuous activation, did not correlate with a cell surface phenotype commonly attributed to an activated T cell. An examination of cell cycle machinery was more informative. Here the phenotype was more consistent with the cell cycle progression data. The presumably “resting” T cells isolated from the animal and tested without further in vitro manipulation expressed low levels of the cyclin dependent kinase inhibitor p27kip (23;26). Kip has been shown to act as the primary regulatory molecule controlling progression through the G1 restriction checkpoint in numerous cells including T cells (27). Reduction in kip protein expression was accompanied by increased basal and serum stimulated gene transcription of the gene products cyclin D1, cyclin A and DFHR which are involved in the progression from G1 to S phase (28).
Kip binding to cyclin prevents T cells from progressing from G1 into S phase and the ubiquitin dependent degradation of kip is held to be accomplished via the receipt of costimulation. As expected, T cells from normal, non-lymphopenic donors expressed high kip levels and were prevented from entering cell cycle without the provision of costimulation. Only in the presence of two signals was kip degraded and cell cycle progression initiated. The T cells from lymphopenic animals also expressed high levels of PCNA a proliferating cell nuclear antigen which functions as a requisite processivity factor for DNA polymerase (29;30). Kip degradation is accompanied by upregulation of several cell cycle proteins that act as effectors downstream of kip in the coordination of cell cycle progression. One such molecule is Rb (31). Rb has been shown to exist in an unphosphorylated, rapidly migrating 110kd species in resting T cells (32). Upon activation two higher molecular weight species were observed that were found to contain multiple phosphorylation sites post-translationally modified by cyclin/cdk complexes that are active in G1 and G1/S phase transition.
We observed increased levels PCNA and hyperphosphorylated Rb, which we interpreted as additional indicators of activation events and cell cycle progression. Therefore the promiscuous activation phenotype, defined by entry into cell cycle without need of costimulation is associated with internal indicators of T cell activation defined by the molecular phenotypes of low Kip, high PCNA and activated Rb. We have coined the phrase “novel activation state” to identify T cells with characteristics of both resting (on the cell surface) but activated (in cell cycle machinery) phenotypes.
An examination of T cells at various stages of maturation indicated that the novel proliferative state originated at the recent thymic emigrant (RTE) stage. Medullary thymocytes but not RTE from lymphopenic donors could respond to costimulation and enter cell cycle comparably to their counterparts in non-lymphopenic animals. Medullary thymocytes from lymphopenic and non-lymphopenic donors also expressed comparable kip levels. The precipitous reduction in kip levels among peripheral T cells from lymphopenic versus non-lymphopenic donors only occurred when RTE were examined. This provided evidence that the promiscuous activation phenotype and novel activation state both had their origins as the medullary thymocytes were leaving the thymus and began entering the peripheral T cell pool.
We propose that promiscuous activation and the novel activation state are related. Cell cycle progression in T cells from lymphopenic donors occurs without the necessity of costimulation because the (normal) costimulation-dependent degradation of the cyclin dependent kinase inhibitor p27kip has already occurred. However, lyp does not appear to be targeting kip directly because kip levels are normal in both B lymphocytes and non lymphoid cells from lymphopenic donors. Also the murine kip gene does not map to the syntenic lyp locus.
If lyp is not directly involved in promiscuous T cell activation and the generation of the novel proliferative state then what is the nature of the stimulus that causes this activation to occur and what role does lyp play in this process that leads to the development of autoimmunity? We believe that the answers to these questions rest with another phenotype of T cells from lymphopenic animals. The majority appear to have an abnormally short life span, measured in days not weeks (33;34). Therefore a widely held view of how the lymphopenic environment is created is that some medullary thymocytes (35) and most recent thymic emigrants die (36) before they have the opportunity to become long lived peripheral T cells. How this lyp gene mediated short life span relates to promiscuous activation and the novel activation state forms the basis around which our proposal is centered.
We reason that the direct effect of lyp involves the shortened life span of recent thymic emigrants and hypothesize that the shortened life span is caused by the lyp gene mediated death of recent thymic emigrants as they enter cell cycle and attempt to transit the G1 to S boundary. The impetus for entering cell cycle comes from two sources. First, we hypothesize that requisite peripheral survival signals (referred to recently as peripheral positive selection) provide an activation impetus. Recently, peripheral T cell homeostasis has received attention. Long term peripheral T cell survival in the absence of conventional antigenic stimulation is dependent on continuous contact with the thymic selecting MHC and peptides (37-42). Several reports have shown that the peptide ligands that mediate thymic positive selection control both peripheral T cell survival and homeostatic proliferation in the peripheral compartments of T cell lymphopenic hosts, in the absence of conventional antigenic stimulation (41;43;44). The proliferation that accompanied homeostatic expansion had two prerequisites, the first being expression of appropriate cognate MHC/peptide ligands and the second being T cell “space” in the periphery (45). Lack of contact induces a short life span in both CD4+ and CD8+ T cell subsets.
However, these signals for survival need not lead to clonal expansion. We propose that the clonal expansion signals are provided by the indirect effect of the lyp gene. Lyp causes T cells to die in cell cycle arrest and thus creates the lymphopenic periphery. The lymphopenic periphery provides the stimulus for clonal expansion; space. It is the presence of space created by lymphopenia that signals the recent thymic emigrants to clonally expand in an attempt to fill up the void in the periphery.
The model for the diabetogenic lyp gene involvement is summarized as follows. The RTE are activated by peripheral positive selection. They attempt to divide as a result of the stimulus of space created by lymphopenia. The lyp gene aborts this process and the RTE die. The cycle continues. Peripheral positive selection provides a T cell survival signal. Lymphopenia provides a stimulus for clonal expansion and lyp perpetuates lymphopenia by interfering with T cell clonal expansion. In essence it is the excitement created by space that may provide an activation stimulus to generate autoreactivity and autoimmune disease.

Reference List - links to PubMed available in Reference List.

For comments, corrections or to contribute teaching slides, please contact Dr. Eisenbarth at: george.eisenbarth@ucdenver.edu

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