Pep19 drives epitope spreading in periodontitis and periodontitis-associated autoimmune diseases.

BACKGROUND AND OBJECTIVE: Epitope spreading is one of valid mechanisms operating in immunopathological processes of infection-induced autoimmune diseases. We hypothesized that the peptide 19 from Porphyromonas gingivalis heat shock protein (HSP) 60 (Pep19) may be the dominant epitope from which epitope-specific immune response to subdominant epitopes may diversify sequentially into autoimmune responses directed at human neoepitopes in P. gingivalis-induced periodontitis and autoimmune diseases. However, the exact feature and mechanism on how Pep19 may drive epitope spreading into human autoantigens in chronic periodontitis or P. gingivalis-induced experimental periodontitis has not been clarified. The present study was performed with the following specific aims: (i) to delineate retrospectively the features of epitope spreading by human cross-sectional analysis; (ii) to demonstrate prospectively the epitope spreading into new antigenic determinants in an ordered, predictable and sequential manner in experimental periodontitis; and (iii) to clarify the mechanism on how immunization with Pep19 may mobilize helper T cells or elicit B-cell responses to human autoantigens and neoantigen. MATERIAL AND METHODS: The study was devised for two independent investigations - a cross-sectional analysis on clinical subjects and a prospective analysis on experimental periodontitis - each being subdivided further into two additional independent observations. Cross-sectional dot immunoblot pattern against a panel of peptides of P. gingivalis HSP60 and human HSP60 was performed among age-dependent healthy subjects and between healthy subjects, patients with chronic periodontitis and patients with autoimmune disease, to identify epitope spreading. A peptide-specific T-cell line was established for phenotype analysis and for proliferation assay to an array of identical peptides. An identical prospective analysis was performed in P. gingivalis-induced experimental periodontitis or in Pep19-immunized mice. Cross-reactivity of anti-Pep19 monoclonal antibody was also investigated. RESULTS: A dominant immune response exclusively to Pep19 prevailed in healthy human subjects (before the age of 40) and mice that persisted in chronic periodontitis and autoimmune diseases without being replaced further by subsequent subdominant epitopes. A sequential epitope spreading provoked by Pep19 to subdominant autoantigen peptide 19 from human HSP60 (Hu19) in most healthy human subjects and mice, and to autoantigen peptide 9 from human HSP60 (Hu9) and neoantigen oxidized low-density lipoprotein (ox-LDL) in P. gingivalis-induced chronic periodontitis and autoimmune diseases could be demonstrated in a reproducible and predictable manner. T-cell proliferative activity to multiple autoantigens Hu19, Hu9 and ox-LDL, and cross-reactivity of anti-Pep19 monoclonal antibody to these epitopes may be proposed as cellular and molecular mechanisms responsible for the phenomenon. Moreover, the predictive value of Pep19 for Hu9 increased remarkably in the disease group when compared with that of the healthy group. CONCLUSION: Taken together, epitope spreading to Hu19, Hu9 and ox-LDL provoked by Pep19 could be proposed as a solid phenomenon observed in P. gingivalis-induced chronic periodontitis and infection-induced autoimmune diseases in a reproducible and predictable manner. T-cell proliferative activity to these peptides and cross-reactivity of anti-Pep19 antibodies to multiple human autoantigens could be proposed as cellular and molecular mechanisms responsible for this phenomenon.

Selectively expanding subsets of T cells in mice by injection of interleukin-2/antibody complexes: implications for transplantation tolerance.

The biological activity of interleukin (IL)-2 and other cytokines in vivo can be augmented by binding to certain anti-cytokine monoclonal antibodies (mAb). Here, we review evidence on how IL-2/anti-IL-2 mAb complexes can be used to cause selective stimulation and expansion of certain T-cell subsets. With some anti-IL-2 mAbs, injection of IL-2/mAb complexes leads to expansion of CD8 T effector cells but not CD4 T regulatory cells (Tregs); these complexes exert less adverse side effects than soluble IL-2 and display powerful antitumor activity. Other IL-2/mAb complexes have minimal effects on CD8 T cells but cause marked expansion of Tregs. Preconditioning mice with these complexes leads to permanent acceptance of MHC-disparate pancreatic islets in the absence of immunosuppression.

T cell memory.

T cell memory induced by prior infection or vaccination provides enhanced protection against subsequent microbial infections. The processes involved in generating and maintaining T cell memory are becoming better understood due to recent technological advances in identifying memory T cells and monitoring their behavior and function in vivo. Memory T cells develop in response to a progressive set of cues-starting with signals from antigen-loaded, activated antigen-presenting cells (APCs) and inflammatory mediators induced by the innate immune response, to the poorly defined subsequent signals triggered as the immune response wanes toward homeostasis. The persistence of the resting memory T cells that eventually develop is regulated by cytokines. This chapter discusses recent findings on how memory T cells develop to confer long-term protective immunity.

NKT cells derive from double-positive thymocytes that are positively selected by CD1d.

CD1d-reactive NKT cells are a separate T cell sublineage. Instructive models propose that NKT cells branch off the mainstream developmental pathway because of their T cell antigen receptor specificity, whereas stochastic models would propose that they develop from precursor cells committed to this sublineage before variable-gene rearrangement. We show here that immature double-positive (DP) thymocytes form the canonical rearranged Valpha gene of NKT cells at nearly equivalent frequencies in the presence or absence of CD1d expression. After interacting with CD1d in the thymus, these cells give rise to expanded populations of NKT cells-including both CD4+ and double-negative lymphocytes in the thymus and periphery-that express this alpha chain. These results confirm the existence of a DP intermediate for CD1d-reactive NKT cells. They also show that the early developmental stages of these T cells are not governed by a distinct mechanism, which is consistent with the TCR-instructive model of differentiation.

Long-term persistence and reactivation of T cell memory in the lung of mice infected with respiratory syncytial virus.

In mice acutely infected with respiratory syncytial virus (RSV), more than 20% of pulmonary CD8(+) T cells, but only 2-3% of CD8(+) T cells in the draining lymph node secreted interferon-gamma in response to a single peptide. Surprisingly, the percentage of virus-specific T cells in the lung remained at these high levels long after the acute infection. Pulmonary memory T cells were further studied in a sensitive adoptive transfer system, which allows visualizing polyclonal CD4(+) and CD8(+) virus-specific memory T cell responses. Fifty days after infection, persisting RSV-specific pulmonary T cells remained CD69(hi) CD62L(lo), but had returned to a resting memory state according to functional criteria. In the absence of neutralizing antibodies reinfection first induced cell division among virus-specific memory T cells 3 days after infection predominantly in the local lymph node. However, divided cells then rapidly accumulated in the lung without significantly increasing in the lymph node. These results suggest rapid export of reactivated cells from the lymph node to the target organ. Thus, although memory T cells can be maintained in the infected organ after a localized virus infection, amplification of a recall response appears to be most effective in organized lymphoid tissue.

IL-7 is critical for homeostatic proliferation and survival of naive T cells.

In T cell-deficient conditions, naive T cells undergo spontaneous "homeostatic" proliferation in response to contact with self-MHC/peptide ligands. With the aid of an in vitro system, we show here that homeostatic proliferation is also cytokine-dependent. The cytokines IL-4, IL-7, and IL-15 enhanced homeostatic proliferation of naive T cells in vitro. Of these cytokines, only IL-7 was found to be critical; thus, naive T cells underwent homeostatic proliferation in IL-4(-) and IL-15(-) hosts but proliferated minimally in IL-7(-) hosts. In addition to homeostatic proliferation, the prolonged survival of naive T cells requires IL-7. Thus, naïve T cells disappeared gradually over a 1-month period upon adoptive transfer into IL-7(-) hosts. These findings indicate that naive T cells depend on IL-7 for survival and homeostatic proliferation.

Generation and maintenance of memory T cells.

Typical immune responses lead to the prominent clonal expansion of antigen-specific T cells followed by their differentiation into effector cells. Most effector cells die at the end of the immune response but some of the responding cells survive and form long-lived memory cells. The factors controlling the formation and survival of memory T cells are discussed. Recent evidence suggests that T memory cells arise from a subset of effector cells. The longevity of T memory cells may require continuous contact with cytokines, notably IL-15 for CD8(+) cells.

Role of self-major histocompatibility complex/peptide ligands in selection and maintenance of a diverse T cell repertoire.

Positive selection has long been thought to be a devise for producing a repertoire of T cells that can efficiently recognize foreign peptides in the context of self-major histocompatibility complex (MHC) molecules. However, in the light of recent evidence that long-term survival of mature T cells requires continuous contact with self-MHC molecules, the possibility for an additional role for positive selection has emerged: to generate a repertoire of T cells that can be maintained in the periphery through contact with self-MHC/peptide ligands. In support of this idea, our recent work suggests that positive selection is highly peptide specific and, more important, that mature T cells require extrathymic contact with the same MHC/peptide ligands that initially induced positive selection in the thymus in order for prolonged survival and to undergo homeostatic proliferation in response to T cell deficiency.

T cells can use either T cell receptor or CD28 receptors to absorb and internalize cell surface molecules derived from antigen-presenting cells.

At the site of contact between T cells and antigen-presenting cells (APCs), T cell receptor (TCR)-peptide-major histocompatibility complex (MHC) interaction is intensified by interactions between other molecules, notably by CD28 and lymphocyte function-associated antigen 1 (LFA-1) on T cells interacting with B7 (B7-1 and B7-2), and intracellular adhesion molecule 1 (ICAM-1), respectively, on APCs. Here, we show that during T cell-APC interaction, T cells rapidly absorb various molecules from APCs onto the cell membrane and then internalize these molecules. This process is dictated by at least two receptors on T cells, namely CD28 and TCR molecules. The biological significance of T cell uptake of molecules from APCs is unclear. One possibility is that this process may allow activated T cells to move freely from one APC to another and eventually gain entry into the circulation.

Transcription factor PU.1 is necessary for development of thymic and myeloid progenitor-derived dendritic cells.

Dendritic cells (DCs) are a heterogeneous population of cells that are specialized for Ag processing and presentation. These cells are believed to derive from both myeloid- and lymphoid-committed precursors. Normal human PBMC-derived, human CD14+ cell (monocyte)-derived, and mouse hematopoietic progenitor-derived DCs were shown to express the hematopoietic cell-restricted, ets family transcription factor PU.1. These populations represent myeloid progenitor-derived DCs. Hematopoietic progenitor cells from PU.1 gene-disrupted (null) mice were unable to generate MHC class IIhigh, CD11c+ myeloid-derived DCs in vitro. Mouse thymic DCs are proposed to be derived from a committed lymphoid progenitor cell that can give rise to T cells as well as DCs. Previously, we showed that CD4 and CD8 T cells developed in PU.1 null mice in a delayed manner and in reduced number. We examined the thymus of 10- to 12-day-old PU.1 null mice and found no evidence of DEC-205+, MIDC-8+ DCs in this tissue. Our findings indicate that PU.1 regulates the development of both thymic and myeloid progenitor-derived populations of DCs, and expand its known role in hematopoietic development.

The peptide ligands mediating positive selection in the thymus control T cell survival and homeostatic proliferation in the periphery.

Positive selection to self-MHC/peptide complexes has long been viewed as a device for skewing the T cell repertoire toward recognition of foreign peptides presented by self-MHC molecules. Here, we provide evidence for an alternative possibility, namely, that the self-peptides controlling positive selection in the thymus serve to maintain the longevity of mature T cells in the periphery. Surprisingly, when total T cell numbers are reduced, these self-ligands become overtly stimulatory and cause naive T cells to proliferate and undergo homeostatic expansion.

Thymic selection by a single MHC/peptide ligand: autoreactive T cells are low-affinity cells.

In H2-M- mice, the presence of a single peptide, CLIP, bound to MHC class II molecules generates a diverse repertoire of CD4+ cells. In these mice, typical self-peptides are not bound to class II molecules, with the result that a very high proportion of H2-M- CD4+ cells are responsive to the various peptides displayed on normal MHC-compatible APC. We show here, however, that such "self" reactivity is controlled by low-affinity CD4+ cells. These cells give spectacularly high proliferative responses but are virtually unreactive in certain other assays, e.g., skin graft rejection; responses to MHC alloantigens, by contrast, are intense in all assays. Possible explanations for why thymic selection directed to a single peptide curtails self specificity without affecting alloreactivity are discussed.

The role of H2-O and HLA-DO in major histocompatibility complex class II-restricted antigen processing and presentation.

The function of major histocompatibility complex (MHC) class II molecules is to sample exogenous antigens for presentation to CD4+ T helper cells. After synthesis in the endoplasmic reticulum, class II molecules are directed into the endosomal system by association with the invariant chain (Ii), which is sequentially cleaved, generating class II dimers loaded with Ii-derived peptides (CLIP). These class II-peptide complexes are physiological substrates for H2-M/HLA-DM, a resident of the endosomal/lysosomal system which facilitates the removal of CLIP from newly synthesised class II alpha beta dimers. Exchange of CLIP for antigenic class II-binding peptides is also promoted by the action of H2-M/HLA-DM, resulting in stable peptide-class II complexes that are transported to the cell surface for presentation to CD4+ T cells. Recent evidence suggests that this H2-M/HLA-DM-mediated 'peptide editing' is influenced by another MHC class II-encoded molecule, H2-O/HLA-DO. This non-polymorphic alpha beta heterodimer is associated with H2-M/HLA-DM during intracellular transport and within the endosomal system of B cells. H2-O/HLA-DO alters the peptide exchange function of H2-M/HLA-DM in a pH-dependent manner, so that H2-M/HLA-DM activity is limited to more acidic conditions, corresponding to lysosomal compartments. Indeed, H2-O/HLA-DO may serve to limit the presentation of antigens after fluid phase uptake by B cells, while augmenting presentation of antigens internalised via membrane Ig receptors. Such a mechanism may maintain the fidelity of the B-cell-CD4+ T-cell interaction, counteracting self reactivity arising from less stringent lymphocyte activation. Here, data evaluating the role of H2-O/HLA-DO shall be reviewed and its putative function discussed.

A role for Fas in negative selection of thymocytes in vivo.

To seek information on the role of Fas in negative selection, we examined subsets of thymocytes from normal neonatal mice versus Fas-deficient lpr/lpr mice injected with graded doses of antigen. In normal mice, injection of 1-100 microg of staphylococcal enterotoxin B (SEB) induced clonal elimination of SEB-reactive Vbeta8+ cells at the level of the semi-mature population of HSAhi CD4+ 8- cells found in the thymic medulla; deletion of CD4+ 8+ cells was minimal. SEB injection also caused marked elimination of Vbeta8+ HSAhi CD4+ 8- thymocytes in lpr/lpr mice. Paradoxically, however, elimination of these cells in lpr/lpr mice was induced by low-to-moderate doses of SEB (

Altered antigen presentation in mice lacking H2-O.

HLA-DM catalyzes the release of MHC class II-associated invariant chain-derived peptides (CLIP) from class II molecules. Recent evidence has suggested that HLA-DO is a negative regulator of HLA-DM in B cells, but the physiological function of HLA-DO remains unclear. Analysis of antigen presentation by B cells from mice lacking H2-O (the mouse equivalent of HLA-DO), together with biochemical analysis using purified HLA-DO and HLA-DM molecules, suggests that HLA-DO/H2-O influences the peptide loading of class II molecules by limiting the pH range in which HLA-DM is active. This effect may serve to decrease the presentation of antigens internalized by fluid-phase endocytosis, thus concentrating the B cell-mediated antigen presentation to antigens internalized by membrane immunoglobulin.

Thymic selection by a single MHC/peptide ligand produces a semidiverse repertoire of CD4+ T cells.

The influence of individual peptides in thymic selection was examined in H2-M- mice, in which positive selection is directed to a single peptide, class II-associated invariant chain peptide (CLIP) bound to H2-A(b). Two sensitive in vivo approaches showed that 70%-80% of CD4+ T cells undergoing positive selection to CLIP+H2-A(b) have self-reactivity to the various peptides expressed on wild-type H2-M+ antigen-presenting cells. When these self-reactive T cells were depleted, the residual CD4+ cells displayed a polyclonal repertoire in terms of alloreactivity, responses to foreign protein antigens, and Vbeta usage. Nevertheless, studies with two T cell receptor transgenic lines suggested that the repertoire of CD4+ cells induced by CLIP was less diverse than the repertoire of CD4+ cells in normal mice. Generation of a fully diverse T cell repertoire thus requires positive selection against multiple peptides.

Rat stem cells developing in irradiated SCID mice fail to become tolerized and cause lethal graft-versus-host disease.

Graft-versus-host disease (GVHD) is prominent in irradiated hosts given whole allogeneic bone marrow cells but is generally undetectable when T-depleted stem cells are transferred; under these conditions, the mature T cells arising from the donor stem cells become tolerant to host antigens and fall to cause GVHD. We show here that a radically different situation can occur when hosts are reconstituted with xenogeneic stem cells. When lightly irradiated, adult C.B-17 SCID mice injected with Lewis rat fetal liver (FL) cells show near-total repopulation with rat-derived lymphohemopoietic cells, including T and B cells. However, in marked contrast to chimeras prepared with allogeneic mouse FL cells, rat FL-->SCID chimeras develop severe and often lethal chronic GVHD. In these rat-->mouse chimeras, the rat T cells show limited tolerance to host mouse antigens as determined by various parameters including mixed lymphocyte reaction and cytotoxic T lymphocyte assays in vitro, adoptive transfer of T cells to secondary SCID hosts, and the lack of V beta deletion to endogenous host mtv antigens. GVHD in irradiated rat-->SCID chimeras is most prominent with Lewis FL but also applies to Fisher 344 and Wistar Furth FL cells. The failure of newly formed rat T cells in rat-->SCID chimeras to become fully tolerant to host mouse antigens appears to be due to depletion of host antigen-presenting cells by irradiation. Thus, rat-->SCID chimeras generated by transplanting rat FL cells into unirradiated neonatal SCID mice fail to develop GVHD, and the rat T cells display self-tolerance. As allogeneic H-2-different mouse FL-->irradiated SCID chimeras display strong self-tolerance, presumably through recognition of host antigens on thymic epithelial cells, the implication is that mouse thymic epithelial cells are tolerogenic only for mouse and not for rat immature T cells.

Antigen presentation in retroviral vector-mediated gene transfer in vivo.

We have examined mechanisms involved in gene transfer, protein expression, and antigen presentation after direct administration of retroviral vectors using a variety of antigen systems. We have identified transduced infiltrating cells at the injection site, and the majority of the infiltrating cells were of the monocyte/macrophage lineage. We found that the splenic dendritic cell fraction contained proviral DNA, expressed antigenic proteins, and was able to present antigens efficiently to the immune system. Furthermore, the dendritic cell fractions from retroviral vector-immunized mice were able to prime naive T cells in vitro, and adoptive transfer of in vitro-transduced dendritic cell fractions elicited antigen-specific cytotoxic T lymphocytes. These data suggest a role for dendritic cells in induction of immune responses elicited by retroviral vector-mediated gene transfer.

Antigen compartmentation and T helper cell tolerance induction.

The process of antigen recognition depends in part on the amount of peptide antigen available and the affinity of the T cell receptor for a particular peptide-major histocompatibility complex (MHC) molecule complex. The availability of self antigen is limited by antigen processing, which is compartmentalized such that peptide antigens presented by MHC class I molecules originate in the cytoplasm, whereas peptide antigens presented by MHC class II molecules are acquired from the endocytic pathway. This segregation of the antigen-processing pathways may limit the diversity of antigens that influence the development and selection of, e.g., CD4-positive, MHC class II-specific T cells. Selection in this case might involve only a subset of self-encoded proteins, specifically those that are plasma membrane bound or secreted. To study these aspects of immune development, we engineered pigeon cytochrome for expression in transgenic mice in two forms: one in which it was expressed as a type II plasma membrane protein, and a second in which it was targeted to the mitochondria after cytoplasmic synthesis. Experiments with these mice clearly show that tolerance is induced in the thymus, irrespective of antigen compartmentation. Using radiation bone marrow chimeras, we further show that cytoplasmic/mitochondrial antigen gains access to the MHC class II pathway by direct presentation. As a result of studying the anatomy of the thymus, we show that the amount of antigen and the affinity of the TCR affect the location and time point of thymocytes under-going apoptosis.