Avidity maturation of memory CD8 T cells is limited by self-antigen expression

Immune tolerance to self-antigens is a complex process that utilizes multiple mechanisms working in concert to maintain homeostasis and prevent autoimmunity. We developed a system that revealed a population of self-specific CD8 T cells within the endogenous T cell repertoire. Immunization of ovalbumin (OVA)-expressing transgenic mice with recombinant viruses expressing OVA-peptide variants induced self-reactive T cells in vivo that matured into memory T cells able to respond to secondary infection. However, whereas the avidity of memory cells in normal mice increased dramatically with repeated immunizations, avidity maturation was limited for self-specific CD8 T cells. Despite decreased avidity, such memory cells afforded protection against infection, but did not induce overt autoimmunity. Further, up-regulation of self-antigen expression in dendritic cells using an inducible system promoted programmed death-1 expression, but not clonal expansion of preexisting memory cells. Thus, the self-reactive T cell repertoire is controlled by overlapping mechanisms influenced by antigen dose.     

Loss of the pro-apoptotic BH3-only Bcl-2 family member Bim inhibits BCR stimulation-induced apoptosis and deletion of autoreactive B cells

During development, the stochastic process assembling the genes encoding antigen receptors invariably generates B and T lymphocytes that can recognize self-antigens. Several mechanisms have evolved to prevent the activation of these cells and the concomitant development of autoimmune disease. One such mechanism is the induction of apoptosis in developing or mature B cells by engagement of the B cell antigen receptor (BCR) in the absence of T cell help. Here we report that B lymphocytes lacking the pro-apoptotic Bcl-2 family member Bim are refractory to apoptosis induced by BCR ligation in vitro. The loss of Bim also inhibited deletion of autoreactive B cells in vivo in two transgenic systems of B cell tolerance. Bim loss prevented deletion of autoreactive B cells induced by soluble self-antigen and promoted accumulation of self-reactive B cells developing in the presence of membrane-bound self-antigen, although their numbers were considerably lower compared with antigen-free mice. Mechanistically, we determined that BCR ligation promoted interaction of Bim with Bcl-2, inhibiting its survival function. These findings demonstrate that Bim is a critical player in BCR-mediated apoptosis and in B lymphocyte deletion.     

Phenotypic and functional analysis of CD8(+) T cells undergoing peripheral deletion in response to cross-presentation of self-antigen

Not all T cells specific for autoantigens are eliminated in the thymus, and therefore alternate mechanisms are required to prevent potentially autoreactive T cells from developing into effectors. Adoptive transfer of CD8(+) T cells from influenza hemagglutinin-specific Clone 4 TCR transgenic mice into mice that express hemagluttinin in the pancreatic islets results in tolerance. This is preceded by activation of Clone 4 T cells that encounter antigen cross-presented in the draining lymph nodes of the pancreas. In this report we compare the phenotype, function, and costimulatory requirements of Clone 4 T cells activated by endogenous self-antigen, with Clone 4 T cells stimulated by influenza virus. The cells undergoing tolerance upregulate both CD69 and CD44, yet only partially downregulate CD62L, and do not express CD49d or CD25. Most importantly, they lack the ability to produce interferon-gamma in response to antigen and show no cytolytic activity. Clone 4 T cells disappear after several cycles of division, apparently without leaving the site of initial activation. Surprisingly, despite the fact that such stimulation occurs through recognition of antigen that is cross-presented by a professional antigen-presenting cell, we find this activation is not dependent on costimulation through CD28. These data demonstrate that the recognition by naive CD8(+) T cells of cross-presented self-antigen results in localized proliferation and deletion, without the production of effector cells.     

T cell receptor-mediated negative selection of autoreactive T lymphocyte precursors occurs after commitment to the CD4 or CD8 lineages

To identify the maturational stage(s) during which T cell receptor (TCR)-mediated positive and negative selection occurs, we followed the development of CD4+8- and CD4-8+ T cells from TCRlo CD4+8+ thymic blasts in the presence of different positive and negative selecting (major histocompatibility complex or Mls) elements. We describe novel lineage-committed transitional intermediates that are TCRmed CD4+8lo or TCRmed CD4lo8+, and that show evidence of having been positively selected. Furthermore, negative selection is not evident until after cells have attained one of the TCRmed transitional phenotypes. Accordingly, we propose that negative selection in normal mice occurs only after TCRlo CD4+8+ precursors have been positively selected into either the CD4 or CD8 lineage.     

CD4+CD25+ regulatory T cells suppress allograft rejection mediated by memory CD8+ T cells via a CD30-dependent mechanism

CD4(+)CD25(+) regulatory T (Treg) cells suppress naive T cell responses, prevent autoimmunity, and delay allograft rejection. It is not known, however, whether Treg cells suppress allograft rejection mediated by memory T cells, as the latter mount faster and stronger immune responses than their naive counterparts. Here we show that antigen-induced, but not naive, Treg cells suppress allograft rejection mediated by memory CD8(+) T cells. Suppression was allospecific, as Treg cells induced by third-party antigens did not delay allograft rejection. In vivo and in vitro analyses revealed that the apoptosis of allospecific memory CD8(+) T cells is significantly increased in the presence of antigen-induced Treg cells, while their proliferation remains unaffected. Importantly, neither suppression of allograft rejection nor enhanced apoptosis of memory CD8(+) T cells was observed when Treg cells lacked CD30 or when CD30 ligand-CD30 interaction was blocked with anti-CD30 ligand Ab. This study therefore provides direct evidence that pathogenic memory T cells are amenable to suppression in an antigen-specific manner and identifies CD30 as a molecule that is critical for the regulation of memory T cell responses.     

HLA-E-dependent presentation of Mtb-derived antigen to human CD8+ T cells

Previous studies in mice and humans have suggested an important role for CD8+ T cells in host defense to Mtb. Recently, we have described human, Mtb-specific CD8+ cells that are neither HLA-A, B, or C nor group 1 CD1 restricted, and have found that these cells comprise the dominant CD8+ T cell response in latently infected individuals. In this report, three independent methods are used to demonstrate the ability of these cells to recognize Mtb-derived antigen in the context of the monomorphic HLA-E molecule. This is the first demonstration of the ability of HLA-E to present pathogen-derived antigen. Further definition of the HLA-E specific response may aid development of an effective vaccine against tuberculosis.     

Bim-mediated deletion of antigen-specific CD8 T cells in patients unable to control HBV infection

HBV-specific CD8(+) T cells are critical for a successful immune response to HBV infection. They are markedly diminished in number in patients who fail to control the virus, but the mechanisms resulting in their depletion remain ill defined. Here, we dissected the defective HBV-specific CD8(+) T cell response associated with chronic HBV infection by gene expression profiling. We found that HBV-specific CD8(+) T cells from patients with different clinical outcomes could be distinguished by their patterns of gene expression. Microarray analysis revealed that overlapping clusters of functionally related apoptotic genes were upregulated in HBV-specific CD8(+) T cells from patients with chronic compared with resolved infection. Further analysis confirmed that levels of the proapoptotic protein Bcl2-interacting mediator (Bim) were upregulated in HBV-specific CD8(+) T cells from patients with chronic HBV infection. Blocking Bim-mediated apoptosis enhanced recovery of HBV-specific CD8(+) T cells both in culture and directly ex vivo. Consistent with evidence that Bim mediates apoptosis of CD8(+) T cells expressing low levels of CD127 (IL-7R), the few surviving HBV-specific CD8(+) T cells were CD127(hi )and had elevated levels of the antiapoptotic protein Mcl1, suggesting they were amenable to IL-7-mediated rescue from apoptosis. We therefore postulate that Bim-mediated attrition of HBV-specific CD8(+) T cells contributes to the inability of these cell populations to persist and control viral replication.     

Major histocompatibility complex class II presentation of cell-associated antigen is mediated by CD8alpha+ dendritic cells in vivo

Antigen-specific B cells express major histocompatibility complex class II and can present antigen directly to T cells. Adoptive transfer experiments using transgenic B and T cells demonstrated that antigen-specific B cells can also efficiently transfer antigen to another cell for presentation to T cells in vivo. To identify the antigen-presenting cell that receives antigens from B cells, a strategy was developed to follow the traffic of B cell-derived proteins in vivo. B cells were labeled with the fluorescent dye CFSE and loaded with antigen, before adoptive transfer into recipient mice. Populations of splenocytes from the recipient mice were later assayed for the presence of fluorescent proteins and for the ability to activate T cells. A small number of CD8alpha+CD4-CD11b(lo) dendritic cells (DCs) contain proteins transferred from B cells and these DCs effectively present antigens derived from the B cells to T cells. The results suggest that CD8alpha+ DCs sample the cells and membranes in their environment for presentation to T cells circulating through the T cell zone. This function of CD8alpha+ DCs may be relevant to the priming of an immune response or the induction of T cell tolerance.     

Generation of human CD8 T regulatory cells by CD40 ligand-activated plasmacytoid dendritic cells

Although CD8 T cell-mediated immunosuppression has been a well-known phenomenon during the last three decades, the nature of primary CD8 T suppressor cells and the mechanism underlying their generation remain enigmatic. We demonstrated that naive CD8 T cells primed with allogeneic CD40 ligand-activated plasmacytoid dendritic cells (DC)2 differentiated into CD8 T cells that displayed poor secondary proliferative and cytolytic responses. By contrast, naive CD8 T cells primed with allogeneic CD40 ligand-activated monocyte-derived DCs (DC1) differentiated into CD8 T cells, which proliferated to secondary stimulation and killed allogeneic target cells. Unlike DC1-primed CD8 T cells that produced large amounts of interferon (IFN)-gamma upon restimulation, DC2-primed CD8 T cells produced significant amounts of interleukin (IL)-10, low IFN-gamma, and no IL-4, IL-5, nor transforming growth factor (TGF)-beta. The addition of anti-IL-10-neutralizing monoclonal antibodies during DC2 and CD8 T cell coculture, completely blocked the generation of IL-10-producing anergic CD8 T cells. IL-10-producing CD8 T cells strongly inhibit the allospecific proliferation of naive CD8 T cells to monocytes, and mature and immature DCs. This inhibition was mediated by IL-10, but not by TGF-beta. IL-10-producing CD8 T cells could inhibit the bystander proliferation of naive CD8 T cells, provided that they were restimulated nearby to produce IL-10. IL-10-producing CD8 T cells could not inhibit the proliferation of DC1-preactivated effector T cells. This study demonstrates that IL-10-producing CD8 T cells are regulatory T cells, which provides a cellular basis for the phenomenon of CD8 T cell-mediated immunosuppression and suggests a role for plasmacytoid DC2 in immunological tolerance.     

CD40Ig treatment results in allograft acceptance mediated by CD8CD45RC T cells, IFN-gamma, and indoleamine 2,3-dioxygenase

Treatment with CD40Ig results in indefinite allograft survival in a complete MHC-mismatched heart allograft model in the rat. Here we show that serial second, third, and fourth adoptive transfers of total splenocytes from CD40Ig-treated recipients into secondary recipients led to indefinite donor-specific allograft acceptance. Purification of splenocyte subpopulations from CD40Ig-treated recipients demonstrated that only the adoptively transferred CD8(+)CD45RC(low) subset resulted in donor-specific long-term survival, whereas CD8(+)CD45RC(low) T cells from naive animals did not. Accepted grafts displayed increased indoleamine 2,3-dioxygenase (IDO) expression restricted in the graft to ECs. Coculture of donor ECs with CD8(+)CD45RC(low) T cells purified from CD40Ig-treated animals resulted in donor-specific IDO expression dependent on IFN-gamma. Neutralization of IFN-gamma or IDO triggered acute allograft rejection in both CD40Ig-treated and adoptively transferred recipients. This study demonstrates for what we believe to be the first time that interference in CD40-CD40 ligand (CD40-CD40L) interactions induces allospecific CD8(+) Tregs that maintain allograft survival. CD8(+)CD45RC(low) T cells act through IFN-gamma production, which in turn induces IDO expression by graft ECs. Thus, donor alloantigen-specific CD8(+) Tregs may promote local graft immune privilege through IDO expression.     

Neonatal tumor necrosis factor alpha promotes diabetes in nonobese diabetic mice by CD154-independent antigen presentation to CD8(+) T cells

Neonatal islet-specific expression of tumor necrosis factor (TNF)-alpha in nonobese diabetic mice promotes diabetes by provoking islet-infiltrating antigen-presenting cells to present islet peptides to autoreactive T cells. Here we show that TNF-alpha promotes autoaggression of both effector CD4(+) and CD8(+) T cells. Whereas CD8(+) T cells are critical for diabetes progression, CD4(+) T cells play a lesser role. TNF-alpha-mediated diabetes development was not dependent on CD154-CD40 signals or activated CD4(+) T cells. Instead, it appears that TNF-alpha can promote cross-presentation of islet antigen to CD8(+) T cells using a unique CD40-CD154-independent pathway. These data provide new insights into the mechanisms by which inflammatory stimuli can bypass CD154-CD40 immune regulatory signals and cause activation of autoreactive T cells.     

Perinatal blockade of b7-1 and b7-2 inhibits clonal deletion of highly pathogenic autoreactive T cells

A number of in vitro studies have suggested that costimulatory molecules B7-1 and B7-2 and their receptor CD28 can promote clonal deletion, and limited in vivo studies have indicated that CD28 is involved in the clonal deletion of some T cells. However, the significance of B7-mediated clonal deletion in preventing autoimmune diseases has not been studied systematically. Here we report that the perinatal blockade of B7-1 and B7-2 substantially inhibits the clonal deletion of T cells in the thymus and leads to an accumulation of T cells capable of inducing fatal multiorgan inflammation. These results reveal a critical role for costimulatory molecules B7-1 and B7-2 in deleting pathogenic autoreactive T cells in the thymus. The critical role of B7-1 and B7-2 in T cell clonal deletion may explain, at least in part, the paradoxical increase of autoimmune disease in mice deficient for this family of costimulatory molecules, such as cytotoxic T lymphocyte associated molecule 4, CD28, and B7-2. The strong pathogenicity of the self-reactive T cells supports a central hypothesis in immunology, which is that clonal deletion plays an important role in preventing autoimmune diseases.     

Detection of autoreactive CD4+ T cells by MHC class II multimers in HLA-linked human autoimmune diseases

Recognition of self-peptides in association with distinct HLA class II alleles by autoreactive CD4⁺ T cells is central for loss of immunological tolerance leading to autoimmune disease. However, identifying immunodominant self-peptides and characterizing autoreactive T cells is challenging. In this issue of the JCI, Falta et al. identify a disease-associated complementarity-determining region 3β motif specific for beryllium-modified C-C motif ligand 4 (CCL4) and CCL3 self-peptides in patients with chronic beryllium disease (CBD), a granulomatous lung disorder with a known HLA class II allelic association. Detection of these antigen-specific CD4⁺ T cells by beryllium-pulsed HLA-DP2 tetramers presenting CCL4/CCL3 confirms these autoantigens in humans and mice and enables monitoring in the progress of disease. Detection of autoreactive CD4⁺ T cells by peptide–MHC class II multimers allows for the detailed characterization of disease-promoting T cells. This knowledge has profound implications for the monitoring and development of targeted therapies in human autoimmune disorders.     

CD28-induced costimulation of T helper type 2 cells mediated by induction of responsiveness to interleukin 4

Type 1 and type 2 cloned T helper (Th) cells are believed to require different antigen-presenting cell (APC)-derived costimuli for proliferation. In the case of Th1-cloned T cells, CD28 signaling costimulates production of autocrine interleukin 2 (IL-2). Th2 cells produce their autocrine growth factor, IL-4, without costimulation, but require APC-derived costimuli, or IL-1, to respond to IL-4. Here we demonstrate that engagement of CD28 on Th2 cells with anti-CD28 antibody or with APC-associated B7 costimulates Th2 responsiveness to IL-4 but does not affect IL-4 or IL-2 production by Th2 cells. Costimulation of Th2 cells via CD28 appears to involve the induction of IL-1 production by Th2 cells, which acts in an autocrine fashion to induce IL-4 responsiveness. These results suggest that CD28-induced costimulation plays an important role in responses mediated by both types of Th cells.     

Direct activation of CD8+ cytotoxic T lymphocytes by dendritic cells

Recent experiments (11-13) have shown that antigen-specific, CD8+, CD4- T lymphocytes can be induced to proliferate and become killer cells in the absence of a second population of "helper" CD8-, CD4+ cells. We have studied early events in the activation of CD4+ and CD8+ T cell subsets in the primary mixed leukocyte reaction. Dendritic cells are a major if not essential accessory cell for the activation of both subpopulations. Antigen-bearing macrophages fail to stimulate unprimed CD8+ cells, but act as targets for the sensitized cytolytic lymphocytes that are induced by dendritic cells. The initial proliferative response is comparable for CD4+ and CD8+ lymphocyte subsets. For both subpopulations, dendritic cells efficiently cluster the responding lymphocytes on the first day and induce the release of IL-2. The data indicate that CD4+ and CD8+ lymphocytes can be activated by a similar mechanism, and illustrate the special role of dendritic cells in the sensitization stage of cell-mediated immunity.     

Inhibition of T cell responses by activated human CD8+ T cells is mediated by interferon-gamma and is defective in chronic progressive multiple sclerosis.

The autologous mixed lymphocyte reaction (AMLR) involves the activation of T cells by autologous antigen presenting cells. Cells are generated during the course of the AMLR that have suppressive properties in vitro. In the present study we investigated the induction of CD8+ T cells in the AMLR with suppressive properties and the mechanism by which these cells downregulate in vitro proliferative responses. Purified CD8+ but not CD4+ T cells activated in the AMLR in conditioned medium inhibited proliferation of autologous T cells by anti-CD3 or PPD. Nonactivated CD8+ T cells did not suppress. The CD8+ T cells activated in the AMLR in the presence of conditioned medium (CD8+ Tact) were CD11b negative and were noncytotoxic. The inhibitory effect of CD8+ Tact cells was completely abrogated by anti-IFN-gamma antibody, but not by anti-IL-4, anti-IL-10, or anti-TGF-beta antibody. The induction of CD8+ Tact cells in the AMLR was blocked by anti-IL-2 or by anti-GM-CSF antibody and the combination of these two recombinant cytokines could support the induction of suppressive CD8+ Tact cells. CD8+ Tact cells were defective in patients with chronic progressive multiple sclerosis (MS) as compared to patients with relapsing-remitting MS or normal controls. Our studies provide a basis for understanding the mechanism of suppression by human CD8+ T cells in terms of specific cytokines, and demonstrate the potential importance of these cells in a human autoimmune disease as their function is defective in patients with progressive MS.     

CD30 expression by CD8+ T cells producing type 2 helper cytokines. Evidence for large numbers of CD8+CD30+ T cell clones in human immunodeficiency virus infection

A large panel of CD8+ T cell clones generated from peripheral blood lymphocytes (PBL) of healthy donors or human immunodeficiency virus (HIV)-infected individuals were assessed for both cytokine secretion profile and CD30 expression and release. The great majority of CD8+ T cell clones generated from healthy individuals showed the ability to produce interferon gamma (IFN-gamma), but not interleukin 4 (IL-4), and none of them either expressed membrane CD30 or released substantial amounts of soluble CD30 (sCD30) in their supernatant. In contrast, high numbers of CD8+ T cell clones generated from HIV-infected individuals, which produced IL-4 (and IL-5) in addition to IFN-gamma or IL-4 (and IL- 5) alone, expressed membrane CD30 and released detectable amounts of sCD30 in their supernatants. Indeed, CD30 expression appeared to be positively correlated with the ability of CD8+ T cell clones to produce IL-4 and IL-5 and inversely correlated with their ability to produce IFN-gamma, whereas no correlation between CD30 expression and production of IL-10 was observed. These data suggest that CD30 is a marker for CD8+ T cells that have switched to the production of type 2 helper cytokines.     

Expression of cutaneous lymphocyte-associated antigen by CD8(+) T cells specific for a skin-tropic virus

Virus-specific CD8(+) T cells traffic to infected tissues to promote clearance of infection. We used herpes simplex virus type 2 (HSV-2) as a model system to investigate CD8(+) T cell trafficking to the skin in humans. Using human leukocyte antigen (HLA) class I tetramers, we observed that HSV-specific CD8(+) T cells in the peripheral blood expressed high levels of cutaneous lymphocyte-associated antigen (CLA). In contrast, CD8(+) T cells specific for non-skin-tropic herpesviruses lacked CLA expression. CLA-positive HSV-2-specific CD8(+) T cells had the characteristics of central memory cells, expressing CCR7, CD62L, and CD28, and they proliferated briskly in response to antigen. CLA is related to a functional E-selectin ligand, and both E-selectin and CLA-positive cells were detected in HSV-2-infected skin. HSV-2-specific T cells adhered to cells transfected with E-selectin. A higher proportion of HSV-specific CD8(+) T cells recovered from herpes lesions express CLA compared with blood, consistent with a role for CLA in skin homing. To our knowledge, this is the first report of expression of tissue-specific adhesion-associated molecules by virus-specific CD8(+) T cells. The evaluation of vaccines for skin and mucosal pathogens should include study of the induction of appropriate tissue-specific homing molecules.