Naturally occurring CD4+ T-cell epitope variants act as altered peptide ligands leading to impaired helper T-cell responses in hepatitis C virus infection

Hepatitis C virus (HCV) has a high rate of replication and lacks RNA-proofreading capabilities, thereby leading to variant or mutant viruses circulating within the host as a quasispecies. Previous work in our laboratory identified viral variants that emerged in a class-II immunodominant epitope NS3(358-375) of the non-structural-3 (NS3) protein region of HCV, the sequence of which is based on genotype 1A, the most prevalent genotype in the United States. Further work suggested that positive immune selection pressure was driving viral variation. Paradoxically, viral variants account for only a small percentage of the circulating virus in human beings and in chimpanzees, suggesting that passive evasion is not the only means of escape by HCV. This observation suggests a unique pathogenesis for HCV as it persists in the host. In the current study, we hypothesize that viral variants are acting as altered peptide ligands (APLs). To test this hypothesis, we used cloned T cells specific for NS3(358-375) peptide, which demonstrated attenuated T-cell and interferon-γ (IFN-γ) responses to individual variant peptides, when compared with the NS3(358-375) stimulated T-cell clones. Furthermore, such variants could act as APLs, based on their ability to antagonize the IFN-γ proliferative responses of clones specific for NS3(358-375). In addition, major histocompatibility complex (MHC) class II tetramer staining demonstrated that variant peptide-MHC complexes were able to specifically bind to NS3(358-375) T-cell clones and that both the variant and NS3(358-375) tetramers were able to bind to the same CD4(+) T cells. Taken together, the results suggest that viral variants may act as APL to effectively blunt the T-cell response to an important HCV epitope.     

Regulation of CD4 T‐cell receptor diversity by vaccine adjuvants

New vaccines based on soluble recombinant antigens (Ags) require adjuvants to elicit long‐lasting protective humoral and cellular immunity. Despite the importance of CD4 T helper cells for the generation of long‐lived memory B and CD8 T cells, the impact of adjuvants on CD4 T‐cell responses is still poorly understood. Adjuvants are known to promote dendritic cell (DC) maturation and migration to secondary lymphoid organs where they present foreign peptides bound to class II major histocompatibility complex molecules (pMHCII) to naïve CD4 T cells. Random and imprecise rearrangements of genetic elements during thymic development ensure that a vast amount of T‐cell receptors (TCRs) are present in the naïve CD4 T‐cell repertoire. Ag‐specific CD4 T cells are selected from this vast pre‐immune repertoire based on the affinity of their TCR for pMHCII. Here, we review the evidence demonstrating a link between the adjuvant and the specificity and clonotypic diversity of the CD4 T‐cell response, and consider the potential mechanisms at play.     

CD4 T cells in tumor immunity

T cell immunity is the key to protective immune responses against tumors. Traditionally, this function has been ascribed to CD8 T lymphocytes with cytotoxic activity, which are restricted by MHC class I molecules. In recent years the realization that CD4 T cells can also play a relevant role in protective anti-tumor responses has received growing attention. Here we will discuss the role of MHC class II-restricted T cells in response to, and in the regulation of, tumor antigens. Emphasis will be placed on four areas: (1) the role of CD4 T cell immunity in tumor protection in animal models and putative mode of action, (2) tumor antigens recognized by human CD4 T cells, (3) the cooperation between two CD4 T cells of different specificity as a new way to jump start the response against sub-immunogenic determinants of tumor antigens in a tolerant environment, and (4) the negative impact of regulatory CD4 T cells on anti-tumor T cell responses. By drawing attention to these four areas, it is our intention to provide the reader with a comprehensive view of issues of contemporary importance for this field, in the expectation that the information will help a better design of therapeutic cancer vaccines.     

"One size fits it all": translocation of foreign antigens by Yersinia type III secretion system (TTSS) leads to concomitant CD4 and CD8 T-cell priming

Live replicating bacteria expressing heterologous antigens are vaccine candidates that are able to induce complex immune responses. Yersinia pseudotuberculosis employs a type III secretion system for translocation of several virulence factors directly to the cytosol of eukaryotic cells. Mice orally inoculated with an attenuated recombinant Yersinia strain translocating a chimeric Yersinia outer protein E (YopE) molecule reveal high numbers of foreign antigen-specific CD4 and CD8 T cells. Thus, cytosolic display of a single hybrid protein results in concomitant CD4 and CD8 T-cell priming. This "one-size-fits-it-all"-feature of Yersinia-translocated heterologous antigens might be advantageous to mount T-cellular immune responses against complex microbes and tumors.     

Immunoprevalence of the CD4+ T-cell response to HIV Tat and Vpr proteins is provided by clustered and disperse epitopes, respectively

Recent studies have suggested including nonstructural proteins as Tat and Vpr in HIV vaccines. However, little is known about the CD4+ T-cell response that these small proteins induce in humans. We have therefore evaluated these responses by in vitro priming experiments of CD4+ T lymphocytes harvested in healthy donors. In the Tat protein, only one peptide primed CD4+ T cells of eight HLA unrelated healthy donors. T cells induced by this peptide recognized immature DC loaded with the native Tat protein and are restricted by multiple HLA-DR molecules, in agreement with its binding capacity. This peptide was therefore processed in an appropriate manner and was highly immunoprevalent. CD4+ T-cell response to Vpr peptides was more disperse and involved six different peptides depending on the HLA-DR molecules of the donors. Two overlapping peptides were T-cell stimulating in at least half of the donors. T-cell response to Vpr in multiple donors is the result of a combination of several CD4+ T-cell epitopes with good to moderate immunoprevalence. Altogether, our results show that the frequency of responders to HIV Tat or Vpr proteins relies on one or multiple CD4+ T-cell epitopes, respectively.     

Phenotypic analysis of perennial airborne allergen‐specific CD4+ T cells in atopic and non‐atopic individuals

Background Accumulating evidence suggests that T cells play an important role in the pathogenesis of atopic dermatitis (AD); yet, little is known of the differentiation status of CD4⁺ T cells specific for common environmental allergens, such as the major cat allergen, Fel d 1. Objective To determine the frequency, differentiation phenotype and function of circulating Fel d 1‐specific CD4⁺ T cells in adult individuals with severe persistent AD in comparison with healthy controls. Methods Using HLA class II tetrameric complexes based on a HLA‐DPB1^*0401‐restricted Fel d 1 epitope, ex vivo and cultured T cell frequency and phenotype were analysed in individuals with AD and healthy controls. Cytokine secretion was measured by ex vivo and cultured IL‐4 and IFN‐γ ELISpots. Results Ex vivo Fel d 1‐specific DPB1^*0401‐restricted CD4⁺ T cells in both atopics and non‐atopics express high levels of CCR7, CD62L, CD27 and CD28, placing the cells largely within the central memory subgroup. However, the functional phenotype was distinct, with greater IL‐4 production from the cells derived from atopics, which correlated with disease severity. Conclusions and Clinical Relevance Circulating Fel d 1‐specific DPB1^*0401‐restricted CD4⁺ T cells in both atopic and non‐atopic donors maintain a central memory phenotype; however in atopics, the cells had greater Th2 effector function, compatible with a disease model of altered antigen delivery in atopic individuals. Cite this as: L. R. Crack, H. W. Chan, T. McPherson and G. S. Ogg, Clinical & Experimental Allergy, 2011 (41) 1555–1567.     

Inhibition of T-cell responses by CD4 on an antigen-presenting cell

In addition to T-helper cells, CD4 is expressed on monocytes, macrophages and dendritic cells. To see whether CD4 molecules on antigen-presenting cells affect T-cell responses, we expressed CD4 on Raji cells, and compared positive and negative cells as stimulator cells for peripheral blood mononuclear cells in mixed lymphocyte reactions. We found that expression of CD4 on Raji had an inhibitory effect on production of IFN-gamma and other cytokines.     

Genome-wide screening of human T-cell epitopes in influenza A virus reveals a broad spectrum of CD4 T-cell responses to internal proteins, hemagglutinins, and neuraminidases

We performed a genome-wide screening for T-cell epitopes using synthetic peptides that encompass all of the influenza A viral proteins, including subtype variants for hemagglutinin (HA; H1, H3, and H5) and neuraminidase (NA; human and avian N1 and N2) proteins, based on the sequence information of recently circulating strains. We identified a total of 83 peptides, 54 of them novel, to which specific T cells were detectable in interferon-γ (IFN-γ) enzyme-linked immunosorbent spot assays using peripheral blood mononuclear cells from four healthy adult donors. The surface glycoproteins, HA and NA, major components of vaccines, expressed many T-cell epitopes. HA and matrix protein 1 expressed more T-cell epitopes than other viral proteins, most of which were recognized by CD4⁺ T cells. We established several cytotoxic CD4⁺ T-cell lines from these donors. We also analyzed H1 and H3 HA-specific T-cell responses using the peripheral blood mononuclear cells of 30 hospital workers. Fifty-three percent of donors gave a positive response to H3 HA peptides, whereas 17% gave a positive response to H1 HA peptides. Our genome-wide screening is useful in identifying T-cell epitopes and is complementary to the approach based on the predicted binding peptides to well-studied HLA-A, -B, and -DR alleles.     

Loss in CD4 T-cell responses to multiple epitopes in influenza due to expression of one additional MHC class II molecule in the host

An understanding of factors controlling CD4 T-cell immunodominance is needed to pursue CD4 T-cell epitope-driven vaccine design, yet our understanding of this in humans is limited by the complexity of potential MHC class II molecule expression. In the studies described here, we took advantage of genetically restricted, well-defined mouse strains to better understand the effect of increasing MHC class II molecule diversity on the CD4 T-cell repertoire and the resulting anti-influenza immunodominance hierarchy. Interferon-γ ELISPOT assays were implemented to directly quantify CD4 T-cell responses to I-A(b) and I-A(s) restricted peptide epitopes following primary influenza virus infection in parental and F(1) hybrid strains. We found striking and asymmetric declines in the magnitude of many peptide-specific responses in F(1) animals. These declines could not be accounted for by the lower surface density of MHC class II on the cell or by antigen-presenting cells failing to stimulate T cells with lower avidity T-cell receptors. Given the large diversity of MHC class II expressed in humans, these findings have important implications for the rational design of peptide-based vaccines that are based on the premise that CD4 T-cell epitope specificity can be predicted by a simple cataloguing of an individual's MHC class II genotype.     

Luminal bacterial antigen-specific CD4+ T-cell responses in HLA-B27 transgenic rats with chronic colitis are mediated by both major histocompatibility class II and HLA-B27 molecules

Rats transgenic (TG) for the human major histocompatibility complex (MHC) class I HLA-B27 and beta2-microglobulin genes develop chronic colitis under specific pathogen-free (SPF) but not sterile (germ-free, GF) conditions. We investigated the role of antigen-presenting molecules involved in generating immune responses by CD4+ mesenteric lymph node (MLN) cells from colitic HLA-B27 TG rats to commensal enteric micro-organisms. All TG MLN cells expressed HLA-B27. A higher level of MHC class II was expressed on cells from TG rats, both SPF and GF, compared to non-TG littermates. In contrast, rat MHC class I expression was lower on TG than non-TG cells. Both TG and non-TG antigen presenting cells (APC) pulsed with caecal bacterial antigens induced a marked interferon-gamma (IFN-gamma) response in TG CD4+ T lymphocytes but failed to stimulate non-TG cells. Blocking MHC class II on both TG and non-TG APC dramatically inhibited their ability to induce TG CD4+ T cells to produce IFN-gamma. Blocking HLA-B27 on TG APC similarly inhibited IFN-gamma responses. When the antibodies against MHC class II and HLA-B27 were combined, no APC-dependent IFN-gamma response was detected. These data implicate both native rat MHC class II and TG HLA-B27 in CD4+ MLN T-cell IFN-gamma responses to commensal enteric microflora in this colitis model.     

Recent insights into CD4+ T-cell specificity and function in Type 1 diabetes

Type 1 diabetes (T1D) is caused by T-cell-mediated destruction of the insulin-producing β-cells in the pancreas. Genetic and immunological evidence from humans and mouse models indicates that CD4⁺ T cells play a crucial role in the development and prevention of T1D. The dichotomy between CD4⁺ T regulatory and effector T cells has encouraged research into the role of these cell subsets in T1D. New antigens and epitopes recognized by CD4⁺ T cells in affected individuals have been identified. Growing knowledge of T-cell specificity and function is helping to develop new assays for analyzing islet antigen-specific CD4⁺ T cells from human blood. Here we discuss, with particular reference to human studies, advances in our understanding of CD4⁺ T-cell responses in T1D.     

CD1d-restricted NKT cell activation enhanced homeostatic proliferation of CD8+ T cells in a manner dependent on IL-4

CD1d-restricted NKT cells are activated by TCR-mediated stimulation via CD1d plus lipid antigens such as alpha-galactosylceramide (alpha-GalCer). These cells suppressed autoimmunity and graft rejection, but sometimes enhanced resistance to infection and tumor immunity. This double-action phenomenon of NKT cells is partly explained by cytokines produced by NKT cells. Therefore, roles of cytokines from activated NKT cells have been extensively examined; however, their roles on T cell homeostatic proliferation in lymphopenic condition have not been investigated. Here, we showed that alpha-GalCer enhanced homeostatic proliferation of CD8+ but not CD4+ T cells and this effect of alpha-GalCer was required for NKT cells. IL-4 was essential and sufficient for this NKT cell action on CD8+ T cell homeostatic proliferation. Importantly, the expression of IL-4Ralpha and STAT6 in CD8+ T cells was essential for the NKT activity, indicating a direct action of IL-4 on CD8+ T cells. Consistent with this, the level of IL-4Ralpha expression on memory phenotype CD8(+) T cells was higher than that on naive phenotype one and CD4+ T cells. Thus, these results showed the 'involvement' of IL-4 that is produced from activated NKT cells for CD8+ T cell homeostatic proliferation in vivo.     

Characterization of CD4+ T-cell–dendritic cell interactions during secondary antigen exposure in tolerance and priming

Despite the recent advances in our understanding of the dynamics of the cellular interactions associated with the induction of immune responses, comparatively little is known about the in vivo behaviour of antigen-experienced T cells upon secondary antigen exposure in either priming or tolerance. Such information would provide an insight into the functional mechanisms employed by memory T cells of distinct phenotypes and provide invaluable knowledge of how a specific tolerogenic or immunogenic state is maintained. Using real-time imaging to follow the in vivo motility of naïve, primed and tolerized CD4⁺ T cells and their interactions with dendritic cells (DCs), we demonstrate that each of these distinct functional phenotypes is associated with specific patterns of behaviour. We show that antigen-experienced CD4⁺ T cells, whether primed or tolerized, display inherently slower migration, making many short contacts with DCs in the absence of antigen. Following secondary exposure to antigen, primed T cells increase their intensity or area of interaction with DCs whereas contacts between DCs and tolerized T cells are reduced. Importantly, this was not associated with alterations in the contact time between DCs and T cells, suggesting that T cells that have previously encountered antigen are more effective at surveying DCs. Thus, our studies are the first to demonstrate that naïve, primed and tolerized T cells show distinct behaviours before and after secondary antigen-encounter, providing a novel mechanism for the increased immune surveillance associated with memory T cells. These findings have important consequences for many immunotherapeutics, which aim to manipulate secondary immune responses.     

Induction and maintenance of self tolerance: the role of CD4+CD25+ regulatory T cells

The immune system responds vigorously to invading pathogens (non-self, foreign), while remaining unresponsive (tolerant) to the body's own components and circulating constituents (self). This indifference to self components is a result of finely orchestrated events of thymic negative selection (central tolerance) of developing T cells that are autoaggressive combined with those operative in the periphery (peripheral tolerance) to control the activity of potentially autoreactive T cells that escaped thymic tolerance. Recently, autoimmune regulator expressed in the thymus has been identified as a critical mediator of central tolerance towards tissue-specific antigens. In the periphery, a variety of regulatory T cells are involved in effecting tolerance. There is immense interest and excitement about the newly identified subset of CD4(+)CD25(+) T cells. This is a unique subset of CD4(+) T cells that bear CD25 (IL-2Ralpha chain) on the cell surface in the na?ve state and express FoxP3 as a unique marker. These cells suppress the activity of autoreactive effector T cells primarily via cell-cell contact. The deficiency and/or altered function of CD4(+)CD25(+) T cells is associated with autoimmunity. Mice deficient in FoxP3 (scurfy mice) bear an autoimmune phenotype, and human males with mutations in the corresponding gene express the phenotype of wide-spread autoimmunity, the immune dysregulation, polyendocrinopathy and enteropathy, and X-linked syndrome. In vitro expansion of antigen-specific CD4(+)CD25(+) T cells and their adoptive transfer into patients suffering from autoimmunity is emerging as a promising new therapeutic approach for these debilitating disorders.     

Chemokines,chemokine receptors and CD4+CD25+ regulatory T cells

The fields of regulatory T (Treg) cells and chemokines/chemokine receptors have progressed rapidly in the last few years. Treg cells, especially CD4⁺CD25⁺ Treg cells, play a critical role in maintaining self-tolerance and immune homeostasis. Chemokines and chemokine receptors are crucial for lymphoid development, homing and immunological regulation. This review will discuss the biological effects of chemokines and chemokine receptors on regulating the migration and development of CD4⁺CD25⁺ Treg cells, and the potential clinical implications of these findings when considering chemokine receptors as therapeutic targets.     

CD4+ T-cell responses and distribution at the colonic mucosa during Brachyspira hyodysenteriae-induced colitis in pigs

The spirochaete Brachyspira hyodysenteriae causes swine dysentery, a severe colitis characterized by mucosal enlargement as a result of crypt elongation and epithelial necrosis. Most efforts to understand the pathogenesis of this disease have focused on the aetiological agent and its virulence factors. However, the host immune response has been considered an important factor in disease development. Previous research has shown that B. hyodysenteriae induces systemic CD4(+) and gammadelta T-cell responses after intramuscular immunization. Here, we have evaluated changes in the CD4(+) and gammadelta T-cell composition and distribution the different compartments of the colonic mucosa of pigs challenged with B. hyodysenteriae. We report that, in infected pigs, gammadelta T cells were significantly depleted from the epithelial layer, although their numbers were maintained in the lamina propria. In addition, CD4(+) T cells aggregated in clusters located in the lamina propria and submucosa. Ex vivo analyses of CD4(+) T-cell responses to B. hyodysenteriae antigens correlated with the changes in the mucosal CD4(+) T-cell distribution observed in infected pigs; CD4(+) T cells recovered from peripheral blood and colonic lymph nodes of infected pigs proliferated to B. hyodysenteriae antigens, whereas no differences were found in the gammadelta T-cell responses between challenged and control groups. In addition, colonic lymph node CD4(+) T cells had a predominant memory/activated phenotype. These results indicate that infection with B. hyodysenteriae induces a mucosal CD4(+) T-cell response and points to CD4(+) T cells being important contributors to the immunopathogenesis of swine dysentery.