Induction and function of virus-specific CD4+ T cell responses

CD4+ T cells - often referred to as T-helper cells - play a central role in immune defense and pathogenesis. Virus infections and vaccines stimulate and expand populations of antigen-specific CD4+ T cells in mice and in man. These virus-specific CD4+ T cells are extremely important in antiviral protection: deficiencies in CD4+ T cells are associated with virus reactivation, generalized susceptibility to opportunistic infections, and poor vaccine efficacy. As described below, CD4+ T cells influence effector and memory CD8+ T cell responses, humoral immunity, and the antimicrobial activity of macrophages and are involved in recruiting cells to sites of infection. This review summarizes a few key points about the dynamics of the CD4+ T cell response to virus infection, the positive role of pro-inflammatory cytokines in the differentiation of virus-specific CD4+ T cells, and new areas of investigation to improve vaccines against virus infection.     

Generation and maintenance of Listeria-specific CD8+ T cell responses in perforin-deficient mice chronically infected with LCMV

Disruption of the perforin gene results in primary immunodeficiency and an increased susceptibility to opportunistic pathogens. Perforin-deficient (PKO) mice fail to clear primary lymphocytic choriomeningitis virus (LCMV) Armstrong, resulting in persistent infection and functional exhaustion of virus-specific CD8+ T cells. CD8+ T cell responses to Listeria monocytogenes (LM) challenge within the first week after LCMV infection were diminished in both WT and PKO mice, and correlated with enhanced bacterial clearance. However, bacterial challenge at later time points generated similar CD8 T cell responses in both groups of mice. The phenotype and function of pre-existing LM-specific memory CD8+ T cells were maintained in persistently infected PKO mice. Thus persistent LCMV infection, as a result of perforin deficiency, results in dysfunction of the virus-specific CD8+ T cell response but does not compromise the host's ability to maintain pre-existing memory CD8+ T cells or to generate new memory CD8+ T cell responses against other pathogens.     

Peripheral CD4 loss of regulatory T cells is associated with persistent viraemia in chronic HIV infection

Chronic HIV infection is associated with T cell abnormalities and altered effector function. Regulatory T cells (Treg) are CD4+ T cells that play a critical role in regulating the immune system. The impact of regulatory T cells on HIV infection and disease progression may be highly significant. We hypothesize that chronic antigenic stimulation from a persistent, high viraemic state may promote a population of Treg that contributes to HIV-associated immune dysfunction. We evaluated the pattern of Treg in chronically infected, HIV-positive individuals over a course of 6 months. Treg are depleted at a distinct rate from that of absolute CD4 cells and loss of Treg is slower in the presence of viral suppression. In vitro depletion of CD25+ CD4+ cells resulted in increased Gag-specific CD4 and CD8 responses. A significant correlation between ex vivo measurement of Treg and Gag-specific CD4 T cell responses was observed (r=-0 x 41, P=0 x 018) with a trend observed with Gag-specific CD8 T cell responses (P=0 x 07). The impact of HIV infection on the Treg population directly complicates the measured effect of Treg on the immune dysfunction although our data support the important role of Treg on modulating the effector T cell response in chronic infection.     

Differential role of IL-2R signaling for CD8+ T cell responses in acute and chronic viral infections

IL-2 is a cytokine with multiple and even divergent functions; it has been described as a key cytokine for in vitro T cell proliferation but is also essential for down-regulating T cell responses by inducing activation-induced cell death as well as regulatory T cells. The in vivo analysis of IL-2 function in regulating specific T cell responses has been hampered by the fact that mice deficient in IL-2 or its receptors develop lymphoproliferative diseases and/or autoimmunity. Here we generated chimeric mice harboring both IL-2R-competent and IL-2R-deficient T cells and assessed CD8+ T cell induction, function and maintenance after acute or persistent viral infections. Induction and maintenance of CD8+ T cells were relatively independent of IL-2R signaling during acute/resolved viral infection. In marked contrast, IL-2 was crucial for secondary expansion of memory CD8+ T cells and for the maintenance of virus-specific CD8+ T cells during persistent viral infections. Thus, depending on the chronicity of antigen exposure, IL-2R signaling is either essential or largely dispensable for induction and maintenance of virus-specific CD8+ T cell responses.     

CD52 is a novel costimulatory molecule for induction of CD4+ regulatory T cells

We previously reported that 4C8 monoclonal antibody (mAb) provides a costimulatory signal to human CD4+ T cells and consequently induces regulatory T (Treg) cells, which are hypo-responsive and suppress the polyclonal response of bystander CD4+ cells in a contact-dependent manner. In this study, we identified the antigen of 4C8 mAb as CD52. Costimulation with Campath-1H, a humanized anti-CD52 mAb, also induced Treg cells. Anti-CD52-induced Treg cells suppressed the proliferation of both CD4+ and CD8+ T cells provided with polyclonal or allogeneic stimulation. When Treg cells were induced from Staphylococcal enterotoxin B (SEB) treated cells, they suppressed the response to SEB more efficiently than that to another superantigen, SEA. Furthermore, anti-CD52-induced Treg cells could be expanded by culture with IL-2 followed by CD52-costimulation, and co-injection of expanded Treg cells suppressed lethal xenogeneic graft versus host disease (GvHD) reactions in SCID mice caused by human peripheral blood mononuclear cells (PBMCs).     

CD8+ T Cell Exhaustion During Persistent Viral Infection is Regulated Independently of the Virus-Specific T Cell Receptor

During chronic viral infections, responses by virus-specific CD8⁺ T cells become marginalized by the acquisition of functional defects and reduced cell numbers in a process defined as T cell exhaustion. Similarly, T cell tolerance to self-antigen is also characterized by impaired effector function and eventual deletion of self-reactive T cells. Induction of both tolerance and exhaustion involve many shared inhibitory mechanisms, thus similar therapeutic approaches have proven effective in these distinct environments. We previously demonstrated that tolerant self-reactive CD8⁺ T cells expressing dual-T cell receptors (i.e., dual-TCR) could be rescued by immunization through a second TCR specific for a foreign antigen. These data revealed that T cell tolerance was regulated at the level of the self-reactive TCR. Here, dual-TCR CD8⁺ T cells were used to examine if exhaustion during persistent viral infection could be rescued by an analogous strategy of immunization through a second TCR not involved in recognition of virus. In direct contrast to the rescue achievable in tolerant CD8⁺ T cells, exhausted T cells were equally impaired through both TCR. These findings suggest that exhaustion is maintained by defects downstream of the virus-specific TCR, and establish that exhaustion and tolerance are distinctly regulated states of T cell dysfunction.     

CD4+ CD25+调节性T细胞在HIV/AIDS患者中的作用及其与HIV病毒载量的相关性研究

目的 研究HIV感染者／AIDS患者外周血CD4^＋ CD25^＋ 调节性T细胞（CD4^＋ CD25^＋ regulatory Tcell，Treg）频率、功能及其临床意义。方法 选择31例HIV感染者／AIDS患者和30例健康对照者，采用流式细胞仪检测各组外周血Treg的表型和频率。采取MACS磁珠分选CD4^＋CD25^＋T细胞，利用[^3H]胸腺嘧啶掺入法检测CD4^＋ CD25^＋T细胞在特异性HIV抗原刺激下对CD4^＋ CD25-T细胞的增殖影响。结果HIV／AIDS患者组与正常对照组相比较，外周血CD4^＋ CD25^＋ T细胞频率在统计学上差异无统计学意义。与正常对照组比较，HIV感染者外周血CD4^＋ CD25^＋ T细胞频率升高，差异有统计学意义（P〈0.01）；与正常对照组比较，AIDS患者者外周血CD4^＋ CD25^＋ T细胞频率降低，差异有统计学意义（P〈0．0001）。HIV RNA病毒载量与患者外周血CD4^＋ CD25^＋ T细胞数量呈正相关性（P〈0．01）。CD4^＋ CD25^＋ T细胞具有抑制HIV特异性的CD4^＋ CD25^- T细胞的增殖作用。结论HIV感染者／AIDS患者的细胞免疫功能紊乱，CD4^＋ CD25^＋ T细胞能抑制HIV感染者／AIDS患者的HIV特异性细胞免疫反应，促进HIV病毒复制，与形成持续HIV感染有关。     

Generating functional CD8+ T cell memory response under transient CD4+ T cell deficiency: implications for vaccination of immunocompromised individuals

Studies based on either MHC class II-knockout or CD4+ T cell-depleted murine models have demonstrated a critical role for CD4+ T cells in the generation of CD8+ T cell memory. However, it is difficult to extend these findings to immunocompromised humans where a complete loss of CD4+ T cells is rarely observed. Here, we have developed a model setting, which allows studies on the generation of CD8+ T cell memory responses in a transient CD4+ T cell-deficient setting similar to that seen in immunocompromised patients. Immunisation with an adenoviral vaccine under transient helpless or help-deficient conditions showed varying degrees of impact on the priming of CD8+ T cell responses. Antigen-specific T cells generated under normal CD4+ T cell help and transient help-deficient conditions showed similar effector phenotype and were capable of proliferation upon secondary antigen encounter. Most importantly, in spite of CD4+ T cell deficiency, the long-term CD8+ T cell memory response remained functionally stable and showed comparable cytotoxic effector function as seen in CD8+ T cells generated with normal CD4+ T cell numbers. These findings provide evidence that in spite of partially impaired activation of a primary CD8+ T cell response, a fully functional and stable memory CTL response can be induced under conditions of severe transient CD4+ T cell deficiency.     

TGF-beta1 production by CD4+ CD25+ regulatory T cells is not essential for suppression of intestinal inflammation

Naturally occurring CD4+ CD25+ regulatory T cells (Treg) are potent suppressors of CD4+ and CD8+ T cell responses in vitro and inhibit several organ-specific autoimmune diseases. While most in vitro studies suggest that CD4+ CD25+ Treg cells adopt a cytokine-independent but cell contact-dependent mode of T cell regulation, their precise mechanism of suppression in vivo remains largely unknown. Here we examine the functional contribution of Treg cell-derived TGF-beta1 and effector T cell responsiveness to TGF-beta in CD4+ CD25+ T cell-mediated suppression of inflammatory bowel disease (IBD). We show that CD4+ CD25+ Treg cells from either TGF-beta1+/+ or neonatal TGF-beta1-/- mice can suppress the incidence and severity of IBD as well as colonic IFN-gamma mRNA expression induced by WT CD4+ CD25- effector T cells. Furthermore, TGF-beta-resistant Smad3-/- CD4+ CD25+ Treg cells are equivalent to WT Treg cells in their capacity to suppress disease induced by either WT or Smad3-/- CD4+ CD25- effector T cells. Finally, anti-TGF-beta treatment exacerbates the colitogenic potential of CD4+ CD25- effector T cells in the absence of CD4+ CD25+ Treg cells. Together, these data demonstrate that in certain situations CD4+ CD25+ T cells are able to suppress intestinal inflammation by a mechanism not requiring Treg cell-derived TGF-beta1 or effector T cell/Treg cell responsiveness to TGF-beta via Smad3.     

CD8 T cell responses to influenza virus infection in aged mice

Influenza is one of the most common infectious diseases afflicting humans, particularly the elderly. The murine model has been widely employed for investigation of immunity to influenza virus infection. In this paper, we review the recent advances in understanding the diminished CD8 T cell immune response to influenza virus infection in aged mice. Possible mechanisms of impaired CD8 T cell responses with aging are addressed, including: (1) the role of dendritic cells (DCs); (2) the effect of age-associated changes in the T cell repertoire; and (3) the interactions with CD4 T cells, including T regulatory (Treg) cells and CD4 T helper cells. The aged murine model of the CD8 T cell response to influenza virus is helping to elucidate the mechanisms of immunosenescence which can lead to therapeutic improvements in the primary CD8 T cell response to new infections, as well as the development of new strategies for immunization to prevent influenza in the elderly.     

Distinct CD4 T-cell effects on primary versus recall CD8 T-cell responses during viral encephalomyelitis

CD4 T-cell help is not a universal requirement for effective primary CD8 T cells but is essential to generate memory CD8 T cells capable of recall responses. This study examined how CD4 T cells affect primary and secondary anti-viral CD8 T-cell responses within the central nervous system (CNS) during encephalomyelitis induced by sublethal gliatropic coronavirus. CD4 T-cell depletion before infection did not impair peripheral expansion, interferon-γ production, CNS recruitment or initial CNS effector capacity of virus-specific CD8 T cells ex vivo. Nevertheless, impaired virus control in the absence of CD4 T cells was associated with gradually diminished CNS CD8 T-cell interferon-γ production. Furthermore, within the CD8 T-cell population short-lived effector cells were increased and memory precursor effector cells were significantly decreased, consistent with higher T-cell turnover. Transfer of memory CD8 T cells to reduce viral load in CD4-depleted mice reverted the recipient CNS CD8 T-cell phenotype to that in wild-type control mice. However, memory CD8 T cells primed without CD4 T cells and transferred into infected CD4-sufficient recipients expanded less efficiently and were not sustained in the CNS, contrasting with their helped counterparts. These data suggest that CD4 T cells are dispensable for initial expansion, CNS recruitment and differentiation of primary resident memory CD8 T cells as long as the duration of antigen exposure is limited. By contrast, CD4 T cells are essential to prolong primary CD8 T-cell function in the CNS and imprint memory CD8 T cells for recall responses.     

Failing immune control as a result of impaired CD8+ T-cell maturation: CD27 might provide a clue

Despite readily detectable virus-specific CD8(+) T cells in most HIV-infected patients, immune surveillance is eventually lost, leading to progression to AIDS. Recently developed insights into human T-cell differentiation have been used to study the phenotype of virus-specific T cells in HIV-infected individuals. Based on these results, we propose that failing immune control in human viral infection could be a result of impaired cytotoxic T-lymphocyte (CTL) maturation into fully differentiated effector T cells. Impaired maturation is not confined to HIV-specific CD8(+) T cells but could also be involved in failing immunity to Epstein-Barr virus and other viral infections. We postulate that CD27(-) effector CD8(+) T cells might be required for adequate control of chronic viral infection and prevention of disease development.     

In vivo stimulation of CD137 broadens primary antiviral CD8+ T cell responses

Given the key role CD8+ T cells play in controlling viral infection, strategies to enhance these responses may have important clinical applications. We found that in vivo CD137 stimulation with an agonistic monoclonal antibody enhanced the primary CD8+ T cell response to influenza type A viral infection in mice. Stimulation of CD137 increased the absolute number of CD8+ T cells to influenza epitopes in the lungs of infected animals, preferentially expanded CD8+ T cells that recognized nondominant epitopes and greatly enhanced direct ex vivo cytotoxicity. CD137 stimulation also restored the CD8+ T cell response to the immunodominant influenza epitope in CD28-/- mice. Thus, in vivo CD137 stimulation enhances and broadens the CD8+ T cell response to influenza virus and can restore the CD8+ T cell response when CD28 costimulation is absent. This suggests that CD137 stimulation may be useful as a strategy to enhance the CD8+ T cell response to viruses.     

The Role of CD4+CD25+ T Regulatory Cells in Autoimmune Diseases

Among the several mechanisms that play role in maintaining peripheral self-tolerance is the existence of a unique CD4+CD25+ population of naturally occurring regulatory T (Treg) cells that actively prevent both the activation and the effector function of autoreactive T cells that have escaped different mechanisms of tolerance. Many studies have shown the benefit of targeting this cell population by restoring self-tolerance. Therapies that could possibly increase the suppressive ability of T regulatory cells were proven to improve that course of autoimmune diseases.     

CD8+ suppressor-mediated regulation of human CD4+ T cell responses to glutamic acid decarboxylase 65

Although potentially autoreactive T cells are present even in healthy subjects, most individuals do not develop autoimmune disease. It has been well demonstrated that CD4+ CD25+ regulatory T cells play a significant role in controlling the expansion of autoreactive T cells in the periphery. However, some healthy individuals exhibit measurable responses to self peptide even in the presence of CD4+ CD25+ regulatory cells. This article describes the regulation of human CD4+ T cell responses to glutamic acid decarboxylase 65 (GAD65), an autoantigen implicated in type-1 diabetes, by autologous CD8+ suppressor T cells. In cells cultured from healthy individuals, the inclusion of autologous CD8+ T cells at physiological levels resulted in a dramatic decrease in the magnitude of in vitro CD4+ T cell responses to GAD65 peptide. Based on transwell experiments, the observed suppression was cell contact-dependent. However, antibody blocking studies indicated that suppression was mediated by IL-10. Cell fractionation studies suggested that CD8+ suppressor T cells originate from the CD45RA+ CD27- population. The suppression of CD4+ T cell responses to GAD65 in healthy individuals raises the possibility that CD8+ suppressor T cells play an important role in controlling potentially autoreactive T cells in the general population.     

Human effector and memory CD8+ T cell responses to smallpox and yellow fever vaccines

To explore the human T cell response to acute viral infection, we performed a longitudinal analysis of CD8(+) T cells responding to the live yellow fever virus and smallpox vaccines--two highly successful human vaccines. Our results show that both vaccines generated a brisk primary effector CD8(+) T cell response of substantial magnitude that could be readily quantitated with a simple set of four phenotypic markers. Secondly, the vaccine-induced T cell response was highly specific with minimal bystander effects. Thirdly, virus-specific CD8(+) T cells passed through an obligate effector phase, contracted more than 90% and gradually differentiated into long-lived memory cells. Finally, these memory cells were highly functional and underwent a memory differentiation program distinct from that described for human CD8(+) T cells specific for persistent viruses. These results provide a benchmark for CD8(+) T cell responses induced by two of the most effective vaccines ever developed.     

Characterization of mouse CD4 T cell subsets defined by expression of KLRG1

The mouse killer cell lectin-like receptor G1 (KLRG1) is an inhibitory receptor known to be expressed on a subset of NK cells and antigen-experienced CD8 T cells. Here, we have characterized expression of KLRG1 on CD4+ T cells from normal mice. While a polyclonal TCR repertoire suggests thymic origin of KLRG1+ CD4+ cells, KLRG1 expression was found to be restricted to peripheral CD4+ T cells. Based on phenotypic analyses, a minority of KLRG1+ CD4+ cells are effector/memory cells with a proliferative history. The majority of KLRG1+ CD4+ cells are, however, bona fide Treg cells that depend on IL-2 and/or CD28 and express both FoxP3 and high levels of intracellular CD152. KLRG1-expressing Treg are contained within the CD38+ subset but are only partially overlapping with the CD25+ CD4+ Treg subset. In functional assays, KLRG1+ CD4+ cells were anergic to TCR stimulation with respect to proliferation, and sorted KLRG1+ CD25+ CD4+ cells were equal or superior to KLRG1+ CD25- CD4+ cells, which were more potent than KLRG1- CD25+ CD4+ cells in suppressing responder cell proliferation. Together, our results demonstrate that KLRG1 expression defines novel and distinctive subsets of senescent effector/memory and potent regulatory CD4+ T cells.     

Definition of the HLA-A2 restricted peptides recognized by human CD8+ effector T cells by flow-assisted sorting of the CD8+ CD45RA+ CD28- T cell subpopulation

In response to antigenic stimulation, naive MHC-class I restricted and antigen-specific CD8+ CD45RA+ CD28+ T cells undergo clonal expansion, differentiate into CD8+ CD45RO+ memory T cells and convert to CD8+ CD45RA+ CD28- T cells displaying potent immune effector functions upon re-encounter with the nominal antigen. We show that the effector CD8+ CD45RA+ CD28- T cell subset is expanded in peripheral blood lymphocytes (PBL) from patients with human papilloma virus (HPV)+ cervical lesions as well as in PBL from patients with pulmonary tuberculosis. Flow-cytometric cell sorted CD8+ CD45RA+ CD28- and CD8+ CD45RA+ CD28- T cells were tested for recognition of HLA-A2 restricted peptides derived either from the human papillomavirus (HPV)16-E7 gene product, or from M. tuberculosis antigens. Mostly CD8+ CD45+ CD28- T cells define antigen/peptide-specific and MHC-restricted responses. These data were confirmed in PBL from patients with tuberculosis using HLA-A2 tetramer-complexes loaded with a peptide from the M. tuberculosis Ag85b antigen by flow cytometry. The sorting of this T cell subset enables to determine the fine specificity of CD8+ effector T cells without the need for in vitro manipulation.     

Induction of immunosuppressive functions of dendritic cells in vivo by CD4+CD25+ regulatory T cells: role of B7-H3 expression and antigen presentation

Suppressive functions of CD4+CD25+ regulatory T cells (Treg) are mainly studied by their interaction with conventional T cells. However, there is evidence that Treg also interact with antigen-presenting cells (APC), leading to suppression of APC function in in vitro coculture systems. Studying the in vivo distribution of Treg after injection, we found that Treg are located in direct proximity to dendritic cells (DC) and affect their functional maturation status. After contact to Treg, DC up-regulate the inhibitory B7-H3 molecule and display reduced numbers of MHC-peptide complexes, leading to impaired T cell stimulatory function. When Treg-exposed DC were used to immunize animals against antigens, the DC failed to produce a robust immune response as compared to control DC. Thus, these data indicate that Treg are able to inhibit DC activation and produce an inhibitory phenotype of DC. Accordingly, Treg may recruit DC for the amplification of immunosuppression by restraining their maturation in vivo and inducing an immunosuppressive phenotype of DC.     

CD4+ T cells clear virus but augment disease in mice infected with respiratory syncytial virus. Comparison with the effects of CD8+ T cells.

Respiratory syncytial (RS) virus-specific T cell lines were derived from the spleens of BALB/c mice primed by intranasal infection with RS virus. The lines were expanded by repeated antigenic stimulation in vitro, and separated into CD4+ and CD8+ T cell-enriched fractions by immunomagnetic adhesion. The effects of passive transfer of these fractions into RS virus infected mice were observed. The most severe immunopathological changes were seen in mice receiving CD4+ cells. Transfer of CD4+, CD8+ or both cell fractions caused RS virus-infected mice to become ill and lose weight. Both cell lines caused an increase in the severity of lung pathology (as monitored by bronchoalveolar lavage) with the appearance of lung haemorrhage and polymorphonuclear cell efflux. In addition, recipients of CD4+ cells developed striking pulmonary eosinophilia. In CD4+ cell recipients, 5 x 10(5) cells were sufficient to decrease lung virus titre, whereas 2 x 10(6) CD8+ cells were needed to produce a similar effect. The unseparated T cell line and the CD4+ cell fraction secreted significant amounts of IL-3, IL-4 and IL-5 (P less than 0.001). High levels of IL-2 were produced only by the unseparated T cell line. The CD8+ cell fraction secreted IL-3 only. The results show that, cell-for-cell, CD4+ cells are more anti-viral and more immunopathogenic than CD8+ cells in RS virus infected mice. Such effects may have contributed to the augmented disease seen in some infants vaccinated against RS virus.