Phenotypic characterization of CD8(+)NKT cells

We describe a novel CD8(+)NKT cell population expressing TCRalpha /beta or TCRgamma /delta. These CD8(+)NKT cells were prominent in the liver, and except for the thymus, virtually absent in other lymphoid organs. CD8(+)NKT cells expressed activation markers and comprised a high proportion of Ly49(+) cells. The development of the majority of CD8(+)NKT cells expressing TCRalpha /beta, but not TCRgamma /delta, depended on classical MHC class I. No CD8(+)NKT cells were detectable in young athymic mice, whereas the cells expressing TCRgamma /delta, but not TCRalpha /beta, appeared randomly in aged athymic mice. CD8(+)NK1(+) TCRalpha /beta cells showed polyclonal TCRVbeta usage and were virtually devoid of TCRValpha14. CD8(+)NK1(+) TCRgamma /delta cells predominantly expressed TCRVgamma1, 2 and 4, and Vdelta4, 5, 6 and 7. CD8(+)NKT cells, in particular those expressing TCRgamma /delta, were a major population in early life. IFN-gamma, but not IL-4, was induced in CD8(+)NKT cells by in vitro stimulation, independent of the TCRalpha /beta or TCRgamma /delta lineage. Hence, these cells represent a unique, though heterogeneous T cell population that shares markers with, but is distinct from, both conventional NKT cells and conventional CD8(+) T cells, and that may play a role in immune regulation.     

Comparative analysis of CD8 expressed on mature CD4+ CD8+ T cell clones cultured with IL-4 and that on CD8+ T cell clones: implication for functional significance of CD8 beta

Interleukin (IL-4) can induce CD8 expression on mature CD4+ T cells. To study this phenomenon in more detail, we characterized CD8 expressed on IL-4-induced CD4+ CD8+ (double positive) T cell clones in comparison with that on CD8+ T cell clones. Using 2ST8-5H7 mAb that detects CD8 beta expression, we found that double positive T cell clones isolated with IL-4 express CD8 alpha but not beta, in contrast to CD8+ CTL cell clones, which express both chains of CD8. Northern blot analysis revealed that these double positive clones expressed CD8 alpha but not beta mRNA, indicating that CD8 alpha and beta are independently regulated at the pre-translational level. Immunoprecipitation experiments showed that CD8 expressed on a representative IL-4-induced double positive T cell clone consists mainly of homodimers of a single 34 kd protein of CD8 alpha. The amount of multimers detected from this clone was much less than that from a CD8+ CTL clone. These results suggest that persistent expression of CD8 beta is specific for the CD8+ lineage and may be involved in polymerization and stabilization of CD8 which enhances the efficiency of class I-restricted antigen recognition.     

Generation and maintenance of autoantigen-specific CD8(+) T cell clones isolated from NOD mice.

The non-obese diabetic (NOD) mouse develops insulin dependent diabetes mellitus (IDDM) spontaneously with a higher incidence in females than in males. There are many similarities to the human disease, making it an ideal model. Our group is examining the role that CD4(+) and CD8(+) T cells play in IDDM in the NOD mouse, as it is known that both T cell subsets are required for onset of disease. Although IDDM has an autoimmune etiology, the initial triggering event is unknown and the autoantigen involved has not been identified. This investigation focussed on one of the potential autoantigens involved, the enzyme glutamic acid decarboxylase (GAD). We raised GAD peptide-specific CD8(+) T cells by immunising NOD mice with the GAD peptide alongside an irrelevant peptide that induced a CD4(+) T cell response. In order to maintain these peptide specific T cells in vitro and generate clones, it was found that antibodies specific to CD4(+) and MHC class II molecules needed to be included in the culture medium. This paper outlines the methods we employed to generate and maintain these CD8(+) T cells in vitro.     

Cognate CD4(+) T cell licensing of dendritic cells in CD8(+) T cell immunity

Several studies have indicated that CD8(+) T cells require CD4(+) T cell help for memory formation. Evidence suggests that such help can be antigen independent, challenging whether the 'licensing' of dendritic cells (DCs) by CD4(+) T cells is ever required for cytotoxic T lymphocyte (CTL) responses. We show here that help is essential for the generation of CTL immunity to herpes simplex virus 1 and that CD4(+) T cells mediate help in a cognate, antigen-specific way. We provide direct in vivo evidence for DC licensing by helper T cells and show that licensing is rapid and essential for the formation of effector and memory CTLs. In situations in which DCs are poorly licensed by pathogen-derived signals, our findings suggest that CTL immunity may be heavily dependent on cognate DC licensing.     

A critical role for IL-12 in CCR5 induction on T cell receptor-triggered mouse CD4(+) and CD8(+) T cells

Despite increasing evidence for the role of the chemokine system in leukocyte trafficking, the mechanism underlying the induction of chemokine receptors is poorly understood. Here, we investigated how CCR5, a chemokine receptor implicated in T cell migration to inflammatory sites, is induced in the T cell. CCR5 mRNA was hardly detected in resting T cells and marginally induced following T cell receptor (TCR) stimulation. However, TCR-triggered T cells expressed IL-12 receptor, and stimulation with recombinant IL-12 resulted in high levels of CCR5 expression on both CD4(+) and CD8(+) T cells. In contrast, IL-2 failed to up-regulate CCR5 expression. The effect of IL-12 was selective to CCR5 because IL-12 did not up-regulate CXCR3 expression. Surface expression of CCR5 was shown by staining with anti-CCR5 monoclonal antibody. Stimulation of these CCR5-positive T cells with the relevant chemokine MIP-1 alpha elicited Ca(2+) influx, showing that IL-12-induced CCR5 is functional. These results indicate a critical role for IL-12 in the induction of CCR5 on TCR-triggered T cells.     

Transient expression of bacterial gene fragments in eukaryotic cells: implications for CD8(+) T cell epitope analysis

CD8(+) T cells are potent effectors of acquired immunity against some viruses and intracellular bacterial pathogens. Antigens recognized by CD8(+) T cells are small, 8-9 amino acid peptides derived from proteins produced by the pathogen. These peptides are presented by MHC class I molecules on the surface of the infected cell. When characterizing the CD8(+) T cell response to a bacterial or viral pathogen, it is often necessary to express an antigenic protein in a eukaryotic host cell that is capable of processing and presenting peptide epitopes to antigen-specific CD8(+) T cells. We describe a system designed to transiently express bacterial polypeptides and MHC class I molecules in eukaryotic cells. Recognition of these peptide-MHC complexes stimulates TNF production by antigen-specific CD8(+) T cell lines. This system should be useful for analysis of CD8(+) T cell epitope-containing bacterial gene fragments when expression of the entire bacterial protein is detrimental to the eukaryotic cell, or when overexpression of the bacterial gene is detrimental to the bacterial cloning strain. Furthermore, this system can be used for the rapid mapping of CD8(+) T cell epitopes within a protein.     

How do cultured CD8(+) murine T cell clones survive repeated ligation of the TCR?

Many murine T cell clones grow continuously in culture despite weekly ligation of their TCR by antigen. To learn how the cultured cells avoid or minimize antigen-induced cell death (AICD), we compared Fas and tumor necrosis factor (TNF) receptors (TNFR) on several long-term cultured CD8(+) T cell clones with those on naive and activated naive cells expressing the same TCR (2C). In contrast to the naive cells, Fas was absent on the cultured clones and the TNFR-II receptor, present initially at high levels on the cultured cells, was rapidly down-modulated in response to TCR ligation and had virtually disappeared by 2 h, when only approximately 10% of the cloned cells had been induced to express TNF-alpha. The extent of AICD of the cultured clones in response to cognate peptide-MHC on the presenting cells used for routine stimulation of the cultures was also considerably less than the massive cell death of the clones following exposure to anti-CD3 antibody plate-bound at high density.     

Phenotypic and functional characterization of CD11c+ dendritic cell population in mouse Peyer's patches

The antigen-presenting cell system in the gastrointestinal tract, one of three main sites (skin and lung being the others) of primary antigen contact, is poorly understood. Our study focused on dendritic cells (DC) as possible candidates for antigen uptake, processing and presentation in mucosal inductive sites, such as Peyer's patches (PP). To investigate the morphology, immunophenotype and stimulatory activity of intestinal DC, a procedure was developed to obtain a cell population by using collagenase digestion of PP, density centrifugation and cell sorting on the basis of CD11c expression. The resultant low-density cell fraction consisted of a nonadherent cell population expressing different intensities of CD11c that could at least be characterized by typical DC morphology (e.g. abundant cytoplasma with veil-like cytoplasmatic dendrites, irregularly shaped nuclei, multivesicular and multilamellar bodies), constitutive levels of surface MHC class II, the presence of macrophage-specific markers, such as F4/80, Mac-I and Fc receptors, respectively, on subpopulations of CD11c⁺ sorted cells and expression of adhesion and co-stimulatory receptors like ICAM-1 and CD44. The capability of this low-density CD11c⁺ fraction to stimulate T cell responses was demonstrated in primary allogeneic mixed-lymphocyte reactions (MLR). Herein, we show that the freshly isolated CD11c⁺ cells showed weak accessory function, but develop this capacity following short-term culture in vitro in the presence of granulocyte/macrophage colony-stimulating factor. Although the nature and functional capacity of the isolated CD11c⁺ needs further clarification, these preliminary results describing phenotype and accessory function provide some evidence that these cells isolated from the PP may be immature forms of DC and play a crucial role as antigen-presenting cells with important implications for understanding the complex network regulating intestinal antigen uptake, processing and presentation.     

Proliferative arrest and cell cycle regulation in CD8(+)CD28(-) versus CD8(+)CD28(+) T cells

CD8(+)CD28(-) T cells have been characterized by oligoclonal expansions, impaired proliferative responses, but preserved cytotoxicity and reduced telomeres. To examine this subset further and define the underlying mechanisms of proliferation arrest, we investigated several features of this cell type compared with CD8(+)CD28(+) controls. We analyzed expression of various activation markers, thymidine incorporation upon activation, T-cell receptor (TCR) zeta-chain phosphorylation, cell cycle characteristics, and cell cycle related gene expression. Flow cytometry revealed higher expression of CD11b, CD29, CD57, and CD94, and lower expression of CD25 in CD8(+)CD28(-) compared with CD8(+)CD28(+) T cells. Sorted CD8(+)CD16(-)CD28(-) cells exhibited decreased phosphorylation of the TCR zeta-chain in three of four probands. Proliferation of these T cells was impaired, even when activated with mitogens that bypass TCR signaling. Cell cycle profiles demonstrated a lower percentage of cycling cells and significantly higher levels of cyclin dependent kinase inhibitor p16(INK4a) in the CD28(-) subset compared with the CD28(+) control. These observations suggest that expanded CD8(+)CD28(-) T cells in normal elderly individuals have reduced proliferation concomitant with increased p16(INK4a) expression. Defects in TCR signaling were associated with altered TCR zeta-chain phosphorylation.     

Heterogeneity of CD4(+) and CD8(+) T cells

There is extensive plasticity in the T-cell response to antigen. Helper CD4(+) T cells, cytotoxic CD8(+) T cells, the progression from na?ve to effector and memory T cells, and differentiation into Th1, Tc1, Th2 and Tc2 subsets have long been recognized. More recently it has become apparent that T-cell populations display additional diversity in terms of phenotype, anatomical distribution and effector function.     

Costimulation in antiviral immunity: differential requirements for CD4(+) and CD8(+) T cell responses

A key step toward improving vaccines is understanding the molecular interactions responsible for inducing antiviral T cell responses. An emerging theme from recent studies is that CD4(+) and CD8(+) T cell responses require distinct costimulatory pathways for activation. In addition, these costimulatory interactions can play a crucial role during the death phase of the T cell response and determine the number of effector T cells that survive to become memory T cells.     

CD40 ligand-positive CD8+ T cell clones allow B cell growth and differentiation

A fraction of activated CD8⁺ T cells expresses CD40 ligand (CD40L), a molecule that plays a key role in T cell-dependent B cell stimulation. CD8⁺ T cell clones were examined for CD40L expression and for their capacity to allow the growth and differentiation of B cells, upon activation with immobilized anti-CD3. According to CD40L expression, CD8⁺ clones could be grouped into three subsets. CD8⁺ T cell clones expressing high levels of CD40L (≥80% CD40L⁺ cells) were equivalent to CD4⁺ T cell clones with regard to induction of tonsil B cell proliferation and immunoglobulin (Ig) production, provided the combination of interleukin (IL)-2 and IL-10 was added to cultures. CD8⁺ T cell clones, with intermediate levels of CD40L expression (10 to 30% CD40L⁺ cells), also stimulated B cell proliferation and Ig secretion with IL-2 and IL-10. B cell responses induced by these CD8⁺ T cell clones were neutralized by blocking monoclonal antibodies specific for either CD40L or CD40. By contrast, CD40L^?? T cell clones (?5 % CD40L⁺ cells), only induced marginal B cell responses even with IL-2 and IL-10. All three clone types were able to activate B cells as shown by up-regulation of CD25, CD80 and CD86 expression. A neutralizing anti-CD40L antibody indicated that T cell-dependent B cell activation was only partly dependent on CD40-CD40L interaction. These CD40L^?? clones had no inhibitory effects on B cell proliferation induced by CD40L-expressing CD8⁺ T cell clones. Taken together, these results indicate that CD8⁺ T cells can induce B cell growth and differentiation in a CD40L-CD40-dependent fashion.     

Regulation of CD8(+) T cell functions by RARgamma

Retinoic acid plays a key role in the development and function of the immune system; however, the contribution of each of the three retinoic acid receptors (RARs) to the T cell immune response is not yet well understood. Of these receptors, both RARalpha and RARgamma are expressed in T lymphocytes. While possible functional redundancy thus complicates understanding of the role of each receptor in T cells, emerging data suggest that RARalpha and RARgamma function differently in thymocyte development and that RARgamma is required for both primary and secondary CD8(+) T cell immune responses.     

CD8+ T lymphocytes specific for glutamic acid decarboxylase 90-98 epitope mediate diabetes in NOD SCID mouse

During the past decade, glutamic acid decarboxylase (GAD) has been considered a crucial beta-cell autoantigen involved in type 1 diabetes in the NOD mouse and human. Recently, the etiological role of GAD has remained controversy. In the NOD mouse, some previous studies argued in favor of a regulatory role for GAD-specific CD4+ T cells, and no diabetogenic CD8+ T cells specific for GAD have been identified so far, discrediting the importance of GAD in beta-cell injury. Here, we identified, in the NOD model, a relevant GAD CD8+ T cell epitope (GAD(90-98)) using immunization with a plasmid encoding GAD, a protocol relying on in vivo processing of peptides from the autoantigenic protein. In pancreatic lymph nodes of na?ve female NOD mice, CD8+ T lymphocytes recognizing GAD(90-98) peptide were detected during the initial phase of invasive insulitis (between 4 and 8 weeks of age), suggesting an important role for these cells in the first stage of the disease. GAD(90-98) specific CD8+ lymphocytes lysed efficiently islet cells in vitro and transferred diabetes into NOD(SCID) mice (100%). Finally, diabetes was accelerated greatly in 3-week-old female NOD mice injected i.p. with GAD(90-98), strengthening the role of GAD-specific CTLs in diabetes pathogenesis.     

Phenotypic characterization of CD8+ T cell populations in HIV disease and in anti-HIV immunity.

The CD8+ T cell population is believed to play an important role in the control of viral infection, both for suppression of viral replication and for cytotoxic activity against viral infected cells. Elevated numbers of CD8+ T cells have been demonstrated in HIV infection, and CD8+ cytotoxic T cell (CTL) activity is associated with the early, asymptomatic stage of disease. We investigated the phenotypic characteristics of the CD8 population, in whole blood, in HIV disease and determined the predominant CD8+ subpopulation involved in anti-HIV CTL activity. We found that CD8+ T cells co-expressing markers of activation (HLA-DR), memory (CD45RO, CD29), and cytotoxic activity (S6F1) were significantly elevated in the early stages of disease, while the numbers of naive (CD45RA) cells remained unchanged. Progression to AIDS resulted in an overall loss of absolute CD8+ T cells, though the percentages of CD8+ HLA-DR+ and CD8+ S6F1+ remained elevated. In contrast to patients in the late stages of disease, anti-HIVgag CTL activity, following in vitro stimulation, was present in most HIV+ asymptomatic subjects and was associated with an expansion of CD8+ HLA-DR+ and CD8+ CD45RO+ cells. The absence of CTL activity was associated with a reduced ability of these populations to expand in vitro and with a significant loss of peripheral CD4+ T cells, independent of clinical stage. We suggest that CD8+ expressing HLA-DR+ CD45RO+ and S6F1+ play an important role in anti-HIV cytotoxicity.     

Viral neuraminidase treatment of dendritic cells enhances antigen-specific CD8(+) T cell proliferation, but does not account for the CD4(+) T cell independence of the CD8(+) T cell response during influenza virus infection

In vitro studies demonstrate that the increased alloreactive T cell response to dendritic cells (DC) that are treated with either live or inactivated influenza virus A/PR/8/34 is due to viral neuraminidase (NA) activity. Since virus-specific cytotoxic T lymphocytes (CTL) play an important role in immunity to heterologous influenza strains, we compared the activation of CD8(+) T cells by untreated and NA-treated DC. Increased CTL activity was induced by NA-treated DC both in vitro and in vivo. Since the generation of CTL in response to influenza virus infection does not require prior "activation" of DC by CD4(+) T cells (as is the case for many antigens), we asked whether NA activity contributed to this unconditional CD8(+) T cell response. This was not the case. Future studies will determine the factors that are responsible for the CD4(+) T-cell-independent influenza virus-specific CTL response.     

Age-related impairment of human T lymphocytes' activation: specific differences between CD4(+) and CD8(+) subsets.

The relevance of physiological immune aging is of great interest with respect to determining disorders with pathologic immune function in aging individuals. In recent years, the relevance of changes in peripheral lymphocytes in age-associated neurologic diseases has become more evident. Due to the lack of immunological studies, covering more than one event after mitogenic activation, we envisaged a new concept in the present study, aiming to investigate several events, starting from T cell receptor (TCR) ligation up to T cell proliferation. In addition, we addressed the question whether changes are present in the subsets (CD4, CD8) with aging. Phosphorylation of tyrosine residues declines with increasing age in CD4(+) cells. Fewer levels of CD69 positive cells after 4 h mitogenic activation, altered expression of cytokines (IL2, IFN-gamma and TNF-alpha; 22 h) and lower proliferation (72 h) were determined in aging. Moreover, it could be shown that CD8(+) lymphocytes react more effectively to mitogenic stimulation with reference to CD69 expression and proliferation in both age groups (<35 and >60 years old). These data indicate that T cell activation, mediated by TCR engagement, is significantly impaired in aging and both subsets are affected. However, bypassing the TCR does not fully restore T cell function, indicating that there are more mechanisms involved than impaired signal transduction through TCR only. The results will be discussed in relation to their relevance in neurodegenerative and psychiatric disorders.     

Distribution and functional analysis of memory antiviral CD8 T cell responses in HIV-1 and cytomegalovirus infections

In the present study, we have investigated the anatomic distribution and the function of different populations of HIV-1- and cytomegalovirus (CMV)-specific memory CD8 T cells. The different populations of virus-specific memory CD8 T cells were distinguished on the basis of the expression of CD45RA and CCR7, and the composition of HIV-1- and CMV-specific memory CD8 T cell pools were compared in subjects with chronic HIV-1 and CMV co-infection. The distribution of HIV-1-specific CD8 T cells was similar between blood and lymph node. However, CMV-specific CD8 T cells were accumulated predominantly in the blood away from the lymphoid tissue. The majority (>70%) of HIV-1- and CMV-specific CD8 T cells in both blood and lymph node had a phenotype, e.g. CCR7-, typical of effector T cells. HIV-1-specific memory CD8 T cells were mostly (>80%) pre-terminally differentiated cells, e.g. CD45RA-CCR7-, in both blood and lymph node while 30-50% of CMV-specific CD8 T cells were terminally differentiated, e.g. CD45RA+CCR7-. Therefore, consistently with studies in mice, antigen-specific effector memory CD8 T cells accumulate predominantly in the target organ of the pathogen in humans, and the differences in the composition of HIV-1- and CMV-specific CD8 T cell pools were also present in the lymphoid tissue. A substantial proportion (30-40%) of virus-specific CD8+CCR7+ T cells produced IFN-gamma. Thus, indicating that the expression of CCR7 does not provide a clear-cut separation of memory CD8 T cells with distinct functional capacities. Taken together, these results provide further advances in the characterization of human memory CD8 T cells.