Homeostasis of naïve, effector and memory CD8 T cells

Homeostatic control of CD8 T cell populations is essential for defense against infectious pathogens. Our understanding of the mechanisms that control na?ve, effector and memory T cell populations in the intact animal has increased significantly over the last several years. There have been some surprises. For example, peripheral tissues have been found to harbor unexpectedly large numbers of effector memory T cells. Also unexpected was the finding of programmed T cell proliferation following very brief exposure to antigen. These and other recent advances are summarized in the following review.     

Antigen-presenting cells recruited by Brugia malayi induce Th2 differentiation of naïve CD4(+) T cells

A key feature of nematode infection is a bias towards a type 2 immune response. To investigate the role that antigen-presenting cells (APC) may play in promoting this bias, we used adherent peritoneal exudate cells (PEC) recruited in response to the filarial nematode Brugia malayi, to stimulate na?ve T cells from pigeon cytochrome c (PCC)-specific TCR transgenic (PCC-tg) mice. Although the proliferation of PCC-tg T cells was inhibited by parasite- induced PEC during primary stimulation, they proliferated normally upon secondary stimulation and were not rendered anergic. However, PCC-tg T cells primed by suppressive APC differentiated into IL-4-producing Th2 cells upon secondary stimulation instead of IFN-gamma-producing Th1 cells, as has been previously described. Studies with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled cells indicated that Th2 differentiation was associated with the inhibition of (or failure to stimulate) IFN-gamma production during primary stimulation. Interestingly, blocking antibodies against TGF-beta (but not IL-10) restored the differentiation of IFN-gamma-producing Th1 cells. Identical results with CFSE-labeled cells were obtained using purified IL-4-dependent F4/80(+) macrophages. These data indicate that T cells exposed to parasite-induced alternatively activated macrophages are driven towards Th2 differentiation. This may be an important factor in the Th2 bias that accompanies nematode infection.     

The capacity of the natural ligands for CD28 to drive IL-4 expression in naïve and antigen-primed CD4+ and CD8+ T cells

The B7/CD28 costimulatory pathway plays a critical role in T cell activation including Th1/Th2 differentiation. However, little is known about whether CD28 costimulation favors polarization of either Th1 and Th2 or both. Here, we show a critical role of the natural ligands for CD28 molecules (B7.2-Ig or B7.1-Ig fusion proteins), particularly in the induction of type 2 T cell polarization. Upon TCR-triggering with suboptimal doses of anti-CD3, costimulation of na?ve CD4+ T cells with anti-CD28 mAb or B7-Ig fusion proteins led to comparable levels of IFN-gamma production. Na?ve T cells could produce IL-4 when CD28 costimulation was done with B7-Ig, but not with anti-CD28. IL-4-selective upregulation was also observed when T cells from anti-OVA TCR transgenic mice were stimulated with OVA in the presence of B7-Ig. Correlating with IL-4 expression, GATA-3 expression was induced much more potently by costimulation with B7-Ig than with anti-CD28 mAb, while T-bet induction by these two costimulatory reagents was comparable. This B7 effect was also applied for na?ve and antigen-primed CD8+ T cells: IL-4-expressing CD8+ T cells were generated when na?ve and alloantigen-primed T cells were stimulated with anti-CD3 and recall antigens, respectively, in the presence of B7-Ig costimulation. Importantly, such CD8+ T cell differentiation required the coexistence of CD4+ T cells during the initial TCR stimulation. These observations indicate that both type 2 CD4 and CD8 T cell polarizations are efficiently induced via costimulation of CD28 with its natural ligands, although the differentiation of CD8+ T cells is dependent on CD4+ cells.     

Age-dependent loss of naïve T cells in TCR transgenic bone marrow chimeras

Study of immune senescence is complicated by low numbers of antigen-specific lymphocytes, particularly na?ve T (Tn)cells which disappear with aging. Although T cell receptor (TCR) transgenic mice facilitate aging research, their large number of Ag-specific T cells renders their T cell pool abnormal, precluding normal in vivo immunity. To create a physiologically relevant model with measurable numbers of TCR transgenic CD4+ T cells in the context of normal lymphocytes, mixed (DO11.10+BALB/c) bone marrow (BM) chimeras were constructed. As found in normal mice, the total number of transgenic T cells and the Tn:memory T cell ratio declined with the aging of the BM chimeras. Although these shifts in T cell subsets were evident in both the lymph nodes and the spleen (SP), they were more pronounced in the SP. Unlike DO11.10 mice, transgenic T cells in the chimera acquired an effector/memory phenotype upon antigen challenge. These results reveal a pliable model to study the impact of the constriction of the Tn cell repertoire upon optimal vaccine responses in the elderly.     

ICOS cooperates with CD28, IL-2, and IFN-gamma and modulates activation of human naïve CD4+ T cells

Several sets of data indicate that ICOS regulates cytokine production in activated T cells, but is less effective on naïve T cells. This work evaluates ICOS function in human naïve CD4⁺ T cells through an assessment of the effect of soluble forms of the ICOS and CD28 physiological ligands on activation driven by anti‐CD3 mAb. ICOS strikingly potentiated secretion of IL‐2, IFN‐γ, IL‐10, and TNF‐α, but not IL‐4, promoted by optimal stimulation of CD3+CD28, and it was the key switching‐factor of activation when cells received suboptimal stimulation of CD3+CD28 or stimulation of CD3 alone in the presence of exogenous IL‐2. In these conditions, blockade of IL‐2 and IFN‐γ showed that ICOS builds up a positive feedback loop with IFN‐γ, which required IL‐2 and was inhibited by IL‐4. By contrast, in the absence of CD28 triggering or exogenous IL‐2, ICOS‐induced costimulation mainly supported expression of TGF‐β1 and FoxP3 and differentiation of regulatory T cells capable to inhibit proliferation of naïve CD4⁺ T cells driven by allogeneic cells. These data suggest that ICOS favors differentiation of Th effector cells when cooperates with appropriate activation stimuli such as CD3+CD28 or CD3+IL‐2, whereas it supports differentiation of regulatory T cells when costimulatory signals are insufficient.     

Response of naïve and memory CD8+ T cells to antigen stimulation in vivo

We studied the influence of memory T cell properties on the efficiency of secondary immune responses by comparing the in vivo immune response of the same numbers of T cell receptor (TCR) transgenic (Tg) na?ve and memory T cells. Compared to na?ve Tg cells, memory cells divided after a shorter lag time; had an increased division rate; a lower loss rate; and showed more rapid and efficient differentiation to effector functions. We found that initial na?ve T cell priming resulted in cells expressing mutually exclusive effector functions, whereas memory T cells were multifunctional after reactivation, with each individual cell expressing two to three different effector functions simultaneously. These special properties of memory T cells ensure the immediate control of reinfection.     

Interleukin-7 mediates the homeostasis of naïve and memory CD8 T cells in vivo

The na?ve and memory T lymphocyte pools are maintained through poorly understood homeostatic mechanisms that may include signaling via cytokine receptors. We show that interleukin-7 (IL-7) plays multiple roles in regulating homeostasis of CD8+ T cells. We found that IL-7 was required for homeostatic expansion of na?ve CD8+ and CD4+ T cells in lymphopenic hosts and for CD8+ T cell survival in normal hosts. In contrast, IL-7 was not necessary for growth of CD8+ T cells in response to a virus infection but was critical for generating T cell memory. Up-regulation of Bcl-2 in the absence of IL-7 signaling was impaired after activation in vivo. Homeostatic proliferation of memory cells was also partially dependent on IL-7. These results point to IL-7 as a pivotal cytokine in T cell homeostasis.     

Different profile of CD8+ effector T cells induced in Der p 1-allergic and naïve mice by DNA vaccination

DNA vaccination holds great promise in both prophylactic and therapeutic vaccines. Recent evidence suggests that DNA vaccines could be powerful therapies countering Th2-mediated disorders such as allergies. Here, we studied the allergen-specific CD4+ and CD8+ T cell populations induced following immunization of allergic and non-allergic mice with DNA vaccine vectors encoding discrete epitopes of the house dust mite (HDM) Dermatophagoides pteronyssinus group I (Der p 1) allergen. Specifically, mice were sensitized to Der p 1 and exhibited a strong Th2/allergic response. Sensitized and non-allergic mice were then compared for their responses to DNA immunization. Using Elispot analysis, we demonstrate that allergic/vaccinated mice generate a mixed Th1/Th2 response against the allergen with high numbers of allergen-specific CD4+ T cells secreting IFN-gamma or IL-4, whereas in non-allergic/vaccinated mice a polarized Th1 response was dominant. Allergen-specific CD8+ T cells secreting IFN-gamma were induced at equal frequencies in both allergic and non-allergic mice. However, the CD8+ T cells from allergic mice were markedly deficient in their cytotoxic potential when compared to their counterparts in non-allergic mice. These results indicate that during an ongoing Th2 response, DNA vaccination leads to the generation of a distinct population of non-cytotoxic/regulatory CD8+ T cells.     

Age-associated changes in the frequency of naïve, memory and effector CD8+ T cells

We investigated age-associated changes in the frequency of CD8+ T cell subsets with different functions. Based on expression of CD45RA and CCR7, na?ve (CD45RA+ CCR7+), central memory (CM, CD45RA- CCR7+), effector memory (EM, CD45RA- CCR7-) and effector (CD45RA+ CCR7-) CD8+ T cells were identified in peripheral blood from healthy young (n = 17) and elderly (n = 17) people using flow cytometry. The elderly had a decreased frequency of na?ve and an increased frequency of EM and effector CD8+ T cells compared to the young. However, both groups had a similar frequency of CM cells. These findings suggest that age-associated changes in CD8+ T cell subsets occur, which could be a potential explanation for altered CD8+ T cell function in the elderly.     

Indirect activation of naïve CD4+ T cells by dendritic cell-derived exosomes

Dendritic cells (DCs) secrete vesicles of endosomal origin, called exosomes, that bear major histocompatibility complex (MHC) and T cell costimulatory molecules. Here, we found that injection of antigen- or peptide-bearing exosomes induced antigen-specific na?ve CD4+ T cell activation in vivo. In vitro, exosomes did not induce antigen-dependent T cell stimulation unless mature CD8alpha- DCs were also present in the cultures. These mature DCs could be MHC class II-negative, but had to bear CD80 and CD86. Therefore, in addition to carrying antigen, exosomes promote the exchange of functional peptide-MHC complexes between DCs. Such a mechanism may increase the number of DCs bearing a particular peptide, thus amplifying the initiation of primary adaptive immune responses.     

CD5 levels define functionally heterogeneous populations of naïve human CD4+ T cells

Studies in murine models show that subthreshold TCR interactions with self-peptide are required for thymic development and peripheral survival of naïve T cells. Recently, differences in the strength of tonic TCR interactions with self-peptide, as read-out by cell surface levels of CD5, were associated with distinct effector potentials among sorted populations of T cells in mice. However, whether CD5 can also be used to parse functional heterogeneity among human T cells is less clear. Our study demonstrates that CD5 levels correlate with TCR signal strength in human naïve CD4⁺ T cells. Further, we describe a relationship between CD5 levels on naïve human CD4⁺ T cells and binding affinity to foreign peptide, in addition to a predominance of CD5^hi T cells in the memory compartment. Differences in gene expression and biases in cytokine production potential between CD5^lo and CD5^hi naïve human CD4⁺ T cells are consistent with observations in mice. Together, these data validate the use of CD5 surface levels as a marker of heterogeneity among human naïve CD4⁺ T cells with important implications for the identification of functionally biased T- cell populations that can be exploited to improve the efficacy of adoptive cell therapies.     

Changes in the expression of CD31 and CXCR3 in CD4+ naïve T cells in elderly persons

So far, very few studies exist on the na?ve T cell population of elderly persons. Only recently an increase in the percentage of long lived CD4(+)CD31(-) na?ve T cells has been claimed to occur with aging. We, therefore, characterised CD31(+) and CD31(-) CD45RA(+) CD4(+) T cells in young and healthy elderly persons. The production of IL-2 and IFN-gamma by the different subpopulations was studied following stimulation with PMA and Ionomycin. The expression of CD28, CD11a, CD62L, CXCR3 and CCR7 was also analysed. The results of this study demonstrate a pronounced increase in the percentage of CD31(-) CD45RA(+) T cells within the CD4 subpopulation of elderly persons. Both, CD31(-) and CD31(+) CD45RA(+) cells expressed CD28, CD62L, were CD11a (dim) and produced IL-2 but no IFN-gamma. This phenotype confirms that they were na?ve T cells. IL-2 production by na?ve T cells was not impaired in elderly persons. Interestingly, CD31(+) as well as CD31(-) na?ve T cells contained a subpopulation of CXCR3(+) cells in elderly individuals, but not in young ones. In spite of expressing this chemokine receptor that enables the cells to migrate into inflammatory tissues, they were still CCR7(+) and CD62L(+). We speculate that due to previous contact with local environmental factors, this subset of na?ve T cells acquires a different chemokine receptor phenotype, resulting in an altered migratory capacity in old age. Aberrant contact with antigen and effector cell differentiation in unorthodox locations may be the consequence. This could also affect Th1/Th2 polarisation, which is known to be impaired in elderly persons.     

Differential regulation of naïve and memory CD4+ T cells by alternatively activated dendritic cells

Promising immunotherapeutic tools for T cell-mediated pathologies are alternatively activated dendritic cells (aaDC), which exert their effect through the regulation and tolerization of T cells. As na?ve and memory T cells have different susceptibilities to tolerogenic signals, it is important to understand the modulatory effects of aaDC on these T cell subsets. We have examined regulation of na?ve and memory CD4+ T cells by human aaDC generated with dexamethasone, the active form of vitamin D3, 1alpha,25-dihydroxyvitamin D3, and LPS. Although aaDC induced low, primary, allogeneic responses by na?ve and memory T cells, aaDC regulated the differentiation of these T cell subsets in a distinct manner. Na?ve T cells primed by aaDC retained a strong, proliferative capacity upon restimulation but were skewed toward a low IFN-gamma/high IL-10 cytokine profile. In contrast, memory T cells primed by aaDC became hyporesponsive in terms of proliferation and cytokine production. Induction of anergy in memory T cells by aaDC was not a result of the presence of CD25hi regulatory T cells and could be partially reversed by IL-2. Both T cell subsets acquired regulatory activity and inhibited primary CD4 and CD8 responses. Addition of exogenous IL-12p70 during T cell priming by aaDC prevented anergy induction in memory T cells and cytokine polarization in na?ve T cells, indicating that the lack of IL-12p70 is a key feature of aaDC. Our finding that aaDC differentially regulate na?ve and memory T cells is important for understanding and maximizing the therapeutic potential of aaDC.     

Yeast-surface expressed BVDV E2 protein induces a Th1/Th2 response in naïve T cells

Yeast species such as Saccharomyces cerevisiae are known to be potent activators of the immune system. S. cerevisiae activates the innate immune system by engaging pattern recognition receptors such as toll like receptor 2 (TLR2) and dectin-1. In the current project, we express the immunogenic envelope protein E2 of bovine viral diarrhoea virus (BVDV) on the surface of S. cerevisiae. After successful expression, components of the innate and adaptive immune response induced by the recombinant S. cerevisiaein vitro were analysed to determine if expression in yeast enhances the immunogenicity of the viral protein. Recombinant S. cerevisiae stimulated production of the chemokine CXCL-8 in primary bovine macrophages, but did no stimulate production of reactive oxygen species (ROS) in the same cells. Additionally, bovine macrophages primed with S. cerevisiae expressing viral envelope proteins had a greater capacity for stimulating proliferation of CD4+ T-cells from BVDV-free animals compared to macrophages primed with envelope protein alone or S. cerevisiae without envelope protein expression. Heat inactivation of recombinant S. cerevisiae increased ROS production and capacity to stimulate CD4+ T-cells in macrophages but did not alter CXCL-8 release compared to the live counter-part. Additionally, heat-inactivation of recombinant S. cerevisiae induced less INFγ and IL-4 but equal amounts of IL-10 compared to live yeast T-cell cultures. Our studies demonstrate a use for S. cerevisiae as a vehicle for transporting BVDV vaccine antigen to antigen-presenting cell in order to elicit cell-mediated immunity even in na?ve animals.     

Activation rather than Foxp3 expression determines that TGF-β-induced regulatory T cells out-compete naïve T cells in dendritic cell clustering

Regulatory T (Treg) cells are critically important for the maintenance of immunological tolerance. Both centrally arising natural nTreg cells and those emerging in the periphery in response to TGF‐β, iTreg cells, play a role in the control of unwanted immune responses. Treg cells adopt multiple mechanisms to inhibit effector T cells, yet it is unclear whether these mechanisms are shared by nTreg cells and iTreg cells alike. Here, we show that iTreg cells, like nTreg cells, are able to out‐compete naïve T cells in clustering around dendritic cells (DCs). However, using both a tamoxifen‐responsive inducible Foxp3 retroviral construct and TGF‐β‐induced iTreg cells from hCD2‐Foxp3 knock in reporter mice, we show that it is prior antigen‐induced activation rather than Foxp3 expression per se that determines the ability of iTreg cells to competitively cluster around DCs. We found no difference in the capacity of iTreg cells to displace naïve T cells around DCs to that of Tr1, Th1, Th2, or Th9 cells. An important difference was, however, that clustering of iTreg cells around DCs, just as for naïve T cells, did not effectively activate DCs.     

Prolonged exposure of naïve CD8+ T cells to interleukin-7 or interleukin-15 stimulates proliferation without differentiation or loss of telomere length

Interleukin (IL)‐7 and IL‐15 are cytokines implicated in homeostatic control of the peripheral CD8 T‐cell pool. We compared the effects of IL‐7 and IL‐15 on survival and proliferation of purified human CD8⁺ T‐cell subsets. Low concentrations of either cytokine reduced the spontaneous apoptosis of all subsets, and enhancement of survival corresponded to the extent of Bcl‐2 up‐regulation. Surprisingly, although minimal proliferation of naïve CD8⁺ T cells was observed during the first week of culture with cytokines, a marked expansion of these cells occurred at later time points, particularly in response to IL‐15. This occurred largely without phenotypic change or acquisition of effector function, indicating a dissociation of differentiation from proliferation. Notably, progression of naïve CD8⁺ T cells through several cell divisions resulted in up‐regulation of telomerase and the maintenance of telomere length. These data show that IL‐7 and IL‐15 induce cell proliferation and rescue from apoptosis in a concentration, time and subset‐dependent manner, and have implications for the homeostatic expansion of the naïve CD8⁺ T‐cell pool.     

Differential sensitivity of naïve and subsets of memory CD4+ and CD8+ T cells to hydrogen peroxide-induced apoptosis

CD4+ and CD8+ memory T cells are identified into central and effector memory subsets, which are characterized by distinct homing patterns and functions. In this investigation, we show that na?ve and central memory CD4+ and CD8+ T cells are sensitive to hydrogen peroxide (H2O2)-induced apoptosis, whereas effector memory CD4+ and CD8+ T cells are relatively resistant to H2O2-induced apoptosis. Apoptosis in na?ve and central memory CD4+ and CD8+ is associated with the release of cytochrome c and activation of caspase-9 and caspase-3, upregulation of Bax and voltage-dependent anion channel (VDAC) expression, and decreased intracellular glutathione (GSH). In vitro GSH and a superoxide dismutase mimetic Mn(III) tetrakis (1-methyl-4-pyridyl) porphyrin inhibited H2O2-induced apoptosis in both na?ve and central memory CD4+ and CD8+ T cells. Furthermore, VDAC inhibitor 4,4'-diisothiocynostilbene-2,2'-disulfonic acid blocked H2O2-induced apoptosis. These data demonstrate that H2O2 induces apoptosis preferentially in human na?ve and central memory CD4+ and CD8+ T cells via the mitochondrial pathway by regulating intracellular GSH and the expression of Bax and VDAC.