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.     

Cytokine-driven proliferation and differentiation of human naïve,central memory and effector memory CD4+ T cells

Memory T lymphocytes divide in vivo in the absence of antigen maintaining a pool of central memory (T(CM)) and effector memory cells (T(EM)) with distinct effector function and homing capacity. We compared human CD4+ na?ve T, T(CM) and T(EM) cells for their capacity to proliferate in response to cytokines, which have been implicated in T cell homeostasis. Interleukin (IL)-7 and IL-15 expanded with very high efficiency T(EM), while T(CM) were less responsive and na?ve T cells did not respond at all. Dendritic cell (DC)-derived cytokines allowed na?ve T cells to respond selectively to IL-4 and potently boosted the response of T(CM) to IL-7 and IL-15 by increasing the expression of the IL-2/IL-15Rss and the common gamma chain (gamma(c)). The ERK and the p38 MAP kinases were selectively required for TCR and cytokine-driven proliferation, respectively. Importantly, in cytokine-driven cultures T(CM) proliferated and some of the proliferating cells acquired effector function and non-lymphoid tissue homing capacity. Ex vivo BrdU incorporation experiments showed that both T(CM) and T(EM) proliferated under steady state conditions in vivo. Altogether these results provide a plausible mechanism for the maintenance of a polyclonal and functionally diverse repertoire of human CD4+ memory T cells and for a sustained antigen-independent generation of T(EM) from a pool of T(CM) 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.     

Reciprocal changes of naïve and effector/memory CD8+ T lymphocytes in chronic hepatitis B virus infection

Persistent viruses, such as cytomegalovirus or human immunodeficiency virus, cause major perturbations of CD8+ T-lymphocyte subpopulations. To test whether chronic infection with hepatitis B virus (HBV) could also be responsible for such modifications, we analyzed the expression of CD27, CD28, CCR7, and perforin in blood CD8+ T lymphocytes. In comparison to healthy subjects and patients recovering from acute hepatitis B, chronic hepatitis B patients showed higher percentages of na?ve CD8+ T lymphocytes (CD45RA+CD27+CD28+), and lower percentages of intermediately-differentiated CD27+CD28?CD8+ T cells. The late differentiated CD45RA+CD27?CD28? subset was also present in a large percentage in some patients, but no statistically significant difference with healthy controls was observed. Removal from the circulation of intermediately-differentiated CD8+ T lymphocytes may occur during chronic HBV infection, favoring the recruitment of na?ve cells. This may result in impairment of the generation of functionally-competent memory cells, and an inability to achieve control of HBV replication.     

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.     

Enolase 1 of Candida albicans binds human CD4+ T cells and modulates naïve and memory responses

To obtain a better understanding of the biology behind life-threatening fungal infections caused by Candida albicans, we recently conducted an in silico screening for fungal and host protein interaction partners. We report here that the extracellular domain of human CD4 binds to the moonlighting protein enolase 1 (Eno1) of C. albicans as predicted bioinformatically. By using different anti-CD4 monoclonal antibodies, we determined that C. albicans Eno1 (CaEno1) primarily binds to the extracellular domain 3 of CD4. Functionally, we observed that CaEno1 binding to CD4 activated lymphocyte-specific protein tyrosine kinase (LCK), which was also the case for anti-CD4 monoclonal antibodies tested in parallel. CaEno1 binding to naïve human CD4⁺ T cells skewed cytokine secretion toward a Th2 profile indicative of poor fungal control. Moreover, CaEno1 inhibited human memory CD4⁺ T-cell recall responses. Therapeutically, CD4⁺ T cells transduced with a p41/Crf1-specific T-cell receptor developed for adoptive T-cell therapy were not inhibited by CaEno1 in vitro. Together, the interaction of human CD4⁺ T cells with CaEno1 modulated host CD4⁺ T-cell responses in favor of the fungus. Thus, CaEno1 mediates not only immune evasion through its interference with complement regulators but also through the direct modulation of CD4⁺ T-cell 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.     

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.     

Commensal microbiota alter the abundance and TCR responsiveness of splenic naïve CD4+ T lymphocytes

The epidemiologic risk of certain systemic immunologic diseases is affected by commensal or environmental microbiota, but the cellular basis of the "hygiene hypothesis" is poorly understood. In this study, we demonstrate that composition of the commensal microbiota affects the functional state of the peripheral na?ve (CD62L(hi)CD44(lo)) T lymphocyte populations. Restricted flora (RF) mice (stably colonized with excess nonpathogenic Clostridium sp., and changes in other bacterial and fungal taxa) were distinguished after the neonatal period by a progressive deficiency in absolute numbers of na?ve CD4+ and CD8+ T lymphocytes. SPF and RF mice had comparable levels of memory CD4+ and CD8+ T cells. This phenotype was attributable to the altered levels of certain commensals and their products, since germ-free mice had normal absolute numbers of splenic CD4+ and CD8+ T cells and their respective na?ve and memory subsets. The na?ve CD4+ T cell subset was functionally distinguished in RF mice versus SPF mice by TCR hyperresponsiveness, pro-inflammatory cytokine production, and increased activation-induced cell death. Biochemically, these traits were associated with higher basal phosphorylation of the TCR signaling proteins ZAP-70, Lck, and LAT. These findings indicate that enteric microbial products, through unknown cellular circuitry, influence steps in CD4 T cell differentiation moderating basal TCR signaling and immune responsiveness.     

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.     

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.     

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.     

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.     

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.     

Stem cell-like plasticity of naïve and distinct memory CD8+ T cell subsets

Most models regarding the ‘clonal’ origin of CD8⁺ T cell effector and memory subset diversification suggest that during the first contact of a naïve T cell with the priming antigen-presenting cell major decisions for subsequent differentiation are made. Data using novel single-cell T cell tracking technologies demonstrate that a single naïve CD8⁺ T cell can give rise to virtually all different subtypes of effector and memory T cells, and direct major determinants of subset diversification to the time period beyond the first cell division. Thereby, some ‘stem cell-like’ characteristics typical for naïve T cells are probably still maintained within distinct subsets of memory T cells. These observations have direct consequences for clinical applications like adoptive T cell therapy.     

Staphylococcal enterotoxin B increases TIM4 expression in human dendritic cells that drives naïve CD4 T cells to differentiate into Th2 cells

Aberrant T helper (Th)2 polarization plays a critical role in the pathogenesis of allergic disorders; the etiology remains unclear. Dendritic cells (DCs) express T cell immunoglobulin mucin domain (TIM)4 that ligates TIM1 on CD4 T cells to drive them to become Th2 cells, but the pathogenic source of TIM4 is unknown. Here we report that a significant increase in TIM4 expression in human DCs was observed in response to Staphylococcal enterotoxin B (SEB) stimulation via Toll-like receptor (TLR)2 and nucleotide-binding oligomerization domain (NOD)1 pathway. Coculture SEB-conditioned DCs with na?ve CD4 T cells induced Th2 responses that could be abolished using TLR2 or NOD1 or TIM4 or TIM1 with counterpart antibodies or RNA interference. The results demonstrate that Staphylococcus aureus derived SEB promotes the TIM4 production in human DCs. The interaction between TIM4 and TIM1 drives na?ve CD4 T cells to develop to Th2 cells.     

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.     

Anti-CD28-induced co-stimulation and TCR avidity regulates the differential effect of TGF-beta1 on CD4+ and CD8+ naïve human T-cells

TGF-beta1 is a powerful regulator of various T-cell functions. However, it has been unclear how the T-cell responsiveness towards TGF-beta1 is affected by its phenotype or signaling intensity. In the present study, we demonstrate that the phenotype and the TCR-signaling intensity of the responding T-cell as well as the presence of anti-CD28 co-stimulation markedly affects how na?ve human cord blood T-cells respond to TGF-beta1. In this report we demonstrate that the strength of the stimulatory signal modifies the T-cell response towards TGF-beta1. Thus, the greatest anti-proliferative effect of TGF-beta1 was observed during weak stimulatory conditions (low dose of anti-CD3 with no co-stimulatory signal). However, such anti-proliferative effect was reduced during strong stimulatory signal (high dose of anti-CD3 with a CD28-directed co-stimulatory signal). In addition, our results indicate that CD8+ T-cells are generally more responsive towards TGF-beta1 than CD4+ T-cells. To our surprise, na?ve T-cells had a skewed Th1/Tc1 cytokine secretion pattern with high amounts of IL-2, IFNgamma and TNFalpha, but low amounts of IL-4, IL-5 and IL-10. TGFbeta1 significantly reduced the secretion of IL-2 and IFNgamma, but such suppression was partially prevented by anti-CD28-induced co-stimulation. In contrast, the inhibitory effect on IL-5 secretion was unaffected by anti-CD28 co-stimulation. Interestingly, TGF-beta1 induced IL-10 and TNFalpha secretion. However, the induction of IL-10 secretion was reduced during optimal stimulatory conditions while TGF-beta1 further induced TNFalpha secretion. These data demonstrate that the duration, intensity and type of signaling alters the sensitivity of T-cells to powerful immunological modifying agents like TGF-beta1.