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 effect of TGF-beta1 on immune responses of naïve versus memory CD4+ Th1/Th2 T cells

The role of TGF-beta1 in the regulation of T cell responses has been perplexing, possibly because it is dependent on the type of T cell being regulated and its cytokine microenvironment. In the present study, we demonstrate that TGF-beta1 has a profound inhibitory effect on naive CD4+ T cell undergoing differentiation under defined neutral, Th1 and Th2 priming conditions. In addition, we show that if CD4+ T cells are primed in the presence of TGF-beta1, they exhibit reduced secondary anti-CD3/anti-CD28-induced and antigen-specific immune responses (even when TGF-beta is absent during the secondary response), which is not due to reduced expression of co-stimulatory molecules or to inadequate IL-2 production. Finally, with respect to the effect of TGF-beta on fully differentiated antigen-specific memory CD4+ T cells, we demonstrate that while antigen-specific activation and cytokine secretion by memory Th1 T cells is inhibited by TGF-beta1, such inhibition is associated with partial down-regulation of IL-12 receptor beta2 chain expression. In contrast, memory Th2 T cells are not subject to TGF-beta1 -mediated suppression. In summary, these studies reveal that TGF-beta1 is a powerful negative regulator of the primary immune response of CD4+ T cells, but only Th1 T cells are subject to such regulation after the memory stage of T cell differentiation has been reached. Thus, these studies define the potential regulatory role of TGF-beta1 in Th1 and Th2 T cell-mediated autoimmunity.     

HIV-1 matrix protein p17 prevents loss of CD28 expression during IL-2-induced maturation of naïve CD8(+) T cells

Na?ve CD8(+) T cells differentiate into effectors secreting various cytokines that modulate immune functions. A striking finding for most HIV-1-infected patients is that they accumulate CD8(+) T cells belonging to early and intermediate differentiated elements. Structural HIV-1 proteins, and among these the matrix protein p17, have been associated with loss of functional competence by different immune cells. We therefore evaluated the influence of p17 on na?ve CD8(+) T-cell activation and maturation. Anti-CD3 mAb preactivation and subsequent IL-2 stimulation are able to drive human naive CD(+) T cells to an effector phenotype characterized, among other features, by downregulation of the co-stimulatory molecule CD28. Strikingly, however, IL-2-induced downmodulation of CD28 was completely prevented by p17, and cells derived from p17-stimulated cultures showed a strong Tc1 polarization that was fourfold higher than that observed in IL-2-stimulated cultures.Moreover, p17 preserved a markedly high proportion of CD8(+) T cells that were able to respond to CD28 triggering with a proinflammatory cytokine storm. Our evidence suggests that p17 has important effects on cytokine polarization and phenotype of terminally differentiated CD8(+) T cells, and that new p17-based therapeutic approaches could control or prevent HIV-1-related immune disorders.     

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.     

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.     

Growth inhibition of Candida albicans by vaginal cells from na?ve mice.

Recurrent vulvovaginal candidiasis (RVVC) is a common idiopathic mucosal infection caused by Candida albicans. Current data suggests that local immunity is more important than that in the peripheral circulation for protection against infection. In the present study, anti-Candida innate resistance at the vaginal mucosa was investigated using a murine model. For this, splenic and vaginal cells were assessed for in vitro growth inhibition (GI) of C. albicans and cytotoxicity of natural killer (NK) cell-sensitive tumour targets (YAC-1). As expected, significant GI of C. albicans by splenic cells was mediated predominantly by polymorphonuclear leucocytes (PMNL) at effector to target (E:T) ratios of 100 and 50:1. From the vaginal mucosa, na?ve unfractionated, but not nylon wool non-adherent (NWN), cells extracted from whole vaginal tissue showed significant GI of C. albicans at E:T ratios as low as 1:1, but only modest killing of YAC-1 targets at all E:T ratios. Subsequent experiments showed significant GI of C. albicans by vaginal epithelioid-enriched cells and with several epithelial cell lines, but not in supernatants collected from the co-cultures. In contrast, lymphoid cell lines had no anti-Candida activity. These results suggest that anti-Candida activity is present at the vaginal mucosa, but unlike that from the spleen, the vaginal activity appears to be predominantly mediated by epithelial 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.     

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.     

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.     

Vγ1+ γδ T cells reduce IL-10-producing CD4+CD25+ T cells in the lung of ovalbumin-sensitized and challenged mice

In OVA-sensitized and challenged mice, gammadelta T cells expressing Vgamma1 enhance airway hyperresponsiveness (AHR) but the underlying mechanism is unclear. These cells also reduce IL-10 levels in the airways, suggesting that they might function by inhibiting CD4(+)CD25(+) regulatory T cells (T(reg)) or other CD4(+) T cells capable of producing IL-10 and suppressing AHR. Indeed, sensitization and challenge with OVA combined with inactivation of Vgamma1(+) cells increased CD4(+)CD25(+) cells in the lung, and markedly those capable of producing IL-10. The cellular change was associated with increased IL-10 and TGF-beta levels in the airways, and a decrease of IL-13. T(reg) include naturally occurring Foxp3(+) T(reg), inducible Foxp3(-) T(reg), and antigen-specific T(reg) many of which express folate receptor 4 (FR4). Although Foxp3 gene expression in the lung was also increased pulmonary CD4(+) T cells, expressing Foxp3-protein or FR4 remained stable. Therefore, the inhibition by Vgamma1(+) gammadelta T cells might not be targeting Foxp3(+) T(reg) but rather CD4(+) T cells destined to produce IL-10.     

Vaccinia virus infection of mature dendritic cells results in activation of virus-specific naïve CD8+ T cells: a potential mechanism for direct presentation

Understanding how vaccinia virus (VV) generates immunity necessitates an appreciation for how this virus interacts with dendritic cells (DC), which are the most potent activators of na?ve CD8(+) T cells. In order to optimally activate na?ve CD8(+) T cells, DC must undergo maturation, during which costimulatory molecules are upregulated and cytokines are produced. In this report, we show that VV infection of immature murine bone marrow-derived DC (BMDC) failed to induce maturation. Similar results were obtained when CD8(+) DC were analyzed, a subset shown previously to be important in vivo in the generation of a vaccinia-specific response. The finding that VV infection of DC resulted in APC that were incapable of initiating T-cell activation was surprising given the previously reported role for direct presentation in the generation of anti-VV CD8(+) T-cell responses in mice. To address the potential mechanism responsible for direct presentation, we tested the hypothesis that previously matured DC were susceptible to vaccinia virus infection and could present newly synthesized VV-derived epitopes for CD8(+) T-cell activation. Our results show, that during VV infection of mature DC, threshold levels of viral protein are produced that promote T-cell activation. These results suggest that, even though VV cannot mature DC, previously matured DC exposed to VV can generate a VV-specific CD8(+) T-cell response providing a potential mechanism by which direct infection results in T-cell activation in vivo.     

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.     

Generation of anti-tumour effector T cells from naïve T cells by stimulation with dendritic/tumour fusion cells

Tumour-draining lymph node T cells are an excellent source of effector T cells that can be used in adoptive tumour immunotherapy because they have already been sensitized to tumour-associated antigens in vivo. However, such tumour-specific immune cells are not readily obtained from the host due to poor immunogenicity of tumours and reduced host immune responses. One obstacle in implementation of adoptive immunotherapy has been insufficient sensitization and expansion of tumour-specific effector cells. In this study, we aim to improve adoptive immunotherapy by generating anti-tumour effector T cells from naïve T lymphocytes. We attempted to achieve this by harnessing the advantages of dendritic cell (DC)-based anti-cancer vaccine strategies. Electrofusion was routinely employed to produce fusion cells with 30–40% efficiency by using the poorly immunogenic murine B16/F10 cell line, D5 cells, and DC generated from bone marrow cells. CD62L-positive T cells from spleens of naïve mice and the fusion cells were cocultured with a low concentration of IL-2. After 9 days of culture, the antigen-specific T cells were identified with an upregulation of CD25 and CD69 expression and a downregulation of CD62L expression. These cells secreted IFN-γ upon stimulation with irradiated tumour cells. Moreover, when transferred into mice with 3-day established pulmonary metastases, these cells with coadministration of IL-2 exhibited anti-tumour efficacy. In contrast, naïve T cells cocultured with a mixture of unfused DC and irradiated tumour cells did not exhibit anti-tumour efficacy. Our strategy provides the basis for a new approach in adoptive T cell immunotherapy for cancer.     

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.     

A B-1a cell subset induces Foxp3? T cells with regulatory activity through an IL-10-independent pathway

Regulatory T (Treg) cells play a critical role in the maintenance of tolerance. B-1a cells belong to a specific and functionally important B-cell subset that exerts its regulatory role through the production of IL-10. While IL-10 has been correlated with the induction of type 1 Treg (Tr1) cells or Tr1-like cells, whether IL-10-producing B-1a cells are able to induce Treg cells, especially the Tr1 lineage, is poorly understood. We have demonstrated that, similar to the reported B-2 cells, B-1a cells are able to convert naïve CD4⁺CD25⁻ T cells into a subset of T cells with suppressive function, which we called ‘Treg-of-B1a'' cells. Treg-of-B1a cells do not express Foxp3, but upregulate the Treg markers OX40, programmed death 1 (PD-1), inducible costimulator (ICOS) and IL-10R. Moreover, Treg-of-B1a cells do not express Foxp3 and produce high levels of IFN-γ and IL-10, but minimal amounts of IL-4; therefore, they resemble Tr1 cells. However, utilizing IL-10^−/− mice, we showed that IL-10 was not involved in the induction of Treg-of-B1a cells. On the contrary, CD86-mediated costimulation was essential for B-1a cells to drive the induction of Treg-of-B1a cells. Finally, we demonstrated that, in contrast to the Treg cells generated by B-2 cells that mediate contact-dependent suppression, Treg-of-B1a cells suppress through secreting soluble factors. While Tr1 cells mediate suppression mainly through IL-10 or TGF-β secretion, Treg-of-B1a cells mediate suppression through an IL-10- and TGF-β-independent pathway. Together, these findings suggest that B-1a cells induce a functionally and phenotypically distinct Treg population that is dissimilar to the reported Foxp3⁺ Treg or Tr1 cells.