Primary response of naive CD4(+) T cells to amino acid-substituted analogs of an antigenic peptide can show distinct activation patterns: Th1- and Th2-type cytokine secretion, and helper activity for antibody production without apparent cytokine secretion

Naive CD4(+) T cells differentiate into two types of helper T cells showing an interferon-gamma-predominant (Th1) or an interleukin-4-predominant (Th2) cytokine secretion profile after repeated antigenic stimulation. Their differentiation can be influenced by slight differences in the interaction between the T cell receptor (TCR) and its ligand at the time of primary activation. However, the primary response of freshly isolated naive CD4(+) T cells to altered TCR ligands is still unclear. Here, we investigated the primary response of splenic naive CD4(+) T cells derived from transgenic mice expressing TCR specific for residues 323-339 of ovalbumin (OVA323-339) bound to I-A(d) molecules. Naive CD4(+) T cells secreted either Th1- or Th2-type cytokines immediately after stimulation with OVA323-339 or its single amino acid-substituted analogs. Helper activity for antibody secretion by co-cultured resting B cells was also found in the primary response, accompanied by either low-level Th2-type cytokine secretion or no apparent cytokine secretion. Our results clearly indicate that dichotomy of the Th1/Th2 cytokine secretion profile can be elicited upon primary activation of naive CD4(+) T cells. We also demonstrate that the helper activity of naive CD4(+) T cells for antibody production does not correspond to the amounts of the relevant cytokines secreted.     

Activation, differentiation, and migration of naive virus-specific CD8+ T cells during pulmonary influenza virus infection

The low precursor frequency of individual virus-specific CD8(+) T cells in a naive host makes the early events of CD8(+) T cell activation, proliferation, and differentiation in response to viral infection a challenge to identify. We have therefore examined the response of naive CD8(+) T cells to pulmonary influenza virus infection with a murine adoptive transfer model using hemagglutinin-specific TCR transgenic CD8(+) T cells. Initial activation of CD8(+) T cells occurs during the first 3 days postinfection exclusively within the draining lymph nodes. Acquisition of CTL effector functions, including effector cytokine and granule-associated protease expression, occurs in the draining lymph nodes and differentially correlates with cell division. Division of activated CD8(+) T cells within the draining lymph nodes occurs in an asynchronous manner between days 3 and 4 postinfection. Despite the presence of Ag for several days within the draining lymph nodes, dividing T cells do not appear to maintain contact with residual Ag. After multiple cell divisions, CD8(+) T cells exit the draining lymph nodes and migrate to the infected lung. Activated CD8(+) T cells also disseminate throughout lymphoid tissue including the spleen and distal lymph nodes following their emigration from draining lymph nodes. These results demonstrate an important role for draining lymph nodes in orchestrating T cell responses during a local infection of a discrete organ to generate effector CD8(+) T cells capable of responding to infection and seeding peripheral lymphoid tissues.     

A new dynamic model of CD8+ T effector cell responses via CD4+ T helper-antigen-presenting cells

A long-standing paradox in cellular immunology has been the conditional requirement for CD4(+) Th cells in priming of CD8(+) CTL responses. We propose a new dynamic model of CD4(+) Th cells in priming of Th-dependent CD8(+) CTL responses. We demonstrate that OT II CD4(+) T cells activated by OVA-pulsed dendritic cells (DC(OVA)) are Th1 phenotype. They acquire the immune synapse-composed MHC II/OVAII peptide complexes and costimulatory molecules (CD54 and CD80) as well as the bystander MHC class I/OVAI peptide complexes from the DC(OVA) by DC(OVA) stimulation and thus also the potential to act themselves as APCs. These CD4(+) Th-APCs stimulate naive OT I CD8(+) T cell proliferation through signal 1 (MHC I/OVAI/TCR) and signal 2 (e.g., CD54/LFA-1 and CD80/CD28) interactions and IL-2 help. In vivo, they stimulate CD8(+) T cell proliferation and differentiation into CTLs and induce effective OVA-specific antitumor immunity. Taken together, this study demonstrates that CD4(+) Th cells carrying acquired DC Ag-presenting machinery can, by themselves, efficiently stimulate CTL responses. These results have substantial implications for research in antitumor and other aspects of immunity.     

Inducible CD4+LAP+Foxp3- regulatory T cells suppress allergic inflammation

Regulatory T cells (Tregs) play a critical role in the maintenance of airway tolerance. We report that inhaled soluble Ag induces adaptive Foxp3(+) Tregs, as well as a regulatory population of CD4(+) T cells in the lungs and lung-draining lymph nodes that express latency-associated peptide (LAP) on their cell surface but do not express Foxp3. Blocking the cytokine IL-10 or TGF-β prevented the generation of LAP(+) Tregs and Foxp3(+) Tregs in vivo, and the LAP(+) Tregs could also be generated concomitantly with Foxp3(+) Tregs in vitro by culturing naive CD4(+) T cells with Ag and exogenous TGF-β. The LAP(+) Tregs strongly suppressed naive CD4(+) T cell proliferation, and transfer of sorted OVA-specific LAP(+) Tregs in vivo inhibited allergic eosinophilia and Th2 cytokine expression in the lung, either when present at the time of Th2 sensitization or when injected after Th2 cells were formed. Furthermore, inflammatory innate stimuli from house dust mite extract, nucleotide-binding oligomerization domain containing 2 ligand, and LPS, which are sufficient for blocking airway tolerance, strongly decreased the induction of LAP(+) Tregs. Taken together, we concluded that inducible Ag-specific LAP(+) Tregs can suppress asthmatic lung inflammation and constitute a mediator of airway tolerance together with Foxp3(+) Tregs.     

Dynamics of CCR5 expression by CD4(+) T cells in lymphoid tissues during simian immunodeficiency virus infection

Early viral replication and profound CD4(+) T-cell depletion occur preferentially in intestinal tissues of macaques infected with simian immunodeficiency virus (SIV). Here we show that a much higher percentage of CD4(+) T cells in the intestine express CCR5 compared with those found in the peripheral blood, spleen, or lymph nodes. In addition, the selectivity and extent of the CD4(+) T-cell loss in SIV infection may depend upon these cells coexpressing CCR5 and having a "memory" phenotype (CD45RA(-)). Following intravenous infection with SIVmac251, memory CD4(+) CCR5(+) T cells were selectively eliminated within 14 days in all major lymphoid tissues (intestine, spleen, and lymph nodes). However, the effect on CD4(+) T-cell numbers was most profound in the intestine, where cells of this phenotype predominate. The CD4(+) T cells that remain after 14 days of infection lacked CCR5 and/or were naive (CD45RA(+)). Furthermore, when animals in the terminal stages of SIV infection (with AIDS) were examined, virtually no CCR5-expressing CD4(+) T cells were found in lymphoid tissues, and all of the remaining CD4(+) T cells were naive and coexpressed CXCR4. These findings suggest that chemokine receptor usage determines which cells are targeted for SIV infection and elimination in vivo.     

Either IL-2 or IL-12 is sufficient to direct Th1 differentiation by nonobese diabetic T cells

Th cell differentiation from naive precursors is a tightly controlled process; the most critical differentiation factor is the action of the driving cytokine: IL-12 for Th1 development, IL-4 for Th2 development. We found that CD4(+) T cells from nonobese diabetic mice spontaneously differentiate into IFN-gamma-producing Th1 cells in response to polyclonal TCR stimulation in the absence of IL-12 and IFN-gamma. Instead, IL-2 was necessary and sufficient to direct T cell differentiation to the Th1 lineage by nonobese diabetic CD4(+) T cells. Its ability to direct Th1 differentiation of both naive and memory CD4(+) T cells was clearly uncoupled from its ability to stimulate cell division. Autocrine IL-2-driven Th1 differentiation of nonobese diabetic T cells may represent a genetic liability that favors development of IFN-gamma-producing autoreactive T cells.     

IL-2 receptor beta-chain signaling controls immunosuppressive CD4+ T cells in the draining lymph nodes and lung during allergic airway inflammation in vivo

IL-2 influences both survival and differentiation of CD4(+) T effector and regulatory T cells. We studied the effect of i.n. administration of Abs against the alpha- and the beta-chains of the IL-2R in a murine model of allergic asthma. Blockade of the beta- but not the alpha-chain of the IL-2R after allergen challenge led to a significant reduction of airway hyperresponsiveness. Although both treatments led to reduction of lung inflammation, IL-2 signaling, STAT-5 phosphorylation, and Th2-type cytokine production (IL-4 and IL-5) by lung T cells, IL-13 production and CD4(+) T cell survival were solely inhibited by the blockade of the IL-2R beta-chain. Moreover, local blockade of the common IL-2R/IL-15R beta-chain reduced NK cell number and IL-2 production by lung CD4(+)CD25(+) and CD4(+)CD25(-) T cells while inducing IL-10- and TGF-beta-producing CD4(+) T cells in the lung. This cytokine milieu was associated with reduced CD4(+) T cell proliferation in the draining lymph nodes. Thus, local blockade of the beta-chain of the IL-2R restored an immunosuppressive cytokine milieu in the lung that ameliorated both inflammation and airway hyperresponsiveness in experimental allergic asthma. These findings provide novel insights into the functional role of IL-2 signaling in experimental asthma and suggest that blockade of the IL-2R beta-chain might be useful for therapy of allergic asthma in humans.     

Murine Peyer's patches favor development of an IL-10-secreting,regulatory T cell population

Peyer's patches (PP) are believed to be the principal sites for induction of tolerance to Ags from food and commensal flora, yet the phenotype of T cells activated within the PP is largely unexplored. We hypothesize that exposure to Ags within the PP promotes differentiation of T cells with immunoregulatory functions. Cytokine production and cell surface marker expression of murine PP mononuclear cells (MC) are compared with those from mesenteric lymph nodes and peripheral lymph nodes (PLN). In response to stimulation through the TCR/CD3 complex, PP MC exhibit vigorous proliferation, modest production of IL-2, and significantly elevated synthesis of IL-10. Exogenous IL-12 enhances both IL-10 and IFN-gamma secretion by activated PP MC. Cell surface marker analysis reveals that PP T cells consist of activated and memory subpopulations compared with the predominantly naive T cells identified in the PLN and mesenteric lymph nodes. Upon stimulation, only CD45RB(low)CD4(+) PP T cells produce IL-10, whereas secretion of IL-2, IL-4, and IFN-gamma was not detected. Furthermore, PP MC, but not PLN MC, stimulated through the TCR/CD3 complex suppress proliferation of purified PLN T cells in vitro, evidence for a regulatory function among PP lymphocytes. We conclude that PP favor differentiation of an IL-10-producing, regulatory CD45RB(low)CD4(+) T cell population and that inhibition of T cell proliferation by activated PP MC may reflect regulatory activity consistent with T regulatory cells.     

IFN-gamma production by Th1 cells generated from naive CD4+ T cells exposed to norepinephrine

During activation in vivo, naive CD4(+) T cells are exposed to various endogenous ligands, such as cytokines and the neurotransmitter norepinephrine (NE). To determine whether NE affects naive T cell differentiation, we used naive CD4(+) T cells sort-purified from either BALB/c or DO11.10 TCR-transgenic mouse spleens and activated these cells with either anti-CD3/anti-CD28 mAbs or APC and OVA(323-329) peptide, respectively, under Th1-promoting conditions. RT-PCR and functional assays using selective adrenergic receptor (AR) subtype antagonists showed that naive CD4(+) T cells expressed only the beta 2AR subtype to bind NE and that stimulation of this receptor generated Th1 cells that produced 2- to 4-fold more IFN-gamma. This increase was due to more IFN-gamma produced per cell upon restimulation instead of more IFN-gamma-secreting cells, as determined by IFN-gamma-specific immunofluorescence and enzyme-linked immunospot. In contrast, Th1 cell differentiation was unaffected when naive T cells were exposed to NE and activated either in the presence of a neutralizing anti-IL-12 mAb or by APC from IL-12-deficient mice. Moreover, the addition of IL-12 to the IL-12-deficient APC cultures restored the ability of NE to increase Th1 differentiation. Taken together, these results indicate that a possible link may exist between the signaling pathways used by NE and IL-12 to increase naive CD4(+) T cell differentiation to a Th1 cell.     

TLR2 stimulation drives human naive and effector regulatory T cells into a Th17-like phenotype with reduced suppressive function

Naturally occurring CD4(+)CD25(+)FOXP3(+) regulatory T cells suppress the activity of pathogenic T cells and prevent development of autoimmune responses. There is growing evidence that TLRs are involved in modulating regulatory T cell (Treg) functions both directly and indirectly. Specifically, TLR2 stimulation has been shown to reduce the suppressive function of Tregs by mechanisms that are incompletely understood. The developmental pathways of Tregs and Th17 cells are considered divergent and mutually inhibitory, and IL-17 secretion has been reported to be associated with reduced Treg function. We hypothesized that TLR2 stimulation may reduce the suppressive function of Tregs by regulating the balance between Treg and Th17 phenotype and function. We examined the effect of different TLR2 ligands on the suppressive functions of Tregs and found that activation of TLR1/2 heterodimers reduces the suppressive activity of CD4(+)CD25(hi)FOXP3(low)CD45RA(+) (naive) and CD4(+)CD25(hi)FOXP3(hi)CD45RA(-) (memory or effector) Treg subpopulations on CD4(+)CD25(-)FOXP3(-)CD45RA(+) responder T cell proliferation while at the same time enhancing the secretion of IL-6 and IL-17, increasing RORC, and decreasing FOXP3 expression. Neutralization of IL-6 or IL-17 abrogated Pam3Cys-mediated reduction of Treg suppressive function. We also found that, in agreement with recent observations in mouse T cells, TLR2 stimulation can promote Th17 differentiation of human T helper precursors. We conclude that TLR2 stimulation, in combination with TCR activation and costimulation, promotes the differentiation of distinct subsets of human naive and memory/effector Tregs into a Th17-like phenotype and their expansion. Such TLR-induced mechanism of regulation of Treg function could enhance microbial clearance and increase the risk of autoimmune reactions.     

CpG methylation of the IFNG gene as a mechanism to induce immunosuppression [correction of immunosupression] in tumor-infiltrating lymphocytes

The execution of appropriate gene expression patterns during immune responses is of eminent importance where CpG methylation has emerged as an essential mechanism for gene silencing. We have charted the methylation status of regulatory elements in the human IFNG gene encoding the signature cytokine of the Th1 response. Surprisingly, human naive CD4(+) T lymphocytes displayed hypermethylation at the IFNG promoter region, which is in sharp contrast to the completely demethylated status of this region in mice. Th1 differentiation induced demethylation of the IFNG promoter and the upstream conserved nucleotide sequence 1 enhancer region, whereas Th2-differentiated lymphocytes remained hypermethylated. Furthermore, CD19(+) B lymphocytes displayed hypomethylation at the IFNG promoter region with a similar pattern to Th1 effector cells. When investigating the methylation status among tumor-infiltrating CD4(+) T lymphocytes from patients with colon cancer, we found that tumor-infiltrating lymphocytes cells are inappropriately hypermethylated, and thus not confined to the Th1 lineage. In contrast, CD4(+) T cells from the tumor draining lymph node were significantly more demethylated than tumor-infiltrating lymphocytes. We conclude that there are obvious interspecies differences in the methylation status of the IFNG gene in naive CD4(+) T lymphocytes, where Th1 commitment in human lymphocytes involves demethylation before IFNG expression. Finally, investigations of tumor-infiltrating lymphocytes and CD4(+) cells from tumor draining lymph node demonstrate methylation of regulatory regions within key effector genes as an epigenetic mechanism of tumor-induced immunosuppression.     

Differential expression of a 70 kDa O-glycoprotein on T cells: a possible marker for naive and early activated murine T cells

We purified a 70 kDa O-glycoprotein that binds to the GalNAc specific lectin from Amaranthus leucocarpus (ALLr) and determined its expression pattern on T lymphocytes from different murine lymphoid organs. High level of ALLr expression was demonstrated in 95-98% of both CD4(+)8(+) and CD4(-)8(+) thymocytes, and in 80-95% of CD8(+) T cells from peripheral blood, lymph nodes, and spleen, whereas a minor fraction of CD4(+)8(-) thymocytes (46-67%) and peripheral CD4(+) T cells (9-40%) showed low ALLr expression. Peripheral CD19(+) B cells were ALLr negative and most of the peripheral ALL(+) T cells showed a CD62L(hi)CD45RB(hi)CD44(lo/-) phenotype, indicating features of naive cells. Mitogenic activation of peripheral T cells increased 3-fold the number of ALL(+)CD4(+) T cells 24 h after stimulation, as opposed to a >80% decrease in CD8(+) T cells 72 h after stimulation. Our results suggest that ALL detects a non-described surface O-glycoprotein selectively expressed by naive CD8(+) T cells and by early activated CD4(+) T cells.     

Differentiation of naive CD4<Superscript>+</Superscript> T cells towards T helper 2 cells is not impaired in rheumatoid arthritis patients

An impaired differentiation of naive CD4+ T cells towards Th2 cells may contribute to the chronic tissue-destructive T-cell activity in rheumatoid arthritis (RA). The differentiation of naive CD4+ T cells into memory Th2 cells by IL-7 in comparison with that by IL-4 was studied in RA patients and in healthy controls. Naive CD4+ T cells from peripheral blood were differentiated by CD3/CD28 costimulation in the absence of or in the presence of IL-7 and/or IL-4. The production of IFN-gamma and IL-4 was measured by ELISA and by single-cell FACS analysis to indicate Th1 and Th2 cell activity. CD3/CD28 costimulation and IL-7 were early inducers of IL-4 production, but primarily stimulated IFN-gamma production. In contrast, in short-term cultures exogenously added IL-4 did not prime for IL-4 production but suppressed IL-7-induced IFN-gamma production. Upon long-term stimulation of naive CD4+ T cells, IFN-gamma production was differentially regulated by IL-7 and IL-4, but IL-4 production was increased by both IL-7 and IL-4. IL-7 and IL-4 additively induced polarization towards a Th2 phenotype. This susceptibility of naive CD4+ T cells to become Th2 cells upon culture with IL-7 and IL-4 was increased in RA patients compared with that in healthy controls. These findings demonstrate that, in RA patients, differentiation of naive CD4+ T cells towards a Th2 phenotype by CD3/CD28 costimulation, IL-7 and IL-4 is not impaired. The perpetuation of arthritogenic T-cell activity in RA therefore seems not to be the result of intrinsic defects of naive CD4+ T cells to develop towards suppressive memory Th2 cells.     

IL-33 induces antigen-specific IL-5+ T cells and promotes allergic-induced airway inflammation independent of IL-4

Type 2 cytokines (IL-4, IL-5, and IL-13) play a pivotal role in helminthic infection and allergic disorders. CD4(+) T cells which produce type 2 cytokines can be generated via IL-4-dependent and -independent pathways. Although the IL-4-dependent pathway is well documented, factors that drive IL-4-independent Th2 cell differentiation remain obscure. We report here that the new cytokine IL-33, in the presence of Ag, polarizes murine and human naive CD4(+) T cells into a population of T cells which produce mainly IL-5 but not IL-4. This polarization requires IL-1R-related molecule and MyD88 but not IL-4 or STAT6. The IL-33-induced T cell differentiation is also dependent on the phosphorylation of MAPKs and NF-kappaB but not the induction of GATA3 or T-bet. In vivo, ST2(-/-) mice developed attenuated airway inflammation and IL-5 production in a murine model of asthma. Conversely, IL-33 administration induced the IL-5-producing T cells and exacerbated allergen-induced airway inflammation in wild-type as well as IL-4(-/-) mice. Finally, adoptive transfer of IL-33-polarized IL-5(+)IL-4(-)T cells triggered airway inflammation in naive IL-4(-/-) mice. Thus, we demonstrate here that, in the presence of Ag, IL-33 induces IL-5-producing T cells and promotes airway inflammation independent of IL-4.     

Polarized development of memory cell-like IFN-gamma-producing cells in the absence of TCR zeta-chain

TCR/CD3 complex-mediated signals play critical roles in regulating CD4(+) Th cell differentiation. In this report, we have examined the in vivo role of a key TCR/CD3 complex molecule zeta-chain in regulating the differentiation of Th cells. We have studied T cells from zeta-chain-deficient mice (zetaKO mice), zeta-chain-bearing mice (zeta(+) mice), and from zetaKO mice expressing a FcRgamma chain transgene (FcRgammaTG, zetaKO mice). Our results demonstrated that, compared with those of control mice, CD4(+) T cells and not CD8(+) T cells from zetaKO mice were polarized into IFN-gamma-producing cells. Some of these IFN-gamma-producing cells could also secrete IL-10. Interestingly, zetaKO mouse T cells produced IFN-gamma even after they were cultured in a Th2 condition. Our studies to determine the molecular mechanisms underlying the polarized IFN-gamma production revealed that the expression level of STAT4 and T-bet were up-regulated in freshly isolated T cells from zetaKO mice. Further studies showed that noncultured zetaKO mice CD4(+) T cells and thymocytes bore a unique memory cell-like CD44(high), CD62L(low/neg) phenotype. Altogether, these results suggest that, in the absence of the zeta-chain, CD4(+) T cells develop as polarized IFN-gamma-producing cells that bear a memory cell-like phenotype. The zeta-chain-bearing T cells may produce a large amount of IFN-gamma only after they are cultured in a condition favoring Th1 cell differentiation. This study may provide important implications for the down-regulation of zeta-chain in T cells of patients bearing a variety of tumors, chronic inflammatory and infectious diseases.     

Differentiation of CD8+ T cells from tumor-invaded and tumor-free lymph nodes of melanoma patients: role of common gamma-chain cytokines

Differentiation of CD8(+) T cells at the tumor site toward effector and memory stages may represent a key step for the efficacy of antitumor response developing naturally or induced through immunotherapy. To address this issue, CD8(+) T lymphocytes from tumor-invaded (n = 142) and tumor-free (n = 42) lymph nodes removed from the same nodal basin of melanoma patients were analyzed for the expression of CCR7, CD45RA, perforin, and granzyme B. By hierarchical cluster analysis, CD8(+) T cells from all tumor-free lymph nodes and from 56% of the tumor-invaded lymph node samples fell in the same cluster, characterized mainly by CCR7(+) CD45RA(+/-) cytotoxic factor(-) cells. The remaining three clusters contained only samples from tumor-invaded lymph nodes and showed a progressive shift of the CD8(+) T cell population toward CCR7(-) CD45RA(-/+) perforin(+) granzyme B(+) differentiation stages. Distinct CD8(+) T cell maturation stages, as defined by CCR7 vs CD45RA and by functional assays, were identified even in melanoma- or viral Ag-specific T cells from invaded lymph nodes by HLA tetramer analysis. Culture for 7 days of CCR7(+) perforin(-) CD8(+) T cells from tumor-invaded lymph nodes with IL-2 or IL-15, but not IL-7, promoted, mainly in CCR7(+)CD45RA(-) cells, proliferation coupled to differentiation to the CCR7(-) perforin(+) stage and acquisition of melanoma Ag-specific effector functions. Taken together, these results indicate that CD8(+) T cells differentiated toward CCR7(-) cytotoxic factor(+) stages are present in tumor-invaded, but not in tumor-free, lymph nodes of a relevant fraction of melanoma patients and suggest that cytokines such as IL-2 and IL-15 may be exploited to promote Ag-independent maturation of anti-tumor CD8(+) T cells.