CD4+ T-cell effectors inhibit Epstein-Barr virus-induced B-cell proliferation.

In immunodeficient hosts, Epstein-Barr virus (EBV) often induces extensive B-cell lymphoproliferative disease and lymphoma. Without effective in vitro immune surveillance, B cells infected by the virus readily form immortalized cell lines. In the regression assay, memory T cells inhibit the formation of foci of EBV-transformed B cells that follows recent in vitro infection by EBV. No one has yet addressed which T cell regulates the early proliferative phase of B cells newly infected by EBV. Using new quantitative methods, we analyzed T-cell surveillance of EBV-mediated B-cell proliferation. We found that CD4+ T cells play a significant role in limiting proliferation of newly infected, activated CD23+ B cells. In the absence of T cells, EBV-infected CD23+ B cells divided rapidly during the first 3 weeks after infection. Removal of CD4+ but not CD8+ T cells also abrogated immune control. Purified CD4+ T cells eliminated outgrowth when added to EBV-infected B cells. Thus, unlike the killing of EBV-infected lymphoblastoid cell lines, in which CD8+ cytolytic T cells play an essential role, prevention of early-phase EBV-induced B-cell proliferation requires CD4+ effector T cells.

IL-4 promotes airway eosinophilia by suppressing IFN-gamma production: defining a novel role for IFN-gamma in the regulation of allergic airway inflammation.

Airway eosinophilia in asthma is dependent on cytokines secreted by Th2 cells, including IL-5 and IL-4. In these studies we investigated why the absence of IL-4 led to a reduction in airway, but not lung tissue, eosinophils. Using adoptively transferred, in vitro-generated TCR-transgenic Th2 cells deficient in IL-4, we show that this effect is independent of IL-5 and Th2 cell generation. Airway eosinophilia was no longer inhibited when IL-4(-/-) Th2 cells were transferred into IFN-gammaR(-/-) mice, indicating that IFN-gamma was responsible for reducing airway eosinophils in the absence of IL-4. Intranasal administration of IFN-gamma to mice after IL-4(+/+) Th2 cell transfer also caused a reduction in airway, but not lung parenchymal, eosinophils. These studies show that IL-4 indirectly promotes airway eosinophilia by suppressing the production of IFN-gamma. IFN-gamma reduces airway eosinophils by engaging its receptor on hemopoietic cells, possibly the eosinophil itself. These studies capitalize on the complex counterregulatory effects of Th1 and Th2 cytokines in vivo and clarify how IL-4 influences lung eosinophilia. We define a new regulatory role for IFN-gamma, demonstrating that eosinophilic inflammation is differentially regulated at distinct sites within the respiratory tract.

Measurement of human and murine interleukin 2 and interleukin 4.

This unit describes protocols employing cell lines or bioassays that can be used for the quantitation of murine total T cell growth factor (TCGF) activity, interleukin 2 (IL-2), and interleukin 4 (IL-4), and of human IL-2 and IL-4. The ability to distinguish between different growth factors is crucial to understanding the regulation of the immune response. The Basic Protocol describes the use of the CTLL-2 line to detect murine IL-2 and IL-4 in supernatants. One alternate protocol describes the detection of IL-2 in samples of human serum or supernatants using CTLL-2 cells, while other alternate procedures describe the detection and quantitation of murine IL-4 using a mutagenized subline of CTLL-2, CT.4S, and the detection of human IL-4 using a derivative of the CT.4S mouse cell line, CT.h4S. Support protocols are provided for the quantitation of CTLL-2, CT4.S, or CT.h4S proliferation using a standard [3H]thymidine incorporation method or by using the 3-(4,5-dimenthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Support Protocol 1). Support protocols also describe the calculation of cytokine units from samples based on DNA synthesis data and procedures for the maintenance of the CTLL-2, CT.4S, and CT.h4S cell lines.

Identification of human lung and skin proteins conjugated with hexamethylene diisocyanate in vitro and in vivo.

Diisocyanates are asthma-causing chemicals used in the commercial production of polyurethane. We have previously shown that human lung epithelial cell proteins can become conjugated with hexamethylene diisocyanate (HDI) and may be biologically important in diisocyanate-induced asthma. The objective of this study was to identify specific human lung and skin proteins that become conjugated with diisocyanate after in vitro and in vivo exposure. Following in vitro exposure of human airway epithelial cells (A549), keratin 18, the 78-kD glucose-regulated protein, trans-1, 2-dihyrobenzene-1,2-diol dehydrogenase, and actin were identified as prominent diisocyanate-conjugated proteins through use of a combination of immunocytochemical and mass spectrometric techniques. Following in vivo inhalation of an HDI aerosol, keratin 18 was also identified as the predominant diisocyanate-conjugated protein in human endobronchial biopsy samples, whereas albumin was the predominant diisocyanate-conjugated protein in bronchoalveolar lavage fluid. Keratin was also identified as a predominant diisocyanate-conjugated protein in human skin biopsy samples after epicutaneous exposure to liquid-phase HDI, although the major skin diisocyanate-conjugated protein (56-kD) differed from the predominant lung diisocyanate-conjugated keratin (47-kD). The data from this study identify keratin and other proteins as potential "carriers" for diisocyanates in vivo, and suggest that HDI conjugation of these proteins may play a role in the pathogenesis of diisocyanate-induced asthma.

Combinatorial effect of T-cell receptor ligation and CD45 isoform expression on the signaling contribution of the small GTPases Ras and Rap1.

By using ligands with various affinities for the T-cell receptor (TCR) and by altering the contribution of the CD45 tyrosine phosphatase, the effects of the potency of TCR-induced signals on the function of small GTPases Ras and Rap1 were studied. T cells expressing low-molecular-weight CD45 isoforms (e.g., CD45RO) exhibited the strongest activation of the Ras-dependent Elk-1 transcription factor and the highest sensitivity to the inhibitory action of dominant negative mutant Ras compared to T cells expressing high-molecular-weight CD45 isoforms (ABC). Moreover, stimulation of CD45RO(+), but not CD45ABC(+), T cells with a high-affinity TCR ligand induced suboptimal Elk-1 activation compared with the stimulation induced by an intermediate-affinity TCR-ligand interaction. This observation suggested that the Ras-dependent signaling pathway is safeguarded in CD45RO(+) expressors by a negative regulatory mechanism(s) which prohibits maximal activation of the Ras-dependent signaling events following high-avidity TCR-ligand engagement. Interestingly, the biochemical activity of another small GTPase, the Ras-like protein Rap1, which has been implicated in the functional suppression of Ras signaling, was inversely correlated with the extent of Elk-1 activation induced by different-affinity TCR ligands. Consistently, overexpression of putative Rap dominant negative mutant RapN17 or the physiologic inhibitor of Rap1, the Rap GTPase-activating protein RapGAP, augmented the Elk-1 response in CD45RO(+) T cells. This is in contrast to the suppressive effect of RapN17 and RapGAP on CD45ABC(+) T cells, underscoring the possibility that Rap1 can act as either a repressor or a potentiator of Ras effector signals, depending on CD45 isoform expression. These observations suggest that cells expressing distinct isoforms of CD45 employ different signal transduction schemes to optimize Ras-mediated signal transduction in activated T lymphocytes.

Alteration at a single amino acid residue in the T cell receptor alpha chain complementarity determining region 2 changes the differentiation of naive CD4 T cells in response to antigen from T helper cell type 1 (Th1) to Th2.

To study whether changes in the structure of a T cell receptor (TCR) at a single peptide-contacting residue could affect T cell priming with antigenic peptide, we made transgenic mice with a point mutation in the TCR alpha chain of the D10.G4.1 (D10) TCR and bred them to D10 beta chain transgenic mice. The mutation consisted of a leucine to serine substitution at position 51 (L51S), which we had already established contacted the second amino acid of the peptide such that the response to the reference peptide was reduced by approximately 100-fold. A mutation in the reference peptide CA134-146 (CA-WT) from the arginine at peptide position 2 to glycine (R2G) restored full response to this altered TCR. When we examined in vitro priming of naive CD4 T cells, we observed that the response to doses of CA-WT that induced T helper cell type 1 (Th1) responses in naive CD4 T cells from mice transgenic for the D10 TCR gave only Th2 responses in naive CD4 T cells derived from the L51S. However, when we primed the same T cells with the R2G peptide, we observed Th1 priming in both D10 and L51S naive CD4 T cells. We conclude from these data that a mutation in the TCR at a key position that contacts major histocompatibility complex-bound peptide is associated with a shift in T cell differentiation from Th1 to Th2.

Function and regulation of memory CD4 T cells.

The development of peripheral naive CD4 T cells is dependent on the success of positive selection of immature T cells in the thymus. Only thymocytes that express a T cell receptor (TCR) capable of recognizing self-MHC with low affinity are selected for survival and differentiation into mature naive T cells. Although the TCR of naive T cells has to maintain self-tolerance, it also propagates naive CD4 T cell proliferation on recognition of appropriate foreign peptide associated with MHC class II on antigen-presenting cells (APCs). Naive CD4 T cells that successfully engage foreign peptide undergo further differentiation that leads to the maturation of a select few into the memory T cell pool. Although the requirements that lead to memory T cell development are currently not known, functional changes have been described that are thought to be associated with the greater efficiency with which memory T cells respond to antigen. This article will discuss differences associated with signaling through the TCR of naive and memory CD4 T cells and describe unique control mechanisms imposed on memory CD4 T cells that are likely to have ari sen to counterbalance the altered TCR signaling.

Biochemical association of CD45 with the T cell receptor complex: regulation by CD45 isoform and during T cell activation.

CD45 is the predominant transmembrane tyrosine phosphatase in lymphocytes and is required for the efficient induction of T cell receptor signaling and activation. However, the regulation of CD45 activity and substrate specificity are poorly understood. In the present study, we demonstrate a basal biochemical association of CD45 with the T cell receptor complex that is regulated in part by CD45 isoform expression. Further, maintenance of CD45/TCR association is differentially regulated following TCR ligation with peptide: a partial agonist peptide induces CD45/TCR dissociation while an agonist peptide promotes sustained association in a CD4-dependent manner. These data suggest that T cell receptor signaling pathways may be modulated by altering access of CD45 to TCR-associated substrates involved in T cell activation.

Survival of naive CD4 T cells: roles of restricting versus selecting MHC class II and cytokine milieu.

The diversity of naive CD4 T cells plays an important role in the adaptive immune response by ensuring the capability of responding to novel pathogens. In the past, it has been generally accepted that naive CD4 T cells are intrinsically long-lived; however, there have been studies suggesting some CD4 T cells are short-lived. In this report, we identify two populations of naive CD4 T cells: a long-lived population as well as a short-lived population. In addition, we identify two factors that contribute to the establishment of long-lived naive CD4 T cells. We confirm earlier findings that MHC class II interaction with the TCR on CD4 T cells is important for survival. Furthermore, we find that MHC class II alleles with the correct restriction element for Ag presentation mediate the peripheral survival of naive CD4 T cells more efficiently than other positively selecting alleles, regardless of the selecting MHC in the thymus. The second component contributing to the survival of naive CD4 T cells is contact with the cytokines IL-4 and IL-7. We find that the physiological levels of IL-4 and IL-7 serve to enhance the MHC class II-mediated survival of naive CD4 T cells in vivo.

STAT5 interaction with the T cell receptor complex and stimulation of T cell proliferation.

The role of STAT (signal transducer and activator of transcription) proteins in T cell receptor (TCR) signaling was analyzed. STAT5 became immediately and transiently phosphorylated on tyrosine 694 in response to TCR stimulation. Expression of the protein tyrosine kinase Lck, a key signaling protein in the TCR complex, activated DNA binding of transfected STAT5A and STAT5B to specific STAT inducible elements. The role of Lck in STAT5 activation was confirmed in a Lck-deficient T cell line in which the activation of STAT5 by TCR stimulation was abolished. Expression of Lck induced specific interaction of STAT5 with the subunits of the TCR, indicating that STAT5 may be directly involved in TCR signaling. Stimulation of T cell clones and primary T cell lines also induced the association of STAT5 with the TCR complex. Inhibition of STAT5 function by expression of a dominant negative mutant STAT5 reduced antigen-stimulated proliferation of T cells. Thus, TCR stimulation appears to directly activate STAT5, which may participate in the regulation of gene transcription and T cell proliferation during immunological responses.

Differential role of CTLA-4 in regulation of resting memory versus naive CD4 T cell activation.

Regulation of peripheral T cell responses is critical for preserving self tolerance. Memory T cells have a lower threshold for activation through the TCR and are thought to be less dependent on costimulation than naive T cells, suggesting a requirement for more stringent regulation of memory T cells. We have recently shown that CD4 engagement apart from the TCR results in the inactivation of memory, but not naive, CD4 T cells. We show here that this inhibition requires ligation of CTLA-4, in that blocking CTLA-4-B7 interactions restores memory CD4 T cell responsiveness. Early signaling through CTLA-4 is possible because resting memory, but not naive, CD4 T cells contain intracellular stores of CTLA-4 that are continuously recycled between the cytoplasm and the cell surface. This mechanism ensures that low intensity TCR engagements, which are thought to be important for peripheral T cell longevity, do not cause memory T cell activation but instead raise their threshold for costimulatory signals. This may give memory T cells an extended lifespan with a reduced risk of inappropriate activation.

Induction of IL-4-producing CD4+ T cells by antigenic peptides altered for TCR binding.

The adaptive immune responses to foreign Ags are primarily regulated by the cytokines produced by CD4 T cells. The generation of distinct cytokine-producing T cell subsets has been shown to be influenced by a number of factors, including cytokines, different types of APCs, and the amounts of priming Ag. We have previously reported that the affinity of an antigenic peptide for its presenting MHC class II molecules and that different doses of Ag peptide affect the outcome of the functional CD4 T cell response. In the current study, we further examined the impact of the affinity of an antigenic peptide for its TCR on CD4 T cell priming. We generated a panel of Ag peptide variants mutated at positions known to be critical for binding to a well-characterized TCR (known as altered peptide ligands, or APLs). Compared with the WT peptide, these APLs are defective in stimulating the proliferative responses of T cells. However, they can effectively prime in vitro naive CD4 T cells for differentiation into both Th1-like and Th2-like cells. In contrast, the WT peptide primes only for IFN-gamma-producing Th1-like cells. Using highly purified dendritic cells as APCs to present the APL or WT peptide leads to the same pattern of priming as using total splenic APCs. These results indicate that priming by APLs for both IL-4 production and IFN-gamma production does not require two different types of APCs. In summary, our data indicate that APL can directly stimulate naive CD4 T cells to become Th2 effector cells.

Reciprocal regulation of CD30 expression on CD4+ T cells by IL-4 and IFN-gamma.

Prior studies have implicated CD30 as a marker for Th2 cells, but the mechanism that underlies this correlation was unknown. We show here that CD30 was expressed on activated CD4+ T cells in the presence of IL-4. In the absence of endogenously produced IL-4, however, even Th2 lineage cells lost CD30 expression. Thus, CD30 is not an intrinsic marker of Th2 cells, but is inducible by IL-4. CD30 was also found to be down-regulated by IFN-gamma. Committed Th1 effector cells do not express CD30, although differentiating Th1 lineage cells temporarily express CD30. The transient expression of CD30 on differentiating Th1 lineage cells was mainly the result of endogenously produced IL-4 induced by IL-12. Culture of IL-12-primed cells under conditions that reverse the phenotype (Ag plus IL-4) resulted in two cell populations based upon their ability to express CD30. One population responded to IL-4 upon restimulation and became a CD30-positive, Th0-like cell population, while the other remained CD30 negative and synthesized only IFN-gamma. Thus, CD30 expressed on CD4+ T cells reflected the ability of CD4+ T cells to respond to IL-4.

Induction of Th1 and Th2 CD4+ T cell responses: the alternative approaches.

T helper lymphocytes can be divided into two distinct subsets of effector cells based on their functional capabilities and the profile of cytokines they produce. The Th1 subset of CD4+ T cells secretes cytokines usually associated with inflammation, such as IFN-gamma and TNF and induces cell-mediated immune responses. The Th2 subset produces cytokines such as IL-4 and IL-5 that help B cells to proliferate and differentiate and is associated with humoral-type immune responses. The selective differentiation of either subset is established during priming and can be significantly influenced by a variety of factors. One of these factors, the cytokine environment, has been put forward as the major variable influencing Th development and is already well reviewed by others. Instead, in the current review, we focus on some of the alternative approaches for skewing Th1/Th2 responses. Specifically, we discuss the effects on Th priming of (a) using altered peptide ligands as antigens, (b) varying the dose of antigen, and (c) altering costimulatory signals. The potential importance of each of these variables to influence immune responses to pathogens in vivo is discussed throughout.

Distinct biochemical signals characterize agonist- and altered peptide ligand-induced differentiation of naive CD4+ T cells into Th1 and Th2 subsets.

We have recently shown that altered peptide ligands influence differentiation of CD4+ T cells into Th1 and Th2 subsets. In the present study, we have examined the biochemical signals in naive CD4+ T cells after priming with altered peptide ligand (APL) that correlate with differences in cytokine expression. Although we observed zeta-chain phosphorylation in APL-stimulated cells, other signaling events such as ZAP70 and Lnk phosphorylation are not initiated. This altered pattern observed in the early phosphorylation events correlates with a distinct Ca2+ mobilization pattern that characterizes APL-stimulated cells. By changing the calcium signaling environment during T cell priming, we present data indicating that qualitative differences in calcium mobilization are associated with differentiation of naive CD4+ T cells into Th1- and Th2-like effector subsets.

Strength of TCR signal determines the costimulatory requirements for Th1 and Th2 CD4+ T cell differentiation.

Differentiation of naive CD4 T cells into cytokine-secreting effector Th1 and Th2 cells is influenced by several factors. We have previously reported that the affinity of antigen for TCR and antigen dose can influence the differentiation of Th1 and Th2 cells. Several in vitro and in vivo models have demonstrated a role for the costimulatory molecules, B7-1 (CD80) and B7-2 (CD86), in the generation of distinct effector T cell responses. To determine whether the strength of TCR signaling controls the involvement of CD28 costimulation in selective CD4 T cell differentiation, naive CD4 T cells bearing a transgenic TCR are primed by a weak or strong TCR signal (signal 1) in the presence or absence of B7 costimulatory molecules (signal 2). In this system, IL-4-producing Th2 cells are generated by priming with a weak but not a strong TCR signal. Th2 cell differentiation is dependent on CD28/B7 interactions in that disruption of CD28/B7 interactions inhibits the priming of Th2 cells and cross-linking CD28 with anti-CD28 antibody augments the priming of Th2 cells. In contrast, however, IL-4-producing Th2 cells cannot be generated by priming with a strong TCR signal even in the presence of strong costimulation or high doses of IL-2. Thus, our results suggest that naive CD4 T cells are receptive to CD28-dependent IL-4 production only if they receive a weak TCR signal.

Role of dendritic cells in the priming of CD4+ T lymphocytes to peptide antigen in vivo.

The contribution of dendritic cells (DC) in the initial priming of CD4+ T lymphocytes in vivo was examined. To assess the capacity of different APC to prime CD4+ T cells, a series of MHC class II I-E transgenic mice that differentially express I-E on APC were utilized. Transgenic mice that express I-E primarily on DC, on macrophages, and on B cells were primed with an I-E restricted peptide in vivo, and the extent of priming was determined by restimulation of CD4+ T cells in vitro with the same Ag. The results indicate that DC are required for priming of CD4+ T cells, and that the extent of priming correlates with the frequency of I-E+ DC. Moreover, DC alone can serve as APC for the peptide Ag, in that priming can be restored in an I-E negative mouse by the transfer of peptide-pulsed I-E+ DC. The potency of DC, compared with B cells and macrophages, to prime naive CD4+ T cells was confirmed with in vitro studies.

Calcitonin gene-related peptide inhibits interleukin 2 production by murine T lymphocytes.

Evidence from the literature suggests that the nervous and the immune systems closely interact via neuromediators, which affect the immune system, and cytokines, which control nerve cell growth and activity. Calcitonin gene-related peptide (CGRP) is a neuropeptide that has been identified in numerous tissues including immune organs and inhibits the proliferation of spleen cells. We investigated whether CGRP altered the function of T lymphocytes. We present evidence that CGRP induces a dose-dependent cAMP accumulation in interleukin 2-producing TH1 cells and inhibits their production of interleukin 2. These effects are prevented by CGRP8-37, a CGRP antagonist that is missing the first 7 amino acids. This CGRP-mediated inhibition of interleukin 2 production is accompanied by a decrease in interleukin 2 mRNA accumulation. CGRP also inhibits the accumulation of mRNA coding for tumor necrosis factor-alpha and -beta and interferon-gamma. Thus, we have identified one mechanism by which CGRP inhibits the proliferation of spleen cells.

A polymerase chain reaction assay for the detection and quantitation of cytokine gene expression in small numbers of cells.

A reverse transcriptase-polymerase chain reaction (RT-PCR) assay is described that allows the rapid detection and quantitation of mRNA encoding the cytokines interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma). Analysis of cytokine production by defined CD4+ T cell clones and the thymoma cell line EL4, demonstrates that the oligonucleotide primers used in this assay are specific for the genes encoding the individual cytokines, generating PCR products of different sizes. This allows the simultaneous and unambiguous detection of all three cytokine mRNAs in the same cDNA sample. The assay is sensitive enough to reproducibly detect cytokine mRNA expressed in as few as ten cells and requires 10,000-fold less cells for the detection of IL-2 production than that required for its detection using a conventional bioassay. Reverse transcribed mRNA is quantitated in the PCR assay by amplifying in the presence of a known amount of competitive genomic DNA (gDNA) template containing a small intron using the same primers. The PCR products obtained form the target cDNA and gDNA templates, which are distinguished by size, are processed by Southern analysis and quantitated by scanning densitometry of autoradiographs. As little as two-fold differences in cytokine mRNA can be reliably detected using this assay. We have demonstrated the successful application of this assay to the quantitation of pg amounts of IL-2 mRNA that is constitutively produced at low levels by fetal thymocytes in vivo during T cell ontogeny. The sensitivity, specificity, reliability and speed of this assay will facilitate the analysis of cytokine production in in vivo-derived or, in vitro propagated cells which are not available in sufficient numbers for analysis using more conventional molecular and biochemical assays.