Deletion of CD4+ T cells and thymocytes by apoptosis in mouse mammary tumor virus (C4)-infected Vβ2 transgenic mice

Mouse mammary tumor virus MMTV(C4) encodes a Vβ2-specific superantigen. In Vβ2 transgenic (TG2) mice more than 98 % of peripheral T cells express Vβ2. Infection of Tg2 mice with MMTV(C4) at birth through their mothers' milk or at 6–8 weeks of age by intravenous injection resulted in massive deletion of peripheral CD4⁺ T cells and suppressed thymopoiesis. The number of peripheral CD8⁺ T cells was not affected in neonatally infected mice. In older mice injected with MMTV(C4), splenic CD8⁺ T cells were significantly elevated. Suppressed thymopoiesis was observed in both neonatally infected and older mice injected with MMTV(C4). Thymocytes which expressed high level CD3 or Vβ2 were deleted. To determine if T cells or thymocytes were deleted through apoptosis, DNA fragmentation was examined by flow cytometry and diphenylamine (DPA) binding assay. Approximately 31 % of CD4⁺ T cells from MMTV(C4)-infected Tg2 mice as compared to 6% from normal Tg2 mice contained fragmented nuclear DNA by flow-cytometric analysis. The DPA binding assay showed significantly increased total soluble DNA in lymph node cells and thymocytes from MMTV(C4)-infected mice. The kinetics of T cell and thymocyte apoptosis correspond to their deletion, supporting apoptosis as the mechanism of T cell and thymocyte deletion. CD4⁺ T cell and thymocyte deletion by MMTV(C4) in Tg2 mice provides a sensitive system for the analysis of retrovirus superantigen-induced apoptosis.     

Down-modulation of CD4+ T helper type 2 and type 0 cells by T helper type 1 cells via Fas/Fasligand interaction

Fas was recently demonstrated to be the major target molecule engaged by CD4⁺ cytolytic T lymphocytes (CTL). We examined Fas expression on various cloned T cell subpopulations and their susceptibility to lysis by CD4⁺ or CD8⁺ CTL. A reciprocal relationship in Fas and Fas-ligand expression was observed in CD4⁺ T helper (Th)1- and Th2-type clones, and Fas mRNA was predominantly detected in Th2 clones, whereas Fas-ligand mRNA was principally found in Th1 clones. The two Th0 clones tested expressed both Fas and Fas-ligand, but only one exhibited cytolytic activity, whereas both were sensitive to CD4-mediated lysis. A functional consequence of the inverse Fas-Fas-ligand expression pattern was that Th2 and Th0 cells were sensitive to lysis by both Th1 CD4⁺ CTL and a CD8⁺ CTL clone in a Fas-dependent manner. These results suggest that cytolytic CD4⁺ Th1 cells may play an immunomodulatory role, regulating a Th2/Th0 response by Fas-mediated lysis.     

Predominance of activated,clonally expanded T helper type 17 cells within the CD4+ T cell population in psoriatic lesions

Recent evidence points to the T helper type 17 (Th17) subset as key in the pathogenesis of psoriasis, but cells of this type in lesions remain to be fully characterized. Here we isolated, enumerated, functionally tested and clonotyped the CD4⁺ Th cell population ex vivo from lesional biopsies and paired peripheral blood samples from psoriasis patients. Th17 cells were over‐represented dramatically in lesions from all patients, representing 49–93% of CD4⁺ Th cells compared with 3–18% in blood. Most lesional Th17 cells produced interleukin (IL)‐17A ex vivo without further stimulation and expressed the CD45RO⁺ phenotype characteristic of activated or memory cells. There was no increase in ‘natural’ [CD25^hiforkhead box protein 3 (FoxP3⁺)] regulatory T cells in lesions versus peripheral blood, but there was enrichment of ‘induced’ IL‐10⁺ regulatory T cell numbers in biopsies from some patients. The lesional Th17 cells exhibited a bias in T cell receptor Vβ chain usage, suggestive of specific expansion by antigen. The therapeutic challenge is to overcome the dominance of overwhelming numbers of such antigen‐specific Th17 cells in psoriatic lesions.     

B cell helper T cells and type 1 diabetes

Type 1 diabetes is an autoimmune disease typically starting in childhood that culminates in the destruction of insulin-producing beta cells in the pancreas. Although type 1 diabetes is considered to be a primarily T cell–mediated disease, B cells clearly participate in the autoimmune process, as autoantibodies recognizing pancreatic islet antigen commonly appear in circulation before the onset of the disease. T cells providing helper functions to B cells have recently been shown to be involved in the pathogenesis of a wide range of antibody-associated immune disorders. These T cells include CXCR5-positive follicular T helper (Tfh) cells, and a recently described closely related CXCR5-negative subset coined peripheral T helper (Tph) cells. Here, we review the current state of knowledge on different B cell helper T cell subsets, focusing on their potential involvement in the development of type 1 diabetes.     

Differential response of CD4+ V7+ and CD4+ V7− T cells to T cell receptor-dependent signals: CD4+ V7+ T cells are co-stimulation independent and anti-V7 antibody blocks the induction of anergy by bacterial superantigen

V7 is a novel cell surface glycoprotein that is expressed on 25% of circulating T lymphocytes. This molecule appears to play a critical role in T cell activation based on the observation that a monoclonal anti-V7 antibody inhibits T cell receptor (TCR)-dependent interleukin-2 (IL-2) production and proliferation of T cells. In the current study, CD4⁺ V7+ and CD4⁺ V7? T cells were separated from one another and their response to various stimuli analyzed. Although there were only minor differences between the two subsets in the expression of activation/differentiation markers, including CD45RA and R0 isotypes, when exposed to immobilized anti-CD3 or anti-TCR antibodies in the absence of APC, CD4⁺ V7+ T cells alone produced IL-2 and proliferated vigorously. By contrast, CD4⁺ V7? cells responded poorly to such stimuli, but they recovered their capacity to respond if antigen-presenting cells (APC) or anti-CD28 antibody were added to the cultures. The enhancement of the V7? T cell response by APC appears to be related to augmentation of TCR signals because the effect could be blocked by antibodies against molecules on APC [major histocompatibility (MHC) class II, CD86] that are known to up-regulate such signals through their interaction with counter-receptors on T cells. To assess the role of V7 in a system independent of co-stimulation, CD4⁺ T cells were stimulated with the bacterial superantigens, staphylococcal enterotoxins A and B. The cells responded by proliferating and then becoming anergic. Addition of anti-V7 antibody at the initiation of culture with superantigen did not inhibit cellular proliferation but prevented T cells from becoming anergic, while addition of anti-CD28 antibody had no effect on either proliferation or anergy induction. These results indicate that V7 and CD28 mediate distinct intracellular signals and suggest that V7 functions to preserve T cell reactivity whether the stimulus is mitogenic or anergizing.     

A Vβ4-specific superantigen encoded by a new exogenous mouse mammary tumor virus

The superantigen (SAg) expressed by mouse mammary tumor virus (MMTV) has been shown to play an essential role in the course of the viral life cycle. In the present study, we describe a Vβ4-specific SAg encoded by a new exogenous MMTV carried by the SIM mouse strain. This is the first report of a viral or bacterial SAg reacting with mouse Vβ4⁺ T cells. Injection of MMTV(SIM) into adult BALB/c mice leads to a rapid and strong stimulation of Vβ4⁺ CD4⁺ T cells, followed by a slow deletion of these cells. Neonatal exposure to the virus also leads to a progressive deletion of Vβ4⁺ T cells. In contrast to other strong MMTV SAg, this new SAg requires the presence of major histocompatibility complex class II I-E molecules to be presented efficiently to T cells. Sequence analysis revealed a new predicted amino acid sequence in the C-terminal polymorphic region of this SAg. Furthermore, sequence comparisons to the most closely related SAg with different Vβ specificities hint at the specific residues involved in the interaction with the T cell receptor.     

Priming with high and low respiratory allergen dose induces differential CD4+ T helper type 2 cells and IgE/IgG1 antibody responses in mice

Sensitization of allergic patients normally takes place over several years and is the result of repeated exposure to low levels of allergen. Most mouse asthma models use a high dose of allergen administered over a short period. We have investigated the role of dose in the immune response to an inhaled respiratory allergen (Blomia tropicalis). We observed the effect of priming dose on the allergic response in mice intranasally immunized with low (0·5 μg) and high (50 μg) doses of B. tropicalis extract and killed 1 day after the last challenge. For both doses of allergen, T helper type 2 (Th2) cells and Th2 cytokines were evident as well as eosinophilic inflammation accompanied by mucus hyper‐secretion. By contrast, IgE and IgG1 antibody responses were normally only detected at high‐dose priming. To investigate the mechanism for these effects, we found group 2 innate lymphoid cells (ILC2s) were increased 48 hr after challenge in the low‐dose‐treated but not the high‐dose‐treated mice. Furthermore, we determined whether repeated low‐dose exposure with different priming protocols could induce an antibody response. Repeated low‐dose exposure to 0·5 μg three times weekly for 4 weeks (cumulative 6 μg) had the same effect as a shorter high‐dose exposure (cumulative 80 μg) and increasing cumulative dose induced antibody responses. These data indicate that low doses of allergen are sufficient to prime Th2 cells and ILC2s, but insufficient to induce antibody responses. Cumulative exposure to small amounts of allergen induces both Th2 and antibody responses and may better reflect natural sensitization.     

Flow cytometric determination of cytokines in activated murine T helper lymphocytes: Expression of interleukin-10 in interferon-γ and in interleukin-4-expressing cells

In an immune response, effector functions are controlled by T helper (Th) 1 cytokines [interferon-γ (IFN-γ), interleukin (IL)-2 and tumor necrosis factor-β] and Th 2 cytokines (IL-4, IL-5 and IL-10). Here we analyze by multiparameter immunofluorescence to what extent IL-2, IL-4, IL-5, IL-10 and IFN-γ are co-expressed in individual normal murine Th cells upon activation in vitro with the bacterial superantigen Staphylococcus aureus enterotoxin B, presented in the context of major histocompatibility complex class II. IL-2 and IFN-γ are co-expressed by some, but not by other Th cells. Expression of IL-4 and IFN-γ is exclusive. IL-10 is co-expressed in individual cells either with IL-4 or with IFN-γ. No IL-5-expressing cells are detected. While IL-10- and IL-4-co-expressing Th cells correspond to classical Th 2 cells, cells co-expressing IL-10 and IFN-γ could be involved in negative-feedback regulation of a Th 1 response. Apart from such functional implications, our results show that IL-2, IL-4, IL-5, IL-10 and IFN-γ are expressed independently of each other in individual murine Th cells.     

Regulation of interleukin 6 production in T helper cells

The cytokine interleukin 6 (IL-6) is a cellular regulatory molecule that is produced by both lymphoid and non-lymphoid cells in response to several stimuli. In this report we present evidence that within the murine T cell compartment T helper type 2 cells (Th2) produce this lymphokine, whereas unprimed CD4+ T cells and a T helper type 1 clone (Th1) do not. Furthermore, IL-6 is not an autocrine growth factor for in vitro cultured Th cells, in contrast to what occurs in freshly isolated CD4+ and CD8+ T cells. We have examined the signal transduction pathways that lead to IL-6 production in activated Th2 cells. We have found that protein kinase C activators, such as PMA, Con A, or IL-1, increase the IL-6 expression in these cells. On the other hand, activation of the cAMP-dependent pathway does not seem to have an effect on the IL-6 production, since forskolin, 8BrcAMP, or TNF-alpha, which in these cells increases the level of intracellular cAMP, do not lead to an accumulation of IL-6 message. These results indicate that the IL-6 gene is more tightly regulated in T cells than in other systems described previously.     

Cytotoxicity and weak CD40 ligand expression of CD8+ type 2 cytotoxic T cells restricts their potential B cell helper activity

Naive CD8⁺ T cells differentiate into distinct cytokine-secreting subsets: T helper (Th)1-like cytotoxic T cells (Tc1) and Th2-like Tc2. Although Th2 cells provide strong B cell help, we show that Tc2 cells secreting the same cytokines provide only modest B cell help for IgM production, and only when large numbers of B cells were stimulated with small numbers of Tc2 cells. Lack of effective B cell help by Tc2 cells was attributable partly to their cytotoxicity towards B cells. Both Tc1 and Tc2 cells killed small resting B cells mainly by a perforin-dependent mechanism. In contrast to normal Tc2 cells, perforin-deficient Tc2 cells failed to kill small resting B cells and induced IgM and IgG1 production, although their B cell help was significantly lower than that mediated by Th2 cells. This may be partly attributable to the ability of Tc2 but not Th2 cells to kill activated B cells even in the absence of perforin. Plate-bound anti-CD3 antibodies inhibited Tc2 killing of B cells and induced substantial immunoglobulin production. Additionally, Tc1 and Tc2 cells failed to express CD40 ligand (CD40L), whereas Th1 and Th2 cells expressed high levels of CD40L. Stimulation of Tc1 and Tc2 cells with plate-bound anti-CD3 antibodies for extended periods resulted in low-level expression of CD40L. Proliferation of small resting B cells correlated with immunoglobulin production: proliferation was promoted strongly by Th1 and Th2, weakly by normal Tc1 and Tc2, and moderately by perforin-deficient Tc1 and Tc2 cells. Thus, Tc2 cells may not contribute significantly to cognate B cell help during normal responses.     

Augmented induction of CD8+ cytotoxic T-cell response and antitumour resistance by T helper type 1-inducing peptide

The effector CD8(+) T cells recognize major histocompatibility complex (MHC) class I binding altered self-peptides expressed in tumour cells. Although the requirement for CD4(+) T helper type 1 (Th1) cells in regulating CD8(+) T cells has been documented, their target epitopes and functional impact in antitumour responses remain unclear. We examined whether a potent immunogenic peptide of Mycobacterium tuberculosis eliciting Th1 immunity contributes to the generation of CD8(+) T cells and to protective antitumour immune responses to unrelated tumour-specific antigens. Peptide-25, a major Th epitope of Ag85B from M. tuberculosis preferentially induced CD4(+) Th1 cells in C57BL/6 mice and had an augmenting effect on Th1 generation for coimmunized unrelated antigenic peptides. Coimmunization of mice with Peptide-25 and ovalbumin (OVA) or Peptide-25 and B16 melanoma peptide [tyrosinase-related protein-2 (TRP-2)] for MHC class I led to a profound increase in CD8(+) T cells specific for OVA and TRP-2 peptides, respectively. This heightened response depended on Peptide-25-specific CD4(+) T cells and interferon-gamma-producing T cells. In tumour protection assays, immunization with Peptide-25 and OVA resulted in the enhancement of CD8(+) cytotoxic cell generation specific for OVA and the growth inhibition of EL-4 thymoma expressing OVA peptide leading to the tumour rejection. These phenomena were not achieved by immunization with OVA alone. Peptide-25-reactive Th1 cells counteractivated dendritic cells in the presence of Peptide-25 leading them to activate and present OVA peptide to CD8(+) cytotoxic T cells.     

Parainfluenza type 1 virus-infected cells are killed by both CD8+ and CD4+ cytotoxic T cell precursors.

The memory cytotoxic T lymphocyte (CTL) response to human parainfluenza type 1 virus (hPIV-1), a prominent cause of respiratory infection in young children, has been analysed for a panel of healthy adults. The CTL response to the parainfluenza viruses has not been investigated previously. Precursor CTL (CTLp) with activity against hPIV-1-infected Epstein-Barr virus (EBV)-transformed B lymphoblastoid target cells were found at a relatively high precursor frequency (approximately 1/2500-1/4700 CD8+ and CD4+ subsets respectively) in peripheral blood. Both CD4+ and CD8+ CTLp were detected by the analysis of individual microcultures set up under limiting dilution conditions from freshly isolated blood, the phenotype of the responder cell from individual wells being determined by flow cytometry. Further characterization of the CTL response demonstrated MHC restriction by the HLA-A2 glycoprotein in 3/4 HLA-A2+ donors. The presence of effective, hPIV-1-directed T cell memory may explain, in part, the protection observed in the adult population.     

Maternal transfer of infectious mouse mammary tumor retroviruses does not depend on clonal deletion of superantigen-reactive Vβ14+ T cells

Female C3H/HeJ mice maternally transmit through their milk an infectious mouse mammary tumor retrovirus (MMTV) which causes clonal deletion of T cell receptor (TcR)Vβ14⁺ T cells reactive to the retroviral superantigen (SAG). To test whether CD4⁺ or CD8⁺ T cells are crucial for intestinal infection and maternal transfer of exogenous retroviruses, newborn mice lacking CD4 or CD8 molecules after gene targetting were raised by surrogate C3H/HeJ mothers. In CD8^?/? mice, clonal deletion of TcRVβ14⁺ cells reactive to the SAG from this exogenous MMTV occured with delayed kinetics. Deletion of TcRVβ14⁺ cells was not observed in CD4^?/? mice up to 12 months after exposure to the retrovirus. In both CD4^?/? and CD8^?/? mice TcRVβ5⁺ and TcRVβ11⁺ T cells were deleted in the presence of genomically integrated endogenous MMTV (Mtv), indicating that the lack of SAG-induced clonal deletion was not due to a general defect in these mutant mouse strains. Although TcRVβ14⁺ T cells were not deleted in CD4^?/? mice, female CD4^?/? mice nursed on C3H/HeJ milk maternally transmitted the retrovirus to their offspring, albeit with delayed kinetics. These data demonstrate that CD4⁺ and CD8⁺ lymphocytes influence clonal deletion events and that the mechanisms responsible for clonal deletion of SAG-reactive TcRVβ14⁺ T cells may be different from mechanisms which allow the mammary tumor virus to enter the mammary gland and complete its infectious cycle.     

A paracrine role for chemoattractant receptor-homologous molecule expressed on T helper type 2 cells (CRTH2) in mediating chemotactic activation of CRTH2+ CD4+ T helper type 2 lymphocytes

Activation of human CRTH2(+) CD4(+) T helper type 2 (Th2) cells with anti-CD3/anti-CD28 led to time-dependent production of prostaglandin D(2) (PGD(2)) which peaked at 8 hr. The production of PGD(2) was completely inhibited by cotreatment with the cyclo-oxygenase inhibitor diclofenac (10 microm) but was not affected by cotreatment with ramatroban, a dual antagonist of both the thromboxane-like prostanoid (TP) receptor and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). Supernatants from activated CRTH2(+) CD4(+) Th2 cells caused a concentration-dependent increase in the migration of naive CRTH2(+) CD4(+) Th2 cells compared to supernatants from unstimulated CRTH2(+) CD4(+) Th2 cells. The level of chemotactic activity peaked at 8 hr after activation, corresponding to the peak levels of PGD(2), but production of chemotactic activity was only partially inhibited by the cyclo-oxygenase inhibitor diclofenac. In contrast, ramatroban completely inhibited the chemotactic responses of naive Th2 cells to supernatants from activated CRTH2(+) CD4(+) Th2 cells collected up to 8 hr after activation, although supernatants collected 24 hr after activation were less sensitive to inhibition by ramatroban. The selective TP antagonist SQ29548 did not inhibit migration of Th2 cells, implicating CRTH2 in this response. These data suggest that CRTH2 plays an important paracrine role in mediating chemotactic activation of Th2 cells. Interestingly, although PGD(2) is produced from Th2 cells and contributes to this paracrine activation, it appears that additional CRTH2 agonist factors are also produced by activated Th2 cells and the production of these factors occurs independently of the cyclo-oxygenase pathway of the arachidonic acid metabolism.