Suppression of B cell MIF production by T cells and soluble T cell-derived factors.

Purified populations of guinea pig B cells from nonimmunized animals may be stimualted by PPD or LPS to produce MIF. Unfractionated lymphocyte suspensions from these animals do not produce MIF under these conditions. Reconstitution of B cells with T cells abolishes their ability to generate detectable MIF. A soluble factor obtained from stimulated T cell cultures (MIFIF) is also capable of suppressing this B cell activity. Thus suppressor T cells can interfere with lymphokine production by B cells and this effect is mediated at least in part by a soluble factor. This previously undescribed capacity of T cells may provide an explanation for the fact that B cells do not appear to play a role in reactions of cell-mediated immunity in vivo.     

Distinct autoreactive T cell responses to native and fragmented DNA topoisomerase I: influence of APC type and IL-2

Systemic sclerosis (SSc) is an autoimmune connective tissue disease of unknown etiology in which T cell responses to various autoantigens, including DNA topoisomerase I (Topo I), have been implicated. We investigated whether dendritic cells, generally considered to be the most potent APCs for the initiation of immune responses, would present either of two forms of Topo I to T cells more efficiently than PBMC APCS: Using cells from healthy controls and SSc patients, several important observations were made. First, neither APC type was able to initiate T cell proliferative responses to full-length native Topo I unless exogenous IL-2 was added. This is in contrast to vigorous T cell proliferation in response to Topo I polypeptide fragments presented by either APC type. Second, T cell responses to the full-length form of Topo I presented by dendritic cells were considerably lower than responses to Ag presented by PBMC APCS: Finally, no secondary T cell responses were observed unless the same Ag/APC combination as that used in the primary stimulation was maintained. These data indicate that different peptides are generated based upon the form of the Topo I and the APC that processes it. Taken together, these results suggest that a very specific combination of antigenic form and APC may be involved in breaking tolerance to Topo I in the early stages of development of SSC:     

The inhibition of autoreactive T cell functions by a peptide based on the CDR1 of an anti-DNA autoantibody is via TGF-beta-mediated suppression of LFA-1 and CD44 expression and function

Systemic lupus erythematosus (SLE), which is characterized by the increased production of autoantibodies and defective T cell responses, can be induced in mice by immunization with a human anti-DNA mAb that expresses a major Id, designated 16/6Id. A peptide based on the sequence of the CDR1 of the 16/6Id (human CDR1 (hCDR1)) ameliorated the clinical manifestations of SLE and down-regulated, ex vivo, the 16/6Id-induced T cell proliferation. In this study, we examined the mechanism responsible for the hCDR1-induced modulation of T cell functions related to the pathogenesis of SLE. We found that injection of hCDR1 into BALB/c mice concomitant with their immunization with 16/6Id resulted in a marked elevation of TGF-beta secretion 10 days later. Addition of TGF-beta suppressed the 16/6Id-stimulated T cell proliferation similarly to hCDR1. In addition, we provide evidence that one possible mechanism underlying the hCDR1- and TGFbeta-induced inhibition of T cell proliferation is by down-regulating the expression, and therefore the functions, of a pair of key cell adhesion receptors, LFA-1 (alphaLbeta2) and CD44, which operate as accessory molecules in mediating APC-T cell interactions. Indeed, T cells of mice treated with hCDR1 showed a TGF-beta-induced suppression of adhesion to the LFA-1 and CD44 ligands, hyaluronic acid and ICAM-1, respectively, induced by stromal cell-derived factor-1alpha and PMA. The latter suppression is through the inhibition of ERK phosphorylation. Thus, the down-regulation of SLE-associated responses by hCDR1 treatment may be due to the effect of the up-regulated TGF-beta on the expression and function of T cell adhesion receptors and, consequently, on T cell stimulation, adhesion, and proliferation.     

Abnormal thymocyte development and production of autoreactive T cells in T cell receptor transgenic autoimmune mice.

Development of a C57BL/6-+/+ TCR transgenic mouse containing the rearranged TCR alpha- and beta-chain specific for the Db + HY male Ag results in production of a nearly monoclonal population of early thymocytes expressing the Db + HY reactive TCR. These thymocytes are autoreactive in H-2Db male mice and undergo clonal deletion and down-regulation of CD8. To study the effect of the lpr gene on development of autoreactive T cells, these transgenic mice were backcrossed with C57BL/6-lpr/lpr mice. T cell populations in the thymus and spleen were analyzed by three-color flow cytometry for expression of CD4, CD8, and TCR. The thymus of TCR transgenic H-2b/b lpr/lpr male mice had an increase in percent and absolute number of CD8dull thymocytes compared to TCR transgenic H-2b/b +/+ male mice. However, there was not a complete defect in clonal deletion, because clonal deletion and down-regulation of CD8 was apparent in both +/+ and lpr/lpr H-2Db HY+ male mice compared to H-2Db HY- female mice. The phenotype of splenic T cells was almost identical in TCR transgenic +/+ and lpr/lpr males with about 50% CD4-CD8- T cells and 50% CD8+ T cells. However, there was a dramatic increase in the SMLR proliferative response of splenic T cells from TCR transgenic lpr/lpr males compared to TCR transgenic +/+ males. To determine the specificity of this response, spleen cells from TCR transgenic lpr/lpr and +/+ mice were cultured with irradiated H-2b/b and H-2k/k male and female spleen cells. T cells from TCR transgenic C57BL/6-lpr/lpr male mice had an increased proliferative response to H-2b/b male spleen cells compared to T cells from TCR transgenic C57BL/6(-)+/+ male mice, but both lpr/lpr and +/+ mice had a minimal response to irradiated H-2b/b female or H-2k/k male or female stimulator cells. The splenic T cells from TCR transgenic lpr/lpr mice also had an increased specific cytotoxic activity against H-2b/b male target cells compared to TCR transgenic +/+ mice. These results demonstrate that there is a defect in negative selection of self-reactive T cells in the thymus of lpr/lpr mice and a defect in induction or maintenance of clonal anergy of self-reactive T cells in the periphery of lpr/lpr mice.     

Proteomic profiling of surface proteins on Th1 and Th2 cells

We utilized mass spectrometry to profile cell surface protein differential expression on primary human T helper (Th1 and Th2) cells with the stable isotope labeling by amino acids in cell culture (SILAC) approach. Proteomic and microarray analyses were done concurrently and results were compared for 38 different genes. Although microarray studies displayed wide variability between donors for mRNA expression, these two approaches were shown to be corroborative for most gene products with the exception of a small subset of uncorrelated protein and message levels. The greatest differing Th1 to Th2 ratios were observed for BST2 (bone marrow stromal protein 2) and TRIM (T cell receptor interacting molecule). Both showed greater Th1 expression by proteomic methods, even though mRNA levels were approximately equal for both. To validate this method, we compared protein expression levels of a recently cloned molecule, B and T cell lymphocyte attenuator (BTLA), on Th1 and Th2 cell populations and showed greater protein expression on Th1 cells, which agrees with a previous analysis of higher BTLA mRNA expression in Th1 cells.(1).     

Dendritic cell modulation by mast cells controls the Th1/Th2 balance in responding T cells

The cytokines secreted by pathogen-activated human dendritic cells (DC) are strongly regulated in vitro by histamine, a major component of mast cell granules, ultimately modulating the capacity of the DC to polarize naive T cells. Because DC and mast cells are located in close proximity in peripheral compartments, we hypothesized that mast cell products would influence the maturation of DC and hence the Th balance of an immune response in vivo. In this study, we show that specific mast cell degranulation stimuli, given s.c. in mice with Ag and adjuvant, produce effector T cells that proliferate to Ag but secrete dramatically reduced levels of IFN-gamma and increased amounts of IL-4 compared with control T cells primed in the absence of a mast cell stimulus. Immunization with Ag and adjuvant in the presence of a degranulation stimulus also resulted in the accumulation of DC in the draining lymph nodes that had reduced capacity to induce Ag-specific Th1 cells, in comparison with DC from mice lacking a degranulation stimulus. Therefore, by acting upon DC at sites of inflammation, mast cells play a critical role in determining the polarity of Ag-specific T cell responses in vivo.     

Cognate interactions between helper T cells and B cells. V. Reconstitution of T helper cell function using purified plasma membranes from activated Th1 and Th2 T helper cells and lymphokines.

Th physically interact with B cells and produce lymphokines that influence B cell growth and differentiation. The respective contribution of cell contact and lymphokines to induction of B cell growth and differentiation was addressed using purified plasma membranes (PM) from resting Th (PMrest) and anti-CD3-activated Th (PMCD3) together with lymphokines. Results show that PMCD3, but not PMrest, induce 10% of resting B cells to enter the G1 phase of the cell cycle, with few B cells entering G1b and S/G2. The inclusion of IL-4, but not IL-2, IL-5, or IFN-gamma, amplifies the B cell response to PMCD3 by increasing the total percentage of activatable B cells to greater than 40% and inducing B cell progression into G1b, S, and G2. Direct comparison between PMrest and PMCD3 purified from Th1 and Th2 indicate that both Th1 and Th2 induce similar levels of B cell proliferation in the presence of IL-4. Further, the lymphokine requirements for B cell proliferation induced by PMCD3 from Th1 and Th2 is indistinguishable. B cell differentiation to IgM, IgG1, and IgG2a synthesis by PMCD3 required IL-4 and IL-5. Using lymphokine conditions that supported B cell differentiation, PMCD3 purified from Th1 and Th2 induced similar levels of IgM, and IgG1. Given the functional data on PMCD3 from Th1 and Th2, the data indicate that there are no substantive differences between Th1- and Th2-derived PMCD3, and that the major differences in the ability of viable Th1 and Th2 to activate B cells is the lymphokines produced by the cells.     

Characterization of an endogenous Lyt2+ T-suppressor-cell population regulating autoreactive T cells in vitro and in vivo

Autoreactive T cells have been defined by their capacity to respond to self-Ia antigens expressed on non-T cells. Several recent studies have suggested that these cells may play important immunoregulatory functions. However, it is not clear what regulates the responsiveness of autoreactive T cells and why such cells are not demonstrably stimulated in vivo, where they are in the constant presence of self-Ia antigens. In the present study we examined the role of T suppressor (Ts) cells in regulating autoreactive T cells. We observed that enhanced autoreactivity occurred in vitro when Lyt2+ T cells were depleted from the responding and/or stimulating spleen cells in a syngeneic mixed-lymphocyte reaction. Similarly, addition of irradiated Lyt2+ T cells but not L3T4+ T cells inhibited the response of L3T4+ T cells to self-Ia antigens. The activity of the suppressor cells was specific to the autoreactive T cells since antigen-specific and alloreactive T-cell proliferation were not inhibited. Furthermore, depletion of Lyt2+ T cells by in vivo treatment of mice with anti-Lyt2 monoclonal antibodies caused enhanced endogenous proliferation of lymph node and splenic T cells and increased the T-cell response to self-Ia antigens in vitro. These studies, therefore, suggest that T-cell tolerance to self-Ia antigens in vivo may be maintained by naturally occurring Lyt2+ Ts. Mice having enhanced autoreactivity may provide a useful tool to address the role of autoreactive T cells in the immune response to foreign antigens and in the pathogenesis of autoimmune diseases.     

Type I IFN promotes IL-10 production from T cells to suppress Th17 cells and Th17-associated autoimmune inflammation

Whereas the immune system is essential for host defense against pathogen infection or endogenous danger signals, dysregulated innate and adaptive immune cells may facilitate harmful inflammatory or autoimmune responses. In the CNS, chronic inflammation plays an important role in the pathogenesis of neurodegenerative diseases such as multiple sclerosis (MS). Our previous study has demonstrated a critical role for the type I IFN induction and signaling pathways in constraining Th17-mediated experimental autoimmune encephalomyelitis (EAE), an animal model of human MS. However, it remains unknown if self-reactive Th17 cells can be reprogrammed to have less encephalitogenic activities or even have regulatory effects through modulation of innate pathways. In this study, we investigated the direct effects of type I IFN on Th17 cells. Our data show that IFNβ treatment of T cells cultured under Th17 polarizing conditions resulted in reduced production of IL-17, but increased production of IL-10. We also found that IFNβ induced IL-10 production by antigen specific T cells derived from immunized mice. Furthermore, IFNβ treatment could suppress the encephalitogenic activity of myelin-specific T cells, and ameliorate clinical symptoms of EAE in an adoptive transfer model. Together, results from this study suggest that IFNβ may induce antigen-specific T cells to produce IL-10, which in turn negatively regulate Th17-mediate inflammatory and autoimmune response.     

Responsiveness of naive CD4 T cells to polarizing cytokine determines the ratio of Th1 and Th2 cell differentiation

The intrinsic features of naive CD4 T cells that affect their ability to respond to polarizing signals for Th cell differentiation are not well understood. In this study, we show that naive CD4 T cells from mice transgenic for the Hlx gene expressed lower levels of IL-4Ralpha. The down-regulation of IL-4Ralpha diminished IL-4 signaling and the Th2 response and enhanced the Th1 response under suboptimal polarizing conditions. In nontransgenic CD4 T cells, blocking IL-4Ralpha with Abs had the same effect in an Ab dose-dependent manner. Conversely, Hlx haploinsufficiency caused higher expression of IL-4Ralpha to favor Th2 cell differentiation. Thus, the IL-4Ralpha level on naive CD4 T cells is genetically controlled by Hlx and determines the ratio of Th1 and Th2 cell differentiation.     

Activated self-MHC-reactive T cells have the cytokine phenotype of Th3/T regulatory cell 1 T cells

In the present study, we show that human self-MHC-reactive (autoreactive) T cell clones are functionally distinct from Ag-specific T cell clones. Self-MHC-reactive T cells exhibited helper function for B cell Ig production when cultured with non-T cells alone, and they exhibit suppressor function when cultured with PWM- or rCD40 ligand (rCD40L)-activated non-T cells, whereas tetanus toxoid (TT)-specific clones exhibited only helper function in the presence of TT with or without PWM or rCD40L. Addition of neutralizing Abs to the cultures showed that the suppression was mediated by TGF-beta but not by IL-10 or IFN-gamma. The self-MHC-reactive clones also inhibited proliferation of primary CD4+ T cells and TT-specific T cell clones, but in this case the inhibition was mediated by both IL-10 and TGF-beta. In further studies, the interactions between self-MHC-reactive T cell clones and non-T cells that led to suppressor cytokine production have been explored. We found that prestimulation of non-T cells for 8 h with PWM or for 48 h for rCD40L results in non-T cells capable of inducing self-MHC-reactive T cell to produce high levels of TGF-beta and IL-10. In addition, these prestimulation times coincided with peak induction of HLA-DR and costimulatory B7 molecule (especially CD86) expression on B cells. Finally, addition of CTLA-4/Fc or blocking F(ab')2 anti-CTLA-4 mAb, plus optimally stimulated non-T cells, to cultures of self-MHC-reactive clones inhibited the induction of TGF-beta but not IL-10 or IFN-gamma production. In summary, these studies show that activated self-MHC-reactive T cells have the cytokine phenotype of Th3 or T regulatory cell 1 and thus may be important regulatory cells that mediate oral and peripheral tolerance and prevent the development of autoimmunity.     

T cell surface molecules regulating noncognate B lymphocyte activation. Role of CD2 and LFA-1.

A central event in humoral responses is the Ag-mediated interaction of Th cells and B cells. This interaction leads to the activation of both cell types and results in cytokine secretion by the T cells and proliferation and secretion of Ig by the B cells. The proliferative and differentiative responses of B cells are dependent on contact-mediated signals and cytokines provided by the activated Th cells. Although the role of cytokines in B cell activation and differentiation is understood, the nature of the signals delivered by the activated Th cells and the molecules involved in this process are not known. In this study we have examined Ag-mediated "cognate" T-B cell interactions as well as B cell activation induced by contact with preactivated and fixed Th lymphocytes. Our results indicate that both the T cell surface molecules lymphocyte function associated Ag-1 and CD2 are important in the activation of T cells by Ag presented by B lymphocytes. This indicates that B cells have similar characteristics as other APC. However, once the T cells are activated, contact-mediated stimulation of resting B lymphocytes (the noncognate phase) is dependent on CD2 but not lymphocyte function associated Ag-1. Two lines of evidence indicate this; first, it is inhibited by blocking of CD2 on the T cells and, second, such stimulation is not efficiently mediated by a CD2- Th cell line. Thus, CD2 plays an obligatory role at several discrete stages of T cell-mediated activation of resting B lymphocytes.     

Prostaglandin I2 analogs inhibit proinflammatory cytokine production and T cell stimulatory function of dendritic cells

Signaling through the PGI(2) receptor (IP) has been shown to inhibit inflammatory responses in mouse models of respiratory syncytial viral infection and OVA-induced allergic responses. However, little is known about the cell types that mediate the anti-inflammatory function of PGI(2.) In this study, we determined that PGI(2) analogs modulate dendritic cell (DC) cytokine production, maturation, and function. We report that PGI(2) analogs (iloprost, cicaprost, treprostinil) differentially modulate the response of murine bone marrow-derived DC (BMDC) to LPS in an IP-dependent manner. The PGI(2) analogs decreased BMDC production of proinflammatory cytokines (IL-12, TNF-alpha, IL-1alpha, IL-6) and chemokines (MIP-1alpha, MCP-1) and increased the production of the anti-inflammatory cytokine IL-10 by BMDCs. The modulatory effect was associated with IP-dependent up-regulation of intracellular cAMP and down-regulation of NF-kappaB activity. Iloprost and cicaprost also suppressed LPS-induced expression of CD86, CD40, and MHC class II molecules by BMDCs and inhibited the ability of BMDCs to stimulate Ag-specific CD4 T cell proliferation and production of IL-5 and IL-13. These findings suggest that PGI(2) signaling through the IP may exert anti-inflammatory effects by acting on DC.     

Effect of suplatast tosilate (IPD-1151T) on cytokine production by allergen-specific human Th1 and Th2 cell lines.

Suplatast tosilate (IPD-1151T) is an antiallergic agent that suppresses airway eosinophil infiltration in asthma. We investigated the effects of IPD-1151T on proliferative response and cytokine production by human antigen-specific T cell lines. Purified protein derivatives (PPD)-specific T helper 1 (Th1) cell lines and Dermatophagoides farinae (Der f)-specific T helper 2 (Th2) cell lines were established from patients with asthma sensitized with house dust mite. Stimulation of PPD-specific and Der f-specific T cell lines with relevant antigens resulted in production of mostly interferon (IFN)-gamma and of interleukin (IL)-4 and IL-5, respectively. IPD-1151T did not inhibit the proliferative responses of either the Th1 or Th2 cell line to antigens. Although IPD-1151T did not inhibit IFN-gamma production by PPD-specific Th1 cell lines, it did inhibit IL-4 and IL-5 production by antigen-stimulated Der f-specific Th2 cell lines in a dose-dependent manner. IPD-1151T directly inhibited cytokine production by Der f-specific Th2 cell lines stimulated with immobilized anti-CD3 antibodies. Although IPD-1151T did not inhibit the clonal expansion of memory T cells among PBMCs into PPD-specific Th1 and Th2 cell lines, it did inhibit IL-4 and IL-5 production by Der f-specific Th2 cell lines but not IFN-gamma production by PPD-specific Th1 cell lines. These results suggest that IPD-1151T selectively inhibits Th2-type cytokine production.     

Effect of T helper 1 (Th1)/Th2 cytokine on chemokine-induced dendritic cell functions

Effects of T helper 2 (Th2) and Th1 cytokines, interleukin (IL)-4, and interferon (IFN)-gamma, respectively, on chemokine-induced DC migration and endocytosis are not well understood. We investigated herein the effects of these cytokines on chemokine-induced functions of murine myeloid DCs. As expected, immature DCs markedly migrated to CCL3 but not CCL19, while mature DCs showed vigorous migration in response to CCL19 but not CCL3. Both IL-4 and IFN-gamma significantly decreased CCL3-induced migration of immature DCs. In contrast, these cytokines exerted no significant effects on CCL19-induced migration of mature DCs. Of note, both IL-4 and IFN-gamma markedly enhanced CCL3-induced endocytosis of immature DCs. The messenger RNA level of CCR5, a CCL3 receptor, in immature DCs was slightly increased by IL-4 or IFN-gamma treatment. These results demonstrate that these Th1/Th2 cytokines can act to either inhibit or enhance chemokine-mediated DC functions, and may play a role in increasing antigen uptake by immature DCs at inflammatory sites.     

Antigen presentation to Th1 but not Th2 cells by macrophages results in nitric oxide production and inhibition of T cell proliferation: interferon-gamma is essential but insufficient

The induction and role of nitric oxide (NO) during antigen presentation by macrophages to T helper (Th) cell subsets was examined. When cultured with Th1 clones, macrophage APC produced NO only in the presence of cognate Ag, which in turn suppressed T cell proliferation. IFN-gamma production by the activated Th1 cells was essential for the induction of NO. Th2 cells presented with the same cognate Ag did not induce NO production and proliferated uninhibited. Coactivation of Th1 and Th2 cells specific for the same Ag indicated that Th2 cells did not inhibit NO production, but were sensitive to NO induced by stimulated Th1 cells. Antigenic activation of Th2 cells in the presence of rIFN-gamma resulted in NO-mediated inhibition of proliferation. Th2 cells provided only a cell-associated cofactor, whereas Th1 cells secreted a soluble cofactor for IFN-gamma as well, i.e., TNF-alpha. Finally, a role for IFN-gamma and NO during immune responses was studied in spleen cells obtained from immunized IFN-gamma(-/-) mice. NO production and subsequent inhibition of Ag-specific proliferation ex vivo was observed only after the addition of rIFN-gamma. These studies suggest an IFN-gamma-dependent regulatory role for NO during Ag-specific Th cell activation involving macrophages, with obvious implications for Th subset-dependent immune responses in general.     

Induction of antigen-specific antibody responses in primed and unprimed B cells. Functional heterogeneity among Th1 and Th2 T cell clones

CD4+ T cells have been recently divided into two subsets. The functions of these subsets are thought to be distinct: one subset (Th1) is responsible for delayed type hypersensitivity responses and another (Th2) is primarily responsible for induction of antibody synthesis. To more precisely define the roles of both subsets in humoral immune responses, we examined the ability of a panel of nominal antigen specific Th1 and Th2 clones to induce anti-TNP specific antibody synthesis in TNP-primed or unprimed B cells. Four of nine Th1 clones induced little or no antibody synthesis with TNP-primed B cells. However, five other Th1 clones were very effective at inducing IgG anti-TNP plaque-forming cell (PFC) responses in primed B cells. One of these Th1 clones was analysed in detail and found to also provide helper function for unprimed B cells. Cognate B-T cell interaction was required for induction of both primary and secondary responses with this clone, indicating that a Th1 clone could function as a "classical" Th cell. The seven IL-4 producing Th2 clones examined were also heterogeneous in their ability to induce antibody secretion by TNP-primed B cells. Although four of the Th2 clones induced IgG and IgM anti-TNP PFC responses, two Th2 clones induced only IgM and no IgG antibody, and another clone failed to induce any anti-TNP PFC. All Th2 clones failed to induce any anti-TNP PFC. All Th2 clones produced high levels of IL-4, but "helper" Th2 clones produced significantly greater amounts of IL-5 than "non-helper" Th2 clones. These studies indicate that some IL-2- and some IL-4-producing T cell clones can induce TNP-specific antibody in cell clones can induce TNP-specific antibody in primed and unprimed B cells, and that Th1 and Th2 clones are heterogeneous in their ability to induce Ig synthesis. Therefore, although T cell clones can be classified as Th1 or Th2 types according to patterns of IL-2, IFN-gamma, or IL-4 synthesis, the functional capacity to induce antibody synthesis cannot be predicted solely by their ability to secrete these lymphokines.