Enhanced proliferation of murine T cell lines following interaction of poly(Glu60,Phe40) (GPhe) and antigen-presenting accessory cells. I. GPhe-stimulated enhancement of antigen-dependent proliferative responses.

Following interaction of the random polymer (Glu60,Phe40)n (GPhe) with antigen-presenting accessory cells (APC), unusual costimulatory activities were noted in several murine T cell systems. When GPhe, in contrast with other random copolymers (GT,GL), was added during "inhibition" and T cell "repertoire" studies as a (negative) control to GLA-reactive nonclonal T cell lines of haplotypes H-2d (DCL-2) or H-2bm12, augmentation of T cell proliferation ([3H]thymidine incorporation ([3HT]) to homologous antigen was observed. Augmentation by GPhe was also observed in the response of a GLPhe-reactive (H-2s X H-2d)F1 T cell line and the allogeneic response of the clonal T cell line D10.G4.1. This augmentation was critically dependent on the concentration of adherent accessory cells. Although the mechanism of action of GPhe remains, as yet, undefined, the GPhe-mediated enhancement of DCL-2 (a TH2, H-2d anti-GLA, T cell line) proliferation was not dependent upon the production of either IL-1 or IL-6 by accessory cells. In addition, enhanced DCL-2 proliferation was not accompanied by a significant increase in detectable IL-4 release.     

Enhanced proliferation of murine T cell lines following interaction of poly(Glu60,Phe40) (GPhe) and antigen-presenting accessory cells. II. The role of accessory cells and cytokines in the activity of GPhe on antigen-independent proliferation.

Our previous study (1) demonstrated the "cytokine-like" activity of poly(Glu60,Phe40)(GPhe) in augmenting the antigen-dependent proliferation of a variety of long-term murine T cell lines, particularly the bulk, BALB/c anti-poly (Glu36,Lys24,Ala40) (GLA), interleukin-4-producing, DCL-2 T cell line. GPhe was found to also augment the antigen-independent proliferation of DCL-2 in response to exogenous cytokines ([interleukin(IL)-2 +/- IL-1] in most experiments). Such exogenous cytokine-driven proliferative responses of DCL-2 were used to investigate further the role of accessory cells and of various soluble factors in the action of GPhe. GPhe did not act as a direct mitogen for T cells, rather it acted in a costimulatory fashion, requiring the presence of plastic-adherent accessory cells and a T cell growth factor (either IL-2 or IL-4). In the presence of accessory cells and exogenous IL-2, augmentation of antigen-independent DCL-2 proliferation by GPhe or by IL-1 depended upon the induction of autocrine IL-4 production. However, GPhe also augmented the response of these cells in the presence of exogenous IL-4 (+/- IL-2, +/- IL-1), and exogenous IL-4 added in combination with exogenous IL-2 (+/- IL-1) failed to mimic the GPhe effect, suggesting that another signal was involved in the mechanism of action of GPhe. The ability of allogeneic accessory cells to interact with GPhe to augment proliferative responses suggested that either a soluble factor or an unusual non-MHC-restricted cell-cell interaction provided this signal. In the presence of uv-irradiated accessory cells, DCL-2 proliferation was enhanced over that observed in the presence of non-uv-treated accessory cells, mimicking the GPhe effect, and interaction of GPhe with uv-irradiated accessory cells did not result in further enhancement of DCL-2 cytokine-driven proliferation. Using monoclonal antibodies which could block the function of IA or CD4 molecules, these cell-surface "adhesion" molecules were shown not to participate in the activity of GPhe. By the addition of recombinant cytokines, neutralizing antibodies, or indomethacin, the mechanism of action of GPhe was also shown not to be dependent upon IL-1, IL-6, IL-7, TNF alpha, or prostaglandin production by accessory cells. However, the presence, individually, of some of these factors (IL-1, IL-7, or prostaglandins) could influence to a variable degree the magnitude of the GPhe effect.     

Enhanced proliferation of murine T cell lines following interaction of Poly(Glu60, Phe40) (GPhe) and antigen-presenting accessory cells. III. Possible mechanisms responsible for activity of GPhe.

The random copolymers (Glu80, Phe20)n (GPhe20), (Glu60, Phe40)n (GPhe), and (Glu50, Phe50)n (GPhe50) were compared for the capacity to augment proliferation of antigen-reactive murine T cell lines. GPhe20, GPhe, and GPhe50 showed "augmenting" activity in order of increasing potency. Phenylalanyl residues constituted a significant portion of the "active" determinant(s) in the GPhe polymers tested. High titer murine anti-GPhe (ascites fluid) inhibited augmentation by GPhe of exogenous (IL-1 + rat-conditioned media (RCM] driven T cell proliferation, indicating that (a) the antibodies by binding to specific active determinant(s) in GPhe may have prevented critical GPhe-APC membrane interaction, and/or (b) "GPhe-anti-GPhe" complexes interfered with necessary "processing" of GPhe by APCs. Time course studies demonstrated that the appearance of increased T cell proliferation after GPhe addition occurred after proliferation to (a) nominal antigen or (b) exogenous (IL-1 + RCM) had reached peak [3H]thymidine incorporation ([3HT]). This suggested that more than GPhe-APC membrane interaction was necessary for GPhe activity. Leupeptin, a lysosomal protease inhibitor, inhibited the augmentation of T cell proliferation by GPhe, which led to the conclusion that GPhe must be "processed" by APCs to exhibit activity.     

Gangliosides: differentiation markers for murine T helper lymphocyte subpopulations TH1 and TH2.

On the basis of the pattern of lymphokines they secrete, murine T helper clones can be divided into two subsets, TH1 and TH2. This concept of two different T helper effector cells helps to explain the diversity of immune reactions occurring in different parts of the body. The in vivo localization of T helper subtypes is of great interest, but up to now no biochemical or surface markers were available to distinguish between them. We analyzed the glycolipids from altogether 12 murine TH1 and TH2 cell lines or clones. A comparison of the gangliosides by thin-layer chromatography showed differences between the TH1 and TH2 cells. Binding studies with specific antibodies to asialo backbone structures after degradation by neuraminidases showed that the main gangliosides from these lymphocytes shared a common GgOse4 backbone and thus differed only in their degree or position of sialylation. Two disialogangliosides appeared to be characteristic. They were isolated from the D10.G4.1 TH2 cell clone and identified by fast atom bombardment mass spectrometry as IVNeuAc,IINeuAc-GgOse4Cer (GD1a) and IVNeuAc,IIINeuAc-GgOse4Cer (GD1 alpha), respectively. GD1a was characteristically only detected in TH2 cells, whereas GD1 alpha was preferably, but not exclusively, expressed by TH1 lymphocytes. Although GD1a was also found in the lung, heart, kidney, and spleen, its expression within the murine immune cells under investigation was unique to TH2 lymphocytes. Scarcely any GD1a was found in thymocytes, B cells, or CD8 positive (cytolytic) T cells, but significant expression was seen in CD4 positive (helper) T cells which include the TH2 subpopulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal T cell responses to two epitopes on a single immunogen controlled by two distinct genes

The fine specificities of immune T cells were studied in a system in which the response to the antigen can involve two immune response (Ir) genes and two epitopes on a single synthetic polypeptide immunogen. The (BALB/c X SJL)F1 (H-2d X H-2s) mice can respond to the random terpolymer poly(Glu55, Lys36, Phe9) (GLPhe) through the H-2d-linked Ir gene (Ir-d) or through the complementing Ir gene (Ir-dxs), which controls the immune response to poly(Glu, Phe), epitopes that are present in GLPhe. Nine groups of monoclonal T cells were obtained from (H-2d X H-2s)F1 mice immunized with GLPhe. These groups were delineated by the differences in major histocompatibility (MHC) restriction on antigen-presenting cells (APC) and the cross-reactions with GPhe or GLT. A unique T cell line was discovered that can react to the three polymers (GLPhe, GLT, GPhe) even though GLT and GPhe immune T cells do not normally show reciprocal cross-reactions. The monoclonal T cells retain helper activities in the Mishell-Dutton culture. Although the activation of T cells is antigen specific and MHC restricted, the subsequent B cell response is nonspecific.     

Heterogeneity in direct cytotoxic function of L3T4 T cells. TH1 clones express higher cytotoxic activity to antigen-presenting cells than TH2 clones

In the process of generating culture supernatant from T cell clones, with anti-CD3 antibodies and the B lymphoma A20 as APC, a striking difference in the stimulation of TH1 and TH2 clones was observed, i.e., TH2 clones produced higher levels of lymphokines than TH1 clones. This prompted us to test the hypothesis that differential killing of APC (thus the removal of stimuli) by T cells led to differential T cell activation. By studying a panel of five TH1 and seven TH2 clones, it was demonstrated that TH1 clones mediated significantly higher levels of cytotoxicity toward A20 cells in the presence of soluble anti-CD3 antibody (as opposed to immobilized anti-CD3). Although T cell clones could, when activated with immobilized anti-CD3, produce lymphokines cytotoxic to A20 cells, experiments in which lymphokine production was blocked indicated that T cell clones, in the presence of soluble anti-CD3, mediated killing of A20 through direct cytotoxicity. A higher level of cytotoxicity, by TH1 compared with TH2 clones, was not restricted to anti-CD3 or a particular target cell type, because it also occurred with Con A- or Ag-dependent killing (a monocyte-macrophage cell line), and LPS blasts. Furthermore, the higher cytotoxic activity of TH1 clones compared with TH2 clones was independent of the stage of T cell activation and was unlikely a result of the length of in vitro culture. High levels of killing of APC led to low levels of T cell activation, the significance of which may be as a negative feedback mechanism in the immune response. Other biologic relevancies of higher cytotoxic activity in TH1 vs TH2 cells were also discussed.     

Evidence implicating utilization of different T cell receptor-associated signaling pathways by TH1 and TH2 clones

We have reported recently that high concentrations of anti-CD3 mAb inhibited IL-2-dependent proliferation of TH1 but not TH2 clones. The selective inhibitory effect on TH1 clones suggested that the two helper T lymphocyte subsets might utilize different TCR-associated signal transduction mechanisms. In the present study, we demonstrate that this distinction was not due to a gross difference in the level of TCR expression by TH1 and TH2 clones. Inhibition of TH1 proliferation by anti-CD3 mAb appeared to depend on calcium for maximal effect, suggesting that a substantial elevation of intracellular free calcium concentration ([Ca2+]i) might not occur after ligation of the TCR complex of TH2 clones. Calcium ionophore inhibited IL-2-dependent proliferation of both subsets, suggesting that receptor/ligand systems which stimulate elevated [Ca2+]i would be expected to inhibit proliferation. Although elevated [Ca2+]i and generation of inositol phosphates were readily detected in TH1 clones, these second messengers were not detected following stimulation of TH2 clones via the TCR complex. In addition, lymphokine production by TH1 clones was more sensitive to inhibition by cholera toxin, 8-bromoadenosine 3':5'-cyclic monophosphate, and cyclosporin A than was lymphokine production by TH2 clones. Collectively, these results suggest that TH1 and TH2 clones utilize distinct TCR-associated signal transduction mechanisms for lymphokine gene expression. The difference in signaling mechanisms suggests a potential pharmacologic target for intervention in situations where inappropriate activation of TH1 or TH2 cells occurs in vivo.     

T cell induction of macrophage IL-1 during antigen presentation. Characterization of a lymphokine mediator and comparison of TH1 and TH2 subsets

We described in a previous study two pathways by which Th cells induced macrophage membrane IL-1(mIL-1) during Ag presentation. One pathway was lymphokine-independent and mediated by cell-cell contact. The second was lymphokine-dependent. We have now characterized this lymphokine and show that it belongs to the TNF family of proteins. All TH1 and most TH2 clones produced the lymphokine, although TH1 levels were markedly greater. Both types of clones also induced macrophage mIL-1 by cell-cell contact (i.e., in the absence of lymphokine release). Examination of macrophages by in situ hybridization showed that both IL-1 alpha and IL-1 beta mRNA were coordinately induced during Ag presentation to either TH1 or TH2 clones.     

Functional and ontogenetic analysis of murine CD45Rhi and CD45Rlo CD4+ T cells

CD4+ murine T cell clones, TH1 and TH2, can be distinguished by both functional responses and by their patterns of lymphokine secretion. Recently, a mAb, 23G2, which reacts with a subset of CD45 molecules (CD45R), has been reported to bind differentially to clones of TH1 and TH2 cells. In the present study, normal splenic T cells were analyzed for differences in 23G2-reactivity and were separated into two populations based on their density of CD45R (CD45Rhi and CD45Rlo). The CD45Rhi cells secrete more IL-2 than IL-4 after stimulation in vitro; the reverse is true for the CD45Rlo cells. Because neither population secretes only IL-2 or IL-4, we were unable to classify cells as TH1 or TH2. In vivo and in vitro analyses of the CD45Rhi and CD45Rlo cells suggest a lineage relationship between the two subsets that correlates with the degree of Ag exposure and the state of maturation of the mice. In newborn mice and mice raised under sterile conditions, splenic CD4+ T cells are predominantly CD45Rhi. Under conditions of increased antigenic exposure and maturation of the mice, CD45Rlo cells develop; after long term priming in vivo, the majority of specific Ag-reactive cells are CD45Rlo. Adoptive transfer studies using BALB/c nu/nu recipients demonstrate that CD45Rhi cells become CD45Rlo cells and that the recall response (IgG) to specific Ag is mediated by CD45Rlo cells. Taken together, these data indicate that the level of expression of CD45R on CD4+ T cells distinguishes virgin (CD45Rhi) from primed/memory (CD45Rlo) T cells in normal mice.     

Two types of murine helper T cell clone. II. Delayed-type hypersensitivity is mediated by TH1 clones

We have previously shown that at least two types of Lyt-1+, Lyt-2-, L3T4+ helper T cell clones can be distinguished in vitro by different patterns of lymphokine secretion and by different forms of B cell help. Evidence is presented here to show that one type of helper T cell clone (TH1) causes delayed-type hypersensitivity (DTH) when injected with the appropriate antigen into the footpads of naive mice. The antigen-specific, major histocompatability complex (MHC)-restricted footpad swelling reaction peaked at approximately 24 hr. Footpad swelling was induced by all TH1 clones tested so far, including clones specific for soluble, particulate, or allogeneic antigens. In contrast, local transfer of TH2 cells and antigen did not produce a DTH reaction, even when supplemented with syngeneic spleen accessory cells. Similarly, local transfer of an alloreactive cytotoxic T lymphocyte clone into appropriate recipients did not produce DTH. The requirements for the DTH reaction induced by TH1 cells were investigated further by using TH1 clones with dual specificity for both foreign antigens and M1s antigens. Although these clones responded in vitro to either antigen + syngeneic presenting cells, or M1s disparate spleen cells, they responded in vivo only to antigen + MHC and did not cause footpad swelling in an M1s-disparate mouse in the absence of antigen. Moreover, in vitro preactivation of TH1 or TH2 cells with the lectin concanavalin A was insufficient to induce DTH reactions upon subsequent injection into footpads. From these results, we conclude that the lack of DTH given by TH2 clones in vivo could be due to the inability of the TH2 cells to produce the correct mediators of DTH, or to a lack of stimulation of TH2 clones in the footpad environment.     

Human bronchial epithelium controls TH2 responses by TH1-induced, nitric oxide-mediated STAT5 dephosphorylation: implications for the pathogenesis of asthma

Increased levels of NO in exhaled air in association with increased NO synthetase (NOS)2 expression in bronchial epithelial are hallmark features of asthma. It has been suggested that NO contributes to asthma pathogenesis by selective down-regulation of TH1 responses. We demonstrate, however, that NO can reversibly limit in vitro expansion of both human TH1 and TH2 CD4+ T cells. Mechanistically, NO induces cGMP-mediated reversible STAT5 dephosphorylation and therefore interferes with the IL-2R activation cascade. Human bronchial epithelial cells (HBEC) up-regulate NOS2 after stimulation with IFN-gamma secreted by TH1 CD4+ T cells and release NO, which inhibits both TH1 and TH2 cell proliferation. This reversible T cell growth arrest depends on NO because T cell proliferation is completely restored after in vitro blocking of NOS2 on HBEC. HBEC thus drive the effector end of a TH1-controlled feedback loop, which protects airway mucosal tissues at the potential lesional site in asthma from overwhelming CD4+ TH2 (and potentially TH1) responses following allergen exposure. Variations in the efficiency of this feedback loop provides a plausible mechanism to explain why only a subset of atopics sensitized to ubiquitous aeroallergens progress to expression of clinically relevant levels of airways inflammation.     

A monoclonal antibody that recognizes an Ly-6-linked antigen inhibits the generation of functionally active T cell subsets

A monoclonal antibody (mAb) generated against the chemically-induced BALB/c Meth A sarcoma, designated HD42, reacts in cytotoxic tests with Meth A as well as with BALB/c peripheral lymph node cells and mitogen-activated spleen cells. The antigen was detected by FACS analysis on BALB/c spleen and lymph node cells, and by absorption assays on all normal lymphoid cells of BALB/c but not B6 mice. The expression of the antigen was not found on normal adult lung fibroblasts, on brain, nor on an extensive panel of tumors of BALB/c and B6 origin. Because the strain distribution of the antigen is reciprocal to that of Ly-6.2 and is not expressed in congenic C3H.Ly-6b mice, we have tentatively defined it as Ly-6.1 and referred to the mAb as alpha-Ly-6.1. The presence of alpha-Ly-6.1 abrogates both the Con A-induced and the IL 2-dependent proliferative response of normal T cells, whereas the response of normal B cells to LPS remains unaffected. alpha-Ly-6.1 is a potent suppressor of the primary in vitro plaque-forming cell (PFC) response to SRBC. Pretreatment of normal splenic T cells with alpha-Ly-6.1 and complement had no effect on the ability of these cells to generate in vitro either T helper cells (TH) or T suppressor cells (TS) to SRBC. However, addition of antibody in the absence of complement during the generation of TH or TS, or posttreatment of these T cell subsets with antibody and complement after in vitro education, completely removed the functional activity of these cell types. Addition of alpha-Ly-6.1 to MLC suppressed the MLR as well as the generation of cytotoxic lymphocytes (CTL), whereas the presence of the antibody during a cell-mediated lympholysis (CML) had no effect. Therefore, it appears that alpha-Ly-6.1 recognizes an antigen that is important for the generation of TH and TS cell subsets.     

Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins

A panel of antigen-specific mouse helper T cell clones was characterized according to patterns of lymphokine activity production, and two types of T cell were distinguished. Type 1 T helper cells (TH1) produced IL 2, interferon-gamma, GM-CSF, and IL 3 in response to antigen + presenting cells or to Con A, whereas type 2 helper T cells (TH2) produced IL 3, BSF1, and two other activities unique to the TH2 subset, a mast cell growth factor distinct from IL 3 and a T cell growth factor distinct from IL 2. Clones representing each type of T cell were characterized, and the pattern of lymphokine activities was consistent within each set. The secreted proteins induced by Con A were analyzed by biosynthetic labeling and SDS gel electrophoresis, and significant differences were seen between the two groups of T cell line. Both types of T cell grew in response to alternating cycles of antigen stimulation, followed by growth in IL 2-containing medium. Examples of both types of T cell were also specific for or restricted by the I region of the MHC, and the surface marker phenotype of the majority of both types was Ly-1+, Lyt-2-, L3T4+, Both types of helper T cell could provide help for B cells, but the nature of the help differed. TH1 cells were found among examples of T cell clones specific for chicken RBC and mouse alloantigens. TH2 cells were found among clones specific for mouse alloantigens, fowl gamma-globulin, and KLH. The relationship between these two types of T cells and previously described subsets of T helper cells is discussed.     

Antigen-specific sIalo B cells do not secrete IgG2a in response to TH1 cells and antigen: responsiveness can be restored by IL-4

The ability of Th cells, type 1 (TH1), to activate and induce differentiation of B cells into antibody-secreting cells is controversial because 1) some clones of TH1 cells provide help while others do not, and 2) by using the same TH1 clone, different laboratories disagree on whether they provide help to B cells. One possible explanation for the latter is the variability in the activation status of the B cells used in different laboratories. In the present studies, we have used Ag-specific B cells from athymic (nu/nu) mice, or sterilely housed nu/+ mice to study the TH1-mediated activation of B cells that had received little or no prior help from T cells and/or antigen in vivo. These B cells express low levels of surface Ia (sIa) Ag, and fail to secrete IgG2a in response to TH1 cells plus Ag; in contrast, responses to TH2 cells plus Ag are normal. To explore this observation further, we prepared "surface(s) Ia1o" B cells from conventionally housed BALB/c mice by sorting spleen cells on the fluorescence-activated cell sorter. This sIalo population also failed to produce IgG2a in response to TH1 cells plus Ag. In contrast, the sIahi, (presumably more mature) B cells, responded to both the TH1 and TH2 cells. The addition of LPS, TH2 cells or the lymphokine, IL-4, to cultures of sIalo B cells from normal or nu/nu mice (plus Ag and TH1 cells), restored IgG2a responses to control levels. Low sIa levels were not the sole cause of nonresponsiveness of the nu/nu B cells because a 24-h pulse with IL-4 restored sIa to control levels without restoring IgG2a production after activation with TH1 cells plus Ag. These data support the conclusion that sIalo B cells are immature and require an activation/maturation signal from IL-4 in vivo in order to respond to TH1 cells and Ag in vitro.     

Genetic control of immune responses to the 18-kDa protein of Mycobacterium leprae. Different TH1 subsets may be involved in proliferative and delayed-type hypersensitivity responses.

In vitro and in vivo responses to the 18-kDa protein of Mycobacterium leprae have been analysed in different strains of mice. Lymphocytes from BALB/cJ (H-2d), BALB.B (H-2b), B10.BR (H-2k), and B10.M (H-2f) mice primed with 18-kDa protein yielded high T cell proliferative responses, while those from C57BL/10J (H-2b) mice yielded lower responses. Both H-2 and non-H-2 genes contributed to the magnitude of responsiveness. F1 mice from high and low responder strains showed high responsiveness to the 18-kDa protein. Supernatants from lymph node cell cultures prepared from 18-kDa protein-immunised BALB/cJ, B10.BR, and C57BL/10J mice contained IL-2 but no IL-4, indicating that activated T cells from both high and low responder mice were of a TH1 phenotype. Cell cultures from low responder C57BL/10J mice produced less IL-2 than those from high responders. The low responsiveness to the 18-kDa protein in proliferative assays might be due to a low frequency of antigen-specific T cells in the C57BL/10J mouse strain. BALB/cJ, C57BL/10J, and F1 (BALB/cJ x B10.BR) mouse strains were tested for in vivo DTH reactions to the 18-kDa protein. All strains, including C57BL/10J, were high DTH responders. Although DTH effector cells and 18-kDa protein-specific proliferative T cells belong to the TH1 subset, our data comparing high and low responder status indicate that distinct TH1 subpopulations are stimulated in response to the 18-kDa protein of M. leprae.     

Interleukin 1 responsiveness and receptor expression by murine TH1 and TH2 clones

Murine Th1 and Th2 T cell lines differ in their responses to interleukin 1 (IL 1). Therefore, we examined two T-cell lines, D10.G4.1 (Th2) and MTg12B (Th1) in an attempt to correlate IL 1 receptor (IL 1R) expression with their IL 1 responsiveness. D10.G4.1 cells, which respond to IL 1, expressed two forms of the IL 1R, with molecular masses of approximately 80 kDa and approximately 60 kDa. In contrast, MTg12B cells failed to respond to IL 1 and only expressed the approximately 60 kDa receptor form. This suggests that the approximately 80 kDa receptor is essential for signaling. Expression of both IL 1R forms on D10.G4.1 cells could be inhibited by the anti-IL 4 antibody, 11B11. Antigen presentation reversibly upregulated both forms of the IL 1R, whereas stimulation with concanavalin A (ConA) and anti-CD3 only upregulated the approximately 60 kDa moiety. Upregulation of the approximately 80-kDa IL 1R by repeated antigenic stimulation resulted in a marked increase in sensitivity of D10.G4.1 cells to IL 1.     

TH2 lymphocytes secrete functional VIP upon antigen stimulation

In addition to the peptidergic innervation, immune cells may also represent a source for VIP in the lymphoid organs. Previous studies reported increased VIP mRNA and protein expression in mitogen-stimulated B and T lymphocytes. To determine whether specific T cell subsets are responsible for VIP production, we derived TH1 and TH2 effector cell lines from T-cell receptor transgenic mice. TH1 and TH2 cells were stimulated with the specific (pigeon cytochrome C peptide) or nonspecific (ovalbumin) antigen presented by MHC class II compatible antigen-presenting cells. Upon stimulation with the specific antigen, TH2, but not TH1 cells express VIP mRNA and intracelllular VIP protein, as determined by Northern blots and FACS analysis. Supernatants harvested from antigen-stimulated TH2 cells contain secreted VIP, as determined by Elisa, and induce cAMP in HEK293 cells transfected with the specific VIP/PACAP receptor VPAC1. These results confirm that TH2, but not TH1 cells, express and secrete functional VIP following specific antigen stimulation. The release of VIP within the lymphoid microenvironment following antigenic stimulation provides a physiological basis for the immunoregulatory effects of VIP on neighboring immune cells, such as downregulation of macrophage activation, effects on lymphocyte migration, on antigen-induced T cell apoptosis, and on T cell differentiation.     

LPS and specific T cell responses: interleukin 1 (IL 1)-independent amplification of antigen-specific T helper (TH) cell proliferation

Lipopolysaccharide (LPS) fraction of endotoxin induces a significant potentiation of the antigen-specific proliferative response of T helper (TH) cell lines. This effect was obtained with LPS from different bacterial sources and reproduced with the lipid A moiety of endotoxin. Purified adherent spleen cells used as antigen-presenting cells (APC) support this LPS-enhanced TH cell proliferation. In addition, the effect of endotoxin on specific TH cell responses was found to be absolutely dependent on the interaction between TH lymphocytes and APC through antigen-specific recognition. Thus, it was not observed in the absence of specific antigen or when monoclonal antibodies against class II MHC products or against L3T4 antigens were used to inhibit the T cell-APC interaction. Similarly, it was found that APC from the B6.CH-2bm12 mutant do not support the LPS-mediated enhancing effect. Furthermore, interleukin 1 (IL 1) appears not to be involved in LPS-mediated enhancement, and this effect is not reproduced by muramyl dipeptide (MDP)-mediated activation of APC.