Antigen-specific helper factor reacts with antibody to the T-cell receptor

Antigen-specific helper factor (ASHF), a soluble product of T helper (Th) cells, binds antigen and can induce B-cell and cytotoxic T-lymphocyte (CTL) differentiation. Its relationship to the T-cell surface antigen receptor (TcR) is unknown. Both have MHC-restricted recognition of nominal antigen, thus they may share very similar combining sites. Using monoclonal anti-TcR to immunoprecipitate partially purified ASHF, we have obtained evidence for shared determinants between ASHF and the TcR. Antigen affinity-enriched supernatants of a Th clone, LB19, are functionally active in antigen-specific, help-dependent CTL assays. FPLC anion exchange salt fractions of these supernatants were 125I-labelled and immunoprecipitated with KJ16.133 monoclonal anti-TcR coupled to Sepharose 4B. Precipitates were analysed by SDS-PAGE. We have obtained clear evidence that functionally active Th culture supernatants contain molecules specifically precipitable by anti-TcR antibody.     

Inhibition of the production of a soluble helper mediator by cyclosporin A results in the failure to generate alloreactive cytolytic cells in mixed-lymphocyte culture

The mechanism of action of cyclosporin A (CsA) in inhibiting the induction of alloreactive cytolytic T lymphocytes (CTL) in mixed-lymphocyte culture (MLC) was investigated. CsA at concentrations of 10(-3) to 10(-1) micrograms/ml completely prevented the generation of CTL. However, the addition of culture supernatants from mitogen-activated lymphocytes to MLC not only significantly reversed the suppressive effect of CsA but also fully restored the reactivity of lymphocytes already treated with CsA. By measuring the presence of a soluble helper mediator (SHF) in MLC supernatants, we found that CsA-treated lymphocytes produced no SHF, possibly interleukin 2 (IL-2). The effect of CsA on receptors for IL-2 was subsequently studied and it was found that the binding capacity of 125I-labeled IL-2 to lymphocytes was not altered by the presence of CsA. These findings suggest that the prevention of helper cells from producing SHF, rather than the inhibition of the response of effector cells to SHF, is a possible explanation for the immunosuppression mediated by CsA.     

An allospecific murine T helper clone which can help both T and B cell responses in vitro and in vivo

Both B lymphocytes and cytotoxic T lymphocytes respond to signals from the T helper (Th) compartment, and such signals are mediated by a number of biochemically distinct factors. This raises the question whether help for B cells and T cells is a function of one or several different kinds of Th cell. Here we describe an in vitro and in vivo study of this problem, using a Th clone, designated MTH-1. The clone carries the cell surface markers Thy-1 and L3T4a, but lacks Lyt-2. It recognizes a minor alloantigen shared by DBA/2, B10.D2 and NZB spleen cells, and such recognition is restricted by H-2Ed. Recognition of antigen in vitro is accompanied by secretion of IL-2. In vivo, both primary and secondary CTL responses to multiple minor alloantigens are enhanced by small numbers (less than or equal to 10(4] of MTH-1 cells. Recognition of alloantigen in a T-depleted B cell population results in the polyclonal activation and maturation of the B cells to secrete immunoglobulin; also, antigen-primed B cells are augmented in their in vivo synthesis of specific antibody to the Thy-1 X 1 alloantigen by around 10(5) MTH-1 cells. Taken together, these results suggest a single Th clone can help both B cells and T cells.     

Generation and characterization of peptide-specific, MHC-restricted cytotoxic T lymphocyte (CTL) and helper T cell lines from unprimed T cells under microculture conditions

We describe a microculture system for the generation of CTL and T helper cells against peptides. Tryptic digest and cyanogen bromide fragments of chicken ovalbumin and synthetic peptides of ovalbumin (323-339) and influenza virus (NP 365-380) were used to generate CTL and T helper lines from unprimed T cells. These lines were both peptide-specific and MHC-restricted. The relative ease of generating peptide-specific, MHC-restricted CTL and helper T cell lines with as few as 10(6) unprimed lymphocytes can be an efficient method of detecting potential immunogenic determinants of an antigen.     

Murine cytomegalovirus independently inhibits priming of helper and cytotoxic T lymphocytes.

Murine cytomegalovirus (MCMV) inhibits antigen-specific cytotoxic T lymphocyte (CTL) priming in vivo (Campbell et al., 1989). To address the mechanism of this immune suppression, two possibilities were considered: (1) MCMV directly interferes with in vivo priming of CTL precursors (CTLp), or (2) MCMV suppresses T helper cell functions necessary for CTL priming. We therefore quantitated T helper cell function in MCMV-infected, SV40-immune mice and assessed dependency of SV40-specific CTLp priming on T helper cell activity. MCMV infection of H-2b mice significantly suppressed the frequency of IL-2 producing T helper cells generated in SV40-immune mice. This suppression was not due to alterations in the number or percentage of CD4 lymphocytes. The helper cell deficiency correlated with suppressed SV40-specific CTL activity. However, CTLp priming in vivo was found to be independent of CD4 T helper cells and IL-2. Therefore, the suppressive effects of MCMV on helper and cytotoxic T cell functions are independent, implying that MCMV directly inhibits an event in lymphocyte priming common to both helper and cytotoxic T cells.     

Immunoregulatory roles of interleukin-13 in cattle.

Interleukin-13 (IL-13) is produced predominantly by helper T lymphocytes of the Th2 phenotype and mediates its effects on several immune cells, including B lymphocytes and macrophages, stimulating their proliferation, differentiation, and effector functions. IL-13 activates human B cells but has no detectable activity on murine B lymphocytes, suggesting that the activity of IL-13 varies among species. Our studies show that IL-13 enhances proliferation and differentiation of bovine B cells and upregulates cell surface major histocompatibility complex (MHC) class II expression. We examined mRNA expression of the putative signaling component of the bovine IL-13Ralpha1 homolog in several peripheral blood populations. After stimulation with calcium ionophore and phorbol ester, IL-13Ralpha1 mRNA levels appeared to be downmodulated in T cells, upregulated in macrophages and B cells, and unchanged in neutrophils. Together, these studies begin to provide insight into the relative importance of IL-13 in immunoregulation in cattle.     

A role for IL-5 in the induction of cytotoxic T lymphocytes in vivo

IL-5 is generally regarded as a Th2 cytokine involved in eosinophil maturation and function and in B cell growth and antibody production, but without any well-established effects on T cells. Early reports suggested that IL-5 could stimulate the production of cytotoxic T lymphocytes (CTL) in vitro, but no evidence has been obtained to date for such a role in studies with IL-5-deficient (IL-5-/-) mice. Here we demonstrate that when oxidized mannan MUC1 fusion protein (M-FP) is used as an antigen in mice, IL-5 is required for the optimal generation of the CTL response. IL-5 was as effective as IL-2 for the induction of CTL from spleen cells in vitro and both CD4+ and CD8+ T cells from M-FP-immunized animals could be shown to secrete IL-5 in culture. In IL-5-/- mice, CTLp frequency was greatly diminished resulting in the inability to reject MUC1- tumors. Clearly, IL-5 is produced by functional T cells, especially the Tc1 type, after M-FP immunization and is required for an optimal CTL response to this antigen.     

Effect of Subhypnotic Doses of Dexmedetomidine on Antitumor Immunity in Mice

Dexmedetomidine, a selective α2 adrenergic receptor agonist, is a drug often used for sedation. Despite the high prevalence of sedating patients with tumors in intensive care settings, little is known about the effect of sedative drugs on tumor growth. We studied the effect of dexmedetomidine on antitumor immunity in mice. Subhypnotic doses of dexmedetomidine decreased interleukin (IL)-12 production from thioglycollate-induced macrophages. The treatment also decreased the ratio of the helper T lymphocytes subsets, Th1 to Th2 (Th1/Th2), in the spleen. Following subcutaneous inoculation of EL4 T-cell lymphoma cells, dexmedetomidine further decreased the splenic Th1/Th2 ratio and activity of EL4-specific cytotoxic T lymphocytes (CTLs). Finally, treatment with dexmedetomidine accelerated EL4 growth in mice. These data show that treatment of mice with subhypnotic doses of dexmedetomidine downregulates antitumor immunity, possibly through the decreased production of IL-12 from antigen presenting cells, resulting in a Th2 shift and decreased CTL activity against EL4 in mice.     

Induction of B cell responsiveness to growth factors by Epstein-Barr virus conversion: comparison of endogenous factors and interleukin-1.

Immortalized B lymphocytes produce a factor(s) that stimulates growth of B cell lines carrying Epstein-Barr virus (EBV). Stimulatory supernatants derived from B cells also exhibit interleukin-1 (IL-1) activity in costimulator assays with the D10.G4.1 helper T cell line. Experiments with purified macrophage-derived IL-1 and recombinant IL-1 beta demonstrate that IL-1 stimulates proliferation of the cell lines that respond to the factors from B lymphocyte lines. One B cell line, Ramos, an EBV-Burkitt's lymphoma, contrasts with other B cell lines in that it is refractory to the growth enhancing effects of B cell conditioned medium and macrophage-derived IL-1. When EBV was introduced into Ramos cells, growth was enhanced by the factor(s) in B cell conditioned medium (six out of seven lines); growth of EBV-converted Ramos lines (six out of seven lines) also was enhanced by IL-1. These findings demonstrate that infection of a non-responsive transformed B lymphocyte by EBV induces cellular responsiveness to factor-mediated growth stimulation.     

Early Changes in Quiescent B Cell Physiology Subsequent to Cognate and Bystander Interaction with Helper T Cells

We have assessed early changes in quiescent B cells following cognate and bystander interaction with cloned helper T cells. Variables monitored include Ia expression, blastogenesis, G0 to G1 transition, and progression through cycle. We have also assessed the antigen specificity, Ia restriction, and dependence on membrane immunoglobulin crosslinking of both generation and delivery of effectors that mediate B cell responses. The results demonstrate that antigen presentation by quiescent B cells to T cells resulting in the generation of effectors that activate B cells is Ia-restricted and dependent on antigen and an (mIgM and mIgD) crosslinking signal. However, once generated, T cell effector functions act independently of Ia haplotype to promote Ia expression, blastogenesis, and G0 to G1 transition by most small B cells. Although these responses can be mediated by T cell supernatants, further progression of B cells through S, G2 and M is only efficient when Th cells are present in cultures. Thus, results suggest that one or more Ia unrestricted, labile and/or cell-associated factors, not active in most conventional T cell supernatants, are necessary to stimulate proliferation of small B cells.     

A vaccine conjugate of 'ISCAR' immunocarrier and peptide epitopes of the E7 cervical cancer-associated protein of human papillomavirus type 16 elicits specific Th1- and Th2-type responses in immunized mice in the absence of oil-based adjuvants.

TraT protein, known as ISCAR (= Immunostimulatory Carrier), is one of a family of integral membrane proteins (Imps) of Escherichia coli representing powerful carrier molecules which when injected into experimental animals generate substantial antibody and T proliferative responses to molecules conjugated to it. We extend these findings to show that ISCAR functions to stimulate Th1- and Th2-type responses, including specific cytotoxic T cells and tumour protection. We report here that by conjugating to ISCAR a 19mer peptide containing linear B epitopes, a T helper (Th) epitope, and a H-2b-restricted T cytotoxic (CTL) epitope of E7 protein of human papillomavirus type 16 (HPV16), and immunizing C57B1/6 (H-2b) mice, we elicited (i) specific IgG2a and IgG1 antibodies; (ii) IL-2 and IL-4 production by specifically recalled lymph node cells in vitro; (iii) cytotoxic T lymphocytes which specifically killed both E7 peptide-pulsed, and whole E7 gene-transfected tumour target cells; and (iv) in vivo protection against an E7 gene-transfected tumour cell inoculum. These findings have implications for the design of vaccines to stimulate immune responses to endogenously processed target antigens (e.g. tumour-associated antigens) without the unwanted side effects of oil-based adjuvants. In addition they support the case for a E7-targeted therapeutic vaccine for HPV-associated human cervical cancer.     

Segregation of effector mechanisms in a tumour-specific CD8+ T-cell clone correlates with CD30 expression

In this study we have analyzed CD30-antigen expression in three melanoma-directed cytotoxic T lymphocyte (CTL) clones with a T helper 0 (Th0)-like cytokine secretion profile (i.e. interleukin (IL)-4, IL-5, and interferon (IFN)-gamma). We show that all CTL clones expressed high levels of CD30 upon contact with the autologous tumour cells. One CTL clone, termed A2 with a monoclonal feature was selected for further analyses and found its CD30 expression dependent on the presence of IL-4. Functionally, a CD30-expressing A2 CTL was capable of producing higher amounts of IFN-gamma (up to 1.5-fold) and IL-4 (up to two-fold) than its CD30- counterpart. Furthermore, CD30-positive A2 CTL displayed an at least three-fold greater proliferative response to the tumour cell stimulation, contrasting with CD30- CTL. However, the antitumour cytotoxic activity of A2 CTL was not modulated by the CD30 expression. These results suggest that CD30 antigen can be inducible on a subset of tumour-directed CD8+ CTL, and that this subset of cells may have profound effector functions, such as cytokine secretion, proliferation, and cytotoxicity.     

Immunoregulation by mouse T cell clones. III. Cloned H-Y-specific cytotoxic T cells secrete a soluble mediator(s) that inhibits cytotoxic responses by acting on both Lyt-2- and L3T4- lymphocytes

In this study we report that cloned Thy-1+, L3T4-, Lyt-1-, Lyt-2+, H-Y-specific and H-2Db-restricted cytotoxic T cell lines (CTLL) when induced by lectin or antigen secrete a soluble mediator(s) (SF) that inhibits proliferation and generation of cytotoxic lymphocytes (CTL) in mixed lymphocyte cultures (MLC). The biological activity was separable by gel filtration and appeared as a broad peak in the molecular mass range between 10 000 and 50 000 kDa. It was found that the suppressive activity released by CTLL neither strictly correlates with their cytotoxic potential nor with their ability to produce immune interferon or lymphotoxin. SF was shown to elicit its activity in an antigen-nonspecific manner in that it suppressed the maturation of T lymphocytes responding to both, the appropriate H-Y antigen as well as to unrelated H-2d alloantigens or to the hapten 2,4,6-trinitrophenyl (TNP). The effect of SF on CTL responses was most pronounced in early phases of primary or secondary MLC. When analyzed for its inhibitory activity on precursor cells in populations selected for either Lyt-2- or L3T4- lymphocytes, it was found that SF interfered with the maturation of both subsets. The inhibition of CTL responses elicited by SF could not be reversed by the addition of exogenous interleukin 2. The finding that SF also inhibited the proliferation of some but not all antigen-dependent cloned T cells with helper or cytotoxic potential provides evidence that the factor also may regulate effector lymphocytes. In addition, the results support the assumption that SF exerts its effect directly on the responder rather than the stimulator population, and demonstrate that the development of CTL from their precursor cells is controlled at least in part by the cytotoxic effector cells themselves via a soluble factor(s) that interferes with distinct stages of T cell maturation. These findings again emphasize the expression of multiple functions by CTL and indicate their possible role during the course of an immune response by their capability to eliminate target cells and to secrete a soluble product(s) that mediates feedback control.     

Role of T cells in the B-cell response: glutaraldehyde-fixed T-helper hybridoma cells synergize with the lymphokine IL-4 to induce B-cell activation and proliferation.

Antigen-unselected helper T-cell hybridomas (Th) which activate normal resting B cells to RNA synthesis and proliferation in the presence of concanavalin A (Con A) have been developed. The response is completely Th cell dependent, and not restricted by the haplotype of the B-cell major histocompatibility complex (MHC). Culture supernatants from the Con A-stimulated Th hybridomas contain interleukin-4 (IL-4) and IL-2, but undetectable level of IL-5. The supernatant alone, however, does not induce B-cell activation or proliferation. Although the Con A-mediated Th cell-dependent B-cell response occurs in an MHC-unrestricted manner, the response of resting B cells can be blocked by monoclonal Ia antibody specific for the surface class II molecules of the responding B cell. The response is also blocked by monoclonal antibody to L3T4. Significant activation and proliferation of resting B cells can also be triggered by glutaraldehyde-fixed Th hybridomas and Con A when exogenous IL-4 is added. The stimulation with fixed Th hybridomas plus IL-4 can be inhibited by monoclonal anti-L3T4 or anti-Ia. These results suggest that maximal B-cell activation requires a direct helper T cell-B cell interaction which depends on availability of Ia on the B cell and L3T4 on the T cell, even when Con A overcomes the requirement for MHC-restricted T-cell recognition. We suggest that this signal, in conjunction with T-cell produced lymphokine IL-4, is responsible for the activation and subsequent proliferation of the B cells which occurs following interaction with T cells.     

Mycobacteria induce CD4+ T cells that are cytotoxic and display Th1-like cytokine secretion profile: Heterogeneity in cytotoxic activity and cytokine secretion levels

Protective immunity against mycobacteria is dependent on antigen-specific T cells. Current evidence suggests that not only helper T cells that activate infected macrophages but also cytotoxic T cells (CTL) that lyse infected macrophages are involved in protection. Mycobacterium-specific CD4⁺ CTL are readily detectable among primary peripheral T cells but what proportion of CD4⁺ T cells display cytotoxic activity is not known. Whether the cytotoxic CD4⁺ T cells are identical to or distinct from those that produce interferon (IFN)-γ is also unknown. In addition, studies on CTL in mycobacterial infections have focused primarily on selected antigens like hsp65 but have not analyzed systematically whether other mycobacterial antigens can activate CTL as well. These issues are relevant not only to a further understanding of protective immunity and immunopathology but also may have implications for the design of effective vaccines. To start addressing these issues, we have studied a large panel of CD4⁺ T cell clones specific for a broad range of mycobacterial antigens, and analyzed their ability to lyse mycobacterium-pulsed target cells and to release IFN-γ and interleukin (IL)-4. Our results show that the vast majority of CD4⁺ T cell clones are able to lyse mycobacterial antigen-pulsed target cells, and that those CTL can be triggered by a wide variety of mycobacterial antigens. CD4+ CTL released high levels of IFN-γ, but low or nondetectable levels of IL-4. In contrast, control tetanus toxoid-specific T cell clones or lines displayed poor or weak cytotoxic activity and released high levels of IL-4. The antimycobacterial clones appeared to be heterogeneous in their levels of cytotoxic activity and IFN-γ release. Interestingly one T cell clone was able to lyse only mycobacterium-pulsed macrophages but not B cells suggesting possible selectivity in target cell recognition for some CTL. These in vitro data have to be interpreted with some caution. Nevertheless they confirm and significantly extend previous observations and suggest that mycobacteria preferentially induce CD4⁺ T helper type 1 (Th1)-like cells that display cytotoxic activity, and release high levels of IFN-γ but no or little IL-4. The induction of such Th1 like cells is specific for mycobacteria since tetanus toxoid induced T cells that were poorly or not cytolytic and secreted high levels of IL-4.     

Effect of subhypnotic doses of dexmedetomidine on antitumor immunity in mice

Dexmedetomidine, a selective alpha2 adrenergic receptor agonist, is a drug often used for sedation. Despite the high prevalence of sedating patients with tumors in intensive care settings, little is known about the effect of sedative drugs on tumor growth. We studied the effect of dexmedetomidine on antitumor immunity in mice. Subhypnotic doses of dexmedetomidine decreased interleukin (IL)-12 production from thioglycollate-induced macrophages. The treatment also decreased the ratio of the helper T lymphocytes subsets, Th1 to Th2 (Th1/Th2), in the spleen. Following subcutaneous inoculation of EL4 T-cell lymphoma cells, dexmedetomidine further decreased the splenic Th1/Th2 ratio and activity of EL4-specific cytotoxic T lymphocytes (CTLs). Finally, treatment with dexmedetomidine accelerated EL4 growth in mice. These data show that treatment of mice with subhypnotic doses of dexmedetomidine down-regulates antitumor immunity, possibly through the decreased production of IL-12 from antigen presenting cells, resulting in a Th2 shift and decreased CTL activity against EL4 in mice.