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In this paper we describe the first in vitro response to the liver alloantigen F. The anti-F response serves as a valuable model for autoimmune phenomena since priming appropriate strains of mice (responders) with allogeneic but not syngeneic type F leads to autoantibody production. The in vitro system is based on the proliferation of T cells, from mice primed in vivo with F, when coincubated with splenic adherent cells (SAC) prepulsed with F in vivo. The system displays two important correlates of the in vivo antibody response to F:1.T cells from mice primed with syngeneic F do not proliferate when incubated with SAC prepulsed with syngeneic F and 2. Mice that do not make antibody responses to allo F in vivo (DBA/2) do not show in vitro proliferative responses. These findings indicate that the proliferative assay is a good in vitro model for the F response. [ABSTRACT FROM AUTHOR]

The number and function of human T cells in the periphery are regulated by homeostatic signals received from antigen-presenting cells (APCs) and the common gamma chain (γc) cytokines interleukin (IL)-7 and IL-15. We found that, in the absence of introduced antigen, blood monocytes or myeloid dendritic cells (MDCs) in the presence of IL-7 and IL-15 (IL-7/IL-15) can regulate CD4+ T memory (Tm) cell numbers by polyclonal cell proliferation. The dynamics of CD4+ Tm cell proliferation, in the presence of IL-7/IL-15, was dependent on contact with MDCs and to a lesser extent on contact with monocytes. IL-7/IL-15 either alone or combined with monocytes or MDCs enhanced the proportion of CD4+ Tm cells with activated and effector phenotype and diminished the helper function of CD4+ Tm cells. These CD4+ Tm cells, preconditioned with IL-7/IL-15 alone or with monocytes or MDCs and IL-7/IL-15, reduced T cell-dependent immunoglobulin M (IgM) and IgG responses. This appeared to be a contact-dependent effect involving a reduction in antibody-producing CD27+ B memory cells, but contact-independent suppression by soluble factors also contributed to the antibody-producing capacity of CD27+ B memory cells. These results indicate that blood monocytes, MDCs and the cytokines IL-7/IL-15 contribute to homeostasis of CD4+ Tm cells by regulating their number, activation state and helper/suppressor (regulatory) function. In healthy individuals, this mode of regulating CD4+ Tm cell homeostasis may provide a basis for the control of autoimmune responses. [ABSTRACT FROM AUTHOR]

To better understand the contribution of T cells to the immunity of pigs to pseudorabies virus (PRV), we examined the lymphoproliferation response to this virus. Depletion studies demonstrated that both CD2+ CD8+ and CD2+ CD4+ cells contributed to lymphoproliferation, but to varying degrees upon stimulation with live and ultraviolet (UV) light-inactivated PRV. Flow cytometric analysis revealed the emergence of both CD2+CD8+ and CD2+CD4+ lymphoblastoid cells. To examine the contribution of specific viral proteins, we prepared immortalized porcine B cells of haplotype d/d that stably expressed a single PRV protein, and used these cells for in vitro stimulation of lymphocytes from PRV-immune miniature pigs of the same haplotype. Cells expressing PRV gB or gC induced proliferation. An immunization/challenge experiment showed that the lymphoproliferation response was stronger upon immunization with the virulent NIA-3 strain than with the attenuated Bartha strain. Upon challenge inoculation, the NIA-3-immunized pigs were almost completely immune, in contrast to the Bartha-immunized pigs. Such poorly protected pigs showed secondary B- and T-cell immune responses upon challenge. In contrast, the better protected NIA-3-immunized pigs did not show a secondary B-cell response. However, they developed a secondary lymphoproliferation response, which was quicker and stronger than in the Bartha-immunized pigs. This dichotomy between secondary B- and T-cell responses indicates that an effective T-cell memory response is able to quickly eliminate challenge virus in immune pigs, so preventing a secondary B-cell response. [ABSTRACT FROM AUTHOR]

In this study, we examined the ability of varying populations of donor cells from B6 mice to induce hyporesponsiveness in T lymphocytes from C3H mice in vitro and in vivo. Small, resting B lymphocytes were inefficient stimulators of T-lymphocyte proliferation compared to splenic mononuclear cells (SMNC) and lipopolysaccharide (LPS)-induced B-cell blasts in vitro (P < 0.05). Pretreatment of SMNC with anti-B7-1 or anti-intracellular adhesion molecule-1 (ICAM-1) monoclonal antibodies (mAb) similarly resulted in inefficient stimulation of T-cell proliferation in vitro (P < 0.05). However, in vivo, only intrahepatic, but not intravenous, injection of donor cells into C3H mice resulted in decreased T-lymphocyte proliferation in response to restimulation by alloantigen. This effect was most pronounced following intrahepatic injection of resting B lymphocytes or SMNC pretreated with anti-ICAM-1 mAb compared to uninjected or intravenously injected mice (P < 0.05). The hyporesponsiveness was associated with an increased production of interleukin-4 (IL-4) by the responder T lymphocytes and correlated with enhanced skin allograft survival. These data demonstrate that intrahepatic injection of donor-derived cells induces T-lymphocyte hyporesponsiveness. The mechanism appears to be modulated by an ICAM-1-mediated signal resulting in expansion of an IL-4-producing T-lymphocyte population. [ABSTRACT FROM AUTHOR]

We have investigated several aspects of γδ T cells in sheep. γδT cells of sheep express a unique transmembrane protein termed T19 but lack the expression of particular cell-surface molecules such as CD2, CD4 and CD8 which are typically associated with αβ T cells. The majority of γδ T cells isolated from animals of all ages examined lacked the expression of CD45RA. A faster rate of activation by γδ T cells compared to either CD4 or CDS T cells was seen in the time-course of IL-2 receptor a chain (CD25) cell-surface expression. All γδ T cells expressed the CD25 protein within 8 hr of activation whereas the majority of CD4 or CD8 T cells did not express CD25 until 24 hr post-concanavalin A (Con A) stimulation. This difference in the rate of expression of activation molecules was not restricted to CD25, as a similar trend was seen with cell-surface expression of major histacompatibility complex (MHC) class II molecules. We have used the distinct phenotypic profile of ovine γδ T cells to purify these cells by positive selection via the T cells molecule to assess their in vitro proliferative response to various antigens. Routinely, cell populations comprising more than 93% γδ T cells with yields of approximately 55% were obtained. Purified γδ T cells were capable of responding to Mycobacterium tuberculosis antigen in a primary and secondary in vitro proliferation assay and to ovalbumin in a secondary response. Ovine γδ T cells showed little, if any, proliferative response to allogeneic stimulator cells. [ABSTRACT FROM AUTHOR]

The T-cell repertoire to a prominent immunogen of Mycobacterium tuberculosis has been investigated on the assumption that differences in epitope specificity could influence the protective and pathogenic host reactions. Proliferative responses of lymph node and spleen cells to overlapping peptides, spanning the entire sequence of the 16,000 MW protein antigen were analysed in C57BL/10 and B10.BR mice. Following footpad priming and in vitro challenge with homologous peptide, 12 out of the 14 peptides tested were found to be immunogenic. However, only two peptides of residues 31-40 and 71-91 stimulated strong proliferative responses of T cells from mice which had been presensitized with either killed or live M. tuberculosis organisms; another three peptides were only weakly stimulatory. These epitopes have been immunodominant in both H-2b and H-2k mouse strains, indicating the genetically permissive nature of their recognition. Furthermore, both major immunodominant epitopes were found to be species specific for the M. tuberculosis complex and therefore potentially suitable for the early diagnosis of tuberculous infection. [ABSTRACT FROM AUTHOR]

After T-cell activation, a switch of leucocyte common antigen (LCA) expression occurs which is detected by the disappearance of CD45R and the appearance of UCHL1 positivity. Upon activation of CD45R+ T cells by phytohaemagglutinin (PHA), the IL-2 receptor (IL-2R) develops at 15 hr and blast cells enter the S phase at 20 hr, while remaining UCHL1-. At 40 hr, mitotic cells continue to express CD45R determinants, but weak UCHL1 reactivity is now detectable on 15-20% of these cells. By inhibiting the separation of daughter cells with cytochalasin D, it can be demonstrated that the UCHL1 antigen is newly synthesized in the Golgi apparatus before insertion into the membrane after the first mitosis. At 65 hr after activation, 80% of proliferating cells are UCHL1+. T cells undergo a similar development during allogeneic activation, and specific alloresponsive cells, tested in secondary mixed lymphocyte reaction (MLR), become greatly enriched among the proliferating UCHL1+ cells. Conversely, after a primary MLR, residual CD45R+ T cells are unresponsive to the original stimulus but can still proliferate in response to unrelated alloantigens. These results clarify both the time-course of the acquisition of UCHL1 antigen during the proliferative cycle of activated T cells, and indicate the shift of antigen responsive T-cell populations from the CE45R+ to the UCHL1+ pool. [ABSTRACT FROM AUTHOR]

In several conditions in the skin, characterized by T-cell infiltration, keratinocytes are induced to synthesize and express class II transplantation antigens. The biological significance of this induced expression is still not understood. In this study, class II antigens were induced on rat ear keratinocytes by local intradermal injections into the ear of rat recombinant interferon-gamma (IFN-γ). Epidermal cell suspensions prepared from these ears contained more than 50% class II-expressing cells, as judged by immunocytochemistry, compared with less than 5% in untreated epidermis. When comparing the capacity of these to different epidermal cell populations to stimulate a syngeneic PPD specific T-helper cell line, it was found that IFN-γ-exposed epidermal cells induced a lower T-cell response to PPD than did normal epidermal cells. This discrepancy could not be explained by either an infiltration of inflammatory cells into the epidermis of IFN-γ-treated ears or by a difference in interleukin-1 production as determined in culture supernatants. The addition of indomethacin to cultures with IFN-γexposed epidermal cells restored the T-cell response to PPD to that of normal epidermal cells, suggesting an inhibitory effect of prostaglandins. Our data indicate that epidermal cells exposed to IFN-γ in vivo can suppress an antigen-specific T-cell proliferation. [ABSTRACT FROM AUTHOR]

Three stimuli were used to compare the signals necessary for interleukin (IL-2) receptor expression and IL-2 production: triggering of the T-cell antigen receptor/CD3 complex (Ti/CD3) by CD3 antibodies, activation of protein kinase C (Pi(C) by phorbol esters, and elevation of intracellular calcium levels by calcium ionophore. The salient observations were that IL2 responsiveness, which reflects IL-2 receptor expression, and Tall proliferation which requires both IL-2 production and IL-2 receptor expression, are not coordinately regulated. Firstly, a low threshold of CD3 activation or a brief (1 hr) exposure of I cells to maximal CD3 stimulation is sufficient to induce IL-2 responsiveness, but higher levels of activation for a prolonged period are necessary to ensure a Tall proliferative response. Secondly, in response to optimal 1-cell stimulation there is a short (2-4 day) period of Tall proliferation followed by a prolonged phase of IL-2 responsiveness (10-14 days). Differences in the kinetics and signalling requirements for IL-2 receptor expression and IL-2 production, regulated at the level of mRNA expression, provide a molecular basis for these observations. A major difference between induction of IL-2 production and IL-2 receptor expression is that the dual signals of calcium and PKC are necessary for IL-2 production, but a sole stimulus of PKC is sufficient for IL-2 receptor expression. Also, a low level stimulation of PKC wilt induce IL-2 receptor expression but higher levels of PKC stimulation are required for IL-2 production. As a consequence, triggering of a single receptor, namely the Ti/CD3 complex, results in IL-2 responsiveness, but an additional signal that activates P1CC is necessary for IL-2 production. These observations suggest that a Ca2+/PKC dual signal model does not explain completely the signal transduction pathways that regulate T-cell growth. Moreover, precise regulatory mechanisms have evolved to control the homeostasis of the autocrine proliferative response of a Tall population. [ABSTRACT FROM AUTHOR]

Utilizing allotype-specific antibodies to immunoprecipitate RT6.2 from DA.68 rat lymphocyte lysates, we have shown this antigen recently to be composed of two related, non-glycosylated polypeptides with apparent molecular weights (MW) of 24,000 and 26,000 (reducing conditions), which evidently are anchored in the cell membrane by covalent linkage to phosphatidylinositol. The present report shows that RT6. 1 allotype-specific antibodies precipitate a more complex pattern of bands from LEW.6A rat lymphocyte lysates. These consist of an endo-F-resistant RT6.2-like 25/ 27,000 MW doublet (reducing conditions) as well as at least five additional endo-F-sensitive, endo-H- resistant polypeptides of 30,000, 32,000, 33,000, 34,000, 35,000 MW. Endo-F treatment seems to convert the additional higher to the lower molecular weight forms. In contrast to the endo-F-resistant 25/27,000 MW doublet, the higher MW forms of RT6. 1 partly bind to lentil lectin and concanavalin A (Con A). Two-dimensional electrophoretic analyses (NEPHGE/SDS-PAGE) reveal similar patterns of charge heterogeneity of the lower and higher MW forms of RT6. 1. Neuraminidase treatment does not affect the pIs of the lower MW forms but shifts the pIs of the higher MW forms to those of the lower ones. All forms of RT6. 1 evidently employ the covalent linkage to phosphatidylinositol for membrane anchorage. Identical patterns of molecular forms-the doublet for RT6.2, the polymorphic pattern for RT6. 1-are observed upon immunoprecipitation of the alloantigens from the lysates of corresponding series of inbred strains of rats with both allotype-specific antibodies and a polyclonal rabbit serum recognizing a common determinant on both alloantigens. [ABSTRACT FROM AUTHOR]

In this report the antigen and restriction specificity of human T-cell clones induced with Dermatophagoides farinae (D.farinae) and isolated from an atopic individual with perennial rhinitis has been investigated. Of the six clones analysed, four were species specific and two showed cross- reactivity for the closely related Dermatophagoides pteronyssinus ((D. pteronyssinus). Inhibition of antigen-dependent proliferation by murine monoclonal antibodies directed against HLA-D-region gene products revealed that all the clones were restricted by HLA-DR molecules. The restriction specificity was investigated further using a panel of histocompatible and allogeneic-presenting cells. Of the clones tested, one appeared to be DR5 restricted while the remainder showed complex patterns suggesting that DRw52 and DRw53 supertypic specificities may be the restriction elements presenting antigen. [ABSTRACT FROM AUTHOR]

Using cloned human T lymphocytes reactive with a 24 amino acid peptide (p20) of the carboxyl terminus of the HA-1 molecule of influenza haemagglutinin (HA), we have investigated the ability of solid-phase antigen to induce antigen-specific T-cell proliferation. The activation by nitrocellulose- bound virus and p20 was accessory-cell dependent and was not caused by immobilized antigen directly cross-linking the specific receptors. Furthermore, we report that separation of complex antigen mixtures such as influenza virus and HA by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) followed by transfer to a nitrocellulose membrane can be used to allow direct screening of individual polypeptides in T-cell proliferation assays. With this immunoblotting procedure the antigenic site recognized by HA-reactive T cells was confirmed to reside in the HA-i molecule of influenza virus of only the appropriate subtype. The general application of this approach is discussed in the case of infections and autoimmune diseases in which the immune response is predominantly T-cell mediated and where antibody studies may fail to identify key antigenic determinants involved in the activation of T cells. [ABSTRACT FROM AUTHOR]

When the serum content of tissue culture medium is reduced from 10% to 1%, the capacity of T cells to proliferate in response to antigen within that medium is dramatically reduced. Physiological concentrations of platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) are able to partially replace the requirement for serum, in that they are able to increase antigen-driven T-cell proliferation at a serum concentration of 1%. Neither growth factor is mitogenic for T cells in the absence of antigen, and neither is able to act synergistically with T-cell growth factor (TCGF) or IL-2) in the absence of antigen. Antigen-presenting cells (APC) pulsed with antigen in the presence of PDGF or EGF are able to stimulate antigen-specific T-cell proliferation to a greater extent than antigen-presenting cells pulsed in the absence of exogenous PDGF or EGF. Both growth factors increase the expression of MHC Class Il antigens on antigen-presenting cells. [ABSTRACT FROM AUTHOR]

Delayed hypersensitivity (DH) response in mice induced by subcutaneous (s.c.) injection of optimal dose of sheep red blood cells (SRBC) (108) was accelerated by s.c. injection of the antigen of 10³ or more doses, given 2 or more days earlier. The accelerated response appeared soon after the injection of optimal antigen dose, that is, 1 or 2 days earlier than the response of non-presensitized control. The acceleration was antigen specific. The accelerated response was generally accompanied by an acceleration and/or enhancement of humoral antibody response. Parallel to the acceleration of DH response, the proliferation of regional lymph node cells in the presensitized mice was induced immediately after the following injection of 108 SRBC, 1 day earlier than that of non-presensitized animals. These results suggest that presensitization of mice with the antigen induces DH-related memory cells which proliferate immediately after the following injection and function as effector cells for DH reactions, and that the development of DH-related memory cells occurs in close relation to that of helper thymus-derived (T) cells for antibody production. [ABSTRACT FROM AUTHOR]

The co-operation of spleen cells of carrier- and hapten-carrier-primed mice in antibody formation against the hapten part of complexes was studied in 550 rad whole body irradiated mice. Hapten-carrier complexes were prepared with the 2,4-dinitrophenyl group (DNP) as a hapten and heterologous bovine serum albumin (BSA) and isologous mouse immunoglobulin (MIg) as carriers. Priming of donor mice with carrier alone did not prepare for a secondary (IgG) response in the recipients of hapten-carrier. Priming of donors and challenge of recipients with the same hapten-carrier complex resulted in high IgG responses. Whereas donor and recipient immunization with complexes differing in the carrier did not give a secondary response, addition of cells of donors immunized with the carrier of the complex used for challenge, resulted in a secondary response. This was only possible when at least one of the complexes had an intermediate hapten:carrier ratio, Only an IgM or a low IgG response was obtained if both complexes had a high hapten:carrier ratio. Three determinants, namely hapten and carrier groups and new antigenic determinant (NAD), are suggested for antibody formation against hapten-protein complexes. In vivo treatment of donor cells with anti-thymocyte serum (ATS) or anti-plasma cell serum (APCS) and complement (C) suggested that: (1) T-cell epitopes are present on the carrier; (2) DNP groups are B-cell epitopes; (3) NAD and possibly DNP are T-cell epitopes; (4) synergism exists in the collaborative antibody response of B cells recognizing DNP, T cells recognizing carrier and T cells recognizing NAD. Mitomycin treatment of donor cells was used to test whether cell division was mandatory. While the B cells were sensitive to mitomycin treatment, no effect of this drug was found on the helper activity of T cells.