Activation of T and B lymphocytes in vitro. I. Regulatory influence of bacterial lipopolysaccharide (LPS) on specific T-cell helper function.

The present studies were undertaken to analyze the nature of the effect of bacterial lipopolysaccharide (LPS) on antibody production in vitro. We have done this by making comparative studies of the effects of LPS on in vitro primary and secondary antibody responses to soluble hapten-protein conjugates and to particulate and soluble sheep erythrocyte antigens. The results obtained demonstrate that the biological action of LPS in vitro may be predominantly manifested on the function of B lymphocytes or T lymphocytes depending on the conditions employed. In the absence of antigen, LPS appears to act primarily on B lymphocytes. In the presence of antigen, however, the data presented here show that LPS significantly influences specific helper T-cell function and it is this latter influence that is predominantly responsible for the adjuvant effects of LPS on antigen-specific antibody responses.

Activation of T and B lymphocytes in vitro. II. Biological and biochemical properties of an allogeneic effect factor (AEF) active in triggering specific B lymphocytes.

The studies presented herein have focused on the biological and biochemical properties of a nonspecific mediator produced by populations of activated T lymphocytes during short-term in vitro reactions with foreign alloantigens. We have analyzed the activity of the unseparated and of chromatographically separated fractions of the supernatants from such cultures on the in vitro responses of mouse lymphocytes to soluble and macrophage-bound DNP-carrier conjugates and also to particulate heterologous erythrocytes. The results demonstrate that a highly active protein moiety, termed allogeneic effect factors (AEF), in the mol wt range of 30,000-40,000, is capable of acting directly on B lymphocytes, in the presence of antigen, probably to effect triggering and subsequent differentiation and proliferation to antibody-forming cells in vitro. The active molecule, although not manifesting specificity for antigen, does exhibit some strain-specific properties suggesting a possible relationship to cell surface antigens or other gene products coded in the major histocompatibility gene complex.

Activation of T and B lymphocytes in vitro. III. Presence of Ia determinants on allogeneic effect factor.

Observations from our own laboratories, as well as those of others, have demonstrated the critical role of histocompatibility gene products in governing the cell-cell interactions concerned with development and regulation of immune responses in several species (8-12). In mice, the relevant genes concerned have been shown to be located in the K end of the H-2 complex, i.e. in the K and/or I See PDF for Structure regions (13, 14). These discoveries have placed histocompatibility gene products on a more complex level of biologic function than was heretofore generally considered (15). Thus, the hypothesis was made from these observations that genes in the H-2 complex coded for products involved in the development of effective cell-cell interactions in the immune response (8, 9, 15). The recent identification of cell surface macromolecules on lymphocytes and macrophages, that may be distinct from immune response gene products but are likewise coded for by genes in the I region, has provided a group of suitable candidate molecules for such a role (2). In our initial studies on the biological and biochemical characteristics of AEF, we were impressed by the apparent preferential activity of the highly purified AEF preparations on B lymphocytes syngeneic to the activated T-cell population from which the AEF was obtained (1). Since a prediction of the aforementioned hypothesis is, of course, that the active molecules involved in regulatory immunocompetent cell interactions are gene products of the H-2 complex, and, accordingly, should be reactive with antisera directed against components of this complex, we were prompted to perform the appropriate analyses on our preparation of AEF. The experiments presented here demonstrate that the enhancing activity of AEF obtained from T cells of the H-2(d) haplotype can be specifically removed by immunoadsorbents prepared from antisera reactive with la molecules of the H-2(d) allele. Identical results were obtained in experiments with both direct and indirect absorption procedures. The possibility that the reaction of AEF with the B10.A anti-B10 (anti-Ia.8) antiserum resulted in release of some components that were in turn toxic to the cultured cells, has been made unlikely in these studies by the use of a direct adsorption method utilizing an immunoadsorbent prepared from thoroughly washed glutaraldehyde-linked antibodies. The results obtained with the (B6A)F(1) anti-B10.D2 antiserum deserve some comment. This antiserum contains antibodies directed predominantly against the H-2K region specificity, H-2.31, but may also be reactive with recently determined Ia(d) specificities (5). The capacity of this antiserum to directly absorb approximately 45% of the AEF activity at the lowest concentration of AEF employed (Fig. 1) could be interpreted to indicate the reactivity of AEF with anti-H-2K antibodies. However, the data presented here are also consistent with the interpretation that partial adsorption by the direct immunoadsorbent and lack of adsorption by the indirect method (in which only a high concentration of AEF was incubated with the alloantisera) reflect reactivity of AEF with anti-Ia(d) antibodies present in this antiserum. We conclude, therefore, that the biologically active enhancing moieties of AEF bear Ia determinants and therefore are most probably gene products of the I region of the H-2 gene complex. Recent data from other investigators have shown that an antigen-specific T-cell product could be specifically adsorbed by immunoadsorbents prepared from antisera directed against the K end of H-2 (16). Since the latter antisera may contain antibodies reactive with specificities of both K and I regions, it is possible that the use of selective anti-Ia sera may yield results consistent with those presented here. Taken collectively, these observations indicate that I-region gene products may be intimately involved in the mechanism of cell-cell interactions and responsible for the regulation of immune responses.

Studies on the interrelation of resistance and immunity in a mouse model system of herpes-simplex type 2 infection.

Intraperitoneal (i.p.) vaccination of mice with attenuated herpes simplex virus type 2 (HSV 2) induced solid protection to i.p. infection with pathogenic virus within two days. Protection was non-virus-specific until day four after sensitization but increased in specificity thereafter. Normal mice could be protected by adoptively transferred spleen cells, serum, and peritoneal fluid from donors vaccinated seven days before. Virus-specific effector cells induced in the spleen by in vivo i.p. sensitization with either live, pathogenic, or attenuated virus and tested in a cytotoxicity assay were exclusively B lymphocytes. No functional B cells, but natural killer (NK) cells, could be detected in the unseparated peritoneal exudate cell (PEC) population. Ability to generate HSV 2 specific antibody responses did not correlate with natural resistance.

Induction of natural killer cells by herpes-simplex virus type 2 in resistant and sensitive inbred mouse strains.

Infection of mice with herpes simplex virus type 2 (HSV 2) stimulated natural killer (NK) cells in a variety of inbred mouse strains including athymic nude mice. Essentially all mouse strains tested exhibited high NK activity on day four after virus inoculation. Assayed 24 hours after infection, SWR/J, AKR/J, SJL/J and C57B1/10J mice were low or negative for these non-virus-specific cytotoxic responses. Whereas the first two mouse strains were most sensitive to the lethal effects of HSV 2, the latter two were highly resistant. Three lines with intermediate susceptibility and three highly resistant strains were all efficient with regard to early NK-cell.

In-vivo modulation of macrophage functions by herpes simplex virus type 2 in resistant and sensitive inbred mouse strains.

Intra-peritoneal (i.p.) infection of mice with herpes simplex virus type 2 (HSV 2) attracted macrophages into the peritoneum. Macrophages from moderately and highly HSV 2 resistant mouse strains expressed elevated phagocytosis activity 24 hours after injection. Stimulation of phagocytosis in low resistant strains was generally less effective or absent. This was, in some experiments, due to the fact that macrophages were already highly activated before the experimental infection. I.p. infection also caused HSV replication in the adherent peritoneal exudate cell (PEC) population. The capacity of macrophages supporting HSV 2 replication was low in three of four resistant mouse strains and high in all moderately and highly susceptible and in one of the resistant (SJL) strains when determined 24 hours after infection. Four different F1 hybrids between resistant and susceptible strains exhibited significantly lower yields of virus-producing macrophages than the HSV-sensitive parent. One hybrid between two HSV-susceptible lines restricted virus replication in the PEC populations better than both parental strains.

In vivo activation of murine natural killer cell functions by human recombinant DNA interleukin 2.

Intraperitoneal (i.p.) injections of purified human recombinant DNA (rDNA)-interleukin 2 (IL 2) resulted in in vivo activation of local natural killer (NK) cell activities in wild-type and congenitally athymic mice. NK cells were identified by short-term cytotoxicity assays against YAC tumor targets and by cell-surface phenotyping. The magnitude of the cytolytic responses was dependent on the IL 2 dose (greater than or equal to 0.1 microgram per injection) and the time period of treatment (the maximum response was on days 3 to 4 after daily treatment). In vivo application of antisera against the murine NK marker asialo GM1 (asGM1) and against interferon-alpha/beta and -gamma (IFN) significantly inhibited NK cell activation. Limiting dilution analysis revealed high frequencies (up to 1 in 1.8 X 10(3)) of in vitro IL 2 reactive mononuclear cells among the peritoneal exudate cells (PEC) of normal mice. rDNA-IL 2 activated non-adherent PEC to proliferate. The majority of these cultures also displayed cytotoxicity against YAC targets. No exogenous IFN was required for either response. Endogenous IFN production did not appear to play an important role for induction of cytotoxicity in this system either. Only a minority of cultures produced measurable levels of IFN without showing excessive cytotoxic activity. In vivo IL 2 treatment resulted in a rapid increase of the total numbers and frequencies of the IL 2 reactive PEC. Hence, IL 2 alone was apparently sufficient for in vitro activation of NK-like activities, whereas IFN-induction by IL 2 was required for in vivo elicitation of similar responses in perhaps the same cell populations.

Induction of IgE and IgG1 in human B cell cultures with staphylococcal superantigens: role of helper T cell interaction, resistance to interferon-gamma.

Non-antigen-specific activation of human B lymphocytes for IgE production in vitro requires the presence of interleukin 4 and non-cognate physical interaction with T cells. The latter can be replaced by antibodies directed against the B cells' CD40 structure. Antigen-specific induction of immunoglobulin responses, including IgE, is difficult in human lymphocyte cultures. Thus, we developed a model system which might resemble physiological B lymphocyte stimulation by antigen. Co-cultures of purified tonsillar B cells from normal donors with non-HLA matched T helper clones obtained from the skin of atopic dermatitis patients produced significant levels of IgE and IgG1 after stimulation with appropriate types of staphylococcal exotoxins, provided that IL-4 was also induced in the T cells. Such responses were further enhanced by addition of low doses of anti-CD40 antibodies. Concentrations of anti-CD40, optimal for stimulation of B cells in the absence of T helper lymphocytes, were less effective in this regard and even inhibitory in some experiments. Most powerful immunoglobulin induction was observed when the cultures were spiked with low amounts of IL-4 and anti-CD40 which did not elicit substantial immunoglobulin production in the absence of the staphylococcal exotoxins. Induction of IL-2 in T/B cell cultures by superantigens without production of appreciable quantities of IL-4 provoked considerable IgG1 titer but no IgE. High amounts of interferon-gamma generated by the T cells in vitro in the presence of superantigens did not appear to interfere with immunoglobulin induction. Addition of recombinant interferon at the beginning of the culture period at doses which effectively suppressed IL-4 plus anti-CD40 induced immunoglobulin responses did not inhibit T helper and superantigen dependent B cell activation. Superantigen mediated B cell stimulation for immunoglobulin production was dependent on cell-cell contact. The experimental results presented suggest that this cellular interaction did not necessarily involve T-B cell bridging by superantigens.

Function, target-cell preference and cell-surface characteristics of herpes-simplex virus type-2-induced non-antigen-specific killer cells.

Wild-type and congenitally athymic nude mice injected with herpes-simplex virus type 2 (HSV 2) responded with a local outburst of non-antigen-specific killer cells masking any virus-specific response. Cytolytic activity could be assayed on mouse-tumor cell lines and on syngeneic or allogeneic non-transformed cells from various sources. Some of the tumor cell lines and proteose-peptone-induced peritoneal exudate cells were lysed more efficiently after infection with either HSV 2, vaccinia or influenza A virus. Preference for virus-infected target cells was already expressed 24 hours after HSV-2 injection. Killing activity was not H-2-restricted, not complement- or immunoglobulin-dependent and did not involve Fc receptors. The cytotoxic cells were non-adherent and could be shown to express Thy1, Quat4, and Quat4 cell-surface antigens. They lacked immunoglobulin and Lyt1: Lyt2,3 determinants. The functional and serological characteristics identify the HSV-2-induced cytolytic cells as natural killer (NK) cells. The potential importance of this cell population for natural resistance will be discussed.

Modulation by cyclosporin A of murine natural resistance against herpes simplex virus infection. I. Interference with the susceptibility to herpes simplex virus infection.

Adult BALB/c mice which are medium-high resistant against intraperitoneal (i.p.) infection with herpes simplex virus type 2 (HSV-2) manifested a drastic increase in susceptibility to the virus when treated locally with cyclosporin A (CyA) during infection. Oral application of the drug had no effect on the natural resistance status. Mice appeared normal 2 weeks after CyA treatment with regard to their ability to resist i.p. infections. CyA did not interfere with established specific immune protection nor with the induction in immune responses to HSV-2.

Modulation by cyclosporin A of murine natural resistance against herpes simplex virus infection. II. Influence on the HSV-induced natural killer cell responses, macrophage activities and interferon levels.

Cyclosporin A (CyA) interfered locally at the site of injection with several resistance functions which are of potential importance in experimental herpes simplex virus (HSV) infections of mice. HSV-induced stimulation of macrophage phagocytosis was reduced by CyA when the mice were infected 5 days before the assay. The in vitro replication of the virus in macrophages, however, was enhanced. Natural killer (NK) cell response were severely impaired. To some extent this could be attribute to the induction of suppressive macrophages by the drug treatment. Interferon levels induced by HSV were not diminished but rather enhanced in some experiments. Inhibitory effects ceased after termination of CyA treatment and could be prevented by presensitization of the mice with attenuated HSV type 2.

Selective induction of immunological tolerance in antiviral T killer cells of inbred mice after treatment with cyclosporin A.

Primary anti-influenza A cytotoxic thymus-derived (T) and bone marrow (B) lymphocyte-dependent responses in inbred mice were used as an in vivo model system to study the effects of the immunosuppressive fungus metabolite cyclosporin A (CyA). Five consecutive daily oral applications of CyA, with the first being given 1 or 2 h before virus inoculation of the animals, caused a complete blockage of induction of anti-influenza T killer cells and a partial reduction of cytotoxic B lymphocyte activities. Adoptive cell transfer experiments revealed that incapability to respond was due neither to humoral factors nor to the generation of suppressor cells. The tolerance state appeared to be specific for influenza A; cytotoxic T lymphocytes against allogeneic cell surface determinants could be stimulated in immunosuppressed mice. CyA treatment abolished virus-specific and cross-reactive anti-influenza killer T cell responses. Suppression was of short duration: less than 1 week for B cell-dependent functions, and between 1 and 2 weeks for T killer cell responses. Animals appeared to be normal with regard to both of these cellular activities for 4 weeks after tolerance induction. Thus, the data indicate that CyA exerted preferential effects on killer T cells. Moreover, evidence was presented that CyA treatment during an ongoing influenza infection did not increase sensitivity to that virus. Mice with no measurable cytolytic anti-influenza T killer cell activities but significant B cell responses, although partially diminished by the drug, were completely protected against the lethal effects of influenza infection.

Phorbol ester, prostaglandin E2, forskolin and okadaic acid differentially modulate interleukin-4-versus interleukin-2-dependent immunoglobulin induction in human cellular models, in contrast to other selected modifiers of cellular activation.

Interleukin 2 (IL2) and 4 (IL4) are the most important mediators for immunoglobulin (Ig) synthesis of human B lymphocytes. There is no obvious difference with regard to Ig isotypes induced by either lymphokine except for IgE: only IL4 induces this allergic antibody type. Monoclonal anti-CD40 antibodies enhance both IL2- and IL4-dependent Ig induction. Searching for drugs which may inhibit induction of IgE but not of rather non-pathogenic immunoglobulins, we selected commercial compounds which are commonly used as probes for transmembrane signalling pathways in other cellular systems. They included modulators of protein kinase C and intracellular calcium, inducers of cAMP, and inhibitors of protein tyrosine kinase, protein serine/threonine phosphatases and phosphodiesterases. The data presented suggest that IL2- and IL4-mediated B cell activation can be differentially modulated. Phorbol ester at non-cell-toxic doses inhibited IL4- but not IL2-dependent Ig induction. Prostaglandin E2 potently enhanced IgE production stimulated with IL4 alone but was inhibitory in the presence of anti-CD40 as a co-stimulatory signal. IgG1 responses elicited with IL2 plus anti-CD40, in contrast, were not affected. All other compounds did not discriminate between IL2- versus IL4-mediated Ig induction.

Induction of cytolytic T- and B-cell responses against influenza virus infections.

Inoculation of mice with live influenza virus results in the induction of cytotoxic thymus-derived (T) lymphocytes and of bone marrow-derived (B) cells producing antiviral antibodies. An assay system was developed to evaluate both types of immune responses on a cellular basis within the same lymphocyte pool with no need to separate out the different effector cell classes. The test system represented a modification of the 51Cr-release assay. T-cell activity was measured exclusively in the absence of active complement using targets that were compatible for determinants encoded by the mouse major histocompatibility gene complex, H-2. H-2-different and even xenogeneic target cells were lysed in the presence of either non-inactivated fetal calf serum or normal rabbit serum as a complement source. Cytotoxicity was mediated in the latter case by direct interaction of B-cell-produced immunoglobulin directed to viral antigens expressed by the target cell and complement. Antibody-dependent cell-mediated cytotoxicity mechanisms did not contribute to cytotoxicity in the test system described. It was demontrated that the cytolytic B-cell responses of one particular strain of mice (BALB/c) against different influenza A viruses were restricted to the immunizing virus on the effector cell level. In another strain of mice (C3H), B cells revealed a broad cross-reactive response resembling that of killer T cells.

Functional cutaneous lymphocyte antigen can be induced in essentially all peripheral blood T lymphocytes.

The cutaneous lymphocyte-associated antigen (CLA) is a skin-homing receptor expressed on a minority of memory-type peripheral blood T (PBT) lymphocytes. Induction of high-level CLA expression in PBT has previously been difficult to accomplish in vitro. Here we report that constitutive CLA expression could be readily induced in virtually all PBT by various polyclonal activators. There was no requirement for accessory cells or addition of other mediators except for IL-2 for maintaining cell survival. Absence of serum in the culture medium was important for optimal induction of CLA. The number of T cells adhering to E-selectin as well as tethering and shear stress resistance under hydrodynamic flow increased in correlation with the level of cell surface CLA expressed. Clonal analysis of CLA induction revealed that in serum-containing medium, which permits the majority of T cells to expand, only a minority of clones did not express CLA. Such T cells could be induced to highly express CLA within 8 days by switching from serum-containing to serum-free medium. This cell-surface phenotype change was closely associated with acquisition of E-selectin ligand activity. Fucosyltransferase VII, which is believed to be important for the generation of the CLA epitope on the P-selectin glycoprotein ligand-1 (PSGL-1) backbone, was shown to be significantly increased in CLA-positive versus CLA-negative T cell populations by PCR analysis. Our findings are consistent with the idea that restriction of CLA expression after activation, rather than positive selection of predetermined T cell subpopulations exposed to restrictive stimulatory conditions in unique microenvironments, may be important in vivo.

Activation of T and B lymphocytes in vitro. IV. Regulatory influence on specific T cell functions by a thymus extract factor.

In the presence of a preparation of purified thymus extract factors (TEF), spleen cells from athymic nude mice develop in vitro immune responses to soluble DNP-protein conjugates, to particulate sheep red blood cells, and to allogeneic cells. TEF has similar effects also on the in vitro antibody responses of normal mouse spleen cells, enhancing antigen-dependent plaque-forming cells responses to dinitrophenyl carrier complexes of unprimed and primed mice. TEF fails to reconstitute helper cell function in cultures of T cell-deprived spleen cells. The thymic factor increases the reactivity or normal thymus cells in the in vitro mixed lymphocyte reaction and has at high doses mitogenic effects. The role of humoral thymus factors on the differentiation process of T cells is discussed.

Killer T cell responses to influenza A during a drift period: studies in mice.

After intravenous immunization of mice with any influenza A H3N2 drift strain attempts to restimulation of cytotoxic T cell (CTL) activities with the same virus or other drift period variants were unsuccessful for up to 6 weeks. Cross-stimulation 4-5 months after primary sensitization yielded, in most situations, positive but lower--as compared to primary--secondary cytotoxic T cell responses. Homotypic challenge was also effective after priming with some influenza A subtypes (A/E/72, A/PC/73, A/T/77) at this time.

The role of histocompatibility gene products in lymphocyte triggering and differentiation.

Studies performed both in vivo and in vitro have demonstrated that genetic restrictions exist in the development of optimal cell-cell interactions in the immune system. Thus, in mice, the presence or absence of gene identities in the I region of the H-2 complex determines the capacity of lymphoid cells to interact. These cell interaction or CI genes appear to code for cell surface molecules integrally involved in regulatory cell interactions. Analysis of biologically active thymus-derived (T) cell factors capable of regulating differentiation events in other lymphoid cells indicates that such factors are around 40,000-50,000 daltons in mass, bear determinants coded for by I region genes, and consist of two covalently or noncovalently linked components--a heavy glycoprotein of around 40,000 daltons and a lighter protein of around 12,000 daltons. Preliminary evidence suggests that the latter component may be beta2-microglobulin.

Experimental infection of inbred mice with herpes simplex virus. IV. Comparison of interferon production and natural killer cell activity in susceptible and resistant adult mice.

Inbred mouse strains differ in susceptibility to infection with herpes simplex virus type 1 or type 2 (HSV-1, HSV-2). In this study interferon production was tested in the peritoneal exudate of mice after intraperitoneal (i.p.) injection of HSV-1 or HSV-2. In HSV-resistant mice (C57 BL/6, C3 H/HeJ) high titers of interferon were already present 2 to 4 hours after injection. In comparison, less resistant mice (DBA/2, AKR) lacked this early response. There was no correlation between interferon titers and resistance at post-infection times later than twelve hours. At twelve hours, however, high titers of HSV were detected in the peritoneum of DBA/2 mice and significantly lower titers in C57 BL/6 mice. In a comparative analysis of eight different inbred mouse strains, again early (2 to 4 hours) interferon production was correlated to resistance. In assays of HSV-stimulated early (24 hours) NK cell responses not only the good interferon producer strains but also one of the less resistant low interferon producers (BALB/c) showed significant cytotoxic activities. Conversely, SJL mice that are very low in HSV-induced NK cell activity are resistant and show high early interferon responses at the local site.

Recombinant interleukin 2 rapidly augments human natural killer cell activity.

Recombinant human interleukin 2 (r-IL-2) rapidly stimulated human natural killer cell activity in vitro. Augmentation of NK activity occurred within 1 hr of preincubation with r-IL-2. Responsive killer cells were typical NK cells as shown by cell fractionation procedures. These included Percoll density gradient separation and depletion of OKT3+ T cells by an indirect rosetting method. Analysis with a panel of polyclonal and monoclonal antibodies against alpha and gamma interferon revealed that this early enhancement of NK activity by r-IL-2 was independent of the production of both types of interferon.