IL-1 acts on T cells to enhance the magnitude of in vivo immune responses.

IL-1 strikingly enhances antigen-driven responses of CD4 and CD8 T cells. It is substantially more effective than LPS and when added to a priming regime of antigen plus LPS, it strikingly enhances cell expansion. The effect is mediated by direct action on CD4 and CD8 T cells; the response occurs when OT-I or OT-II cells are transferred to B6 IL-1R1-/- recipients and only cells that express IL-1 receptors can respond. The major mechanism through which IL-1 enhances responses is by increasing survival of responding cells. IL-1 enhances the proportion of responding CD4 T cells that differentiate into Th17 cells and increases the proportion of responding CD8 cells that express granzyme B. Of a wide range of cytokines tested, only IL-1α and IL-1ß mediate this function. The potency of IL-1 as an enhancer of T cell responses suggests that it could act to enhance responses to weak vaccines and that the pathway utilized by IL-1 might be considered in the design of new generations of adjuvants.

Alloantiserum-induced inhibition of migration inhibition factor production in immune response gene-controlled immune systems.

We have previously demonstrated that alloantisera prepared by reciprocal immunization of strain 2 and strain 13 guinea pigs specifically block stimulation of in vitro DNA synthesis in genetically controlled systems. In order to determine whether this blockade extends to other T-lymphocyte functions, we examined the effect of alloantisera on the production of migration inhibition factor (MIF). (2 x 13)F(1) guinea pigs were immunized with a DNP derivative of the copolymer of L-glutamic acid and L-lysine (DNP-GL) and with DNP guinea pig albumin (GPA). The response to the former is controlled by a 2-linked Ir gene while that to the latter is mainly controlled by a 13-linked Ir gene. MIF production was assayed by an indirect procedure in which the migrating cell population lacked the histocompatibility antigen against which the alloantiserum was directed. Our results showed that anti-2 serum blocked MIF production by F(1) cells in response to DNP-GL but not DNP-GPA while anti-13 serum had the opposite effect. These experiments show that expression of a second major T-cell function is specifically blocked by alloantisera and suggest that Ir-gene products may act as antigen recognition substances on more than one type of T cell.

Generation of interleukin 4 (IL-4)-producing cells in vivo and in vitro: IL-2 and IL-4 are required for in vitro generation of IL-4-producing cells.

T cell populations derived from naive mice produce very small amounts of interleukin 4 (IL-4) in response to stimulation on anti-CD3-coated dishes. IL-4 production by such cells is mainly found among large- and intermediate-sized T cells and is dependent upon IL-2. Injection of anti-IgD into mice, a stimulus that leads to striking increases in serum levels of IgG1 and IgE, causes a striking increase in the IL-4-producing capacity of T cells. This increase is first observed 4 d after injection of anti-IgD. IL-4 production by T cells from anti-IgD-injected donors is mainly found among large- and intermediate-sized T cells. Small, dense T cells are poor producers of IL-4. The capacity of T cells from anti-IgD-injected donors to produce IL-4 is enhanced by addition of IL-2 and is largely, but not completely, inhibited by neutralization of in situ produced IL-2. These results indicate that the control of IL-4 production in T cells from naive and anti-IgD-injected donors is similar. However, it is possible that a portion of the IL-4-producing activity of T cells from activated donors is IL-2 independent. Although small T cells from naive donors have a very limited capacity to produce IL-4 in response to stimulation with anti-CD3, even in the presence of added IL-2, they can give rise to IL-4-producing cells upon in vitro culture on plates coated with anti-CD3 if both IL-2 and IL-4 are added. This leads to the appearance of IL-4-producing cells within 2 d. When analyzed after 5 d of culture by harvesting and re-exposure to anti-CD3-coated culture wells and IL-2, these cells have increased their IL-4-producing capacity by approximately 100-fold. The development of IL-4-producing cells in response to anti-CD3, IL-2, and IL-4 is not inhibited by interferon gamma (IFN-gamma), nor does IFN-gamma diminish IL-4 production by these cells upon challenge with anti-CD3 plus IL-2.

In vitro selection and extended culture of antigen-specific T lymphocytes. I. Description of selection culture procedure and initial characterization of selected cells.

Specific selection of antigen-responsive guinea pig peritoneal exudate lymphocytes (PELs) was achieved by a selection culture procedure. This procedure involved the addition of PELs from immune donors to monolayers of antigen-pulsed adherent peritoneal exudate cells from nonprimed syngeneic donors. PELs which failed to adhere were discarded at 24 and 48 h; after 1 wk of culture, lymphocytes were obtained which were highly responsive to the antigen for which they were selected but which demonstrated little or no response to other antigens to which the original donor of the lymphocyte was immune. These selected cells were largely T lymphocytes and could be maintained in culture for 2-5 wk in an antigen-responsive state and, in 20-30% of cases, for 8-10 wk in an antigen-independent state.

The specificity of cellular immune responses in guinea pigs. I. T cells specific for 2,4-dinitrophenyl-o-tyrosyl residues.

Guinea pigs immunized with the hapten 2,4-dinitrophenyl (DNP) coupled directly to Mycobacterium tuberculosis of strain H37Ra (DNP-H37) show a variety of cell-mediated immune responses to DNP coupled to protein carriers. The cells responsible for this specific response are thought to be T lymphocytes for the following reasons: Guinea pigs immunized with DNP-H37 displayed delayed hypersensitivity reactions to several DNP-proteins and contact sensitivity to dinitrofluorobenzene. Peritoneal exudate lymphocytes (PELs) obtained from DNP-H37 immune animals respond to DNP-proteins with DNA systhesis and cause inhibition of macrophage migration. PELs are highly enriched in T lymphocytes and contain few immunoglobulin-bearing cells. Further depletion of immunoglobulin-bearing cells from this population does not diminish the in vitro proliferative response to antigen. Nitrophenyl conjugates of proteins lacking a paranitro group stimulated DNA synthesis poorly or not at all, indicating the importance of the paranitro group of DNP in antigen recognition by T cells in this system. In this respect, the specificity of T cells resembles that of DNP-specific antibody from the same animals. On the other hand, DNP conjugates of copolymers of glutamic acid and lysine and DNP conjugated to proteins via an interposed beta-alanyl-glycyl-glycyl spacer failed to stimulate DNA synthesis, although such compounds bind very efficiently to anti-DNP antibody. By contrast, DNP conjugates of synthetic polypeptide carriers containing as little as 7% tyrosine strongly stimulated DNA synthesis in DNP-H37 immune PELs. That the determinant responsible for this stimulation was DNP coupled to the hydroxyl group of tyrosine was shown by selective removal of DNP from tyrosine by thiolysis with 2-mercaptoethanol, which abolished their ability to stimulate T cells.

Infection with Nippostrongylus brasiliensis or injection of anti-IgD antibodies markedly enhances Fc-receptor-mediated interleukin 4 production by non-B, non-T cells.

Non-B, non-T cells from spleen and bone marrow of naive mice produce IL-4 upon stimulation by plate-bound IgE or IgG2a in the presence of IL-3. Infection of mice with Nippostrongylus brasiliensis (Nb) or injection of anti-IgD antibodies, treatments known to cause striking polyclonal IgE responses, increase the number of splenic non-B, non-T cells and cause 10-30-fold increase in IL-4 production by a standard number of these cells. In Nb-infected mice, IL-4 producing non-B, non-T cells can be found in the lungs, a site through which Nb larvae migrate. Non-B, non-T cells from anti-IgD-injected mice produce IL-4 in response to anti-IgE antibodies, indicating that these cells have been sensitized in vivo with IgE and that crosslinkage of such IgE can lead to stimulation of lymphokine production. Similarly, non-B, non-T cells from Nb-infected mice produce IL-4 upon stimulation with Nb-antigen, indicating that antigen can also crosslink receptors on in vivo sensitized non-B, non-T cells and stimulate lymphokine production. The striking increases in the IL-4-producing capacity of the splenic non-B, non-T cell population in anti-IgD-injected and Nb-infected mice and the in vivo sensitization of these cells strongly suggests that they may have an important role in lymphokine production in helminthic infections and other situations marked by striking elevations of serum IgE levels.

Neural precursor cells inhibit multiple inflammatory signals.

Intravenous neural precursor cell (NPCs) injection attenuates experimental autoimmune encephalomyelitis by reducing autoreactive T cell encephalitogenicity in lymph nodes in vivo. Here we examined NPC-lymphocyte interactions in vitro. NPCs inhibited the induction of T cell activation marker IL-2-Receptor alpha, ICOS, PD-1 and CTLA-4 and inhibited T cell proliferation. NPCs inhibited T cell activation and proliferation in response to Concavalin-A and to anti-CD3/anti-CD28, which are T cell receptor (TCR)-mediated stimuli, but not in response to phorbol myristate acetate/ionomycin, a TCR-independent stimulus. The suppressive effect was not mediated via downregulation of CD3epsilon or induction of apoptosis. We next examined NPCs effects on inflammatory-cytokine signaling. NPCs impaired IL-2-mediated phosphorylation of JAK3 in lymphocytes, and inhibited IL-6 mediated proliferation of B9 murine hybridoma cells. In conclusion, NPCs ameliorate TCR-mediated T cell activation and inhibit inflammatory cytokines' signaling in immune cells. These findings may underlie the broad anti-inflammatory effects of NPCs in vivo.

Antigen-induced proliferation of murine T-lymphocytes in vitro. I. Characterization of the lymphocyte culture system.

The present report describes further improvements in the methodology for antigen-specific T-lymphocyte activation in vitro. Following the experimental protocol, large numbers of enriched T-lymphocytes can be obtained from the draining lymph nodes of immunized mice after purification on a nylon column. These T-cells respond better to stimulation by the soluble immunizing antigen than do unfractionated lymph node cells. The magnitude of response, measured by the incorporation of [3H]thymidine, was dependent on culture conditions and percentage of macrophages added to the column-purified cells. The optimal specific proliferative response was achieved when lymphocytes were incubated with 50% spleen cells and then cultured in RPMI-1640 supplemented with 2-mercaptoethanol (5 x 10(-5) M), sodium pyruvate (1 mM), AB+ human serum (5%) and syngeneic mouse serum (0.5%). Under the optimal culture conditions the lymphocytes undergo two successive cycles of proliferation as a result of antigen or mitogen stimulation. Thus, our studies have defined the culture conditions which can support extensive antigen-induced proliferation of T-cells easily obtained in large numbers. This in vitro system of antigen-specific T-cells from lymph nodes of immunized mice is most suitable for studies on the mode of T-cell activation.

Antigen-induced proliferation of murine T-lymphocytes in vitro. II. The effect of different macrophage populations on the antigen-induced proliferative response.

Various macrophage-containing preparations were tested for their ability to increase the antigen-specific proliferative response of murine T-lymphocytes. The preparations examined included: peritoneal exudate cells (PEC) from mice injected with mineral oil or thioglycolate; fresh bone-marrow cells; bone marrow cells grown in culture for up to 11 days; normal spleen cells, and spleen cells from mice injected with mineral oil. The best proliferative response was obtained when the lymphocytes were supplemented with 30% spleen cells from mice injected with mineral oil. When spleen cells from mineral oil injected mice are compared with those of spleen cells from normal mice, it is evident that mineral oil given i.p. activates the spleen macrophages. Although the number and percentage of macrophages in the spleen does not increase following mineral oil injection, the activities of some of their enzymes (acid phosphatase and beta-glucuronidase) increase while others do not change (Cathepsin D and lysozyme). Furthermore, the Fc-dependent phagocytic activity of spleen macrophages and their spreading on plastic culture dishes is increased after mineral oil treatment. We conclude that the activation of spleen macrophages caused by an i.p. injection of mineral oil also induces the changes in their antigen-presenting apparatus. Consequently, macrophages from spleens of mineral oil-injected mice are most suitable cell preparations for antigen presentation to T-lymphocytes.

IL-1ß strikingly enhances antigen-driven CD4 and CD8 T-cell responses.

Protective immune response requires massive expansion of antigen-triggered naïve cells, extensive differentiation into effector cells, migration of effectors into the periphery, and generation of a functional memory compartment. IL-1ß strikingly enhances expansion of antigen-primed CD8 and CD4 T cells in vivo. Its T-cell expansion in lymph nodes and spleen was direct, requiring that the stimulated T cells express IL-1R1. Immunization in the presence of IL-1ß increases the frequency of IL-17- and IFN-γ-producing cells among primed CD4 cells and the frequency of granzyme B-expressing and IFN-γ-producing cells and of cytotoxic cells among primed CD8 cells. IL-1ß-induced increase in the number of the activated CD4 and CD8 cells and augmented differentiation of the antigen-triggered T cells is very pronounced in liver and lungs. CD4 and CD8 cells primed in the presence of IL-1ß display augmented cell number and enhanced cytokine production when rechallenged 2 mo after priming with antigen and lipopolysaccharide (LPS). In five in vivo models, IL-1ß enhanced the protective value of weak vaccines. Preliminary analysis of in vivo gene expression in CD4 cells stimulated with IL-1ß revealed that IL-1ß caused gene expression changes consistent with the up-regulation of pathways involved in cell replication, cell survival, and enhanced energy metabolism. Thus, IL-1ß enhances antigen-primed CD4 and CD8 T-cell expansion, differentiation, and migration to the periphery and memory, the specific functions required for generation of effective protective immune responses.

IL-6 increases primed cell expansion and survival.

Cytochrome c-specific CD4 T cells from transgenic donors transferred to syngeneic B10.A mice expand more vigorously upon immunization if exogenous IL-6 is provided during the initial phase of immunization. The resultant increase in the frequency and number of Ag-specific cells is observed in the blood, lymph nodes, spleen, liver, and lung and persists for at least 3 mo. Treatment of immunized recipients with anti-IL-6 or use of IL-6 knockout recipients reduced the frequency of Ag-specific CD4 T cells during a comparable period, indicating that IL-6 is physiologically involved in the expansion of memory and/or effector cells and thus in the persistence of memory. IL-6 did not alter the duration of Ag-presenting activity. Both CFSE dilution studies and labeling with BrdU indicated that IL-6 does not effect proliferative rates of responding CD4 T cells. By contrast, annexin V staining was diminished in responding cells from the IL-6-treated animals, particularly among those cells that had undergone five or more divisions. These results indicate that IL-6 reduces the level of apoptosis among Ag-stimulated cells; thus, it plays a central role in determining numbers of memory and/or effector CD4 T cells in response to immunization over extended periods.

Antigen-specific helper factor production by immortalized clone of OVA-specific T cells. I. In vitro activity.

Immortalized clones of virally transformed OVA-specific T cells produce antigen-specific helper factor upon stimulation in vitro. The helper factor activate DNP-primed B cells to multiply and synthesize IgG anti-DNP antibodies. The trigger of the helper clone is antigen specific and the B cell-stimulating hapten must be coupled to the specific T cell carrier in order to transfer the help signal from the activated T clone to the B lymphocytes. Activation of the helper clone is performed by antigen-pulsed macrophages and cannot be achieved by the free soluble antigen. However, cell-free supernatant of the antigen-pulsed macrophages can stimulate the helper cells. Thus the antigenic determinant must be presented to the helper cell in the form of macrophage-processed antigen. These requirements for antigenic stimulation and the activity of the secreted helper factor demonstrate that the immortalized helper clone preserved the cellular components which control the antigen-specific immune function of the normal T lymphocyte.

In vivo helper activity of enriched populations of antigen-specific T lymphocytes.

Enrichment of murine antigen-specific T cells was achieved by stimulation of primed lymph node cells with macrophages containing the immunizing antigen. After a week in culture, the in vitro-sensitized lymphocytes had an increased helper activity. Inoculation of 10(7) egg albumin (OVA)-enriched T cells together with hapten coupled to OVA, elevated the number of splenic antihapten-producing cells of primed or unprimed mice. Augmentation of the hapten specific B-cell response could be observed as early as 4 days following the injection of the enriched population and peaked at Day 7. The enhancing effect of the enriched population of antigen-specific T cells was carrier specific since it occurred only in the presence of hapten coupled to the T-cell sensitizer [2,4-dinitrophenol (DNP)-OVA]. When given with the hapten conjugated to an irrelevant carrier [DNP-human gamma globulin (HGG)], the enriched lymphocytes caused a depression in the anti-DNP response. The capacity of in vitro enriched lymphocytes to promote a substantial antigen-specific helper activity (up to 1 anti-DNP producing cell per 10(3) spleen cells) upon adoptive transfer to nonirradiated mice, provides an experimental system for studying B-T collaboration in vivo under the normal physiological conditions.

Selection and enrichment of antigen-specific T lymphocyte populations.

Antigen-specific T lymphocytes bind in vitro to cells carrying alloantigens or to macrophages bearing antigen to which the lymphocytes were sensitized. This phenomenon is the basis of present procedures for the isolation and enrichment of antigen-specific T lymphocytes. The antigen-mediated adherence permits the removal of T cells with other specificities, which do not bind to the antigen-containing monolayer. The subsequent activation and multiplication of the antigen-bound cells further increases the proportion of the antigen-specific lymphocytes in culture. The selected cells, which are derived from a small fraction of the initial lymphocyte population, proliferate until they comprise up to 30% of the original total cell number. Another factor favoring enrichment is that unactivated cells do not survive well in culture whereas antigen-specific activated cells maintain their viability. Indeed, the selected cells can be maintained for a few weeks in culture. The activity of the selected lymphocytes is elevated toward the antigen used for enrichment, but lowered, or absent, toward other antigens. The progeny of the antigen-adherent lymphocytes are thus seen to be enriched for one antigen-specific subpopulation, and deprived of lymphocytes committed to other antigens. Cells obtained by this procedure should prove to be very useful for the functional analysis of T lymphocytes.

IL-4 increases IL-2 production by T cells in response to accessory cell-independent stimuli.

Freshly prepared, highly purified T cells from naive mice failed to produce IL-2 in response to soluble anti-CD3 antibody or to Con A and produced only small amounts of IL-2 in response to anti-CD3 coated on the surface of microwells. IL-2 production in response to soluble anti-CD3 or to Con A required the addition of accessory cells. By contrast, the addition of IL-4 strikingly enhanced the production of IL-2 by plate-bound anti-CD3-stimulated T cells in the absence or the presence of added accessory cells. Furthermore, anti-IL-4 mAb inhibited IL-2 production by anti-CD3-stimulated T cells, which indicates that endogenously produced IL-4 was important in IL-2 production by T cells to plate-bound anti-CD3. The capacity of IL-4 to enhance and of anti-IL-4 to inhibit IL-2 production in response to plate-bound anti-CD3 was also observed with both unstimulated T cells and with T cells that had been previously stimulated with anti-CD3 antibody. When activated T cells were restimulated with anti-CD3, the effect of IL-4 in enhancing IL-2 production was detectable within 6 to 8 h after restimulation. The effect of IL-4 on IL-2 production was not due to prolongation of survival or to enhanced proliferation of T cells. Northern blot analysis showed that T cells treated with anti-CD3 plus IL-4 had more than 10-fold more IL-2 mRNA than did T cells treated with anti-CD3 plus anti-IL-4; this was observed within 6 h of stimulation under certain circumstances. The increased level of IL-2 mRNA by IL-4 was achieved without any change in message half-life, suggesting that IL-4 enhances transcriptional activation of the IL-2 gene in such cells. These results lead to the conclusion that IL-4 has a critical role in IL-2 production in response to accessory cell-independent stimuli (plate-bound anti-CD3 antibody), although it is not essential to IL-2 production in response to accessory cell-dependent stimuli (soluble anti-CD3 and Con A).