Shlomo Z. Ben-Sasson

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 challenge leads to in vivo activation and elimination of highly polarized TH1 memory T cells

TH1 memory T cells derived from T cell receptor transgenic mice, in which the T cell antigen receptor is specific for a cytochrome C peptide in association with I-Ek, were transferred into normal B10.A mice and allowed to adopt a resting phenotype. When challenged, 30–60 days after transfer, with i.v. cytochrome C, the transgenic cells rapidly became activated, expressed mRNA for IFNγ, and began to divide. However, after 48 h, the frequency of the cells fell progressively, reaching levels only slightly above the limit of detection by day 8 and thereafter remain depressed for up to 90 days. The remaining cells were anergic as shown by limitation in proliferation and IFNγ production in response to in vitro antigen stimulation. Even if challenged with antigen emulsified in complete Freunds adjuvant, the overall pattern was similar, except that in the draining lymph nodes, the surviving antigen-specific cells were not anergic, although spleen cells were still strikingly anergic. Thus, antigenic challenge of mice possessing resting memory TH1 CD4 T cells leads to the unanticipated loss of most of the specific cells and an apparent depletion rather than enhancement of immunologic memory.

Antigen-specific murine T-cell lymphomas: Functional heterogenicity

Abstract Two ovalbumin (OVA)-specific helper lymphomas (designated ROT/6.1 and ROT/6.2) were established by transformation of enriched, OVA-immune T cells with the radiation leukemia virus (RadLV). Shortly after establishment these lymphomas provided carrier (OVA)-specific help for anti-hapten antibody response. However, 5 months later ROT/6.1 lost its OVA specificity and could augment anti-hapten antibody response in the presence of an unrelated carrier. ROT/6.2 retained its antigen-specific helper function over 10 months of repeated passaging. This OVA-specific helper line inhibited anti-hapten antibody response when given together with an unrelated carrier. Cloning of ROT/6.2 by limiting dilution revealed that only 3 of 10 clones tested had OVA-specific helper activity. None of the clones could induce antigen-specific DTH reaction. The interrelationship between the functional heterogenicity, specificity, and stability of the helper lines is discussed.

In vitro selection of murine antigen-specific T-lymphocytes. I. Description of selection procedure

Abstract Enrichment of antigen-responsive murine T-lymphocytes was achieved by two in vitro procedures through the preferential adherence of the antigen-specific cells to the antigen-pulsed macrophages and their consequent multiplication. The first procedure involved the addition of column-purified T-enriched lymph node lymphocytes from immunized mice to a monolayer of antigen-pulsed adherent spleen cells from non-primed syngeneic donors. Lymphocytes which failed to adhere to the antigen-pulsed monolayer were removed after 4 h of incubation. The adherent cells were cultured for a week and the lymphocytes obtained after that period from the selection plate were highly responsive to the antigen for which they were selected and for a T-cell mitogen (Con A). On the other hand, these selected cells demonstrated little or no response to other antigens, to which the original donor of the lymphocytes was immune, and to a B-cell mitogen (LPS). The same preferential response to the selecting antigen and T-cell mitogen was obtained in lymphocytes enriched in the alternate procedures on ‘supernatant cultures’. The enriched population from ‘supernatant culture’ was derived from cells that did not adhere to the antigen-pulsed monolayers during 4 h of incubation. The non-adherent lymphocytes which still contained antigen-specific lymphocytes were transferred to a fresh monolayer of antigen-pulsed adherent spleen cells to be grown for a week in culture. The improvement in the response to the selecting antigen and the decreased reaction to other immunizing antigens show that the cells harvested from either ‘selected’ or ‘supernatant’ cultures were enriched for a given antigen — the selecting antigen. In most individual experiments the enrichment was better in the selected cultures. The enrichment procedures were dependent upon effective antigen presentation to the lymphocytes. Spleen cells from mineral oil-injected mice, which are the most effective antigen-presenting cells, formed the most efficient monolayers for the enrichment of the antigen-specific lymphocytes, both in the ‘selected’ and ‘supernatant’ cultures.

Backbone cyclic helix mimetic of chemokine (C–C motif) receptor 2: A rational approach for inhibiting dimerization of G protein-coupled receptors

The transmembrane helical bundle of G protein-coupled receptors (GPCRs) dimerize through helix-helix interactions in response to inflammatory stimulation. A strategy was developed to target the helical dimerization site of GPCRs by peptidomimetics with drug like properties. The concept was demonstrated by selecting a potent backbone cyclic helix mimetic from a library that derived from the dimerization region of chemokine (C-C motif) receptor 2 (CCR2) that is a key player in Multiple Sclerosis. We showed that CCR2 based backbone cyclic peptide having a stable helix structure inhibits specific CCR2-mediated chemotactic migration.

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.

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.

Radiation leukemia virus-transformed immunocompetent T cells: II. Antigen-induced macrophage migration inhibition factor and leukocyte migration inhibition factor production

OVA-specific T cells were immortalized by infection with radiation leukemia virus (RadLV). Some clones derived from such population were shown to exhibit helper activity. We then tested clones without such function and found among them some that secreted macrophage migration inhibition factor (MIF) and leukocyte migration inhibition factor (LIF) upon exposure to the antigen in vitro. The lymphokine-producing clones, which were Thy-1+, Ly-1+ and Ly-2-, did not secrete MIF and LIF constitutively. Like other antigen-specific T cells, the immortalized clones could not be stimulated by free soluble antigen but required macrophages for presentation and for triggering the lymphokine production. The antigen-activated clones exclusively produced MIF and LIF, but not interleukin 2 or colony-stimulating factor. They neither provided helper activity nor induced delayed-type hypersensitivity. The data suggest that the T-cell clones carry the antigen receptors and that their antigen-inducible biological function is restricted to the migration inhibitory factor production.

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.