SJL Spleen Cells Research Articles

Syngeneic B Lymphoma Cells Provide a Unique Stimulus to Natural Killer (NK) Cells in Genetically Low-NK SJL/J Mice

SJL/J mice are a genetically low-NK strain, and their cytotoxic activity cannot be augmented with conventional NK inducers. In contrast, effector cells taken from the lymphoid tissues of SJL mice bearing a syngeneic B cell lymphoma (RCS) show variable, but significant levels of cytotoxic activity against NK-susceptible targets, such as YAC-1. Previous results suggested that the RCS cells themselves contributed to this cytotoxicity. However, results presented here indicate the most, if not all of the activity present within the lymphoid tissues of RCS-bearing mice is mediated by RCS-activated, host NK cells. These results were confirmed by in vitro studies, which demonstrate that both gamma irradiated (gamma-) RCS cells and gamma-allogeneic spleen cells induce cytotoxic activity in SJL spleen cells against YAC targets. However, the cytotoxicity induced by gamma-allogeneic cells is mediated largely by lymphokine-activated killer (LAK) cells, since these effectors also lyse NK-resistant target cells, such as L1210. In contrast, the cytotoxic effector cells that are induced by syngeneic gamma-RCS cells cause lysis of YAC targets, but not L1210 target cells. These data indicate that the syngeneic B cell lymphomas of SJL mice are a unique stimulus for host NK cells in this strain. Since activated NK cells produce a variety of lymphokines, RCS stimulation of host NK cells in SJL mice may provide some of the growth-promoting lymphokines that are known to be necessary for progressive growth of these lymphoma cells.

Immunologic differences in murine glial cells and their association with susceptibility to experimental allergic encephalomyelitis

We tested the hypothesis that glial cells from mice resistant or susceptible to the autoimmune disease experimental allergic encephalomyelitis (EAE) may differ in their abilities either to express Ia antigens and/or stimulate anti-class II (Ia)-specific T-cells. Ia antigens were induced on glial cells from EAE-susceptible (SJL/J) and resistant (B10.S and DBA/2) strains of mice by culture with lymphokines from activated T-cells (2° SN). Ia antigen expression was quantified with an enzyme-linked immunosorbent assay (ELISA) in which glia were exposed to monoclonal anti-Ia antibodies and alkaline phosphatase-labeled anti-mouse Ig antibodies. The ability of glial cells to stimulate anti-Ia T-cells was quantified by culturing irradiated glial cells with anti-Ia-specific T-cell lines and measuring the amounts of [ 3H]thymidine incorporated by these lines. Glial cells from all strains of mice could be induced to express Ia antigens and upon exposure to high concentrations of lymphokines, amounts of expressed Ia antigen were equivalent. However, at limiting lymphokine concentrations, glia from the EAE-resistant strain B10.S expressed greater amounts of Ia antigen than did glia from SJL/J mice ( p<0.05), suggesting that B10.S glia were more sensitive to the Ia-inducing effects of T cell lymphokines. In contrast to the above results, glia from EAE-susceptible SJL mice consistently demonstrated an increased ability to induced T-cell proliferation in lines specific for Ia s antigen, compared to glia from EAE-resistant mice, even those of the same Ia haplotype (i.e. B10.S). Spleen cells from resistant strains had equivalent and frequently greater ability to induce anti-Ia-specific T-cell proliferation than did SJL spleen cells. These data suggest (a) that there are differences in the sensitivity of glia from different strains of mice to the Ia antigen-inducing effects of T-cell lymphokines, (b) that expression of Ia antigen does not necessarily correlate with the ability to stimulate Ia-specific T-cells, (c) that there are organ-specific differences in the ability to stimulate Ia antigen-specific T-cells, and (d) that an additional variable involved in determining resistance or susceptibility to an organ-specific autoimmune disease may be the ability of the target organ to stimulate anti-Ia-specific T-cells.

Antigen Presentation by Non-Immune B-Cell Hybridoma Clones: Presentation of Synthetic Antigenic Sites Reveals Clones that Exhibit no Specificity and Clones that Present Only One Epitope

Recently, we reported the preparation and antigen-presenting properties of hybridoma B-cell clones obtained after fusing non-secreting, non-antigen presenting Balb/c 653-myeloma cells with non-immune SJL spleen cells. It was found that antigen presentation at the clonal level can be specific or non-specific, depending on the particular B-cell clone. In the present work, one specific and one general presenter B-cell clones were tested for their epitope presentation ability to SJL T-cells that were specific to lysozyme or myoglobin. B-cell clone A1G12, a general presenter which presented both lysozyme and myoglobin to their respective T-cell lines, was found to present all five myoglobin epitopes while clone A1L16, a lysozyme specific presenter presented only one of the three epitopes of lysozyme. The latter reveals a hitherto unknown submolecular specificity (to a given epitope within a protein) for antigen presenting cells at the clonal level. Therefore, the specificity of T-cell recognition does not only derive from the T-cell but may also be dependent on the epitope specificity of the antigen-presenting B-cell.

Low frequency of NK‐cell progenitors and development of suppressor cells in IL‐2‐dependent cultures of spleen cells from low NK‐reactive SJL/J mice

Studies were performed to determine the basis for low NK activity in the spleens of SJL/J mice. In contrast to substantial boosting by IL-2 or IFN of NK activity of spleen cells from high NK-reactive strains of mice, no detectable effect of these cytokines on SJL spleen cells was seen. When SJL spleen cells were cultured for 7 days in the presence of IL-2, the frequency of proliferating cells was comparable to that of other strains of mice. However, the SJL spleen cells showed a frequency of NK-cell progenitors which was at least 50 times lower than that of the CBA/J spleen cells. In addition to having no detectable effector-cell activity, the cultures of SJL spleen cells contained suppressor cells which were able to inhibit the NK activity of spleen cells from other strains of mice. These suppressor cells did not adhere to plastic or nylon wool and were found in low-density fractions after Percoll density gradient centrifugation. The cultured SJL spleen cells had a high percentage of cells capable of binding to NK-susceptible target cells, which was similar to that seen in lytic cultures from other strains of mice. Thus, the suppressor activity may be attributable to competitive inhibition of the interaction of effector cells with target cells. Although several of the characteristics of suppressor cells were similar to those of cultured effector cells, they may not represent inactive or pre-NK cells since their progenitors were Thy1+ and AsialoGM1- whereas the progenitors of cultured effector cells in high NK strains were Thy1- and GM1+. The ability to eliminate the progenitors of the suppressor cells by pretreatment of spleen cells with anti-Thy1 plus C' also suggested that the low or undetectable NK activity of the cultured SJL cells is not attributable to suppression but may be due to an inherent deficit of NK cells.

Characterization of the molecules on SJL/J lymphomas which stimulate syngeneic T cells.

Abstract I-A antigens isolated from SJL reticulum cell sarcoma (RCS) cells show greater heterogeneity with respect to charge and size of the A alpha chains than do I-A antigens isolated from normal SJL spleen cells. The relevance of these structural changes in RCS I-A to the previously shown syngeneic T cell stimulatory properties of RCS was investigated. It was shown that RCS cells expressed acidic forms of the A alpha chain on their cell surface which were not present on SJL spleen cells, peritoneal cells, or dendritic cells. The only normal cells which showed A alpha chain heterogeneity approaching that of RCS cells were LPS-induced B cell blasts. Treatment with tunicamycin completely abolished the RCS A alpha chain heterogeneity, whereas exposure to neuraminidase removed the charge heterogeneity, but not the size heterogeneity. Parallel studies of the syngeneic T cell proliferative response to RCS showed that tunicamycin abolished, while neuraminidase enhanced, the ability of RCS cells to stimulate syngeneic T cells. It is suggested that polysaccharide chains on RCS I-A molecules are particularly important for the biologic properties of these lymphoma cells. The possibility that highly glycosylated I-A antigens on normal B cell blasts are also involved in the stimulation of syngeneic T cells is discussed.

Characterization of SJL T cell clones responsive to syngeneic lymphoma (RCS): RCS-specific clones are stimulated by activated B cells.

To gain insight into the nature of the syngeneic T cell-stimulating molecules on SJL lymphoma cells (RCS), a panel of eight Ly-1+2- T cell clones that are specific for transplantable RCS has been generated. All of these clones proliferate vigorously in response to two independent RCS lines and to LPS-activated syngeneic or F1 B cell blasts, but not to unstimulated SJL spleen cells or to allogeneic B cell blasts. Only one RCS-specific clone displays a proliferative response to (SJL X BALB/c) resting spleen cells, suggesting that I-E molecules are not the source of stimulation of RCS-responsive cells. Responses of the T cell clones to both RCS and syngeneic LPS-activated B cells are inhibited by monoclonal antibodies to I-A antigens, and not by antibody to I-E antigens. These findings suggest that RCS-responsive T cells are stimulated either by syngeneic I-As alone, in a form expressed on activated B cells, or by I-As in combination with X, where X is a cell surface antigen present on B cells at certain stages of differentiation.

ENHANCEMENT OF T CELL PROLIFERATION AND DIFFERENTIATION BY 8-MERCAPTOGUANOSINE: 70

Studies were undertaken to investigate the ability of the C8-substituted guanine ribonucleosides to modulate the proliferative and/or differentiative activity of T cells. This family of substituted nucleosides has been found to induce B cells to undergo proliferation and differentiation. However, T cells and thymocytes alike do not proliferate in response to 8MGuo. Moreover, this nucleoside does not modulate T cell proliferation evoked by either the mitogenic lectin Con A or by IL-2. 8MGuo can, however, modulate the T cell proliferative response to allogeneic cells. Because of earlier, vigorous B cell proliferation elicited by the nucleoside, modulation of T cell allogeneic responses must be observed in the absence of B cell responses. This can be done by stimulating thymocytes with irradiated cells and 8MGuo in the presence of supplemental IL-2, or by stimulating SJL spleen cells (whose B cells are hyporesponsive to 8MGuo) with irradiated allogeneic cells in the presence of 8MGuo. An analogous situation pertains to T cell function in that only certain functions can be modulated by 8MGuo. That is, T cells can not generate T cell growth or B cell differentiation activity in the presence of 8MGuo alone. Moreover, this nucleoside does not induce polyclonal cytotoxicity in T cell populations. It does, however, enhance generation of H-2-restricted CTL induced by allogeneic cells. Thus, in contrast to the situation pertaining to B cells, 8MGuo acts as an adjunct, but not as an initiator for T cell proliferation and differentiation.

IgE-binding factors from mouse T lymphocytes. II. Strain differences in the nature of IgE-binding factor.

Normal spleen cells from high IgE responder, BDF1 mice, formed IgE-potentiating factor upon incubation with mouse IgE, whereas those from low IgE responder, SJL mice, formed IgE-suppressive factor. The majority of the factors from BDF1 spleen cells had affinity for lentil lectin; those from SJL mice lacked affinity for the lectin. BDF1 spleen cells could be switched, however, to form IgE-suppressive factor if the cells were incubated with IgE in the presence of lipomodulin , a phospholipase inhibitory protein. In contrast, SJL spleen cells could form IgE-potentiating factor in the presence of lysolecithin, which enhances the glycosylation of IgE-binding factors. When the two strains were primed with alum-absorbed ovalbumin and their spleen cells were stimulated with homologous antigen, IgE-binding factors were detected in culture filtrates. The factors formed by BDF1 spleen cells selectively potentiated the IgE response, however, whereas those formed by SJL spleen cells selectively suppressed the response. Analysis of the cellular mechanisms for the selective formation of IgE-potentiating factor by antigen-primed BDF1 spleen cells revealed that antigenic stimulation of Lyt-1+ T cells resulted in the formation of two T cell factors, i.e., an "inducer" of IgE-binding factor and a glycosylation-enhancing factor, and that these factors induced the selective formation of IgE-potentiating factor. In SJL spleen cells, antigenic stimulation of Lyt-1+ T cells resulted in the formation of an "inducer" and a glycosylation-inhibiting factor, and those factors in turn stimulated the formation of IgE-suppressive factor. An additional difference between the two strains was found in Fc gamma R+ Ly-1+ T cells that were the cell source of IgE-binding factors. Upon stimulation with IgE or "inducer", BDF1 Lyt 1+ cells formed IgE-potentiating factor, whereas the same subset of T cells from SJL mice formed IgE-suppressive factor. The results indicate that the genetic differences between the two strains with respect to the nature of IgE-binding factors appear to be expressed in the process of glycosylation of IgE-binding factors.

Ia-Restricted interaction between normal lymphoid cells and SJL lymphoma (RCS) leading to lymphokine production: I. Enhancement and suppression of antibody formation to TI antigens by supernatants and cells from cocultures of RCS and lymphoid cells

Addition of γ-irradiated reticulum cell sarcoma (RCS) cells causes suppression of the antibody response of trinitrophenyl (TNP)-keyhole limpet hemocyanin (KLH)-primed syngeneic SJL spleen cells to TNP-polyacrylamide (PAA) in vitro. The response of anti-brain antigen (BAT) + C-treated spleen cells is not suppressed by γ-RCS, but is suppressed by cells from 48-hr SJL lymph node or thymus + γ-RCS cultures. Addition of as few as 2.5 × 10 5 cultured (anti-I-A + C treated to remove γ-RCS) cells causes significant inhibition of the responses of both syngeneic and allogeneic spleen cells. Treatment of γ-RCS-induced suppressor cells with anti-BAT + C reduces their suppressive activity. In contrast to the cells, supernatants (SN) from (lymph node (LN) + γ-RCS) cultures greatly enhance, in an antigen-dependent fashion, the responses of untreated or anti-BAT + C-treated Sephadex G 10-passed spleen cells to TNP-PAA, TNP-SIII polysaccharide, or TNP-Ficoll, but not as much to TNP-KLH. Addition of SN as late as Day 3 of culture still causes about half as much enhancement as leaving SN in throughout the culture period, but it has no effect if left with the spleen cells for only the first day of culture. SN contains high levels of IL-2 and IFN-γ; absorption with cells from an IL-2-dependent cytotoxic T-cell line removes the enhancing activity, while treatment with pH 2 to remove the IFN-γ has no effect. SN from an IL-2-producing T-cell line (LBRM-33) has a similar effect on antibody production to TI antigens as does SN of (LN + γ-RCS). The results suggest a marked dependency of PFC responses to TI antigens on IL-2 in all strains examined, including SJL, LAF 1, DBA/2Ha, and CBA/N, probably through a direct activation of B cells. The findings also suggest that suppressor T cells, induced by γ-RCS in syngeneic lymphoid cells, absorb the IL-2 needed for responses to TI antigens in vitro.

IgE-binding factors from mouse T lymphocytes. I. Formation of IgE-binding factors by stimulation with homologous IgE and interferon.

Incubation of normal mouse spleen cells with homologous IgE resulted in the formation of soluble factors that inhibited rosette formation of mouse Fc epsilon R+ cells with IgE-coated ox erythrocytes. The soluble factors could be absorbed with mouse or rat IgE coupled to Sepharose and recovered from the beads by acid elution. However, the factors had no affinity for either human IgE or mouse IgG. The IgE-binding factors were derived from T cells. Production of the factors required Lyt1+ T cells and Fc gamma R+ cells, which suggests that the factors are derived from Fc gamma R+ Lyt 1+ T cells. The molecular size of IgE-binding factors was approximately 15,000 daltons. When IgE-binding factors were formed by BALB/c spleen cells, nearly one-half of the factors had affinity for lentil lectin, and the remaining half of the factors failed to bind to the lectin. The proportion of the two species of IgE-binding factors differed depending on mouse strains. The majority of the factors formed by B6D2F1 spleen cells had affinity for lentil lectin, but those formed by SJL spleen cells failed to bind to the lectin. The IgE-binding factors were also induced by incubation of normal spleen cells with polyinosinic-polycytidylic acid (pI:pC). The nucleotide stimulated splenic adherent cells to form "inducers" of IgE-binding factors, which in turn induced normal lymphocytes to form IgE-binding factors. The inducers of IgE-binding factors were inactivated (or neutralized) by antibodies specific for mouse Type I interferon. It was also found that purified mouse beta interferon could induce the formation of IgE-binding factors. IgE-binding factors induced by pI:pC consisted of two different molecules: one had a m.w. of 15,000 daltons, and another had a m.w. of between 40,000 and 60,000 daltons.

Properties of reticulum cell sarcomas in SJL/J mice. VIII. Prominent role of RCS cell I-A antigens in the stimulation of syngeneic T cells.

While T cells from SJL and from F1 hybrids of SJL that do not express I-E antigens give strong proliferative responses to RCS, T cells from F1 hybrids expressing surface I-E do not. The nature of the stimulating antigen on the RCS cell surface was examined using monoclonal antibodies. Complete inhibition of the T-cell proliferative response was obtained with antibodies to I-A antigens, whereas antibodies to I-E antigens did not inhibit at all. This inhibition was mediated via an effect of the antibodies on the stimulating cells. Biochemical characterization of immunoprecipitated 125I-and 35S-labeled RCS antigens was performed using two-dimensional gel electrophoresis. Using this technique, I-A antigens were readily detected. However, neither Ia.7-specific antibodies nor antibodies specific for E alpha: E beta complexes precipitated any E alpha or E beta chains. Comparison of I-A antigens from RCS and normal SJL spleen cells revealed minor mobility differences in the gels, possibly due to differences in glycosylation, the significance of which needs to be further evaluated. Examination of RNA extracted from RCS, using E alpha and A alpha cDNA probes showed that RCS cells do not transcribe the E alpha gene as has been shown previously for normal H-2s cells. Furthermore, DNA from RCS cells showed a defect in the E alpha gene similar to that known to exist in normal H-2s cells. Our findings exclude the presence of E alpha on RCS cells and suggest a major role for I-A, either alone or in conjunction with another as yet unidentified cell surface antigen, in the stimulation of T cells.

A common idiotype on SJL and C57BL/6 anti-(4-hydroxy-3-nitrophenyl) acetyl antibodies and its relationship with lambda chain production.

Hybridoma cell lines secreting antibodies specific to (3-nitro-4-hydroxyphenyl) acetyl (NP) were generated by fusion of NP-immunized SJL spleen cells with the SP2/0 cell line. One hybridoma (N-hybridoma) anti-NP antibody (mu, lambda2) was found to partially inhibit (35-40%) the binding of the predominant idiotype in primary C57BL/6 anti-NP antibodies (NPb). Iodinated hybridoma antibody could be completely bound with anti-idiotypic antiserum made against either specifically purified C57BL/6 anti-NP antibodies, SJL anti-NP antibodies, or N-hybridoma antibody. The idiotypic specificities defined with anti-idiotypic antiserum made against N-hybridoma antibody were termed NP-1 idiotype. Strain distribution and genetic mapping studies indicate that the gene(s) controlling the production of NP-1 idiotype is closely associated with Igh-1b and Igh-1e alleles and mapped within the same chromosomal segment that controls the synthesis of NPb idiotype. However, unlike NPb idiotype, the expression of NP-1 idiotype is not influenced by the gene(s) that control lambda1 chain synthesis. Thus, SJL mice that produce low or undetectable levels of NPb idiotype due to a defect in lambda1 chain production express high levels of NP-1 idiotype. Specifically purified C57BL/6 and SJL anti-NP antibodies fully express NP-1 idiotype, the level of which correlates with the level of lambda2 chain-bearing molecules. Nonetheless, further experiments indicate that lambda1-bearing anti-NP antibodies can express extremely weak NP-1 idiotypic cross-reactivity.

Suppression of IgE antibody production in SJL mice. III. Characterization of a suppressor substance extracted from normal SJL spleen cells.

SJL mice were immunized with 1 microng dinitrophenylated keyhole limpet hemocyanin in 1 mg Al(OH)3. The mice were infected 21 days later with 750 third stage larvae of Nippostrongylus brasiliensis. On day 35, 14 days after infection, they were injected with 1 microng DNP-N, brasiliensis extract (Nb) in 1 mg Al(OH)3. In order to obtain high titer and persistent anti-DNP IgE antibody the mice were irradiated (540 R) 1 day after injection of DNP-Nb. Suppression of anti-DNP IgE antibody production was induced by spleen cells from normal SJL mice. Suppression of IgE antibody response is also obtained by an extract from normal SJL spleen cells. The suppressor substance from normal SJL spleen cell extract is a heat-labile protein, and is not absorbed by anti-mouse immunoglobulin. The mol wt of this substance is larger than 300,000 daltons as determined by gel filtration on Sephadex G-200, but after ultracentifugation, the supernate still has suppressive activity on IgE antibody production.

Suppression of IgE antibody production in SJL mice. I. Nonspecific suppressor T cells.

High titer and persistent antihapten IgE production in SJL mice can be obtained using appropriate immunization and radiation. Nonirradiated mice rapidly terminate this antihapten IgE production. Radiation was not necessary to prolong antihapten IgE production in other strains of mice. Termination can be obtained even in irradiated SJL mice by transferring normal SJL spleen cells. That the suppressor cells are T cells is shown by using thymocytes or cells treated with anti-Thy 1.2 and complement. No appreciable suppressive effect by normal spleen cells could be demonstrated on IgG1 production in SJL mice. The characteristic of low and transient IgE antibody response in SJL mice is inherited as a recessive trait controlled by a single Mendelian autosomal gene and is not linked to the H-2-gene complex. This characteristic does not depend on the infectivity of Nippostrongylus brasiliensis, the effect of anticarrier antibody, or the recognition of antigen.

Cellular basis of the genetic control of immune responses to synthetic polypeptides. II. Frequency of immunocompetent precursors specific for two distinct regions within (Phe, G)-Pro--L, a synthetic polypeptide derived from multichain polyproline, in inbred mouse strains.

DBA/1 mice are high responders to the (Phe, G) determinant of the synthetic polypeptide (Phe, G)-Pro--L, whereas SJL mice respond well to the Pro--L region of this macromolecule (6). In order to determine whether the phenomenon described above is related to the number of antigen-sensitive units detected for both specificities, and whether responses to these determinants can be transferred independently, graded and limiting inocula of spleen cells from SJL, DBA/1, and F(1) donors were injected into X-irradiated, syngeneic, recipient mice with (Phe, G)-Pro--L. By this approach, one antigen-sensitive unit specific for (Phe, G) was detected in 1.7 x 10(6) and 8.5 x 10(6) spleen cells from immunized and nonimmunized DBA/1 donors, respectively. In contrast, one (Phe, G) relevant precursor was detected in 20 x 10(6) SJL spleen cells, irrespective of whether the donors had been immunized. On the other hand, for the Pro--L specificity, one limiting splenic precursor was found in 1.3 x 10(6) and in 3.4 x 10(6) cells for immunized and nonimmunized SJL donors, respectively; whereas one response unit was estimated for this determinant in 9.4 x 10(6) and in 38 x 10(6) spleen cells from immunized and nonimmunized DBA/1 mice. The findings reported here indicate that the phenotypic expression of the genetic control(s) for immune responsiveness to different immunopotent regions of (Phe, G)-Pro--L is directly correlated with the number of immunocompetent response units detected in two inbred mouse strains. In the spleens of immunized F(1) donors, similar frequencies of one limiting precursor in 3.0 x 10(6) and in 2.8 x 10(6) cells were detected for (Phe, G) and Pro--L, respectively. The results of a chi-square test for independence of (Phe, G) and Pro--L responses in F(1) animals is compatible with the hypothesis that the transferred spleen cells limiting the response to (Phe, G)-Pro--L are restricted to generate antibodies specific for only one of the two determinants of this macromolecule.