Hapten-specific murine colony-forming B cells. II. Delineation of a tolerogen-sensitive subpopulation of colony-forming B cells

B cell subpopulations were studied by using B cell cloning procedures and an in vitro tolerance induction model. Fluorescein- (FL) specific B cells from normal spleens were isolated by using FL gelatin plates and were then cultured in semisolid agar in the presence or absence of tolerogen. Hapten-specific cells grew in soft agar to form discrete colonies. Colony growth is dependent on "mitogens" present in agar, sheep red blood cells (SRBC), and lipopolysaccharide (LPS). For example, SRBC plus LPS potentiate the growth of an increased number of colony-forming B cells (CFU-B) compared to either additive alone. These CFU-B could be triggered by a specific antigen to yield plaque-forming cells (PFC). With tolerogen (FL-sheep gamma-globulin) present in the agar, the number of FL-specific CFU-B was reduced by 25 to 50%. The ability of the remaining colonies to form PFC upon antigenic stimulation was also reduced. This reduction in CFU-B numbers, however, was observed only when the agar contained both SRBC and LPS as mitogenic potentiators of growth; no effect of tolerogen on CFU-B numbers was seen when cells were grown with either additive alone. Interestingly, the effect of tolerogen on CFU-B numbers was abrogated when peritoneal macrophages, in addition to SRBC plus LPS, were present during cloning. It is postulated that unique subpopulations of B cells form colonies under varied cloning conditions and that those CFU-B grown with SRBC plus LPS display an increased sensitivity to growth inhibition by tolerogen.     

The NKR-P1B gene product is an inhibitory receptor on SJL/J NK cells

The mouse NKR-P1 family includes at least three genes: NKR-P1A, -B, -C. Neither surface expression nor function of the NKR-P1B gene product has previously been shown. Here, we demonstrate that the SJL/J allele of the NKR-P1B gene product is expressed on SJL/J NK cells, and is recognized by PK136 mAb. Interestingly, the same mAb does not recognize the NKR-P1B gene product of C57BL/6. We have also generated a novel mAb, 1C10, that recognizes an activation receptor on SJL/J NK cells. Activation of the NKR-P1B receptor-inhibited 1C10 mAb induced redirected lysis and recruited SHP-1, indicating that NKR-P1B is an inhibitory receptor. Therefore, the mouse NKR-P1 gene family, like the Ly49 family, includes both activation and inhibitory receptors.     

Characterization of T lymphocyte colony-forming cells in the mouse. The colony-forming cell is confined to Lyt-1,2,3+ cell subsets in the thymus and the lymph nodes

When cell populations from the thymus were studied with FACS, it was found consistently that the brightly labeled Thy-1.2+ populations contained very few T colony-forming cells (CFC), while these latter cells were numerous in the cell populations showing lower Thy-1.2 antigen density. This was paralleled by findings after peanut agglutinin (PNA) separation that showed enrichment of CFC in the PNA-negative medullary population, and by sorting based on TL, T-200, and H-2 determinants or light scatter properties of the cells. By FACS sorting of Lyt-labeled thymic cells, it was also shown that CFC were predominantly present in cell populations that were brightly Lyt-1+, and exclusively in populations that were Lyt-2+ and Lyt-3+. After FACS sorting of lymph node cells, no major differences in colony formation were found between dully- and brightly-labeled Thy-1.2+ or Lyt-1+ populations, or between lymphoid cells showing different light scatter characteristics. In addition, it was shown that CFC--like thymic CFC--were of the Lyt-1,2,3+ phenotype. It is concluded that the CFC may be present in several differentiation steps of Lyt-1,2,3+ cell lines, and that the frequency of these cells increases from the thymic cortex via the medulla and to peripheral lymphoid tissues.     

Opsonization of tumor-reactive T cells in SJL/J mice bearing syngeneic tumors

Summary The role of antigen-reactive cell opsonization (ARCO) in a syngeneic tumor system and its effect on tumor progression was investigated. Thus, anti-tumor reactive T cells were prepared in vivo by immunization of normal SJL/J mice with mitomycin C-inactivated tumor cells of the syngeneic transplantable reticulum cell sarcoma (RCS) line LA-6. Dividing cells were subsequently labeled by injecting iodo-2-deoxyuridine (¹²⁵IUdR) into the same animals 3 days later. Antigen-reactive cells (*ARC) present in the radiolabeled, nylon wool-fractionated spleen cell population taken from these mice on day 4 and injected IV into syngeneic SJL/J mice bearing LA-6 tumors were diverted to the liver and away from the spleen. The effect was maximal by 8 days following inoculation of tumor cells, and was specific inasmuch as ¹²⁵IUdR-labeled cells prepared by immunization with allogeneic spleen or tumor cells which were not opsonized in day-8 LA-6 tumor-bearing mice. Opsonization of *ARC in day-8 LA-6 tumor-bearing mice was completely abrogated by either prior injection of heat-aggregated immunoglobulin into the mice or preincubation of the *ARC in solubilized tumor antigen before injection into tumor-bearing mice, demonstrating the involvement of Fc receptors in the host and antigen-specific receptors on the *ARC, respectively, in the opsonizing process. When anti-LA-6 reactive T cells were incubated in serum from LA-6 tumor-bearing mice and then injected IV into normal syngeneic SJL/J mice, a similar liver diversion was observed. Serum from cyclophosphamide-pretreated mice injected with LA-6 or serum from mice given mitomycin C-inactivated LA-6 cells did not cause opsonization of tumor-reactive T cells, while a mixture of these two sera did have some *ARC opsonizing activity. Further experiments with SJL/J mice bearing spontaneous RCS tumor indicate that tumor-reactive T cells are also opsonized in these mice. The above studies and others suggested that ARCO may play an important role in vivo in the survival of tumors. Abbreviations used in this paper are: ARC, antigen-reactive cells; ARC, radiolabeled antigen-reactive cells; ARCO, antigen-reactive cell opsonization; LA-6 tumor line derived in our laboratory; L.I., localization index; PEG, polyethylene glycol; RCS, reticulum cell sarcoma; STA, soluble tumor antigen; TBS, tumor-bearer serum     

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

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.     

Mapping genetic loci that regulate lipid levels in a NZB/B1NJxRF/J intercross and a combined intercross involving NZB/B1NJ, RF/J, MRL/MpJ, and SJL/J mouse strains

The NZB/B1NJ (NZB) mouse strain exhibits high cholesterol and HDL levels in blood compared with several other strains of mice. To study the genetic regulation of blood lipid levels, we performed a genome-wide linkage analysis in 542 chow-fed F2 female mice from an NZBxRF/J (RF) intercross and in a combined data set that included NZBxRF and MRL/MpJxSJL/J intercrosses. In the NZBxRF F2 mice, the cholesterol and HDL concentrations were influenced by quantitative trait loci (QTL) on chromosome (Chr) 5 [logarithm of odds (LOD) 17-19; D5Mit10] that was in the region identified earlier in crosses involving NZB mice, but two QTLs on Chr 12 (LOD 4.7; D12Mit182) and Chr 19 (LOD 5.7; D19Mit1) were specific to the NZBxRF intercross. Triglyceride levels were affected by two novel QTLs at D12Mit182 (LOD 8.7) and D15Mit13 (LOD 3.5). The combined-cross linkage analysis (1,054 mice, 231 markers) 1) identified four shared QTLs (Chrs 5, 7, 14, and 17) that were not detected in one of the parental crosses and 2) improved the resolution of two shared QTLs. In summary, we report additional loci regulating lipid levels in NZB mice that had not been identified earlier in crosses involving the NZB strain of mice. The identification of shared loci from multiple crosses increases confidence toward finding the QTL gene.     

An age dependent decline in suppressor capacity of SJL/J mice.

Age dependent changes in immune regulation of SJL/J mice have been described by us, in terms of tolerance inducibility. We have now found evidence of the same type of change in the response to sheep erythrocytes. Thymus cells from young donors interfere in a reconstitution system with the plaque forming response of lymph node cells. This suppressive capacity disappears at the age of about 4 months and is gradually replaced by an enhancing effect of thymus cells. Similarly, low molecular suppressive factors are found in the serum of very young SJL/J mice and enhancing factors in the serum of older mice.     

Oligodendrocyte-specific protein peptides induce experimental autoimmune encephalomyelitis in SJL/J mice.

Oligodendrocyte-specific protein (OSP) is a recently isolated and cloned, 207-aa, hydrophobic, four-transmembrane protein found in CNS myelin. It represents approximately 7% of total myelin protein. The OSP cDNA sequence has no significant homology with previously reported genes, but the predicted protein structure suggests that OSP is a CNS homologue of peripheral myelin protein-22. We previously reported the presence of anti-OSP Abs in the cerebrospinal fluid of relapsing-remitting multiple sclerosis (MS) patients, but not control patient groups. In this study, we tested the ability of a panel of 20-mer peptides with 10-aa overlaps, representing the sequence of murine OSP, to induce experimental autoimmune encephalomyelitis (EAE), an animal model for MS. SJL mice challenged with murine OSP peptides 52-71, 82-101, 102-121, 142-161, 182-201, and 192-207 exhibited clinical EAE. OSP:52-71 elicited severe relapsing-remitting EAE in some individuals. All other encephalitogenic peptides elicited, at most, a loss of tail tonicity from which the mice most often completely recovered. Mononuclear cell infiltrates and focal demyelination characteristic of EAE were evident. T cell proliferative responses were seen with all encephalitogenic peptides except 142-161 and 182-201. OSP peptides 72-91 and 132-151 did not cause clinical EAE, but did elicit robust proliferative responses. B10.PL and PL/J mice challenged with the same OSP peptide doses as SJL mice did not exhibit clinical EAE. These results in the SJL EAE model, together with the results from MS patient clinical samples, make OSP a promising candidate for autoantigenic involvement in MS.     

Administration of dehydroepiandrosterone suppresses experimental allergic encephalomyelitis in SJL/J mice.

Experimental allergic encephalomyelitis (EAE) is a Th1-mediated inflammatory demyelinating disease in the CNS, an animal model of multiple sclerosis. We have examined the effect of dehydroepiandrosterone (DHEA) on the development of EAE in mice. The addition of DHEA to cultures of myelin basic protein-primed splenocytes resulted in a significant decrease in T cell proliferation and secretion of (pro)inflammatory cytokines (IFN-gamma, IL-12 p40, and TNF-alpha) and NO in response to myelin basic protein. These effects were associated with a decrease in activation and translocation of NF-kappaB. In vivo administration of DHEA significantly reduced the severity and incidence of acute EAE, along with a decrease in demyelination/inflammation and expressions of (pro)inflammatory cytokines in the CNS. These studies suggest that DHEA has potent anti-inflammatory properties, which at least are in part mediated by its inhibition of NF-kappaB activation.     

Properties of reticulum cell sarcomas in SJL/J mice

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 's S-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_α : E _β 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-2 ^s cells. Furthermore, DNA from RCS cells showed a defect in the E alpha gene similar to that known to exist in normal H-2 ^s 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.     

In vitro generation of natural killer cells from SJL/J bone marrow precursors

The development of natural killer (NK) cells from undifferentiated bone marrow (BM) precursors of low-NK-reactive SJL/J mice was studied. Results indicate that BM cells of untreated mice are not able to generate NK effector cells in cultures supplemented with recombinant interleukin-2 (IL-2). On the other hand in the presence of IL-2, NK cells are generated in cultures of BM from mice pretreated with 5-fluorouracil (5-FU, 150 mg/kg iv 4 days before harvesting), a treatment which has been shown to eliminate more differentiated but spare less differentiated BM precursors. The 5-FU resistant BM progenitor is asialoGM1-, Thy.1+, Lyt.1- and Lyt.2-. The cells generated by culturing with IL-2 are asialoGM1+, Thy.1+, Lyt.5+, Lyt.1-, Lyt.2- and lyse only NK-susceptible targets. Generation of NK cells is blocked by addition of anti-IL-2 receptor (IL-2/r) antibodies. These studies demonstrate that it is possible to generate NK effectors from SJL/J BM cells by in vitro culturing with IL-2.     

Prevention of experimental allergic encephalomyelitis (EAE) in the SJL/J mouse by whole body ultraviolet irradiation

The cellular requirements for the in vivo induction of experimental allergic encephalomyelitis (EAE) were investigated in the SJL/J mouse. Exposure of mice to whole body ultraviolet (UV) irradiation, a treatment that has been shown in other systems to interfere selectively with antigen-presenting cell function, prevented the development of clinical and pathologic signs of acute EAE. Splenic T cells from UV-treated animals did not adoptively transfer resistance to EAE, making it unlikely that UV irradiation resulted in the generation of a specific suppressor cell population responsible for protection from EAE. UV irradiation was effective in preventing EAE when administered before initial immunization; UV irradiation was ineffective in modifying ongoing EAE or in preventing relapses of EAE induced by reimmunization. In additional experiments, adult thymectomized, lethally x-irradiated mice reconstituted with syngeneic marrow cells depleted of mature T lymphocytes were found to be resistant to the induction of EAE. Susceptibility was restored by the addition of splenic T cells, demonstrating that EAE induction is T cell-dependent in the mouse. The prevention of an experimental autoimmune demyelinating disease by whole body UV irradiation suggests that interference with the function of Ia-bearing accessory cells may represent an approach for immunotherapy in autoimmune disorders.     

Properties of reticulum cell sarcomas in SJL/J mice. VII. Nature of normal lymphoid cells proliferating in response to tumor cells.

Treatment of SJL lymph node, spleen, and thymus cells with anti-Ly 1.2 serum and C caused a marked reduction in the usually observed T-cell proliferation in response to syngeneic X-irradiated transplantable reticulum cell sarcoma cells. By contrast, treatment of lymphoid cells with anti-Ly 2.2 serum and C either failed to affect or increased the proliferative response. It is concluded that the Ly 1 cell is the major T-cell subpopulation which proliferates in response to RCS.     

Adoptively transferred experimental autoimmune encephalomyelitis in SJL/J, PL/J, and (SJL/J x PL/J)F1 mice. Influence of I-A haplotype on encephalitogenic epitope of myelin basic protein

Guinea pig basic protein (GPBP)-immune lymph node cells (LNC) from SJL, PL, and SJL x PL (F1) mice proliferated to whole GPBP and GPBP fragments 1-37, 43-88, and 89-169. All three strains of mice developed experimental allergic encephalomyelitis (EAE) by active immunization with whole GPBP or by passive transfer of LNC cultured with whole GPBP. SJL (H-2s) and PL (H-2u) mice developed EAE by active immunization with fragments 89-169 or 1-37, respectively, or by passive transfer of LNC cultured with the same Ag. F1 mice developed EAE by active immunization only with fragment 1-37 or by passive transfer of LNC cultured with either of the above fragments. Removal of macrophages (MO) from immune-F1 LNC resulted in the loss of a proliferative response and the ability to transfer EAE. Reconstitution of MO-depleted immune F1 T cells with either F1-, SJL-, or PL-MO restored the proliferative responses to whole GPBP and the three fragments. Cultures of immune F1 T cells reconstituted with any of the three MO populations and incubated with whole GPBP passively transferred EAE into naive F1 mice. Immune F1 T cells cultured with F1 MO in the presence of either fragment 1-37 or 89-169 transferred EAE. F1 T cells cultured with SJL MO were able to transfer EAE only if the Ag was fragment 89-169, whereas F1 T cells cultured with PL MO were able to transfer disease only if incubated in the presence of fragment 1-37. F1 mice are passively susceptible to EAE induced by adoptive transfer of cells reactive to either the N-terminal or C-terminal fragment and that the encephalitogenic determinant of GPBP is related to the genome of MO present in vitro.     

Failure of (SJL/J x PL/J)F1 hybrid mice to develop immunologic tolerance to V beta 17a+ T cells results in F1 anti-SJL mixed lymphocyte reaction.

It has been reported previously that spleen cells from (SJL x PL) F1 hybrid mice are not tolerant to SJL parental cells as assessed by a one-way MLR. The possibility that the F1 anti-SJL reaction was due to the effect of lymphokines produced by the irradiated SJL T cells in response to I-Eu expressed on the F1 hybrid cells was eliminated since inclusion of anti-I-E mAb was without effect. Cell separations showed the responder cells to be plastic and nylon wool nonadherent Ia- T cells. Separation of the SJL spleen cells showed that the stimulator cells were nonadherent, passed through a nylon wool column, and were Ia-. the F1-anti-SJL MLR was blocked 70 to 90% by inclusion of mAb KJ23a in the culture medium that indicated that the stimulatory cell population was V beta 17a+ T cells. This was confirmed by the use of V beta 17a+ and V beta 17a-T cell clones as stimulators. To determine whether failure to develop tolerance to this T cell subset in F1 hybrid mice might be responsible for the F1-anti-parent MLR, (SJL x PL)F1 mice were treated at birth and 48 h thereafter with anti-I-E mAb for 7 wk. Spleen cells from antibody-treated F1 mice were nonreactive with irradiated SJL parental cells in contrast to spleen cells from control mice which indicated that V beta 17a+ T cells were eliminated by negative selection before the development of tolerance.     

Adjuvant actions of polyclonal lymphocyte activators. V. Proliferation of macrophage colony-forming cells in the draining lymph node

We investigated the action of various polyclonal lymphocyte activators (PLA) on the proliferation of macrophage colony-forming cells in vivo at the local site. As PLA, Klebsiella pneumoniae 03 lipopolysaccharide (K03 LPS), Escherichia coli 0111 lipopolysaccharide (E. coli LPS), dextran sulfate (DS), concanavalin A (Con A), phytohemaggulutinin (PHA), polyadenylic-polyuridylic acid (poly(A:U], polyinosinic-polycytidylic acid (poly(I:C], and pokeweed mitogen (PWM) were used. All PLA tested acted to proliferate macrophage colony-forming cells in the draining lymph node at a late stage after subcutaneous injection. The order of strength of this action of PLA was K03 LPS greater than E. coli LPS greater than Con A greater than DS greater than PHA, PWM, poly(I:C), and poly(A:U), which corresponded to the order of strength of their adjuvant action in initiating helper-T-cell response to subcutaneous injection of aggregate-free bovine gamma-globulin. The detailed relationship between the proliferation of macrophage colony-forming cells and the adjuvant action of PLA is discussed.