Immortalization of cloned mouse splenic macrophages with a retrovirus containing the v-raf/mil and v-myc oncogenes

The recombinant retrovirus J2, which contains the v-raf/mil and v-myc oncogenes, was used to immortalize mouse splenic macrophages that had been cloned in soft agar. When added to freshly harvested colonies, J2 failed to yield cell lines but it immortalized up to 30% of the clones if they had been maintained for at least 4 months in medium containing colony-stimulating factor 1 (CSF-1). All of the cell lines grew in agar in a CSF-1-independent manner, and they produced tumors in nude and syngeneic mice. The cell lines were judged to be macrophage based on morphological criteria and because they secreted lysozyme, were phagocytic for antibody-coated particles, and expressed both the Mac-1 antigen and the CSF-1 receptor. The cell lines could be divided into three groups based on their expression of Ia and their ability to present an antigen to a T-cell hybridoma. The majority of the lines did not constitutively express Ia or present antigen, but a lymphokine did induce Ia in all of the lines, with most of them also acquiring antigen-presenting activity. However, a small proportion of lymphokine-treated lines continued to lack antigen-presenting activity despite their ability to express Ia. The third and smallest group of cell lines constitutively expressed both Ia and antigen-presenting activity. These results show that the J2 recombinant retrovirus is a useful means of immortalizing functionally distinct populations of cloned splenic macrophages.     

Functional analysis of macrophage hybridomas. III. Inhibition of lymphocyte blastogenesis and tumor proliferation

Four cloned macrophage hybridoma cells prepared by fusion of splenic adherent cells with a P388D1 macrophage tumor markedly inhibited the growth of lymphocytes and tumor cells. Macrophage clones 5, 8, 63, and 64 are strong inhibitors of B-cell blastogenesis, T-cell blastogenesis, and tumor proliferation, while the P388D1, tumor line and clones 13, 59, and 67 demonstrated little inhibitory activity in all three systems. The inhibitory effect of macrophage hybridomas can be detected within 8 hr, although greater inhibition was noted following longer incubation. The correlations among these three assay systems suggest that similar mechanisms may be involved. The data indicate that the inhibition of cell proliferation was not due to cell lysis. Furthermore, the inhibition of lymphocyte proliferation in Con A-stimulated cultures was not accompanied by inhibition of lymphokine production in the same cultures. Neither prostaglandins nor hydrogen peroxide appear to be primarily responsible for growth inhibition. The inhibitory properties of these macrophage hybridoma lines represent a stable phenotype and provide a homogeneous source of cells for further analysis of the phenomenon.     

Functional analysis of cloned macrophage hybridomas. V. Induction of suppressor T cell responses

It has been suggested that macrophage-like accessory cells are involved in suppressor T cell (Ts) induction. To further analyze this issue, we obtained several cloned macrophage hybridoma cell lines by somatic cell fusion of the macrophage tumor P388D1 of DBA/2 (H-2d) origin with splenic adherent cells of CKB mice (H-2k). Several cloned lines displayed the serological and functional characteristics of macrophages. We evaluated the ability of these hybridomas to induce third order or effector Ts (Ts3) to suppress the contact sensitivity response against the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). In contrast to the parental P388D1 and two other macrophage hybridomas, one macrophage hybridoma clone, termed 63, when conjugated with NP, induced Ts3, which suppressed contact sensitivity responses against NP but not DNFB, showing that the Ts3 were antigen specific. Macrophage hybridoma 63 could specifically induce Ts3 activity in either H-2k, H-2d, or H-2k/H-2d heterozygous hosts. Thus, macrophage hybridoma 63 functionally expressed major histocompatibility complex-related restricting determinants, and the fusion with cells from a H-2k macrophage donor caused a functional complementation of H-2d-related, Ts-inducing elements. The genetic restriction governing induction of Ts3 was controlled by genes that mapped to I-J region. Furthermore, NP-conjugated macrophage hybridoma 63 could serve as a target for elicitation of suppressor responses after administration of I-Jk, but not I-Jb, restricted suppressor factor. The data suggest that macrophage hybridomas represent a means to dissect heterogeneity within the macrophage population. The data also imply that the I-J determinants expressed on macrophages represent a ligand for the antigen receptor of Ts.     

Functional analysis of cloned macrophage hybridomas. VI. Differential ability to induce immunity or suppression

We previously screened a series of macrophage hybridomas derived from fusion of P388D1 (H-2d) tumor cells with CKB (H-2k) splenic adherent cells for their ability to induce I-J restricted Ts cell responses. One Ia+ macrophage clone (63) consistently induced Ag-specific, I-J-restricted Ts. To evaluate whether macrophage hybridoma 63 also induced delayed-type hypersensitivity (DTH) immunity, mice were immunized with hapten-coupled macrophage hybridoma cells. Hapten-coupled splenic adherent cells and control macrophage hybridomas induced significant primary DTH responses, whereas hapten-coupled macrophage 63 induced little or no immunity when injected into H-2 compatible hosts. However, macrophage hybridoma 63 specifically activated I-Ak, I-Ad, or I-Ed restricted T cell hybridomas/clones, in vitro in the presence of appropriate Ag. Three different strategies designed to eliminate suppressor cell activity were successfully used to demonstrate that hapten-coupled macrophage 63 could also induce in vivo immunity. First, after immunization with hapten-coupled macrophages, mice were treated with cyclophosphamide. Second, macrophage 63 was treated with anti-IJ idiotype antibody before 4-hydroxy-3-nitrophenyl acetyl hapten (NP) coupling. Finally, haptenated macrophages were injected into I-A compatible but I-J incompatible recipients. These protocols are known to inhibit the induction of Ts activity, thus these results indirectly suggest that there is stimultaneous generation of Ts activity in vivo. The latter hypothesis was tested in adoptive transfer experiments. Transfer of lymph node cells from NP-63 primed B10.BR (H-2k) mice induced immunity in naive 4R animals, whereas the same number of immune cells suppressed NP-induced DTH responses in 5R mice. The combined results indicate that a cloned macrophage line can activate both Th and Ts cells. Macrophages which induce Ts activity may be responsible for maintaining the balance of immunity vs suppression. The data support the hypothesis that IJ interacting molecules (IJ-IM) expressed on macrophages are critical for induction of suppressor cell activity.     

Functional analysis of cloned macrophage hybridomas. VII. Modulation of suppressor T cell-inducing activity

We have previously characterized a macrophage hybridoma clone, termed clone 59, which induced immunity but consistently failed to induce Ts responses. Macrophage 59 cells were cultured with supernatants from several activated T cell clones to determine if lymphokines could modulate the activity of this macrophage hybridoma to generate effector Ts. Culture supernatants from Th1 clones and from one atypical IL-4 and IFN-gamma-producing T cell clone successfully modulated clone 59 cells to induce effector Ts cells. In contrast, supernatants from activated Th2 cells failed to generate Ts-inducing activity in macrophage 59 cells. Culture with recombinant derived IFN-gamma was sufficient to cause modulation of Ts-inducing activity in macrophage 59 cells. The data imply that the differential functional activities ascribed to various macrophage hybridoma clones reflect macrophage heterogeneity instead of independent macrophage lineages. The suppression induced by clone 59 macrophages was genetically restricted to the putative I-J region. The ability of IFN-gamma containing supernatants to endow macrophage 59 with the capacity to induce effector suppressor cells was specifically abrogated by addition of an anti-IJk-idiotype antibody, which also reacts with IJ-interaction molecules, indicating that the mechanism of modulation most likely involves expression of IJ-interaction molecule determinants on antigen presenting cells.     

Production of T-T hybrids from T cell clones. Direct comparison between cloned T cells and T hybridoma cells derived from them

It has been assumed, without direct evidence, that T cell hybridomas and non-transformed T cell clones are both good models of normal Ag-specific T cells. To compare directly the difference in activation of cloned normal T cells and T hybridoma cells with the same TCR, cloned T hybridoma cells were obtained by fusing pre-established, myoglobin-specific, Iad-restricted T cell clones (14.5 and 9.27) with BW5147 cells. T cell clones were pre-activated with IL-2 as well as specific Ag before fusion. Cloned T hybridoma A3.4C6 was derived from Lys 140-specific and I-Ed-restricted clone 14.5. The other cloned T hybridoma, C7R14, was a fusion product of Glu 109-specific and I-Ad-restricted clone 9.27. Both T hybridomas showed the same Ag specificity and Ia restriction as the parental cloned T cells. However, C7R14 showed higher apparent affinity and broader cross-reactivity than 9.27. Clone 14.5, but not hybridoma A3.4C6, appeared to stimulate splenic cells to secrete cytokines inhibiting HT-2A cell proliferation. The most striking difference between the clones and hybridomas was that both clones, but neither of the matched hybridomas, were induced to synthesize IL-1 on stimulation with Ag. Finally, both cloned T cells and T hybridomas killed Ag-pulsed Iad-bearing B lymphoma target cells. This evidence suggests that killing function can be inherited from clones to hybridomas. However, the clones were much more efficient at killing than the hybridomas, and the hybridomas were more efficient at IL-2 production than the clones. Thus, matched pairs of clones and hybridomas differ in their capacity to mediate the two functions or may tend to be selected differently during cloning. Thus, although our results generally support the validity of T cell hybridomas as faithful models of the corresponding T cell clones, a number of subtle and not-so-subtle differences indicate that caution must be used in such an extrapolation.     

The processing and presentation of the self-antigen hemoglobin. Self-reactivity can be limited by antigen availability and costimulator expression.

APC do not distinguish between self- and foreign proteins. Previous studies from our laboratory demonstrated that most endogenous host APC constitutively processed and presented the self-Ag, hemoglobin (Hb), as detected by the Hb-specific T cell hybridoma, YO1.6. We have now examined APC in organs known to be involved in RBC degradation (liver Kupffer cells and splenic small resting B cells) for the presence of Hb/Ia complexes and for the expression of the costimulation necessary to trigger proliferation of T cell clones. We detected Hb/Ia complexes not only on splenic small resting B cells, but also on liver Kupffer cells. Interestingly, complexes were not present on lymph node small resting B cells. Splenic small resting B cells expressed costimulatory activity and efficiently stimulated the Th2 clones only. The opposite pattern was observed with liver Kupffer cells, which expressed costimulatory activity for Th1 clones only. However, if costimulatory activity was provided for the Th2 clones (IL-1 beta) and Th1 clones (allogenic spleen cells), the clones did proliferate in response to Kupffer cells and small resting B cells, respectively. In this report we have demonstrated that 1) endogenously formed self Hb/Ia complexes are expressed on splenic small resting B cells and liver Kupffer cells but not on lymph node small resting B cells and 2) these APC are also able to limit the expression of costimulatory activity for Th2 and Th1 T cell clones. Thus, endogenous APC not only constitutively process and present the self-Ag Hb, but also limit expression of the costimulatory activity necessary to trigger T cell proliferation against a self-Ag. The constitutive processing and presentation of self-Ag, as well as the regulation of costimulatory activity on APC, is likely an important feature of the maintenance of self-tolerance.     

Functional analysis of cloned macrophage hybridomas. IV. Induction and inhibition of mixed lymphocyte responses

A series of macrophage (M phi) hybridomas were generated by fusion of drug-marked P388D1 (H-2d) tumor cells with CKB (H-2k) splenic adherent cells. The ability of this panel of cloned M phi hybridomas expressing various levels of surface Ia antigens to induce allogeneic mixed lymphocytes responses (MLR) was examined. All MLR stimulatory M phi hybridomas expressed surface Ia antigens. However, some Ia+ and all Ia- M phi hybridomas were unable to induce vigorous MLR responses. Furthermore, even after induction of surface Ia antigen expression with Con A supernatants (Con A Sn) or purified interferon-gamma, the nonstimulatory M phi hybridomas remained ineffective at inducing strong MLR proliferative responses. Furthermore, addition of the latter M phi hybridoma clones (both with and without Con A Sn treatment) to conventional MLR cultures resulted in inhibition of MLR responses. The series of inhibitory M phi hybridomas secreted normal levels of IL 1 upon stimulation with lipopolysaccharide. After surface Ia induction with Con A Sn, the inhibitory M phi hybridomas could stimulate secretion of IL 2 and expression of IL 2 receptors. Moreover, although they inhibited conventional MLR responses, IL 2 production and IL 2 receptor expression were not significantly inhibited. Addition of these M phi hybridomas 24 to 48 hr after initiation of MLR response also inhibited MLR proliferation. The results indicated that the group of inhibitory M phi hybridomas can inhibit MLR responses after IL 2 secretion and acquisition of IL 2 receptors. Finally, this inhibitory activity has been maintained during 1 yr of continuous in vitro culture, and the hybridomas represent a stable "homogeneous" subpopulation of inhibitory macrophages. Thus, the inhibitory phenotype appears to reflect arrest at a distinct differentiation stage.     

Immortalization of murine leukocytes by oncogenes. II. Phenotypic characterization of transformants immortalized by v-src or Ha-ras oncogenes: expression of B220, a B-cell lineage specific antigen

In the course of study to obtain murine dendritic cell lines using oncogenic retroviruses, we have established several immortalized cell lines with characteristics different from those of dendritic cells. The transformants were mainly round nonadherent cells, capable of growing in soft agar, and negative for nonspecific esterase activity. Profiles of cell surface antigens were examined by indirect immunofluorescence technique. The cell lines were positive for Fc receptor (2.4G2), J11d (J11d.2), and B220 (RA3-3A1/6.1) antigens and negative (or dull positive in small percentages) for Ia (M5/144.15.2), IL-2 receptor (3C7), Thy-1 (B5-5), Mac-1 (M1/70.-15.11.5), and macrophage (F4/80) antigens. They were negative for both surface and cytoplasmic immunoglobulins. Several clones were established from these transformant cell lines and cell surface antigens were examined. Antigenic profiles of these clones were very similar to those of the parental cell lines. Some of these clones, however, seemed to increase their Ia antigen expression. The results suggest that the transformants originated from early B-lineage cells.     

Inhibition of lymphoid cell growth by a lipid-like component of macrophage hybridoma cells

Previous studies have shown that plasma membranes of murine lymphocytes and lymphoid tumor cells can reversibly inhibit the growth of both normal and transformed lymphocytes. The inhibitor can be extracted with organic solvents and has properties consistent with it being a lipid or lipid-like component of the membrane. This report identifies a series of cloned macrophage hybridoma cell lines, obtained by fusion of splenic adherent cells and the P388D1 line, which have very high levels of lipid-like growth-inhibitory molecules. Furthermore, a survey of seven cloned lines indicated that the macrophages fell into two distinct groups with regard to their level of growth-inhibitory activity. Group 1 lines had little or no inhibitory activity when cells were examined for their effect on a B lymphocyte proliferative response. Organic extracts from these macrophages had inhibitory activity (on a per cell basis) comparable to that seen with extracts of the P388D1 parental cell line and lymphoid tumor cells. In contrast, relatively low numbers of Group 2 macrophages could profoundly inhibit B macrophage proliferation. The growth-inhibitory activity was quantitatively recovered in organic extracts of the macrophages. Although the precise nature of the lipid moiety remains undefined, the data argue against the involvement of oxidized cholesterol. These findings indicate that lipid-like inhibitors of cell growth are present and functional in these macrophage cell lines. In addition, the results demonstrate that the inhibitory activity found in plasma membranes and liposomes is present and active in the membranes of intact cells, which is in contrast to the possibility that the inhibitor is an artifact generated during subcellular fractionation. Thus, the inhibitor is likely to have a physiologic role in growth control and in macrophage-mediated immunoregulation, probably acting via a mechanism involving cell-cell contact.     

Macrophage-hybridomas: generation, structure, and function

We report the generation of macrophage-hybridomas, obtained by somatic cell fusion between macrophage-enriched C3H.eB spleen cell population, and a drug-resistant MPC-11 myeloma cell line, designated as 4T00.1L1 clone. Screening for hybridomas possessing macrophage properties was carried out by assaying the presence of two macrophage-specific enzymes: lysozyme and nonspecific esterase. Two hybridomas, E2-7 and E2-10, were selected for further studies. We found that clones of E2-7 (E2-7.7) did not express Fc receptors but possessed cell-surface Ia molecules. In contrast, clones of E2-10 (E2-10.20) possessed Fc receptors but were devoid of Ia molecules. E2-7.7 did, however, express Fc receptors after mitomycin treatment, whereas E2-10.20 eliminated the expression of Fc receptors after treatment with mitomycin C. Opsonized erythrocytes were phagocytized by E2-10.20 cells, but not by E2-7.7. Phagocytosis was thus correlated with the possession of Fc receptors. Testing the response of KLH-primed lymph node cells to KLH-pulsed hybridoma cells, we found that E2-7.7 cells caused antigen-specific lymphoproliferative response, whereas E2-10.20 did not. Thus, antigens could be presented by E2-7.7 but not by E2-10.20 cells. The response was shown to be mediated by T but not by B lymphocytes. The difference in antigen-presenting capacity could not be attributed to differences in antigen uptake by the different hybridomas, because the two hybridomas manifested the same level of pinocytosis. Both hybridomas produced IL1. The differences in the properties of the two hybridomas may indicate that the normal partners represent two distinct subpopulations of macrophages. The segregation of functional properties among the hybridoma clones may lead to a clarification of the dependence of distinct functions on defined molecular structures.     

DNA-mediated transfer of major histocompatibility class II I-Ab and I-Abm12 genes into B lymphoma cells: molecular and functional analysis of introduced antigens

A20.2J B lymphoma cells have been co-transfected with the A alpha b, A beta b or with the A alpha b, A beta bm12 and neomycin resistance genes. The transfected cell lines constitutively express the I-Ab or I-Abm12 class II molecules at a level comparable with that of the endogenous I-Ad antigen. The I-Ab antigens expressed on three independently transfected B cell clones (A20.Ab.1, A20.Ab.2, and A20.Ab.3) are serologically and functionally indistinguishable from the I-Ab molecules expressed by control H-2bxd B hybridoma cells (LB cells). These transfected cell lines were potent I region-restricted antigen-presenting cells to a large panel of antigen-specific, autoreactive and alloreactive T cell hybridomas, as well as normal T cell clones. There were not significant differences in the efficiency of antigen presentation by the Ia molecules encoded by the transfected, as compared with the endogenous, I-A genes. The expression of a functional I-Ab antigen on the surface of cells transfected with A beta bm12 and A alpha b genes is consistent with previous work that implicated the A beta-chain alone in the bm 12 mutation. Furthermore, because the transfected A20.Ab and A20.Abm12 cells display the serologic and functional properties of normal spleen cells from the wild-type and mutant mouse strains, respectively, it is clear that class II genes do not undergo unexpected and unpredictable alterations after transfection in this system. This system permits us to investigate the structural requirements for interactions between class II major histocompatibility complex antigens, a foreign antigen, and the T cell receptor by in vitro site-directed mutagenesis coupled with DNA-mediated gene transfer.     

Homologous human macrophage hybridomas that produce a novel cytotoxic factor in their culture supernatants

Homologous human macrophage hybridoma cell lines were obtained by somatic cell fusion between peripheral blood monocyte-derived macrophages and a subclone of the myelomonocytic cell line, U937-F9. The hybridoma cell lines grown in vitro for more than a year were confirmed by manifestations of phagocytosis, adherence, nonspecific esterase, acid phosphatase, chromosome numbers and other cell surface antigens. Cell surface antigens on hybridomas were detected by flow cytometry analysis with monoclonal antibodies. With interclonal differences, a typical phenotype of hybridoma cells was CDw14+, OKM5+, Mac-1+ (equivalent to OKM1 and Mol), OKT9+, HLA-DR- and CD20+. After stimulation with lipopolysaccharide and calcium ionophore A23187, culture supernatants of clones c18A and c29A showed cytotoxic activity against human melanoma A375 Met-Mix and other cell lines which were resistant to the tumor necrosis factor, lymphotoxin and interleukin 1. This cytotoxic factor was found to be distinct from the tumor necrosis factor, lymphotoxin and interleukin 1 using the anti-tumor necrosis factor, anti-lymphotoxin and anti-interleukin 1 antisera.     

Characterization of an antigen expressed by human natural killer cells

A monoclonal antibody, anti-N901, was produced by fusing NS-1 myeloma cells with spleen cells of a mouse immunized with human CML cells. This antibody was reactive with a subpopulation of peripheral blood LGL, including the natural killer cells. Monocytes, granulocytes, B cells, T cells (T3+ cells), erythrocytes, and platelets were nonreactive. The N901-positive cells in the peripheral blood were heterogeneous with respect to expression of other cell surface antigens. The majority of N901+ cells co-expressed T11, Mo1, and HNK-1, whereas a smaller percentage expressed T8. Ia, T3, T4, Mo2, or B1 antigens were very uncommon on N901+ cells. The heterogeneity of the N901+ LGL was further investigated by examining the expression of N901 antigen on a series of cloned normal human NK cell lines. N901 antigen was expressed by each of the NK cell lines tested, and by a minority of cloned T cell lines without NK activity. Anti-N901 does not block NK activity and can be used to rapidly purify functional NK cells for further study.     

Requirement of down-regulation of NAD+ ADP-ribosyltransferase for the interferon-gamma-induced activation process of murine macrophage tumor cells.

Expression of the NAD+ ADP-ribosyltransferase gene is depressed during interferon-gamma-induced activation of murine macrophage P388D1 tumor cells [Taniguchi, T., Yamauchi, K., Yamamoto, T., Tokushima, K., Harada, N., Tanaka, H., Takahashi, S., Yamamoto, H. & Fujimoto, S. (1988) Eur. J. Biochem. 171, 571-575]. In order to study the role(s) of NAD+ ADP-ribosyltransferase in interferon-gamma-induced activation of P388D1 cells, we transfected an cloned synthetase gene into P388D1 cells and examined the effect of exogenous transferase gene expression on the induction of the Ia antigen, one of the major histocompatibility gene products, by interferon-gamma. The transferase activity of the transfected cells was twice that of control cells, and Southern blot analysis revealed that characteristic restriction sizes of cDNA were detected in the clones. RNA blot analysis using a cDNA for the transferase as a probe showed that the level of mRNA for the transferase in transfected cells was higher than that in control cells, and mRNA for the exogenous transferase was still detectable 2 days after the transfected cells were treated with interferon-gamma. This indicates that the exogenous transferase gene was expressed in transfected cells. RNA blot analysis with a cDNA for the Ia antigen and flow-cytometric analysis showed that the Ia antigen was induced much less in the transfected cells by interferon-gamma, in terms of the mRNA and the Ia antigen. The results suggest that down-regulation of the transferase is required for the induction of the Ia antigen in P388D1 cells by interferon-gamma.     

Antigen presenting ability of thymic macrophages and epithelial cells: evidence for defects in the antigen processing function of thymic epithelial cells

We compared the antigen presenting ability of cloned thymic macrophage and epithelial cell lines using T cell hybridomas with well-characterized activation requirements. A cloned thymic epithelial cell line (3D.1), preinduced with interferon-gamma (IFN-gamma) activated the T cell hybridoma 3DO-18.3 but not the T cell hybridoma DO-11.10. Analyses using preprocessed antigen suggest that the failure of 3D.1 to activate DO-11.10 is due to its inability to process chicken ovalbumin to produce a peptide recognized by the Ag:MHC T cell receptor of DO-11.10. The epithelial cell line 3D.1 was able to activate DO-11.10 if the superantigen staphylococcal enterotoxin B was used for activation instead of ovalbumin. These observations indicate that IFN-gamma-induced 3D.1 expresses sufficient I-Ad molecules to activate DO-11.10 but is unable to produce the peptide of ovalbumin recognized by DO-11.10. Furthermore, 3D.1 appears to be representative of nonmacrophage thymic stromal cells cultured in vitro, since heterogeneous cultures containing epithelial cells exhibited the same selective T cell activation characteristics. In contrast, thymic macrophage cell lines activated all T cells studied. These results suggest that there is a functional difference between the capacity of thymic epithelial cells and macrophages to process and present antigen to T cells.     

Constitutive and mitogen-induced production of T cell growth factor by stable T cell hybridoma lines

Stable T cell growth factor- (TCGF; IL 2) producing cloned T cell hybridoma lines were constructed by fusing murine alloantigen-activated T cells with the 8-azaguanine-resistant lymphoma line, BW5147. Many, but not all, clones of one of these hybridomas, i.e., hybridoma 24, secreted TCGF constitutively, but production was markedly enhanced by stimulation with T cell mitogens. Large numbers of TCGF-secreting hybridoma cells in a stable functional state could be obtained from histocompatible mice inoculated with cloned T cell hybridomas. Moreover, such in vivo-derived hybridoma cells could be stimulated sequentially with mitogen at least twice to secrete their biologically-active product, resulting in larger TCGF yields from the same cells. The secreted product of these T cell hybridoma lines resembled TCGF isolated from other cellular sources in that it: a) supported the growth of a TCGF-dependent T cell line; b) provided help for the induction of alloantigen-reactive cytotoxic T lymphocytes from thymocyte precursors; c) facilitated concanavalin A-induced mitogenic responses of low thymocyte numbers; d) had an apparent m.w. of 30,000 to 40,000 by gel filtration chromatography; and e) was eluted from DEAE-Sephacel ion-exchange chromatography columns by salt concentrations of 30 to 150 mM NaCl. The ability of these T cell hybridomas to grow in vivo and retain their functional characteristics in a stable form should prove useful in terms of providing large numbers of TCGF-secreting cells and studying in vivo aspects of the production of TCGF as well as other immunoregulatory mediators.     

Induction and functional characterization of class II MHC (Ia) antigens on murine keratinocytes

To induce Ia molecules on the surface of murine keratinocytes (KC), healthy mice were treated daily with i.p. injections of rIFN-gamma at a dose of 50,000 U/day for 6 days. This resulted in strong Ia expression by KC as determined by immunofluorescence of epidermal sheets or cell suspensions with anti-class II mAb. To obtain a population of Ia-bearing KC devoid of Langerhans cells, a method of depleting Langerhans cells from such suspensions was developed. Although Ia+ KC were unable to stimulate allogeneic T cells in a primary epidermal cell-lymphocyte reaction (less than 5% control), they did induce a proliferative response in an allospecific T cell line. Ia+ KC were unable to present native peptide molecules to class II restricted, Ag-specific T cell hybridomas. However, Ia+ KC were able to present a peptide fragment of pigeon cytochrome c to a hybridoma, suggesting that although these cells cannot process native protein Ag, they can present antigenic peptides. Ia+ (but not Ia-) KC also served as targets for class II restricted cytolytic T cell clones. These data indicate that the Ia expressed by KC is a functional molecule, and that Ia+ KC can participate in some immunologic reactions.     

Origins of macrophage diversity: functional and phenotypic analysis of cloned populations of mouse splenic macrophages

Soft-agar colonies of mouse splenic macrophages were examined for surface and functional characteristics that might prove useful in studying the origin(s) of macrophage diversity. Flow cytoflurometric analysis revealed that essentially all cells in all of the colonies bore the Mac-1 and Mac-3 antigens. The colonies did not differ appreciably in their phagocytic activity or in their secretion of lysozyme, but did show different patterns of Mac-2 antigen expression. In most colonies, the cells expressed low levels of the antigen, and in the remainder they expressed a high or an intermediate level of Mac-2. The colonies also differed in their ability to present keyhole limpet hemocyanin (KLH) to an antigen-specific H-2-restricted T-cell hybridoma. About 6% of the colonies gave rise to subcultures with antigen-presenting activity. This presentation was always associated with subcultures containing a high proportion of Ia-bearing macrophages, but not all cultures with similarly high proportions of Ia-bearing cells presented KLH to the hybridoma. Indeed, the induction of Ia on all cells in all cultures increased the proportion of KLH-presenting subcultures only about twofold. The results show that not all splenic macrophages have the ability to process KLH and present it to a T-cell hybridoma. This suggests the presence of functionally specialized subpopulations of macrophages, possibly derived from distinct progenitors, in the spleens of mice.     

T cell responses to select Ia determinants using the I-A mutant mouse strain B6.C-H-2bm12

The T cell repertoire of B6.C-H-2bm12 mice (an I-A mutant mouse strain) to wild-type Iab antigens was investigated using both secondary proliferative cultures and cloned T cell lines. Because bm12 mice have a gain-loss mutation of their gene encoding the Ia beta-chain polypeptide, bm12 anti-B6 T cell responses are specific for the select component of Iab specificities that was lost as a result of the mutation. Although stimulator cells bearing Iab antigens elicited the strongest responses, Iaq, d, and s antigens also resulted in reproducible stimulations of these bm12 anti-B6-primed T cells. Cloned T cell lines isolated from bm12 anti-b6 cultures revealed similar findings, with most clones recognizing determinants unique for Iab antigens; however, clones showing cross-reactions with Iad and/or q were also selected. Using F1 hybrid responder T cells (mutant x cross-reactive strain), we further dissected this cross-reactivity into several distinct cross-reactive determinants. Because bm12 mice lack the serologically defined Ia differentiation antigen W39, T cell recognition of this determinant was investigated by using bm12 anti-B6-primed cells. Stimulation by Ia.W39+ cells was appreciably better than by Ia.W39- (Xid-defective) cells, suggesting that bm12 T cells recognize an Xid-regulated, W39-like Ia differentiation antigen.