Bone marrow-derived macrophage expression of endogenous and transfected class II MHC genes during differentiation in vitro

C57BL/6 (H-2b) mice fail to express I-E molecules on the surface of their cells and thus are unable to respond to I-E-restricted antigens such as GL phi and cytochrome c. Previous experiments in our laboratory have involved developing a system for studying differentiation of bone marrow cells into mature macrophage to gain a better understanding of class II MHC gene expression and function. In this study, we have used this system to transfect the E alpha d gene (cosmid 17.2) into C57BL/6 bone marrow cells and subsequently observed I-E expression on bone marrow-derived macrophages (BMDM) after differentiation in vitro. By using a modified calcium phosphate protocol, we found that the optimal period for transfection of the bone marrow cells was after 2 days of culture in vitro. By using the anti-I-E monoclonal antibody (Ia.7) derived from hybridoma 14-4-4, we detected the I-E molecule on the surface of transfected macrophages by a radiobinding assay and immunoprecipitation. BMDM expressed the I-E product maximally at 5 days of differentiation, and expression then declined. Furthermore, we have found that the expression of the I-E molecule on transfected macrophage was dependent upon exposure to interferon-gamma. Expression of I-E molecules was also detected by the generation of an allogeneic response. Transfected BMDM were compared with (CB6)F1 BMDM for their ability to stimulate C57BL/6 T cells and they were found to be equally effective. By using these initial findings, we hope to further optimize the conditions for insertion and expression of class II MHC genes in bone marrow cells.     

1 alpha, 25-Dihydroxyvitamin D3 augments clonal growth of macrophages from rat bone marrow progenitor cells and modulates their function

1 alpha, 25-Dihydroxyvitamin D3 (1,25(OH)2D3) was shown to enhance (approximately 2 fold) the colony-stimulating factor-dependent clonal growth of macrophage colonies and clusters from rat bone marrow progenitor cells. The proliferative capacity of macrophage progenitors in liquid cultures was likewise augmented (2-3 fold). Mononuclear phagocytes (macrophages, for simplicity) developing in the presence of 1,25(OH)2D3 showed a reduced capacity of migration. 1,25(OH)2D3 administered at bone marrow culture initiation led to augmentation of the phagocytic capability of macrophages in four-day cultures and to its suppression in macrophages in seven-day cultures. The observed patterns of modulation of differentiation and function by 1,25(OH)2D3 differ from the patterns we found for mouse bone marrow cells. The results suggest that the differential response to hormones observed in different species may include responses to 1,25(OH)2D3.     

Expression of a binding structure for sialic acid-containing glycoconjugates on rat bone marrow-derived macrophages and its modulation by IFN, TNF-alpha, and dexamethasone

Rat macrophages express a binding structure for sialic acid-containing glycoconjugates (sialic acid-binding receptor, SAR) which can be detected by a rosette assay utilizing SRBC coated with bovine brain gangliosides (E-G). Freshly isolated rat bone marrow cells (BMC) contain about 5% SAR-positive cells. Rat BMC cultured for 1 wk with tissue culture media containing CSF-1 differentiate into a virtually pure population of bone marrow-derived macrophages (BMDM phi). All BMDM phi bound E-G coated with an optimal concentration of gangliosides (100 micrograms/ml). When BMC were cultured for 1 wk with murine recombinant granulocyte-macrophage CSF, irrespective of the dose of GM-CSF, approximately 90% of the cells were identified as rat macrophages, and practically all expressed SAR. Only about 50% of BMDM phi bound SRBC coated with a suboptimal concentration of gangliosides (20 micrograms/ml). However, this percentage increased markedly after 8 to 72 h incubation with 1 to 10,000 U/ml purified murine IFN-alpha or IFN-beta, whereas murine or rat rIFN-gamma at doses above 10 U/ml led to a decrease of E-G binding. Human and murine rTNF-alpha enhanced rosette formation in a dose-dependent manner. These effects could be blocked by the respective anti-cytokine antibodies. Treatment of BMDM phi with dexamethasone also augmented E-G rosetting. The enhancement of E-G binding was abolished by pretreatment of BMDM phi with cycloheximide and actinomycin D but not with mitomycin C, suggesting that de novo synthesis of protein and RNA, but not DNA, is required. Our results demonstrate that all rat BMDM phi constitutively bear SAR, and that murine IFN-alpha, IFN-beta, and TNF-alpha, as well as dexamethasone, may augment SAR expression.     

The role of macrophages in anti-idiotypic antibody and T suppressor factor induction of timothy grass pollen antigen B-specific T suppressor cells

Cultures of normal spleen cells with anti-idiotypic antibody (anti-Id) or antigen B (AgB)-specific T suppressor factor (Tsf1) in mini-Marbrook chambers for 4 days at 37 degrees C lead to the in vitro induction of AgB-specific T suppressor (TS) cells. These TS cells significantly suppress a secondary AgB-specific IgE response, but they do not affect a secondary AgB-specific IgG response. Depletion of both B cells and macrophages from normal spleen cells by panning on anti-Ig-coated petri dishes provides an enriched T cell population. These enriched T cells when cultured with anti-Id or Tsf1 in mini-Marbrook chambers do not produce AgB-specific TS cells, and mice treated with cells harvested from the mini-Marbrook chambers have normal secondary AgB-specific IgG and IgE responses. The addition of as few as 1000 bone marrow-derived macrophages (BMDM) to cultures of the enriched T cells with anti-Id, or Tsf1 restores the ability of these cultures to produce significant levels of AgB-specific TS cells. Further studies reveal that the macrophage population must be histocompatible and express a cell surface I-J antigen. Attempts to pulse BMDM with anti-Id or Tsf1 at 4 degrees C and to culture in mini-Marbrook chambers 10(3) pulsed BMDM with enriched T cells were unsuccessful in producing AgB-specific TS cells. However, pulsing BMDM with anti-Id or Tsf1 at 37 degrees C, and adding 10(3) of these pulsed BMDM to enriched T cells in culture led to the formation of significant levels of AgB-specific TS cells.     

Expression of I-A by macrophages from Bcgr and Bcgs mice. Transient expression of I-A is due to degradation of MHC class II glycoproteins

Murine peritoneal macrophages from mice that are resistant to Mycobacterium bovis (strain BCG) can be induced to continuously express MHC class II (Ia) glycoproteins. In contrast, macrophages from BCG susceptible mice will only transiently express Ia. The purpose of this investigation was to determine the biochemical basis of continuous or transient Ia expression. We therefore compared Ia biosynthesis by macrophages from C.D2Bcgr mice to that by macrophages from congenic BALB/c.Bcgs mice. We show that macrophages from both strains of mice synthesize Ia initially and that very little synthesis occurs after 5 days of in vitro culture. No differences in the amount of Ia synthesized by the macrophages was apparent as determined by quantitative immunoprecipitation and by ELISA. Despite the lack of synthesis, macrophages from C.D2Bcgr mice continue to express Ia. The results of pulse-chase experiments indicate that macrophages from BALB/c.Bcgs mice degrade newly synthesized MHC class II glycoproteins whereas the majority of Ia remains associated with macrophages from the C.D2Bcgr mice. The addition of chloroquine to cultures of macrophages from BALB/c.Bcgs mice prevented the degradation and prolonged the expression of Ia. The results of this investigation show that there are no differences in the synthesis of Ia by macrophages from the two congenic strains of mice. The transient expression of Ia by macrophages from BALB/c.Bcgs mice is due to its degradation.     

A stromal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis

The production of B lymphocytes and myeloid cells occurs in the bone marrow in association with a supporting population of stromal cells. To determine whether these processes are dependent upon the same or different populations of stromal cells, stromal cell lines were generated from the adherent layer of a Dexter type long-term bone marrow culture. These cultures support myeloid cells and their precursors, a B cell precursor, and the adherent layer cells with support B cell differentiation under appropriate conditions. Two of the lines examined, S10 and S17, express class I histocompatibility antigens but not other hemopoietic cell surface determinants such as Thy-1, Lyt-1, Ig, Ia, Mac-1, or BP-1. Both lines could support myelopoiesis under Dexter conditions upon seeding with nylon wool-passed bone marrow. The nylon wool passage depletes stromal cells capable of forming adherent layers in vitro but retains hemopoietic precursors. The number of cells and colony-forming units-granulocytes/macrophages in the nonadherent cell population recovered 3 wk post-seeding had increased 19-fold and 10-fold, respectively, in the reseeded cultures of S10 and S17. After 3 wk of growth in Dexter conditions, the reseeded cultures were transferred to conditions optimal for B cell differentiation described by Whitlock and Witte. After 4 wk of growth, hemopoietic cells were consistently recovered from S17 cultures but not those of S10. A proportion of these cells from S17 cultures expressed the 14.8 antigen and were surface IgM positive. Surviving hemopoietic cells present in cultures of S10 were primarily macrophages. These findings indicate that S17 but not S10 can support both myelopoiesis and B lymphopoiesis and suggest that one stromal cell population has the capacity to form a hemopoietic microenvironment for both lineages.     

The intestinal microflora regulates cytokine production positively in spleen-derived macrophages but negatively in bone marrow-derived macrophages.

Besides its role as a barrier against potential pathogens, intestinal flora is presumed to protect the host by priming the immunological defense mechanisms. In this respect, the influence of intestinal flora on macrophage precursors was examined, and its modulating effect was compared on LPS-induced cytokine production by macrophages derived from bone marrow and spleen precursors (BMDM and SDM respectively). The regulation of IL-1, IL-6, TNF-alpha and IL-12 production in macrophages from germ-free and from three groups of flora-associated mice, conventional, conventionalized and E. coli-mono-associated mice, was investigated. The whole flora inhibited IL-1, TNF-alpha and IL-12 secretion by BMDM, whereas it had a stimulatory effect on IL-12 secretion by SDM. Implantation of E. coli alone enhanced cytokine secretion by BMDM but had a more limited effect than whole flora on SDM, enhancing only TNF-alpha and IL-12 secretion. Study of expression of mRNA showed a correlation with protein secretion for IL-6 but not for TNF-alpha and IL-1. IL-12 enhancement in BMDM seemed to be dependent on regulation of p35 mRNA expression while it was correlated to increased p40 mRNA expression in SDM. The results demonstrated that intestinal flora modulated bone marrow and spleen macrophage cytokine production in a differential manner and suggested a role for bacteria other than E. coli among the whole flora. The contrasting effects exerted by the intestinal flora on bone marrow and spleen precursors are an interesting observation in view of the different functions of these organs in immunity. The finding that intestinal flora enhanced IL-12 production in spleen is also potentially important since this cytokine is implicated in the determination of the relative levels of Th1 and Th2 responses and plays a pivotal role in host defense against intracellular microorganisms.     

Bone marrow-derived macrophages express functional CGRP receptors and respond to CGRP by increasing transcription of c-fos and IL-6 mRNA.

Calcitonin gene-related peptide (CGRP) is a sensory neuropeptide with inflammatory and immunoregulatory properties. CGRP inhibits IL-7 responses by B cell precursors by direct and indirect mechanisms. We recently found that CGRP induces IL-6 and TNF-alpha in long-term bone marrow cultures and that IL-6 and TNF-alpha also inhibit IL-7 responses. Because these are heterogeneous cultures, it was not clear which cells produced IL-6 and TNF-alpha. To determine whether bone marrow-derived macrophages (BMDM) were the source, we did studies to determine whether BMDMs express mRNAs for CGRP receptors and whether CGRP induces c-fos, IL-6, and TNF-alpha mRNA. We found that BMDMs express mRNAs for CRLR and RAMP1, the minimal components for CGRP receptors. CGRP also stimulated dose- and time-dependent increases in c-fos and IL-6. In contrast, CGRP did not induce TNF-alpha in BMDMs. These results suggest that BMDMs are a source of CGRP-induced IL-6 in bone marrow.     

Polycomb group protein Bmi1 negatively regulates IL-10 expression in activated macrophages

Macrophages exert a wide variety of functions, which necessitate a high level of plasticity on the chromatin level. In the work presented here, we analyzed the role of the polycomb group protein Bmi1 during the acute response of bone marrow derived macrophages (BMDM) to lipopolysaccharide (LPS). Unexpectedly, we observed that Bmi1 was rapidly induced at the protein level and transiently phosphorylated upon LPS treatment. The induction of Bmi1 was dependent on MAP-kinase signaling. LPS treatment of BMDM in the absence of Bmi1 resulted in a pronounced increase in expression of the anti-inflammatory cytokine interleukin-10 (IL-10). Our results identify Bmi1 as a repressor of IL-10 expression during macrophage activation.     

Bone marrow-derived macrophages: development and regulation of differentiation markers by colony-stimulating factor and interferons

To investigate the role of specific cytokines in the development of the fully mature macrophage, we have employed murine bone marrow cells that were grown in the presence of CSF-1, a colony-stimulating factor that has been shown to induce the proliferation and differentiation of macrophages from their precursor cells. The CSF-1 employed in these studies was partially purified to ensure removal of contaminating interferon (IFN) from the preparations. After 1 to 2 wk in the presence of the partially purified CSF-1, the adherent macrophages were removed from flasks enzymatically and were recultured at known densities in the absence of CSF-1. Cell surface antigens (Mac-1 and Ia) and Fc receptor capacity (as assessed by Fc-mediated phagocytosis) were examined as markers of macrophage differentiation. Basal levels of Fc receptor capacity and Mac-1 antigen were markedly influenced by exposure to CSF-1, and appear to be modulated by CSF-induced, macrophage-derived IFN. When the bone marrow-derived macrophages were exposed to exogenous IFN in the absence of CSF-1, they proved to be extremely inducible with respect to Fc-mediated phagocytosis (IFN-beta and rIFN-gamma) and Ia antigen expression (rIFN-gamma) when compared with thioglycollate-elicited macrophages. Thus, macrophage growth factors, such as CSF-1, promote macrophage maturation by inducing the production of autostimulatory signals, such as macrophage-derived IFN. In addition, exogenous cytokine stimuli, such as IFN-gamma, further amplify the differentiative potential of these cells. Bone marrow-derived macrophages, propagated under well-defined conditions and never exposed to eliciting agents, provide a powerful model for studying the role of cytokines, such as CSF-1 and IFN, in the differentiative pathway of macrophages.     

Regulation of macrophage populations. V. Evaluation of the control of macrophage Ia expression in vitro

In response to a lymphokine (LK) produced by activated T cells, macrophages can be induced to express Ia in vitro. This appears to be a complex process comprised of a number of discernible events. Peritoneal macrophages elicited by different means, and unstimulated macrophage and monocyte populations, each had a distinct kinetic profile of Ia induction. This response was characterized most noticeably by a latent period before actual Ia expression. The latent period varied from 3 to 7 days, depending on the target population, and was correlated with the state of activation of the macrophage as reflected by 5' nucleotidase activity. In spite of the protracted time course for Ia expression, all macrophage populations could be "triggered" (committed to a subsequent program of Ia expression) by exposure to the LK for as little as 2 hr. To be triggered, however, macrophages first had to go through a period of culture as adherent cells. Both the spontaneous loss and LK-dependent acquisition of Ia correlated with the functional capacity of these macrophages as antigen-presenting cells, indicating that these events are functionally significant.     

A method for generation of bone marrow-derived macrophages from cryopreserved mouse bone marrow cells

The broad use of transgenic and gene-targeted mice has established bone marrow-derived macrophages (BMDM) as important mammalian host cells for investigation of the macrophages biology. Over the last decade, extensive research has been done to determine how to freeze and store viable hematopoietic human cells; however, there is no information regarding generation of BMDM from frozen murine bone marrow (BM) cells. Here, we establish a highly efficient protocol to freeze murine BM cells and further generate BMDM. Cryopreserved murine BM cells maintain their potential for BMDM differentiation for more than 6 years. We compared BMDM obtained from fresh and frozen BM cells and found that both are similarly able to trigger the expression of CD80 and CD86 in response to LPS or infection with the intracellular bacteria Legionella pneumophila. Additionally, BMDM obtained from fresh or frozen BM cells equally restrict or support the intracellular multiplication of pathogens such as L. pneumophila and the protozoan parasite Leishmania (L.) amazonensis. Although further investigation are required to support the use of the method for generation of dendritic cells, preliminary experiments indicate that bone marrow-derived dendritic cells can also be generated from cryopreserved BM cells. Overall, the method described and validated herein represents a technical advance as it allows ready and easy generation of BMDM from a stock of frozen BM cells.     

Macrophage endothelial nitric-oxide synthase autoregulates cellular activation and pro-inflammatory protein expression

Expression of inducible nitric-oxide (NO) synthase (iNOS) and "high-output" production of NO by macrophages mediates many cytotoxic actions of these immune cells. However, macrophages have also been shown to express a constitutive NOS isoform, the function of which remains obscure. Herein, bone marrow-derived macrophages (BMDM?s) from wild-type and endothelial NOS (eNOS) knock-out (KO) mice have been used to assess the role of this constitutive NOS isoform in the regulation of macrophage activation. BMDM?s from eNOS KO animals exhibited reduced nuclear factor-kappaB activity, iNOS expression, and NO production after exposure to lipopolysaccharide (LPS) as compared with cells derived from wild-type mice. Soluble guanylate cyclase (sGC) was identified in BMDM?s at a mRNA and protein level, and activation of cells with LPS resulted in accumulation of cyclic GMP. Moreover, the novel non-NO-based sGC activator, BAY 41-2272, enhanced BMDM? activation in response to LPS, and the sGC inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one attenuated activation. These observations provide the first demonstration of a pathophysiological role for macrophage eNOS in regulating cellular activation and suggest that NO derived from this constitutive NOS isoform, in part via activation of sGC, is likely to play a pivotal role in the initiation of an inflammatory response.     

Evidence for effects of interleukin 4 (B cell stimulatory factor 1) on macrophages: enhancement of antigen presenting ability of bone marrow-derived macrophages

We have studied the effects of recombinant mouse interleukin 4 (IL 4) (previously known as B cell stimulatory factor 1) on the antigen-presenting ability of murine splenic B cells and bone marrow macrophages. Our assay is based on the induction of antigen-presenting ability in these cells after incubation with IL 4 for 24 hr. The presenting cells were then used to stimulate IL 2 production by antigen-specific, I-Ad-restricted T cell hybridomas, a response mainly dependent on the induction of Ia antigens. Consistent with our previously published data using partially purified natural IL 4, we show here that recombinant IL 4 (but not interferon-gamma (IFN-gamma) or IL 1) induces antigen-presenting ability in B cells. Recombinant IL 4 was also found to induce antigen-presenting ability in a cloned, bone marrow derived-macrophage cell line (14M1.4), and in normal bone marrow-derived macrophages. These macrophage populations also respond to IFN-gamma showing enhanced antigen-presenting ability (mediated by increased Ia antigen expression). A small but significant increase in Ia antigen expression was also detected in 14M1.4 macrophages induced with IL 4. However, additional analysis suggested that the effect of IL 4 on 14M1.4 is different from that of IFN-gamma, because IL 4 (but not IFN-gamma) is able to maintain the viability and increase the size of and metabolic activity of bone marrow macrophages. However, IL 4 may not affect all macrophages because the macrophage cell line P388D1, which responds to IFN-gamma, failed to show enhanced antigen-presenting function after stimulation with IL 4. These observations indicate that IL 4, a lymphokine previously considered to be B cell lineage specific, has effects on macrophages and may be involved in their activation.     

Membrane vesicles shed by murine melanoma cells selectively inhibit the expression of Ia antigen by macrophages

We previously demonstrated that membrane vesicles shed by the F10 variant of the murine B16 melanoma cell line inhibited the induction by interferon-gamma (IFN) of murine macrophage immune response region-associated (Ia) antigen expression. In this paper we present evidence that the inhibition of macrophage Ia antigen expression is a selective effect of vesicles and characterize its temporal requirements. Membrane vesicles shed from F10 cells did not affect the expression of macrophage H-2K or H-2D antigens under conditions shown to profoundly inhibit Ia antigen expression. Similarly, the induction of plasminogen activator and interleukin 1 from macrophages was not inhibited by the vesicles. The vesicles did not measurably decrease total cellular RNA or protein synthesis. Macrophages were sensitive to the inhibitory effects of the vesicles during the induction and maintenance phases of Ia expression. Pretreatment of macrophages with vesicles before culture with IFN did not reduce the induction of Ia. The rate of decline of Ia expression after removal of IFN was unaffected by the presence of vesicles. Removal of vesicles from cultures of IFN-treated macrophages resulted in only a partial recovery of Ia expression, suggesting that the inhibition of Ia expression may be a slowly reversible process. The selective and partially reversible inhibition of Ia expression by vesicles shed from the plasma membrane of tumor cells is a possible mechanism whereby tumor-bearing hosts may become immunocompromised.     

Combined effects of tumor necrosis factor-alpha, prostaglandin E2, and corticosterone on induced Ia expression on murine macrophages

Ia expression is an important marker of macrophage functional capacity. IFN-gamma induces Ia expression on perhaps all murine macrophages, whereas IL-4, granulocyte-macrophage CSF, and CSF-1 induce Ia on restricted sets of macrophages. Inhibitors of expression include PGE2, glucocorticoids, and IFN-beta. TNF has been found to augment Ia expression on several macrophage lineage cell lines but to inhibit expression on murine peritoneal macrophages. Our study shows that TNF can have opposite effects on Ia expression (induced by IFN-gamma) on thioglycollate-elicited peritoneal macrophages, depending on the length of time cells are treated and on the presence of other modulators. In particular, TNF augmented early expression induced by IFN-gamma but inhibited later expression. And although TNF synergized with PGE2 to markedly inhibit Ia induction on these cells, it partially antagonized the inhibition by corticosterone and IFN-beta. TNF and PGE2 also synergized to inhibit Ia expression induced on bone marrow-derived and splenic macrophages by either IFN-gamma or IL-4. In contrast to their effect on Ia expression, TNF and PGE2 had opposite effects on expression of gamma 2a FcR in macrophages. TNF blocked the increase in FcR expression due to any combination of PGE2, IFN-gamma, and IFN-beta. However, TNF and PGE2 both increased expression of gamma 2a FcR on WEHI-3 cells. If the different effects of TNF reflect the differentiation states of macrophages, its effects on Ia and FcR expression may vary with the progression of an immune response.     

1,25-Dihydroxyvitamin D3 modulates bone marrow macrophage precursor proliferation and differentiation. Up-regulation of the mannose receptor

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), the biologically active form of vitamin D3, has been shown to inhibit proliferation and promote monocytic differentiation of leukemic cell lines. In the present communication, we extend these observations to normal bone marrow macrophage precursors, and 1) identify the stage of monocytic maturation wherein the steroid exerts its antiproliferative effect, and 2) demonstrate that 1,25-(OH)2D3 promotes bone marrow macrophage differentiation as manifest by specific up-regulation of the lineage-specific membrane protein, the mannose-fucose receptor. In these experiments, the 1,25-(OH)2D3-mediated inhibitory effect on colony formation was shown to be independent of attendant levels of colony stimulating factor-1 and targeted through the adherent bone marrow macrophage precursor. Examination of this steroid-sensitive adherent precursor population demonstrates that its specific binding of 125I-mannose bovine serum albumin spontaneously and progressively increases with time in culture. Whereas adherent bone marrow macrophages cultured for 2 days express 3 X 10(4) mannose receptors/cell, the number of binding sites increases to 7 X 10(4)/cell by day 4. When bone marrow macrophage precursors are exposed to 1,25-(OH)2D3, an additional stepwise enhancement of 125I-mannose bovine serum albumin obtains with time. Four days of culture with the steroid results in 1.6 X 10(5) mannose receptors/cell, a 100% increase as compared to control cells. Neither duration of culture nor exposure to 1,25-(OH)2D3 alters the KD of 125I-mannose bovine serum albumin which approximates 3-5 X 10(-9) ml-1. Finally, the "specificity" of vitamin D-mediated up-regulation of the mannose receptor was established by demonstrating that the steroid does not alter binding of 125I-alpha-thrombin by bone marrow-derived macrophage precursors.     

Unusual distribution of Ia-like antigens on canine lymphocytes

The murine monoclonal antibody 7.2, specific for a framework determinant of human Ia antigens, cross-reacts with canine cell membranes recognizing a bimolecular complex (29 000 and 34 000 daltons) similar to that described in man. We investigated the distribution of these Ia-like antigens on mononuclear cells in peripheral blood, thoracic-duct lymph, marrow, alveolar lavage fluids, lymph nodes, and thymuses from normal dogs. By complement-mediated cytotoxicity and indirect immunofluorescence, virtually all lymphocytes expressing surface immunoglobulin (B lymphocytes), monocytes/macrophages, dendritic cells, and many thymus-epithelial cells were la-positive. Furthermore, most non-B-lymphocytes in peripheral blood, thoracic-duct lymph, and lymph nodes expressed Ia antigens. Alveolar (T) lymphocytes and most thymocytes were la-negative. Generally, fluorescence intensity was higher on monocytes/macrophages and B lymphocytes than on non-B-lymphocytes. In mixed leukocyte cultures and concanavalin A-induced blastogenesis assays, treatment of responder cells with antibody 7.2 and complement abolished proliferation. Proliferative responses could not be restored by adding untreated accessory cells, indicating that cytolytic treatment had eliminated responder T-lymphocytes. However, addition of antibody alone to cultures had no significant effect. These studies indicate that most mature canine T-lymphocytes express la-like antigens. Whether this is an intrinsic property of canine cells or possibly related to continuous in vivo stimulation remains to be determined.