Rat Spleen Cell Cultures Research Articles

Molecular action mechanism of anti-inflammatory hydrolysates obtained from brewers' spent grain.

Brewers' spent grain (BSG) is a relevant, protein-rich by-product of the brewing process. Protein hydrolysates from different sources exert immune-regulatory actions activating toll-like receptors (TLRs), nuclear factor kappa B (NFκB), and mitogen-activated protein kinases (MAPKs). Effects of gastrointestinal digestion have been poorly studied. Here, we studied the immune-regulatory effect of BSG hydrolysates, and their in-vitro-digested products, on rat splenocytes, macrophages, and T lymphocytes RESULTS: In primary cultures of rat spleen cells, BSG hydrolysates induced interleukin 10 and tumor necrosis factor production in basal conditions. Under stimulation with lipopolysaccharide or concanavalin A, hydrolysates further induced interleukin 10 production. Tumor necrosis factor and interferon-γ were inhibited in lipopolysaccharide- and concanavalin-A-stimulated cells respectively. In vitro gastrointestinal digestion attenuated the observed effects. Splenic macrophages and T lymphocytes behaved in a similar fashion. In spleen cells from TLR2-/- and TLR4-/- mice, immune-regulatory effects were greatly reduced or abrogated. The study of signal transduction pathways indicated a major involvement of NFκB, and the contribution of MAPKs p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinases 1 and 2. BSG hydrolysates, like those obtained from other food sources, regulate the immune response, involving TLR2 and TLR4 and the activation of NFκB and MAPKs, an effect partly maintained after in vitro gastrointestinal digestion. Our data support the hypothesis of a shared, rather unspecific, mechanism of action of protein hydrolysates. © 2020 Society of Chemical Industry.

Serotonin and Apoptosis: Studies on Rat Lymphocytes

Objective: The apoptogenic potential of serotonin (5-hydroxytryptamine) on mitogen-stimulated lymphocytes was studied in primary cultures of rat spleen cells, in two sets of experiments. Methods: Apoptosis was measured by morphologic criteria and flow cytometry. Results: In both assays, serotonin, at a concentration producing half-maximal inhibition of lymphocyte proliferation, significantly increased the basal levels of apoptosis. Conclusion: An apoptotic mechanism should be taken into account when considering immunoinhibition induced by serotonin.

Serotonin and Immune Response: Effect of the Amine on in vitro Proliferation of Rat Lymphocytes

Objective: The effect of serotonin (5-hydroxytryptamine; 5HT) on the in vitro proliferation of mitogen-stimulated lymphocytes was studied in primary cultures of rat spleen cells. Methods: 5HT was added to the cultures 1 h prior to the mitogen, at final concentrations from 10^–13 up to 10^–2M. T and B cell mitogens (concanavalin A, pokeweed mitogen and lipopolysaccharide) were used at suboptimal and optimal concentrations. The cell proliferation was measured 24–72 h after the addition of mitogen. The effect of each 5HT concentration was studied on a group of 6–12 animals and was expressed as a percentage of the control values obtained with mitogen alone. Results: No significant effect of 5HT at concentrations from 10^–13 to 10^–5M was found. At concentrations of ≧10^–4M, a regular dose-dependent inhibition of the lymphocyte proliferation appeared, the concentration producing the half-maximal effect being 6 × 10^–4M. The observed suppression was not due to 5HT cytotoxicity toward spleen cells. Conclusion: With the experimental system used, we failed to confirm an immunostimulatory effect of 5HT in the range of concentrations of its receptor sensitivities or lower, but found a clear-cut immunoinhibitory effect at higher concentrations.

Molecular cloning of htra2-beta-1 and htra2-beta-2, two human homologs of tra-2 generated by alternative splicing.

A yeast two-hybrid screen was performed to find new factors involved in pre-mRNA splicing. Using SC35 as a bait, we isolated a human cDNA bearing high homology to the Drosophila transformer-2 (TRA-2) protein. This cDNA was named htra2-beta1. htra2-beta1 is a nuclear protein that colocalizes with SC35 in a speckled pattern. It interacts with several SR proteins tested in yeast. A second form named htra2-beta2 is generated by alternative splicing. This isoform gives rise to a truncated protein without an SR domain. Both isoforms are evenly distributed throughout adult rat tissue. The ratio of these two isoforms changes after stimulation of primary human T-cell and primary rat spleen cell cultures, indicating that alternative splicing is involved in regulation of htra2-beta activity.

Antibody-dependent neutrophil-mediated killing of Acanthamoeba castellanii

Neutrophils from naive rats lysed low numbers of Acanthamoeba castellanii in the presence of normal rat serum and significantly higher numbers of the parasite in the presence of serum from immunized rats. With normal rat serum, neutrophils from rats immunized with crude parasite extract or from naive rats killed similar percentages of A. caslellanii. However, neutrophils from immunized rats killed a significantly greater percentage of parasites in the presence of serum from immunized rats than was seen with any other combination of serum and neutrophils. The addition of supernatant from cultures of concanavalin A-stimulated rat spleen cells to incubations of the parasite in the presence of neutrophils from naive or immunized rats and immune serum resulted in the highest levels of amoebolysis seen in this study. This study has shown that neutrophils from naive rats or from rats immunized with A. castellanii antigen display a very limited amoebolytic capability which is significantly augmented in the presence of serum from immunized rats and further boosted by the addition of supernatant from Con A-stimulated rat spleen cell cultures.

Target cells are sensitized for cytotoxic T-lymphocyte-mediated destruction by estradiol and tamoxifen.

The effect of estradiol (E2) and tamoxifen (TX) on cytotoxic T-lymphocyte (CTL)-mediated target-cell lysis was studied. CTL was generated in mixed cultures of rat spleen cells, using female Fischer 344 rat cells as responders and female Wistar rat cells or Nb2 rat lymphoma cells as stimulators. Concanavalin-A-stimulated Wistar lymphoblasts or Nb2 cells were used as targets. CTL harvested on day 5 exerted 16-25% cytotoxicity when used at 1:12-1:50 target:effector cell ratios. Day-6 CTL had no cytotoxic activity. Treatment of target cells with either TX or E2, or both, at 1-microM concentrations for 4 hr prior to cytotoxicity testing raised the target-cell killing to 100%. Highly significant enhancement of cytotoxicity was also observed when the drugs were used at 100-, 10-, or 1-nM concentrations. Treatment of effector cells under similar conditions led to inhibition of cytotoxicity at 1-microM concentration, some enhancement at 100 nM and no effect at 10 and 1 nM. When treated target and effector cells were combined, the amplification of target-cell lysis was similar in magnitude to that seen in tests with treated targets only. Drug treatment of target cells had no influence on their resistance to lysis in hypotonic solutions or on total and spontaneous 51Cr release. The inhibition of DNA and protein synthesis in target cells interfered with both basal cytolysis and drug-induced enhancement. The release of the nuclear label, [3H]-thymidine, from Nb2 targets by CTL, was also enhanced by target treatment with TX. These results illustrate that CTL-mediated cytotoxicity is amplified by physiological concentrations (1 and 10 nM) of E2 and equimolar concentrations of TX.

In vitro development of lymphocytes that function as progenitors for mucus-secreting lymphokine-activated killer (LAK) cells.

Two different types of cultures developed when two different interleukin 2 (IL-2) preparations were introduced into cultures of lymph node cells of nu/nu (nude) mice maintained on an embryonic fibroblast monolayer. In the first, human recombinant IL-2 (rIL-2) stimulated the generation of colonies of large cytotoxic cells identified as lymphokine-activated killer (LAK) cells that, when grown on mesenchyme fibroblastoid monolayers prepared from 16- to 18-day embryos, could be triggered to synthesize and secrete flowing mucoid material. In the second culture, crude supernatant from cultures of rat spleen cells stimulated by concanavalin A stimulated the appearance and multiplication of blast cells that, after 20 days, differentiated into lymphocytes. This population was homogeneously composed of "wandering" lymphocytes and could be kept in a stable resting form for at least 2 months without loss of viability. When exposed to rIL-2, this whole lymphocyte population underwent a transformation into blast cells that, on the fourth day, generated granules, became cytotoxic, and differentiated into granular mucus-secreting LAK cells. When low numbers of these transformed premitotic blast cells were plated on mitomycin C-treated embryonic fibroblast monolayers, one cell out of 15 to 20 generated a clone of LAK cells. The study demonstrates that both effector and "memory" arms of differentiation can be stimulated in vitro.

Immunogenicity of human T cell leukemia virus type-I (HTLV-I) antigens for cytotoxic T lymphocytes in the rat system.

We showed previously that WKA (rat MHC, RT-1k) and DA (RT-1a) rat CTL specific for human T cell leukemia virus type-I (HTLV-I) recognized the gag and pX gene-encoded Ag. In the present study, we explored HTLV-I Ag recognized by CTL from other MHC genotype (RT-1l) rats, LEW and F344, and examined whether HTLV-I Ag expressed by recombinant vaccinia viruses (rVV) could prime WKA and LEW rats for HTLV-I-specific CTL. Upon priming in vivo and repetitive stimulation in vitro with HTLV-I+ syngeneic T cells, HTLV-I-specific CD8+ CTL were demonstrated in LEW and F344 rat spleen cell cultures. Interestingly, these CTL were directed against HTLV-I env and pX gene products. Immunization of LEW and WKA rats with the env and gag gene-expressing rVV, respectively, resulted in generation of HTLV-I-specific CD8+ CTL. However, a pX-gene expressing rVV failed to prime either rat strain. In addition, HTLV-I-specific CD8+ CTL from F1 hybrid (WKA x LEW) rats generated by immunization and restimulation with HTLV-I+ syngeneic T cells showed gag and pX Ag specificity on WKA rat cells and env and pX Ag specificity on LEW rat cells. These results suggest that immunogenicity of HTLV-I gag and env Ag in induction of HTLV-I-specific CTL varies depending on MHC, and that the pX Ag expressed by rVV is not a potent immunogen for rats.

Assessment of the inhibitory effect of immunosuppressive agents on rat T cell interferon-γ production using an ELISPOT assay

The immunospot (ELISPOT) assay has proven to be an efficient and sensitive method for the enumeration of single cells secreting antibodies or cytokines. Here we show that the generation of interferon-γ producing cells(IFN-γ pc) in rat spleen cell cultures stimulated with concanavalin A (ConA) is dose-dependently inhibited by a wide variety of immunosuppressants such as cyclosporin A, FK506, hydrocortisone, dexamethasone, azathioprine and ART-18, a monoclonal antibody (mAb) with established immunosuppressive activity in organ transplantation and autoimmunity. The minimal inhibitory concentration (m.i.c.) correlated well with the reported m.i.c. in the mixed lymphocyte reaction (MLR) assay and the therapeutically effective plasma levels in vivo. Our data indicate that the IFN-γ-specific immunospot assay is a powerful tool for determining the potency of immunosuppressive agents in vitro and provides a simple and accurate method for screening large numbers of agents with suspected immunosuppressive properties. The assay may additionally prove to be of value for determining the therapeutically effective doses of immunosuppressants that should be administered in vivo.

The production of growth hormone by subpopulations of rat mononuclear leukocytes

In this study we analyzed the production of GH mRNA and secretion of GH by purified subpopulations of rat lymphoid cells. The data demonstrate that mononuclear leukocytes from various tissues, including spleen, thymus, bone marrow, Peyer's patches, and peripheral blood, all have the ability to produce GH mRNA and secrete GH. Data obtained with cells separated by adherence, nylon wool columns, and positive and negative sorting with monoclonal antibodies that define B, monocyte, T helper and T cytotoxic cells show that several different cell types have the ability to produce GH mRNA. The results suggest that B cells, macrophages, and T helper cells produce more GH mRNA and protein than that of T cytotoxic cells. Natural killer (NK) cells also produce detectable levels of GH mRNA and protein. To validate that leukocyte GH RNA produced in vitro was similar in structure to pituitary GH RNA, we studied the RNA by reverse transcription and the polymerase chain reaction. A sample of the PCR reaction products, analyzed by gel electrophoresis, showed a single major DNA band corresponding in length (600 base pairs) to the distance between the 5′ ends of the two GH-specific primers. The DNA band was specifically labeled with a GH-specific probe after Southern transfer to nitrocellulose. Leukocyte GH purified by immunoaffinity chromatography from culture fluids was shown to be bioactive based on its ability to stimulate the incorporation of tritiated thymidine in primary rat spleen cell cultures. The bioactivity could be blocked with specific antibodies to rat GH. Taken together, the data suggest that there is heterogeneity within lymphocytes regarding their ability to produce GH and are consistent with the idea that GH may be active in local immune responses.

Lack of optimal activation of natural killer levels by interleukin-2 in rat spleen cells: Evidence for suppression

The effect of human recombinant IL-2 on the levels of natural killer (NK) activity in spleen cells derived from BALB/c mice and different strains of rats was studied. Enhancement of murine NK activity in response to IL-2 was readily demonstrable. Levels of NK activity in control as well as IL-2-treated spleen cells from Wistar, Sprague-Dawley, Fischer, and Long-Evans rats increased initially and peaked on Day 1 or 2 of culture. No significant difference between the control and the IL-2-treated cultures was found in this duration. The subsequent fall in NK activity was slower in IL-2-treated cultures of Fischer and Long-Evans rat spleen cells resulting in a significant difference between the NK levels in control and IL-2-treated cells on Day 5 and Day 7 of the culture. In the case of Wistar and Sprague-Dawley rats, the boosting effect of IL-2 on NK levels was either poor or nonexistent. Even though NK activation was poor, spleen cells of all strains of rats did proliferate in response to IL-2, indicating that the IL-2 preparation used was biologically active on rat spleen cells. Rat spleen cells cultured alone or with IL-2 released a factor(s) in the culture supernatant which could suppress the IL-2-induced NK activation in mouse spleen cells. Indomethacin could block the release of suppressor factor by cultured rat spleen cells. Moreover the NK levels in rat spleen cells could be augmented by IL-2 in the presence of indomethacin. These results indicate that the subdued IL-2-induced NK activation in bulk cultures of rat spleen cells could be due to spontaneous release of prostaglandins by the cultured cells.

Properties and characterization of a rat spleen cell-derived factor that induces resistance to natural killer cell lysis in YAC lymphoma cells.

Supernatants of Con A-stimulated rat spleen cell cultures contain a factor that induces relative resistance to NK cell-mediated cytotoxicity in the YAC cell line, a line that is otherwise highly susceptible to murine NK cell-mediated lysis. This NK-lysis resistance-inducing factor (LRIF) has a Mr of 12,600 Da, as determined by gel filtration chromatography, and an isoelectric pH of 4.8. NK-LRIF is heat labile and is de-activated by treatment with proteolytic enzymes. Unlike immune-IFN (IFN-gamma), NK-LRIF is not inactivated by pH 2 treatment, and antibodies capable of neutralizing IFN-alpha and IFN-gamma do not abrogate the effect of NK-LRIF. Highly purified IL-2 preparations lack NK-LRIF activity. NK-LRIF does not induce a general resistance to lysis in YAC cells, because control and NK-LRIF-treated YAC cells were equally susceptible to alloimmune cytotoxic T cells. YAC cells treated with NK-LRIF showed a marked enhancement (5- to 10-fold) in the expression of class I MHC Ag. This observation supports the proposition that the NK susceptibility of target cells could be inversely related to the expression of class I MHC Ag.

A novel cell-surface antigen expressed on most leukocytes and a minor cortisone-resistant population of thymocytes in rats: characterization by monoclonal antibodies.

A cell-surface antigen on rat lympho-hemopoietic cells was determined by using a monoclonal antibody, R2-1B3 (1B3). The 1B3 antibody, when tested for its reactivity with different hemopoietic cells by cytofluorography with a FACS analyzer, labeled more than 80% of lymph node, spleen, and bone marrow cells and 10-20% of thymus cells. Cytofluorographic analysis performed on purified rat T cells, B cells, macrophages, and granulocytes demonstrated that the antigen defined by 1B3 was readily detectable on all of these cell types, with the greatest expression on B cells. A minor population of thymocytes that were labeled by 1B3 appeared to be cortisone-resistant and were located mainly in the thymic medulla. These 1B3 positive thymic cells seemed to be functionally more mature than 1B3-negative thymus cells as suggested by the fact that the cytotoxic treatment of thymus cells with 1B3 antibody and complement (C) resulted in significant reduction of their responsiveness to phytomitogens and lymphokines derived from concanavalin A (con A) activated rat spleen cell cultures. Immunochemical data showed that 1B3 antibody recognized the broad ill-defined band with a molecular weight of 32K to 47K daltons as estimated by SDS-polyacrylamide gel electrophoresis. These data indicate that the 1B3 defined antigen is distinct from other, previously reported, antigens on rat lymphoid cells including leukocyte-common (L-C) and MRC OX-22 antigens, and that this 1B3 antibody is a useful reagent for analyzing the intrathymic differentiation of T cells in rats.

Tumour inhibitory effects of TCGF/IL-2/-containing preparations.

Supernatants from ConA-stimulated rat spleen cell cultures and from cultures of PMA-stimulated murine lymphoma subline EL-4TF were found to contain TCGF and to inhibit growth of a transplantable, MC-induced sarcoma MC11 in syngeneic mice. Tumour-inhibitory effects of the supernatants were dependent on local and repeated administration. Prior to use of the supernatants obtained from PMA-stimulated EL-4TF cell cultures, the dialysable PMA had to be removed; contamination with PMA was found to abolish the tumour-inhibitory effect of the supernatants and to produce enhancement of tumour growth. A significant tumour-inhibitory effect has also been obtained with partially purified TCGF prepared from culture supernatants of cloned EL-4TF cells by ammonium sulphate precipitation, ion-exchange (FPLC) chromatography, and AcA 44 Ultrogel filtration.

Mouse B‐ and T‐Cell Colony Formation in Vitro

Rat spleen cell cultures exposed for 24 h to concanavalin A (Con A-CM) contain, in addition to interleukin 2 (IL-2), factors that promote colony formation in vitro by mouse T cells (TCPA) and B cells (BCPA). TCPA and BCPA are separable on a Sephadex G-75 column. TCPA has a molecular weight of 15,000 daltons and shows the same elution profile as IL-2. Absorption studies with Con A-activated T cells suggested that TCPA and IL-2 are the same entity. BCPA has an apparent molecular weight of 45,000 daltons and stimulates colony formation by B lymphocytes seeded at very low cell density (10(4) - 5 X 10(4) cells/ml). In contrast to TCPA, BCPA can only be demonstrated in gel-filtered material owing to the presence of B colony suppressor activities in crude Con A-CM. Two B colony inhibitory activities were demonstrated by AcA 34 chromatography of crude Con A-CM with molecular weights of 80,000-130,000 and about 50,000, respectively. Because of the specificity, simplicity and sensitivity of B and T colony formation these assay systems should be valuable tools for in vitro testing of biological activities regulating the immune system.

Clonal analysis of helper and effector T-cell function in neonatal transplantation tolerance: clonal deletion of helper cells determines lack of in vitro responsiveness.

Mice rendered tolerant at birth of H-2 alloantigens display concordant in vivo and in vitro phenotypes: they fail to reject skin grafts bearing the tolerated antigens, and their lymphoid cells fail to participate in tolerogenspecific mixed lymphocyte reactions (MLRs) and cell-mediated lympholysis (CML). Tolerant animals normally reject third-party skin allografts and develop positive MLRs and CML to third-party antigens. It has been suggested that clonal deletion of antigen reactive cells is the basis for this spectrum of responses. To investigate further the basis for the lack of in vitro alloreactivity, we conducted limiting dilution studies with lymph node cells from adult mice tolerant of various H-2 disparities. When the frequencies of (a) cells responding to the tolerogen in MLR and (b) interleukin-2-producing cells against the tolerogen were determined, it appeared that both types of cells were functionally deleted, that is, the frequency of cells responding to tolerogen-bearing stimulator cells was identical with that of cells stimulated with syngeneic cells. On the assumption that cells from H-2 tolerant mice are deficient in helper cell activity toward the tolerogen, we performed CML cultures under conditions in which exogenous help was provided in the form of supernatants derived from concanavalin A stimulated rat spleen cell cultures. Lymphoid cells from H-2 tolerant mice generated significant cytotoxicity toward the tolerogen under these conditions, although the absolute level of killing was reduced compared with that of cells from normal mice. Limiting dilution assays confirmed that Tc precursors were present in tolerant mice, and that they were reduced to less than 10% of normal numbers; however, tolerogen-specific Tc precursors were present in frequencies significantly greater than self-reactive Tc precursors. These data indicate that a deletion mechanism operates in neonatal transplantation tolerance to reduce the clone size of all three categories of functional T cells assayed, but that its efficiency is greatest among cells destined to provide specific T-cell help. The absolute functional deletion of helper cells determines the in vitro CML unresponsiveness of lymphoid cells from tolerant mice, and may be a crucial factor in promoting the in vivo phenotype of skin allograft tolerance.

Immunoregulation in the rat: requirements for in vitro B cell responses to classical TI-1 and TI-2 antigens.

The presence of suppressor cells and their mediators has made it difficult to induce B cell mitogenic or immune responses in rat spleen cell cultures. In the present study, we have defined culture conditions required for induction of in vitro thymic independent (TI) immune responses in the rat. Rat spleen cell cultures support low responses to various trinitrophenyl (TNP) haptenated antigens including TNP-Brucella abortus (TNP-BA), TNP-lipopolysaccharide [LPS; either phenol (Ph)- or butanol (Bu)-water extracted], TNP-Ficoll, and TNP-dextran. However, all of these antigens induced good splenic anti-TNP PFC responses when given at appropriate doses in vivo. When spleen cells were depleted of adherent cells and cultured with TI antigens in vitro, good anti-TNP PFC responses were seen with TNP-BA, whereas, lower responses were induced by TNP-LPS (Ph or Bu). No responses were observed in cultures incubated with either TNP-Ficoll or TNP-dextran. Purified splenic B cell cultures [prepared by panning on plates coated with anti-rat F (ab')2] supported good responses to TNP-LPS (Ph or Bu) and TNP-BA. The addition of irradiated splenic adherent cells (macrophages, M phi) to either M phi-depleted or purified B cell cultures completely abrogated in vitro responses to TNP-BA or TNP-LPS (Ph or Bu). Purified splenic B cell cultures generally responded poorly to TNP-Ficoll or TNP-dextran. Addition of indomethacin (IM) to spleen cell cultures abrogated suppression and allowed anti-TNP PFC responses to TNP-BA, TNP-LPS (Ph or Bu), TNP-Ficoll, and TNP-dextran. Furthermore, nude spleen cell cultures treated with IM, also allowed significant TNP-Ficoll and TNP-dextran immune responses; however, untreated cultures did not respond to these antigens. Our studies indicate that rat splenic B cell cultures are responsive to TI antigens, and highest responses occur with the murine TI-1 class, e.g., TNP-BA and TNP-LPS. Inhibition of suppression with IM restored splenic B cell responses to the murine TI-2 class, i.e., TNP-Ficoll and TNP-dextran.

Growth inhibition of an MC-induced mouse sarcoma by TCGF (IL 2)-containing preparations. Preliminary report.

Supernatants from Con A-stimulated rat spleen cell cultures containing T cell growth factor inhibited growth of a transplantable 3-methylcholanthrene-induced sarcoma in syngeneic mice. The tumour-inhibitory effects were dependent on the concentration of T cell growth factor and repeated injections of the supernatants.

The analysis of an anti-fluorescein cytotoxic response.

The detailed kinetics of the appearance of clones of anti-fluorescein cytotoxic T lymphocytes (CTL) in a primary limiting dilution response have been examined. The addition of factors from the supernatants of concanavalin A-stimulated rat spleen cell cultures was necessary to obtain linear dose-response curves and strong primary responses. In a standard primary response the maximum detectable number of specific clones was found, on day 5, to be derived from precursors having a frequency of 1 in 10 000 spleen cells. When limiting dilution cultures were restimulated on day 3, responses on day 5 were unaffected but new clones appeared on day 7 with a frequency of 1 in 1000. The emergence of the day-7 response depends on the addition of factor(s) on day 3. It is proposed that the early low frequency precursors detected on day 5 and the high frequency precursors detected on days 7-9 are derived from two stages in the differentiation lineage of CTL precursors. Restimulation of limiting dilution cultures involves not only the continued clonal expansion of primary responses but also the emergence of new "clones" of specific CTL.

Freezing Of Rat Lymphocytes

Spleen lymphocyte proliferation, as measured by 3H-thymidine incorporation induced by phytohaemagglutinin (PHA), was increased after freeze-thawing with 10% dimethyl sulphoxide. Depletion or intoxication of macrophages in fresh spleen cell preparations also increased lymphocyte proliferation in response to PHA. On the other hand, freezing of macrophage-depleted spleen cell suspensions lowered 3H-thymidine uptake of stimulated cultures. At concntrations above 3%, macrophages added to cultures of fresh purified lymphocytes showed a dose-dependent inhibitory effect on the PHA response, and fresh macrophages were more inhibitory than frozen-thawed macrophages. Purified lymphocytes mixed with 10% macrophages showed a higher response after freeze-thawing. It is concluded that macrophages suppress the lymphoproliferative response to PHA in rat spleen cell cultures, and that these macrophages are more sensitive than lymphocytes to the present freeze-thaw process.