Lymphocyte Proliferation In Cultures Research Articles

Canine Bone Marrow-Derived Mesenchymal Stromal Cells Suppress Alloreactive Lymphocyte Proliferation in Vitro but Fail to Enhance Engraftment in Canine Bone Marrow Transplantation

Stable mixed hematopoietic chimerism has been consistently established in dogs who were mildly immunosuppressed by 200 cGy of total body irradiation (TBI) before undergoing dog leukocyte antigen (DLA)-identical bone marrow (BM) transplantation and who received a brief course of immunosuppression with mycophenolate mofetil (28 days) and cyclosporine (35 days) after transplantation. However, when TBI was reduced from 200 to 100 cGy, grafts were nearly uniformly rejected within 3-12 weeks. Here, we asked whether stable engraftment could be accomplished after a suboptimal dose of 100 cGy TBI with host immunosuppression enhanced by donor-derived mesenchymal stromal cells (MSCs) given after transplantation. MSCs were cultured from BM cells and evaluated in vitro for antigen expression. They showed profound immunosuppressive properties in mixed lymphocyte reactions (MLRs) in a cell dose-dependent manner not restricted by DLA. MSC and lymphocyte contact was not required, indicating that immunosuppression was mediated by soluble factors. Prostaglandin E2 was increased in culture supernatant when MSCs were cocultured in MLRs. The addition of indomethacin restored lymphocyte proliferation in cultures containing MSCs. MSCs expressed CD10, CD13, CD29, CD44, CD73/SH-3, CD90/Thy-1, and CD106/VCAM-1. For in vivo studies, MSCs were injected on the day of BM grafting and on day 35, the day of discontinuation of posttransplantation cyclosporine. MSCs derived from the respective BM donors failed to avert BM graft rejection in 4dogs who received DLA-identical grafts after nonmyeloablative conditioning with 100 cGy TBI in a time course not significantly different from that of control dogs not given MSCs. Although the MSCs displayed in vitro characteristics similar to those reported for MSCs from other species, their immunosuppressive qualities failed to sustain stable BM engraftment in vivo in this canine model.

Involvement of graft-derived interleukin-15 in islet allograft rejection in mice

The possibility that islets play a role in graft rejection during islet transplantation for type-1 diabetes patients holds promise for ex vivo islet manipulation and for specific anti-rejection therapy. Interleukin (IL)-15 is a T cell growth factor and chemoattractant that is expressed by non-T cells. Intragraft expression of IL-15 is elevated during acute rejection in patients and in mice, and systemic blockade of IL-15 in mice prolongs allograft survival. However, the source of IL-15 in these conditions is undetermined. Since epithelial cell-derived IL-15 promotes lymphocyte proliferation in culture, we sought to determine whether islet-derived IL-15 promotes rejection in mice.We designed antisense oligodeoxyribonucleotide molecules that target mouse IL-15. Uptake of FITC-labeled antisense molecules and efficacy of IL-15 inhibition in IFNγ-stimulated islets were evaluated. Islets exhibited typical cytoplasmatic distribution of antisense molecules and produced IL-15 levels that were comparable to non-stimulated cells. Antisense-treated islet allografts, that were transplanted across multiple minor-histocompatibility-antigen mismatched strains of mice, were accepted at a higher rate than control-antisense treated islets or untreated islets (88.9% vs. 37.5% and 20%, respectively). Our results suggest that islet-derived IL-15 may be involved in acute islet allograft rejection.

Target organ localization of memory CD4+ T cells in patients with chronic beryllium disease

Chronic beryllium disease (CBD) is caused by exposure to beryllium in the workplace, and it remains an important public health concern. Evidence suggests that CD4+ T cells play a critical role in the development of this disease. Using intracellular cytokine staining, we found that the frequency of beryllium-specific CD4+ T cells in the lungs (bronchoalveolar lavage) of 12 CBD patients ranged from 1.4% to 29% (mean 17.8%), and these T cells expressed a Th1-type phenotype in response to beryllium sulfate (BeSO4). Few, if any, beryllium-specific CD8+ T cells were identified. In contrast, the frequency of beryllium-responsive CD4+ T cells in the blood of these subjects ranged from undetectable to 1 in 500. No correlation was observed between the frequency of beryllium-responsive bronchoalveolar lavage (BAL) CD4+ T cells as detected by intracellular staining and lymphocyte proliferation in culture after BeSO4 exposure. Staining for surface marker expression showed that nearly all BAL T cells exhibit an effector memory cell phenotype. These results demonstrate a dramatically high frequency and compartmentalization of antigen-specific effector memory CD4+ cells in the lungs of CBD patients. These studies provide insight into the phenotypic and functional characteristics of antigen-specific T cells invading other inaccessible target organs in human disease.

Target organ localization of memory CD4+ T cells in patients with chronic beryllium disease

Chronic beryllium disease (CBD) is caused by exposure to beryllium in the workplace, and it remains an important public health concern. Evidence suggests that CD4(+) T cells play a critical role in the development of this disease. Using intracellular cytokine staining, we found that the frequency of beryllium-specific CD4(+) T cells in the lungs (bronchoalveolar lavage) of 12 CBD patients ranged from 1.4% to 29% (mean 17.8%), and these T cells expressed a Th1-type phenotype in response to beryllium sulfate (BeSO(4)). Few, if any, beryllium-specific CD8(+) T cells were identified. In contrast, the frequency of beryllium-responsive CD4(+) T cells in the blood of these subjects ranged from undetectable to 1 in 500. No correlation was observed between the frequency of beryllium-responsive bronchoalveolar lavage (BAL) CD4(+) T cells as detected by intracellular staining and lymphocyte proliferation in culture after BeSO(4) exposure. Staining for surface marker expression showed that nearly all BAL T cells exhibit an effector memory cell phenotype. These results demonstrate a dramatically high frequency and compartmentalization of antigen-specific effector memory CD4(+) cells in the lungs of CBD patients. These studies provide insight into the phenotypic and functional characteristics of antigen-specific T cells invading other inaccessible target organs in human disease.

Interleukin-15 is the main mediator of lymphocyte proliferation in cultures mixed with human kidney tubular epithelial cells.

Interleukin- (IL) 15 is a potent non-T cell-derived cytokine with IL-2-like activities. Elevated levels of IL-15 expression were observed in biopsies of acutely rejected human kidney grafts. We tested the role of IL-15 in mixed lymphocyte kidney reaction (MLKR) and the effects of immunosuppressive drugs on this reaction. Primary cultures of human tubular epithelial cells (TEC) were stimulated by interferon-gamma and treated with cyclosporin A (CsA, 10-1000 ng/ml), rapamycin (Rapa, 2.5-10 ng/ml), and dexamethasone (Dex, 10-10-10-7 M). IL-15 levels were measured by ELISA. To induce MLKR, we seeded OKT3-prestimulated allogenic human peripheral blood mononuclear cells (PBMC) on a monolayer of TEC in the presence of CsA (25-250 ng/ml), Rapa (0.25-1 ng/ml), and Dex (10-10-10-7 M). PBMC proliferation was quantified by 3H-thymidine incorporation. CsA, Dex, and Rapa had no effect on IL-15 production by TEC. The presence of TEC induced marked proliferation of PBMC. Pretreatment of TEC with IFN-gamma enhanced MLKR in direct correlation with the increased IL-15 levels. MLKR was blocked by anti-IL-15, but not significantly by anti-IL-2 monoclonal antibody. Contrary to Rapa and Dex, CsA failed to inhibit MLKR CONCLUSIONS: IL-15 is a major mediator of lymphocyte proliferation in MLKR. Its production by TEC was unaffected by CsA, Rapa, and Dex. However, IL-15 activity is effectively inhibited by Rapa and Dex but not by CsA. The diversity in the effects of the various drugs is probably related to the different mechanisms. Our results support the possible involvement of renal IL-15 in graft rejection and suggest that resistance to CsA treatment is related to its failure to decrease IL-15 activity.

Novel role of extracellular carbon dioxide in lymphocyte proliferation in culture.

CO(2)/HCO(3)(-) buffering system is indispensable to maintain the pH of culture media for long-term cell culture. Now-a-days, the zwiterionic hydrogen buffer HEPES is widely used as an additional buffer in the commonly used culture media. There are reports on the successful use of HEPES-buffered media, under CO(2)/HCO(3)(-) free conditions, for long-term cell cultures. However, still CO(2)/HCO(3)(-) buffering system is widely used. We aimed at investigating the reason for this. We found that lymphocytes proliferate in response to concanavalin A only in HCO(3)(-)-buffered medium in the presence of 5% CO(2), but not in the HEPES-buffered medium in the absence of CO(2). However, lymphocyte proliferation was observed in HEPES-buffered medium in the presence of 5% CO(2) and in the absence of HCO(3)(-). On the other hand, a low level proliferation was observed in HEPES-buffered medium supplemented with HCO(3)(-) in the absence of CO(2). Supplementation of the culture medium with TCA cycle intermediates and the precursors for the salvage pathway of nucleotide synthesis did not support the lymphocyte proliferation at all. Based on these findings and other reports, we suggest that extracellular CO(2) plays a novel role in cell proliferation.

Garlic Extracts Stimulate Proliferation of Rat Lymphocytes in Vitro by Increasing IL-2 and IL-4 Production

Garlic components are known to modulate certain immune functions. However, mechanisms of their action are not sufficiently elucidated. This study was, therefore, undertaken to examine the effects of aqueous and ethanolic extracts prepared from a garlic powder sample on proliferation of rat spleen Iymphocytes in culture. Cells were stimulated with the combination of phorbol myristate acetate (PMA) and a Ca ionophore (A23187) or R73 monoclonal antibody (mAb) directed to the α β chain of T cell receptor. It has been shown that both extracts significantly stimulated proliferation of lymphocytes. The effect correlated with upregulation of the Interleukin 2 receptor a (IL-2Rα) expression and the increase in IL-2 production. Stimulation of IL-2 production by the extracts was higher in cultures with PMA / Ca ionophore than in cultures with R73 mAb. In contrast, both extracts stimulated production of IL-4 by splenocytes triggered by R73 mAb. The complete dependence of lymphocyte proliferation in cultures with R73 mAb and garlic extracts on IL-2 and IL-4 was demonstrated using neutralising mAbs to IL-2Rα and IL-4. These results suggest that the potentiating effect of garlic extracts on lymphocyte proliferation in vitro differs depending on specific stimulators of cell proliferation and probably on the type of responding cells.

Somatostatin antisense oligodeoxynucleotide-mediated stimulation of lymphocyte proliferation in culture

We have previously shown that somatostatin (SRIF) is synthesized in B and T lymphocytes of rat spleen and thymus and released into the medium of cultured lymphocytes. To determine the role of SRIF in the control of lymphocytes proliferation, the expression of SRIF in normal lymphocytes was inhibited using a 3'-terminal phosphorothioate-modified antisense oligonucleotide complementary to a sequence that includes the translation start site of the rat SRIF messenger RNA. Spleens were obtained from adult male rats, and their lymphocytes were cultured for 24 or 72 h to measure SRIF content and cell proliferation, respectively. For the proliferation studies, [3H]thymidine was incorporated during the final 18 h. The lymphocytes were incubated with 15-30 micrograms/ml SRIF antisense and control antisense. SRIF antisense (25 micrograms/ml) increased lymphocyte proliferation 15-fold (P < or = 0.001), reaching a plateau (25- to 30-fold increase) between 25-30 micrograms/ml SRIF antisense. SRIF was extracted from lymphocytes and measured by RIA. Levels of SRIF content were almost undetectable with 10 micrograms/ml antisense and were significantly lower (P < or = 0.01) with 25 micrograms/ml antisense. When RC 160 (10(-5) M), a SRIF agonist analog, was used in the incubation, the stimulation of cell proliferation exerted by the SRIF antisense was completely abolished. Control antisense had no effect on proliferation or SRIF content. These findings indicate that 1) lymphocytes in culture are able to incorporate SRIF antisense; and 2) SRIF antisense inhibits the expression of lymphocytic SRIF, which leads to lymphocyte proliferation. In conclusion, cell proliferation is dramatically increased by eliminating the expression of SRIF from the lymphocytes, which indicates that in vitro SRIF is acting in a paracrine and/or autocrine fashion to inhibit lymphocyte proliferation.

Somatostatin antisense oligodeoxynucleotide-mediated stimulation of lymphocyte proliferation in culture.

We have previously shown that somatostatin (SRIF) is synthesized in B and T lymphocytes of rat spleen and thymus and released into the medium of cultured lymphocytes. To determine the role of SRIF in the control of lymphocytes proliferation, the expression of SRIF in normal lymphocytes was inhibited using a 3'-terminal phosphorothioate-modified antisense oligonucleotide complementary to a sequence that includes the translation start site of the rat SRIF messenger RNA. Spleens were obtained from adult male rats, and their lymphocytes were cultured for 24 or 72 h to measure SRIF content and cell proliferation, respectively. For the proliferation studies, [3H]thymidine was incorporated during the final 18 h. The lymphocytes were incubated with 15-30 micrograms/ml SRIF antisense and control antisense. SRIF antisense (25 micrograms/ml) increased lymphocyte proliferation 15-fold (P < or = 0.001), reaching a plateau (25- to 30-fold increase) between 25-30 micrograms/ml SRIF antisense. SRIF was extracted from lymphocytes and measured by RIA. Levels of SRIF content were almost undetectable with 10 micrograms/ml antisense and were significantly lower (P < or = 0.01) with 25 micrograms/ml antisense. When RC 160 (10(-5) M), a SRIF agonist analog, was used in the incubation, the stimulation of cell proliferation exerted by the SRIF antisense was completely abolished. Control antisense had no effect on proliferation or SRIF content. These findings indicate that 1) lymphocytes in culture are able to incorporate SRIF antisense; and 2) SRIF antisense inhibits the expression of lymphocytic SRIF, which leads to lymphocyte proliferation. In conclusion, cell proliferation is dramatically increased by eliminating the expression of SRIF from the lymphocytes, which indicates that in vitro SRIF is acting in a paracrine and/or autocrine fashion to inhibit lymphocyte proliferation.

Synergism between corticosteroids and Rapamycin for the treatment of intraocular inflammation

The authors studied the combination effect of corticosteroids with the new immunosuppressant Rapamycin for the treatment of intraocular inflammation. A median-effect analysis, of the combined effect of Rapamycin and dexamethasone, was performed on the inhibition of lymphocyte proliferation in culture. The mathematical formulas allowed for the calculation of combination indices and dose-reduction factors. On the basis of these in-vitro results, treatment with reduced doses of drugs in combination was evaluated in the rat model of experimental autoimmune uveoretinitis. The results showed that Rapamycin and dexamethasone were synergistic over a wide concentration range. The calculated dose reduction factors indicated that an equivalent inhibition of lymphocyte proliferation was achieved with a combination of Rapamycin and dexamethasone reduced by 5-7 and 3-6 fold respectively, compared to the concentration required when each drug was used alone. In animals, there was a significant reduction of the incidence and severity of ocular inflammation with low doses of the drugs in combination. The authors conclude that the observed synergistic effect between Rapamycin and dexamethasone suggests that the use of this drug combination might be advantageous in the treatment of patients with severe uveitis.

A role for transforming growth factor-beta 1 in regulating natural killer cell and T lymphocyte proliferative responses during acute infection with lymphocytic choriomeningitis virus.

The role of transforming growth factor beta 1 (TGF-beta 1) in regulating NK and T cell proliferation during acute viral infections was investigated. After infection of mice with lymphocytic choriomeningitis virus, NK cell proliferation peaks on day 3 and subsides by days 5 to 7 postinfection, whereas T cell proliferation peaks on day 7 and declines by days 9 to 14 postinfection. As TGF-beta 1 has been shown to inhibit lymphocyte proliferation in culture, the production and function of TGF-beta 1 during infection was evaluated in this model. Northern blot analysis demonstrated that the accumulation of TGF-beta 1 transcripts remained relatively constant in total splenic leukocytes during infection. The Mv 1 Lu mink lung cell bioassay was modified and used to evaluate the production of biologically active TGF-beta 1 during infection. Media conditioned with splenic leukocytes isolated from infected mice contained factors that inhibited DNA synthesis by the Mv 1 Lu cells. Low levels of inhibition were observed with conditioned media prepared on day 3 postinfection and high levels of inhibition were observed with conditioned media prepared on days 5 through 14 postinfection. Neutralization with antibodies specific for TGF-beta 1 demonstrated that TGF-beta 1 contributed to the inhibitory activity. As TGF-beta 1 was produced at times coinciding with the decline in NK cell proliferation, the TGF-beta 1 sensitivity of in vivo-elicited NK cells was evaluated. In vitro and in vivo studies demonstrated that NK cell proliferation was extremely sensitive to inhibition by TGF-beta 1. In culture, TGF-beta 1 had an ID50 of 8 pg/ml for inhibiting DNA synthesis by blast NK cells. In vivo, administration of a total of 0.18 micrograms of TGF-beta 1 resulted in a 93% inhibition of NK cell-mediated lytic units per spleen on day 3 postinfection. The inhibition was a result of a block in NK cell proliferation, as administration of TGF-beta 1 profoundly suppressed the appearance of blast size NK cells and the incorporation of [3H] thymidine by NK cell-enriched, blast lymphocyte populations on day 3 postinfection. In contrast to NK cell proliferation, T cell proliferation was not inhibited by up to 100-fold higher concentrations of the factor in vitro or in vivo. Taken together, these data demonstrate that TGF-beta 1 is an important regulator of NK cell proliferation in vivo. Furthermore, the results indicate that differential sensitivity to TGF-beta 1 may contribute to the coordination of NK and T cell responses during viral infections.

Infection with the Intracellular Protozoan Parasite Theileria parva Induces Constitutively High Levels of NF-κB in Bovine T Lymphocytes

The intracellular protozoan parasite Theileria parva causes a lymphoproliferative disease of T cells in cattle and uncontrolled lymphocyte proliferation in culture. We have identified and characterized in infected cells the transcriptional activator, NF-kappa B, whose recognition motifs have been identified in several gene enhancers important for lymphocyte-specific gene expression. NF-kappa B is normally constitutively activated in nuclear extracts derived from B cells and can be induced in T cells and nonlymphoid cells by phorbol esters. Theileria-infected lymphocytes contained constitutively high levels of activated NF-kappa B in nuclear fractions and inactive NF-kappa B in cytoplasmic fractions. The inactive cytoplasmic precursor could be activated by treatment of extracts with deoxycholate, which was shown previously to dissociate NF-kappa B from an inhibitor, I kappa B. Treatment of lymphocyte extracts with 3 mM GTP stimulated NF-kappa B binding to its recognition motif in vitro, thereby distinguishing it from a related nuclear factor, H2-TF1. Selective killing of the parasite, which left the host cells intact, resulted in a rapid loss of NF-kappa B from the nuclear fractions and a slower loss from the cytoplasmic fractions. In parasitized cells, NF-kappa B could not be further stimulated by treatment with 12-O-tetradecanoylphorbol-13-acetate whereas in cells treated to remove the parasite, this compound stimulated elevated levels of NF-kappa B. We propose that high levels of activated NF-kappa B are maintained by the presence of the parasite in infected T cells. Similarly, we propose that the high levels of inactive cytoplasmic precursor are a result of increased synthesis due to the presence of the parasite.

Infection with the intracellular protozoan parasite Theileria parva induces constitutively high levels of NF-kappa B in bovine T lymphocytes.

The intracellular protozoan parasite Theileria parva causes a lymphoproliferative disease of T cells in cattle and uncontrolled lymphocyte proliferation in culture. We have identified and characterized in infected cells the transcriptional activator, NF-kappa B, whose recognition motifs have been identified in several gene enhancers important for lymphocyte-specific gene expression. NF-kappa B is normally constitutively activated in nuclear extracts derived from B cells and can be induced in T cells and nonlymphoid cells by phorbol esters. Theileria-infected lymphocytes contained constitutively high levels of activated NF-kappa B in nuclear fractions and inactive NF-kappa B in cytoplasmic fractions. The inactive cytoplasmic precursor could be activated by treatment of extracts with deoxycholate, which was shown previously to dissociate NF-kappa B from an inhibitor, I kappa B. Treatment of lymphocyte extracts with 3 mM GTP stimulated NF-kappa B binding to its recognition motif in vitro, thereby distinguishing it from a related nuclear factor, H2-TF1. Selective killing of the parasite, which left the host cells intact, resulted in a rapid loss of NF-kappa B from the nuclear fractions and a slower loss from the cytoplasmic fractions. In parasitized cells, NF-kappa B could not be further stimulated by treatment with 12-O-tetradecanoylphorbol-13-acetate whereas in cells treated to remove the parasite, this compound stimulated elevated levels of NF-kappa B. We propose that high levels of activated NF-kappa B are maintained by the presence of the parasite in infected T cells. Similarly, we propose that the high levels of inactive cytoplasmic precursor are a result of increased synthesis due to the presence of the parasite.

Immunosuppressive effects of danazol in vitro

The effects of danazol on leukocyte activation in vitro were compared with those of other known immunosuppressive steroids. Danazol at a concentration of 10(-6) M, the systemic level in women taking 600 mg/day, significantly inhibited lymphocyte proliferation in cultures activated by T-cell mitogens (Concanavalin A and phytohemagglutinin) and allogeneic mixed lymphocyte culture (MLC). Comparable immunosuppressive effects were achieved in these assay systems with dexamethasone at a concentration of 10(-8) M, and with cortisol and progesterone at a concentration of 10(-6) M. Physiologic concentrations of estradiol and testosterone did not significantly affect these immunologic test systems. These studies provide evidence that danazol is immunosuppressive. It is possible that immunosuppressive effects contribute to the efficacy of this drug in the treatment of endometriosis and autoimmune disorders.

Differences in the effect of indomethacin and preincubation on the lymphoproliferative response to concanavalin A of spleen cells from low responder C57BL/6 and high responder BALB/c mice

In vivo treatment with 100 μg of indomethacin each 48 h for 2 weeks enhanced the proliferative response to concanavalin A (Con A) of spleen cells from mice of the C57BL/6 (B6) strain, low responder to T cell mitogens, but did not modify the response of spleen cells from mice of the high responder strain BALB/c (C). The enhancing effect of in vivo indomethacin treatment was more marked in cultures of B6 splenocytes stimulated with high, moderately supraoptimal doses of Con A than in cultures stimulated with optimal mitogen doses. Addition of indomethacin to cultures of spleen cells from untreated donors induced greater increase of the lymphoproliferative response of cells from low responder B6 than from high responder C mice. The enhancing effect of indomethacin added in vitro was observed in cultures stimulated by optimal but not by supraoptimal doses of Con A. The addition of indomethacin did not enhance the response of B6 spleen lymphocytes depleted of adherent cells. Preincubation for 24 h prior to mitogen stimulation increased the response to high Con A doses of spleen cells from low responder B6 mice whereas this procedure did not enhance lymphocyte proliferation in cultures of spleen cells from high responder C mice. Supplementation with indomethacin in vitro combined with preincubation induced additive enhancing effects on DNA synthesis by B6 spleen lymphocytes, suggesting that each treatment acts through different mechanism(s). The results indicated that spleen cells from low responder B6 strain mice are more sensitive than cells from high responder C mice to the potentiating effect of indomethacin and preincubation on the proliferative response to Con A. These observations suggest that mechanisms sensitive to indomethacin and to preincubation contribute to the depression of mitogen induced DNA synthesis in low responder B6 mice.

Functional properties of the 50 kd protein associated with the E-receptor on human T lymphocytes: suppression of IL 2 production by anti-p50 monoclonal antibodies.

Monoclonal antibody 9.6 is specific for a 50 kd T cell surface protein (p50) associated with the sheep erythrocyte (E)-receptor on human T lymphocytes. This antibody interferes with many T cell functions. We have examined the effect of antibody 9.6 on lymphocyte proliferation and interleukin 2 (IL 2) production triggered by mitogens, soluble antigens, and alloantigens to elucidate the mechanism(s) of its immunosuppressive action. At concentrations as low as 50 ng/ml, 9.6 suppressed lymphocyte proliferation and the elaboration of IL 2 by T cells stimulated by PHA, alloantigens, or low concentrations of the phorbol ester TPA (less than or equal to ng/ml). Furthermore, in cultures stimulated by a combination of PHA plus TPA, 9.6 did not inhibit the acquisition of IL 2 receptors but inhibited proliferation and IL 2 production. Immunoaffinity-purified IL 2 completely restored lymphocyte proliferation in cultures inhibited by 9.6. Studies of kinetics of inhibition by 9.6 showed that this antibody inhibited lymphocyte proliferation induced by PHA, alloantigen, and PPD even when added at 24, 48, and 72 hr, respectively, after the initiation of these cultures, suggesting that 9.6 does not block lectin binding or antigen recognition by T cells and that it can inhibit lymphocyte proliferation even after cells have undergone one or more rounds of cell division. A dose-response analysis of lymphocyte proliferation induced by PHA or by TPA demonstrated that the degree of inhibition by 9.6 decreased with increasing concentrations of these mitogens. Antibody 9.6 did not inhibit lymphocyte response induced by optimal concentrations of PHA (50 to 100 micrograms/ml; PHA-M) but inhibited proliferation of maximally induced lymphocytes by using a synergistic combination of low concentrations of PHA (5 micrograms/ml, PHA-M) plus TPA (1 ng/ml). Taken together, these findings indicate that 1) 9.6 inhibits lymphocyte proliferation by affecting IL 2 production, 2) 9.6 does not inhibit the acquisition of 9.6 receptors induced by a synergistic combination of PHA plus TPA, and 3) p50 molecules may be involved in multiple pathways of T cell activation.

Cell cycle kinetics in human lymphocyte cultures.

Short-term cultures of phytohaemagglutinin (PHA)-stimulated human lymphocytes are widely used to detect chromosome-damaging agents, possible human exposure to mutagenic carcinogens and the immune response of blood. Because the results are affected by the number of cell divisions before sampling, an accurate knowledge of lymphocyte proliferation in culture is essential for these studies. Unfortunately, the information available on the lymphocyte proliferative characteristics is quite conflicting. For instance, although after stimulation of blood lymphocytes with PHA the cultures soon contain cells that have divided different numbers of times: this heterogeneity has been explained variously as a difference in cell-cycle times or in the times when the cells start blastogenesis by responding to PHA. Prolonged treatment with high concentrations of 3H-thymidine (TdR) have often been used to investigate lymphocyte proliferation. Incorporated 3H-TdR can, however, affect cell kinetics. The differential staining of sister chromatids in cells dividing for different numbers of times in the presence of bromodeoxyuridine (BUdR) can be used to study cell kinetics. In experiments combining sister chromatid differential staining and autoradiography, we show here that 3H-TdR labelling at more than 0.1 microCi ml-1 slows lymphocyte cycling, and that the heterogeneity of different generations of cells is caused by a difference in the times when they start their first DNA synthesis in response to PHA.

The use of harlequin staining to measure delay in the human lymphocyte cell cycle induced by in vitro X-irradiation

Samples of human peripheral blood were given X-ray doses of 1, 2, 3 or 4 Gy at 37°C with a further sample remaining unirradiated. Lymphocytes were then stimulated to divide in cultures containing BrdU for 40–72 h. After harlequin staining the metaphases were recorded as being in their 1st, 2nd or 3rd post-irradiation division. It was confirmed that irradiation delays the proliferation of lymphocytes in culture. A linear relationship between dose and mitotic delay of approximately 1 h per Gray was obtained. This finding of a small effect on cell proliferation is particularly important for biological dosimetry. All in vivo exposures are more or less non-uniform and the lymphocytes in a blood sample therefore possess a spectrum of induced delay characteristics. However, in the great majority of overdose investigations it should not be necessary to increase the normal culture time for the most highly irradiated cells to reach metaphase. The trend towards using harlequin preparations to ensure that only first-division cells are analysed is briefly discussed and it is noted that in this experiment 2nd-cycle metaphases accounted for a maximum of 14% of the cells scored after 48 h in culture.

Impaired bone marrow lymphocyte proliferation in acute myelogenous leukemia

Bone marrow cells were cultured in liquid media with and without phytohemagglutinin (PHA) to test bone marrow lymphocyte response during the remission period of acute myelogenous leukemia (AML). Mitogen-stimulated cultures from nine leukemic patients while in complete remission showed high percentages of morphologically transformed lymphocytes, but proliferation of these cells was significantly decreased (p less than 0.02) in cultures from six patients who subsequently relapsed (mean remission duration 10 months) and died. In contrast, lymphocyte proliferation in cultures from three AML patients with long remission (greater than 30 months) was comparable to controls. Parallel cultures without added PHA showed a progressive decrease in total viable cell number with time, but an increasing percentage of macrophages in both control and AML cultures. These studies suggest that bone marrow lymphocyte proliferation, but not morphologic transformation, is impaired in some patients with AML, and that identification of this defect may be of prognostic value.

Proliferation of PHA-stimulated lymphocytes measured by combined autoradiography and sister chromatid differential staining

Lymphocyte proliferation in culture was studied by combined [ 3H]TdR incorporation and sister chromatid differential staining. The majority of 1st division metaphases in a 72 h culture commenced DNA synthesis after 48 h and had a cell cycle of less than 24 h. A small proportion of cells from some donors commenced DNA synthesis between 24–30 h and had cell cycle times of up to 48 h. Although many cells entered DNA synthesis at the same time, they showed marked asynchrony in the length of their cell cycle, with some completing one, some two and others three cell cycles in the 72 h culture period. The time taken for cells to enter S following stimulation with PHA ranged from 24 to 48 h and there was considerable variation between donors in the number of fast and slow responding cells.