Stimulation of guinea-pig T cells by concanavalin A (Con A) requires Ia-antigen expressing accessory cells. Such functional accessory cells were identified among the normal epidermal cells and could be fractionated and enriched by centrifugation on discontinuous Percoll density gradients. Epidermal cells collecting at a density of 1.08 g/ml were the most effective in mediating Con A-dependent T cell proliferation, induced the highest activity of T cell growth factors (TCGF) and were enriched fivefold for Ia antigen expressing cells. A monolayer immunosorbent technique yielded a 35-fold enrichment of Ia antigen expressing epidermal cells. This cell fraction induced high levels of TCGF in the culture supernatants. Since the only cells in the normal epidermis expressing Ia antigens are the Langerhans cells, we conclude that the Langerhans cells may act as accessory cells for Con A stimulation of T cells.

Staphylococcal Protein A (SpA) induces T cell growth factor (TCGF) production in cultures of human peripheral blood mononuclear cells (PBMC) with titers equivalent to those induced by PHA. The optimal time and cell concentration for TCGF production were found to be the same for SpA and PHA . TCGF induction by SpA was blocked by addition of human AB-serum or human IgG, as was the mitogenic effect of SpA. An easy and inexpensive procedure is described for the quantitative removal of SpA from TCGF without loss of TCGF activity.

Culture supernatants of murine thymocytes or spleen cells responding in a secondary syngeneic mixed leukocyte reaction (SMLR) were studied for their biologic effects on cell-mediated immune responses in vitro. Such supernatants contained helper factor(s) that facilitated the development of alloantigen-specific cytotoxic T lymphocyte (CTL) responses from thymocyte precursors. Thymocytes, but not spleen cells, required activation by allogeneic effect factor (AEF) in primary culture in order to proliferate and produce biologically active mediator(s) during a secondary SMLR. The same culture supernatants possessed, in some instances, weak T cell growth factor (TCGF; IL 2) activity. However, TCGF activity could be dissociated from helper factor(s) active in the CTL induction assay because some culture supernatants that had potent helper activity were devoid of TCGF activity. This lack of TCGF activity was not due to a lower degree of sensitivity of the TCGF assay or to the presence of a selective TCGF inhibitor in the SMLR-derived supernatants, indicating that the helper factor(s) studied is distinct from TCGF. Production of immunoregulatory lymphokines during the SMLR may serve as a physiologically relevant model for studying the role of T cell-derived lymphokines in immunoregulation.

NK activity of human peripheral blood lymphocytes (PBL) for cells of the human myeloid line K562, and antibody-dependent cellular cytotoxicity (ADCC) of PBL for cells of human lung adenocarcinoma line PC-9 were determined by the human tumor clonogenic assay (HTCA). Incubation of K562 cells or anti-PC-9 serum treated PC-9 cells with PBL before plating inhibited the formation of colonies of these tumor cells. The percent inhibition of tumor cell colony formation was dependent on the effector/target ratio, the incubation time before plating and, in the case of PC-9 cells, on the dilution of anti-PC-9 serum. PBL activated with human T-cell growth factor (TCGF), lymphokine-activated killer (LAK) cells, significantly augmented the inhibition of colony formation of K562 cells, compared to the control lymphocytes. The increase in colony inhibition was dependent on the concentration of TCGF and the time of incubation of PBL with TCGF. The HTCA determining the colony inhibition of K562 cells incubated with LAK or PBL correlated with the 51Cr-release assay (p less than 0.001). The HTCA determining the colony inhibition of anti-PC-9 serum-treated PC-9 cells incubated with PBL also correlated with the 51Cr-release assay (p less than 0.001). We found that the NK activity and ADCC of lymphocytes on K562 and PC9 tumor lines could be detected with HTCA.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on human T lymphocyte lymphocyte colony formation in vitro were investigated. The number of T lymphocyte colonies was increased 4-5 times over that of controls by the addition of TPA (10(-7) - 10(-9) M) to phytohemagglutinin (PHA)-containing cultures. Few colonies were observed when stimulated with TPA in the absence of PHA. In the cultures containing a sufficient amount of exogenous T cell growth factor (TCGF), the enhancement of T lymphocyte colony formation by TPA was not observed. TPA enhanced TCGF production by peripheral lymphocytes stimulated with PHA. The optimal concentrations of TPA for T lymphocyte colony formation were similar to those for TCGF production. These findings suggest that TPA enhanced T lymphocyte colony formation by stimulating endogenous TCGF production. Interestingly, T lymphocyte colony formation was not inhibited even at high concentrations of TPA that usually inhibit myeloid and erythroid colony formation. This difference may be due to different sensitivities to TPA between T lymphocyte colony-forming cells and myeloid and erythroid colony-forming cells.

The receptor for T-cell growth factor (TCGF) is an activation antigen that is present in low amounts on a small fraction of resting T lymphocytes. The TCGF receptor on human T cells can be detected with the anti-Tac monoclonal antibody within 7-12 h of stimulating the cells with phytohemagglutinin (PHA). In the current studies, we examined human lymphocytes cultured alone, with PHA, or with PHA plus sufficient actinomycin-D to inhibit RNA synthesis. After varying intervals, aliquots of the lymphocytes were stained with acridine orange (AO) or pyronin-Y(PY) to measure RNA and/or with anti-Tac plus FITC goat anti-mouse Ig. Tac expression began to increase after 6-8 h incubation with PHA, whereas increases in PY or AO staining were not detected until 12 h or later. Furthermore, the initial increase in Tac expression was not affected by sufficient actinomycin-D to block all detectable nucleic acid synthesis. Therefore, it appears that the initial expression of TCGF receptors detected after lymphocyte activation does not require de novo production of RNA.

Several different types of retroviruses have been shown to inhibit the lectin-driven blastogenesis of co-incubated mouse spleen cells. This abrogation of responsiveness is infection-independent, and can be obtained using live as well as UV-inactivated virus particles. Levels of both interleukin-1 (IL-1) and T-cell growth factor (TCGF) activities are greatly reduced in cultures of virus co-incubated cells. However, the inclusion of indomethacin in the culture medium, together with virus, enables responsiveness to Concanavalin A to occur at moderately efficient levels. The addition of exogenous IL-1 activity to virus co-incubated cells is also partially restorative to Con A-driven lymphocyte responsiveness, while at the same time restoring levels of detectable TCGF activity to near-control levels. Finally, the inclusion of exogenous TCGF activity also leads to efficient lymphocyte mitogenesis but does not result in increased levels of IL-1.

Using conditioned media (CM) from phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBL) we observed long-term selective growth of T-cells from normal human donors. This T-cell growth was continuously dependent on addition of a factor called T-cell growth factor (TCGF). The optimal method for preparing highly active CM from single donor PBL involves the addition of mitomycin C-treated B-lymphoblastoid cell lines to the mixture of PBL and PHA. A number of different cell lines greatly augmented the production of TCGF in 18/18 cases. Preparation of plasma membranes from the Daudi cell line could replace the intact cells in the production of TCGF but those from the cell line, Molt-4, could not. Since the cell surface of Daudi possesses HLA-D antigens but not HLA-A, B, and C, and Molt-4 has HLA-A and B and not HLA-D, it is possible that the Ia antigens (HLA-DRw in man) are important in the release of TCGF. Using this method for growth factor production, an analysis was made concerning the events necessary for lymphocyte activation and the requirements for production and release of TCGF. Removal of PHA 12 h after incubation had no effect on lymphocyte transformation but decreased TCGF release by 90%. In addition, colchicine and cytosine arabinoside inhibited DNA synthesis but had no effect on TCGF release. Little or no TCGF activity was present after cellular protein synthesis was inhibited by puromycin and cycloheximide. These results suggest that TCGF production: a) requires protein synthesis; b) requires binding of the stimulating agent; c) can occur in a non-dividing cell, probably a terminally differentiated T-cell, without the need for cellular proliferation; and d) needs the assistance of an adherent cell which probably is a monocyte-macrophage. The ability to produce TCGF from single human donors will allow better understanding of the nature and action of TCGF.

Treatment of EL-4 lymphoma cells with tetradecanoylphorbol-acetate (TPA), a well-known activator of protein kinase C, induces the production of the T cell growth factor interleukin-2 (IL-2) and the expression of IL-2-specific mRNA within 4-8 h. This system is an ideal model for studies on the induction of a differentiated function in a homogeneous lymphoid cell population by a defined signal. TPA induces also an increase of ornithine decarboxylase (ODC) activity and elevates the intracellular concentrations of putrescine and polyamines within 4-8 h. A similar increase of intracellular putrescine and polyamine concentrations can be achieved by administration of 2 mM putrescine to the culture medium. However, putrescine cannot induce the production of IL-2 in the absence of TPA and cannot reconstitute the IL-2 production in cultures with PGE2 or cyclosporine A, i.e., two well-known immunosuppressive substances which inhibit ODC activity. Putrescine has rather a counter-regulatory effect as concluded from the observation that the TPA-induced TCGF production and IL-2-specific mRNA expression are augmented (superinduced) by the ODC inhibitor D,L-alpha-difluoromethylornithine (DFMO) and again suppressed after the administration of putrescine or polyamines to DFMO-treated cultures. The glycolytic activity, general protein synthesis [( 3H]leucine incorporation), and the cell cycle progression from G2/M to G1, in contrast, are inhibited by DFMO and reconstituted by putrescine. This demonstrates that the cells are able to sacrifice to a large extent several vital functions including their general protein synthesis and to devote themselves at the same time to a fulminant production of their functionally most relevant protein IL-2. This process is downregulated by ODC and its product putrescine. A correlation between increased IL-2 production and accumulation of cells in the G2/M phase was also observed in cultures treated with hydroxyurea or with a combination of amethopterin and adenosine.

Human peripheral blood or tonsil lymphocytes produce T cell growth factor (TCGF), when activated with neuraminidase (NA) and galactose oxidase (GO). Partial purification of NAGO-TCGF on Sepharose G-100 columns gave a TCGF-active fraction within the same molecular weight range as the conventional lectin-induced TCGF (approximately 15,000 Da). Human T cells, activated in mixed lymphocyte culture (MLC) with irradiated allogeneic EB-virus transformed B-cells (LCL) could be maintained in continuous culture for several months with retained functional activities. The cells showed similar growth patterns when cultured in the presence of either NAGO-TCGF or PHA-TCGF. The growing cells were characterized by means of monoclonal antibodies. After 4 weeks of culture 98% of these were OKT3+ and 87% were also OKT8+. The cytolytic activities of the cultures were tested in cell-mediated lympholysis (CML) against allogeneic LCL as target cells, in natural cytotoxicity (NK) against K562 cells and in antibody dependent cytotoxicity (ADCC) against bovine erythrocytes. Cultures displaying one or several of these functions were obtained. The results indicate, that TCGF obtained from supernatants of NAGO-activated lymphocytes is as potent as the T cell growth promoting factor obtained by lectin stimulation. One major advantage of using NAGO-generated TCGF is that contamination with lectin is avoided.

Melanoma represents the single best example of a human tumor that has been shown to elicit specific T-cell reactivity. The responsiveness of some patients with metastatic melanoma to treatment with the prototypic T-cell growth factor (TCGF), interleukin-2 (IL-2), indicates that T cells play a role in antitumor immunity. Interleukin-4 (IL-4), another TCGF that has been administered clinically to humans, was not associated with tumor response in our trials conducted at the Surgery Branch of the National Cancer Institute. Combination trials of IL-2 with IL-4 have shown no increase in responsiveness of melanoma or other tumors when compared to IL-2 alone. However, enhanced expansion of tumor-infiltrating lymphocytes (TILs) in vitro has been observed with combinations of low-dose IL-2 and IL-4. We have begun a study evaluating the trafficking of such expanded lymphocytes following their adoptive transfer in association with systemic administration of IL-2 and IL-4. We have established several TIL cultures from fresh tumor samples, maintained them in long-term culture, and marked them with the neomycin phosphotransferase gene using the LNL6 retroviral vector. Such TILs appear to demonstrate no notable alterations in phenotype or cytolytic activity when compared to their nontransduced counterparts. In addition to IL-2 and IL-4, there are a variety of other novel TCGFs that are now available for evaluation in preclinical and clinical trials. IL-7 induces proliferation and lymphokine-activated killer (LAK) cell activity from human peripheral blood mononuclear cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Fourteen days' culture of human spleen cells with recombinant interleukin-2 (rIL-2) or T-cell growth factor (TCGF) results in the generation of lymphokine-activated killer (LAK) effector cells that have the unique property of lysing natural killer (NK)-resistant human tumor cells, Daudi, and NK-sensitive K562 cells. LAK cells were generated from patients with advanced cancer or liver cirrhosis. The splenic LAK-effector cell types were analyzed by two-color flow cytometry. The rIL-2-induced LAK cells showed an increased proportion of CD8+CD11- and CD57+CD16- and a decreased proportion of CD4+Leu-8- cells. In contrast, TCGF-induced LAK cells revealed a significantly increased proportion of CD8+CD11- and CD4+Leu-8- cells and a decreased proportion of CD57+CD16- cells. Thus, splenic LAK cells with different surface phenotypes were induced by the cultivation with rIL-2 or TCGF. Furthermore, TCGF-induced LAK cell activities in patients with cancer were found to be lower than the rIL-2-induced LAK cell activities. It was noted that the TCGF-activated splenic lymphoid cells did not inhibit the effector process of tumor cell lysis by LAK cells that had been activated by rIL-2. Other mechanisms of lower LAK cell activities of TCGF-activated splenic lymphoid cells from patients with cancer were discussed. The findings suggest that spleens of examined patients with gastric or hepatocellular carcinoma do not seem to be responsible for suppression of cell-mediated antitumor immunity.

T cells from the peripheral blood of patients with chronic myeloid leukemia (CML) were cultured with phytohemagglutinin and T-cell growth factor (TCGF) in agar culture. These T-cell colonies were pooled and expanded further in liquid culture with TCGF and then simultaneously analysed for the E-rosette receptor with the monoclonal antibody OKT11 and for the presence of the Philadelphia (Ph1) chromosome. OKT11 analysis showed these populations to be composed 99.5% or more of T cells. In four of the seven patients the T-cell suspension showed 7/50 (14%), 3/36 (8%), 2/34 (6%), and 4/44 (9%) Ph1 metaphases. Furthermore, Ph1 metaphases were demonstrated in T-cell cultures in two patients when bone marrow metaphases simultaneously showed 90 and 100% Ph1 negative metaphases secondary to human leukocyte interferon therapy or combination chemotherapy. A minority of T cells in benign phase CML have the Ph1 abnormality despite reduced number of Ph1 metaphases in bone marrow from therapy.

A CTL line (CTLL-D4) mediating specific cytolytic activity against radiation-induced leukaemia RL male 1 has been established and maintained on a long-term basis without the addition of exogeneous TCGF. This line was originally selected by the limiting dilution of MLTC cells from RL male 1-immune (BALB/c X C57BL/6) F1-nu/+(CB6F1-nu/+) spleen cells (500 cells/well) in the presence of 5% rat TCGF, 2000 rads-irradiated normal CB6F1-nu/+ spleen cells as the feeder cells, and 10,000 rads-irradiated RL male 1 tumour cells as the stimulator. After expansion only with the feeder and tumour cells, CTLL-D4 shows highly specific cytotoxic activity against RL male 1 by in vitro CMC assay, since cells such as RL male 6, RL female 8, RL female 9, P815, MOPC-315 (H-2d), EL-4 (H-2b) and YAC (H-2a) are not killed. Microcytoxicity assay of this line has revealed that CTLL-D4 comprises three subsets of T lymphocytes (100% Thy-1.2+): 15-25% Lyt-1+23-, 60-75% Lyt-1+23+ and 10-15% Lyt-1-23+. The proliferation of this line seems to depend largely upon the syngeneic MLR-like responsiveness of the Lyt-1+23- subsets of CTLL-D4 to the Ia-positive cells in CB6F1-nu/+ splenic feeder cells, and has been restricted to the H-2d-haplotype of the feeder cells. In spite of the vigorous cell proliferation by coculturing with the feeder cells alone, the cytolytic activity of this line begins to decrease after some 7 days of culture in the absence of the stimulator RL male 1 cells which have no capacity to stimulate by themselves. Thus, by long-term culture of CTLL-D4 with the syngeneic feeder cells alone, a new non-cytolytic line (D4f) was established. Mechanisms enabling the long-term maintenance of CTL activity and subset composition have been discussed in terms of cellular cooperation between the subsets of this line.

We developed a monoclonal antibody (mAb) 211, which recognizes the precursors in peripheral blood of lymphokine-activated killer cells (LAK) induced by recombinant interleukin-2 (rIL-2). In conjunction with complement mAb 211 also eliminates natural killer cells (NK) and a majority of the cytotoxic T lymphocytes. B cells and monocytes do not express the 211 antigen. Since mAb 211 recognized such a large percentage of peripheral blood lymphocytes we examined which 211+ subpopulation was the predominant precursor of rIL-2-induced LAK cells using two-color fluoresence-activated cell sorting (fluorescein-conjugated 211 mAb plus phycoerythrin-CD11b). This method identified the 211+/CD11b+ population as the predominant phenotype of the rIL-2-induced LAK precursor. In addition, we directly compared the phenotype of the LAK precursor induced by delectinated T-cell growth factor (TCGF) to that induced by rIL-2. The 211-depleted population, which was devoid of NK cells and LAK precursors (inducible by rIL-2), was capable of generating LAK activity when TCGF was used as the source of lymphokine. LAK cells induced by TCGF from the 211-depleted population lysed a fresh sarcoma and an NK-resistant cultured melanoma tumor target but not the Daudi cell line, which was lysed by rIL-2-induced LAK cells. Lymphoid subpopulations, depleted using NKH1a mAb, behaved similarly, generating high levels of lysis against the two solid tumor targets when cultured with TCGF but not with rIL-2. CD 3-depleted populations showed enrichment for LAK precursors using either rIL-2 or TCGF. These results indicate that while rIL-2-induced LAK precursors cannot be separated from cells with NK activity, TCGF-induced LAK cells can be generated from populations of peripheral blood mononuclear cells without NK activity.

H-Y-specific and H-2Db-restricted, Lyt-1-2+ T-cell clones ( CTLL ) with graded specific cytotoxic activities on male C57BL/6 (B6) target cells ( 1E3 , ; 2C5 , ++; 2A5 , +, 3E6 , +/-) were tested for their capacity to inhibit the generation of H-Y-specific cytotoxic T lymphocytes (CTL) in vitro. Addition of irradiated lymphocytes of CTLL 1E3 and CTLL 3E6 but not those of CTLL 2A5 or CTLL 2C5 abolished the generation of CTL from in vivo primed H-Y-specific precursor cells (CTLP) when added to fresh mixed-lymphocyte cultures (MLC). Exogenous sources of T-cell growth factors (TCGF) did not overcome suppression. Rather the presence of TCGF resulted in a further enhancement of suppressive activities in CTLL 1E3 and 3E6 and the induction of similar activities in cells from CTLL 2A5 and 2C5 , which by themselves were not inhibitory. Moreover when added to similar MLC on Day 1 instead of Day 0, only irradiated cells of CTLL 3E6 but not those of the other three CTLL were suppressive. Induction of suppressive activities in H-Y-specific CTLL was independent of the appropriate male stimulator cells since it was also observed in MLC induced by irrelevant antigens (H-2, trinitrophenol). Furthermore at low cell numbers, irradiated lymphocytes from any of the CTLL consistently enhanced CTL activities generated from H-Y-specific CTLP. This augmenting activity, which was not TCGF, could be transferred by soluble mediators present in antigen-sensitized CTLL cultures. Thus, these data indicate (i) that cytotoxic effector cells can function as suppressor cells in the generation of CTL, (ii) that the cytotoxic activity of cloned CTL does not correlate with their capacity to suppress CTL responses, (iii) that the inhibition of CTL responses by CTLL is not due to simple consumption of T-cell growth factors produced in MLC, and (iv) that different CTL clones may interfere with the generation of CTL at different stages of their maturation. Moreover, the experiments suggest an antigen-independent enhancement of suppression by the interaction of CTL with lymphokines. Together with the augmenting activity evoked by cloned CTL the data provide strong evidence for the expression of multiple immunological functions by one particular subset of T cells and suggest that cytotoxic effector cells can differentially regulate the maturation and/or clonal expression of their precursor cells.

Research into the composition and function of the immune response in domesticated ruminants has tended to focus on the ovine and bovine systems. With the recent domestication of deer, health problems have developed which require a fundamental knowledge of the immune function in exotic ruminants. In this report it is shown that although recombinant human and mouse interleukin-2 (IL-2) were capable of stimulating cervine T-cell proliferation, optimal proliferation was only achieved using recombinant bovine IL-2. While some phylogenetic restriction of IL-2 cross-reactivity was found, in some cases this could be overcome by using high concentrations of recombinant IL-2. Using cervine T-cell blasts it was possible to assay in vitro T-cell growth factor (TCGF) production by lymphocytes isolated from deer naturally exposed to tuberculosis Mycobacterium bovis). Differences were found in the amount of TCGF present in the supernatants of antigen-activated cells isolated from severely diseased animals, those with limited disease and non-diseased animals.

Approximately 20% of normal blood lymphocytes expressing the T-helper (Leu 3/T 4+) surface phenotype display natural killer (NK)-like features such as cytoplasmic granules and the ability to bind NK-cell targets. In this study, we have assessed the frequency, phenotypic features, and functional capabilities of such cells in a variety of lymphoid malignancies or solid tumors. In each patient group, the percentage of granular lymphocytes within the Leu 3/T 4+ T-helper subset was significantly increased. A large percentage of these cells coexpressed the Leu 7 or Leu 15 marker. When Leu 3+ cells from patients with high proportions of such NK-like cells (or Leu 3+-Leu 15+ cells from selected patients) were isolated with a fluorescence-activated cell sorter, these cells did not proliferate in response to allogeneic cells or T-cell mitogens, nor did they provide help for B-cell differentiation. They also did not suppress T-cell proliferative responses or B-cell differentiation. Freshly prepared Leu 3+ granular lymphocytes did not display NK-cell cytotoxic functions. However, after short-term culture in the presence of phytohemagglutinin (PHA), Leu 3+-Leu 15+ cells expressed T-cell growth factor (TCGF) receptors, had a detectable proliferative response to exogenous TCGF, and acquired the ability to lyse NK-cell targets. These studies demonstrate that, in a variety of malignancies, the lymphocyte subpopulation expressing the T-helper (Leu 3/T 4+) phenotype may be comprised largely of cells with NK-like features and functional capabilities distinct from those of classical helper T cells.

Cortisone-resistant (CR) thymocytes did not generate cytolytic activity toward H-2 K or D alloantigen unless they were also stimulated by H-2 I or non-H-2 alloantigens, even though spleen cells generated brisk cytolytic activity toward H-2 K or D alone. Backstimulation by stimulating strain T lymphocytes accounted for neither the response of spleen cells toward H-2 K or D alloantigen nor the response of CR thymocytes to a full set of alloantigens. In addition, lack of non-T accessory cells did not account for the CR thymocyte pattern of reactivity. Rather, CR thymocytes appeared to be relatively deficient in helper T lymphocytes (HTL). CR thymocytes generated specific cytolytic activity toward H-2 D alloantigen when T cell growth factors (TCGF) or cloned alloreactive helper T lymphocytes were added to culture. CR thymocytes contained fewer HTL precursors detected at limit dilution than spleen cells did. Thus spleen cells generated cytolytic activity toward class I alloantigens alone, but under the same culture conditions CR thymocytes had to be stimulated by both class I and class II alloantigens. Class II alloantigens may be required to stimulate cytolytic activity only under culture conditions in which class I-reactive HTL are not sufficient to provide a minimal threshold of help.

We examined the contribution of MHC class II-restricted T cells (CD4+), MHC class I-restricted T cells (CD8+), gamma/delta T cell receptor (TCR)+ T cells, B cells and macrophages to the development and control of in vitro proliferative responses of bovine lymphocytes to ovalbumin (OA). Cell populations for in vitro assay were obtained from peripheral blood (peripheral blood leukocytes, PBL) of OA-primed cattle. Specific cell populations were depleted or purified from PBL by staining with monoclonal antibodies (MAbs) against the appropriate differentiation antigens and sorting on a Fluorescence Activated Cell Sorter (FACS). OA-specific in vitro responses of in vivo primed PBL were dependent on the presence of CD4+ T cells. Their presence could not be replaced by the inclusion of T cell growth factor (TCGF) in the culture system, indicating that CD4+ T cells probably actively proliferate in response to antigenic stimulation. Bovine CD8+ T cells and gamma/delta TCR+ T cells appeared to exert a suppressive effect on proliferative responses. No proliferation was observed in PBL after the depletion of MHC class II+ cells. In this case, the response could be restored by the addition of macrophages or LPS-activated B cells to the MHC class II- population.

Previous observations in our laboratory indicated that rat serum samples being bioassayed for PRL with the use of Nb2 lymphoma cells produced mitogenic responses greater than maximally effective doses of purified rat PRL. The present studies were conducted to confirm these observations and to determine the possible serum factor or factors responsible for the potentiated response of serum. Twenty five rat serum pools prepared from blood samples obtained from lactating, ovariectomized, and steroid-treated rats were assayed in duplicate aliquots of 0.3-50 microliter against NIDDK-RP-1 rat PRL (11 IU/mg) by the Nb2 bioassay in Fischer's Medium containing 10% horse serum and by conventional double antibody RIA. The mitogenic response of 18 of the 25 pools were clearly greater than the response to standard rat PRL when included in the bioassay at 10-50 microliter/ml of cells. When less than 10 microliter serum pools were assayed, parallelism to the standard was observed; but the concentrations of PRL determined by bioassay were 82 +/- (SE) 4% of the levels determined by RIA in samples from ovariectomized, steroid-treated rats stored for two or four weeks and 62 +/- 2% in samples from lactating rats stored for 3-6 months. Horse serum and hypophysectomized rat serum also potentiated the mitogenic responses to 1 or 5 ng standard rat PRL when added at 10-150 microliter/ml of culture medium that already contained 100 microliter (10%) horse serum. No potentiation of the RIA was observed with rat or horse serum. Insulin, proinsulin, or C peptide of proinsulin (0.02 ng-2 micrograms/ml); fibroblast growth factor, nerve growth factor, epidermal growth factor, insulin-like growth factors I or II (0.002-200 ng/ml), T cell growth factor (TCGF) (0.0002-20 half-maximal units/ml) or platelet-derived growth factor (0.002-10 half-maximal units) did not potentiate the responses to 1 or 5 ng/ml standard rat PRL. Of the growth factors tested, only TCGF was mitogenic to Nb2 cells when placed alone in the medium, but this mitogenic effect of TCGF was not potentiated by horse serum. We conclude that serum can potentiate the mitogenic effect of PRL on Nb2 cells grown in Fischer's medium.

This study was designed to test the process of selecting encephalitogenic T cell lines in the Lewis rat using recombinant human IL-2 (rhIL-2) in comparison to TCGF. The lines were tested for growth, antigen induced proliferation, cytokine production, V-beta 8.2 expression and pathogenicity. We now report that rhIL-2 and TCGF were equally effective in supporting short-term pathogenic T-cell lines with similar proportion of V-beta 8.2 usage. For the maintenance of long term lines, however, TCGF was superior to IL-2. The concentration of rhIL-2 influenced the cultures: 10 units/ml led to more T-cell proliferation than either 2 or 50 units/ml. However, 50 units/ml of IL-2 led to enhanced Th1 polarization. Thus, the type and concentration of growth factors can influence both the propagation of T cells and their phenotype.

The process of activation of T lymphocytes by ionophoric substances was analysed according to current concepts of T-lymphocyte activation. Proliferation induced by the ionophore A23-187 was found to be strictly dependent on the exogenous availability of growth factors. Lack of production of growth factors in spleen cell cultures containing the ionophore A23-187 explained the failure to directly stimulate mouse T-lymphocyte proliferation. It was concluded that the main action of A23-187 is the induction of expression of growth receptors, and that the increase in Ca++ uptake is, in itself, not sufficient to induce mitotic events.

A biphasic curve was observed when surviving allogeneic lytic activity was plotted as a function of irradiation delivered before sensitization. Flow cytometry analysis demonstrated that the number of cells was reduced in response to increasing dose and that subset precursors Lyt 1+2+ were proportionally more radiosensitive than the other subsets. Paradoxically, the presence of exogenous T-cell growth factor (TCGF) in limiting dilution analysis changed the shape of the survival curve, and the mere addition of TCGF or Lyt 2- TCGF-producing cells abrogated the irradiation effect even though they were not shown to be the target of low dose irradiation in flow cytometry analysis. It is proposed that TCGF acted by enhancing the proliferation of surviving cells. This effect may be responsible for the relative radioresistance at higher doses due to enhanced availability of TCGF for the remaining cells.