Activities of T cell growth factor (TCGF) of conditioned media from PHA stimulated cultures of peripheral mononuclear cells (PHA-MNC-CM) were compared among patients with cancer, patients with non-malignant disease and normal persons. In nine out of 10 cases of cancer with metastasis (including extensive infiltration to surrounding tissues), the activities were much lower than normal persons, whereas in four out of five cases of cancer without metastasis, the activities were almost the same as those of normal persons. The low activities of PHA-MNC-CM from patients with metastatic cancer were not due to the older age of patients, nor did it seem to be attributed to the malnutritional state of patients. When we examined the activity using non-adherent MNC instead of the whole MNC, the activities in patients with metastatic cancer rose up to almost the normal levels. In normal persons the decline of the activities after 3 days of culture were not observed after the removal of adherent cells. Our data indicate that the adherent cells have a suppressive effect on TCGF production in both normal persons and cancer patients, but that the effect may be exaggerated in patients with metastatic cancer.

Influenza virus immune human T-lymphocyte clones maintained in continuous culture in TCGF were analysed for helper activity and interleukin-2 (IL-2) production. The clones that functioned as helper cells in the production of specific antibody failed to release detectable amounts of IL-2. Conversely, the T cells that produced IL-2 were unable to provide either specific or non-specific helper function. These findings indicated the IL-2 is not an essential component for helper activity. However, phenotypic analysis revealed that both the functional subsets of T-cell clones expressed the helper phenotype in that they were T4+, T3+ and T11+. Nevertheless analysis with other antibodies revealed differences in that the IL-2 releasing clone showed greater staining with the anti-T-cell subset antibodies 9.3 and Leu 8, confirming that there is phenotype as well as functional heterogeneity within the helper inducer T-cell population.

Human T lymphocytes obtained as blasts on day 4 from a primary mixed leukocyte culture (MLC) were cloned in the presence of T cell growth factor (TCGF) and feeder cells. Parameters important in producing higher-specific-activity TCGF were evaluated; irradiation of the responding cells as well as removal of adherent cells or inclusion of indomethacin in the culture was important. In addition, the presence of an irradiated lymphoblastoid cell line (LCL) cell in the TCGF-producing system enhanced activity in the supernate. The long-term maintenance of progeny from clones was achieved by utilizing the LCL autologous with either the responding or sensitizing cells from the initial MLC as feeder cells. Under those conditions, clones could be expanded for 7 or more wk with the maintenance of PLT reactivity. Had all the cells in each clone been maintained for the full 7 wk, more than 1 X 10(10) cells could have been developed in each clone. The cloned reagents provide a higher degree of antigen-specific reactivity than do normal PLT cells. It is to be anticipated that as the requirements for cloning are made more stringent, including the recloning of the cells, these reagents will aid greatly in the dissection of the complexity attendant to HLA-D.

A novel growth factor for bone marrow derived murine mucosal type mast cells has been isolated from the conditioned medium of the Mlsa-reactive mouse Th cell line MLS-4.2. In proliferation assays this growth factor synergizes, like IL-4, with IL-3 on established mast cell lines and was therefore termed MEA: mast cell growth enhancing activity. MEA was characterized as a glycoprotein with a Mr range between 37,000 and 43,000. Apparent homogeneity was obtained by using a four-step purification scheme including cation exchange chromatography, Procion red affinity chromatography, IEF, and gel filtration. Inasmuch as MEA was N-terminally blocked during automated Edman-degradation, peptide fragments after digestion with trypsin were used for partial amino acid sequence determination. All evaluable MEA peptide fragments showed complete sequence homology to a recently purified and cloned novel T cell growth factor (P40/TCGF III), the mouse homologue of human IL-9.

Various retroviruses, including human T-cell lymphotropic virus (HTLV-I) and avian sarcoma virus (ASV), can prevent coincubated human peripheral lymphocytes from responding efficiently to phytohaemagglutinin. Addition of high concentrations of T-cell growth factor (TCGF) activity usually overcomes this inhibition. However, such restoration of responsiveness does not occur in the case of HTLV-coincubated cells.

IL-21 is a key T-cell growth factor (TCGF) involved in innate and adaptive immune response. It contributes to the proliferation of naive, but not memory T lymphocytes. However, the full spectrum of IL-21 activity on T cells remains unclear. Here, we demonstrate that IL-21 primarily maintains the expression of specific naive cell surface markers such as CD45RA, CD27, CD62L and CCR7 on human CD4(+) T lymphocytes and that the expression of CCR7 induces cell migration by means of CCL21 chemoattraction. These effects contrast with those of IL-2 which induced the marked proliferation of CD4(+) T lymphocytes, leading to an activated-memory phenotype. Nevertheless, IL-21 maintained cell cycle activation and expression of proliferation markers, including proliferating cell nuclear antigen and Ki-67, and triggered T-cell proliferation via TCR and co-stimulation pathways. Unlike IL-2, IL-21 decreased the expression of the anti-apoptotic Bcl-2 protein, which correlated with the absence of activation of the phosphatidylinositol 3'-kinase/Akt signaling pathway. Thus, IL-21 is a TCGF whose function is the preservation of a pool of CD4(+) T lymphocytes in a naive phenotype, with a low proliferation rate but with the persistence of cell cycling proteins and cell surface expression of CCR7. These findings strongly suggest that IL-21 plays a part in innate and adaptive immune response owing to homeostasis of T cells and their homing to secondary lymphoid organs.

The proliferative response of nylon wool purified, primed lymph node cells to L. tropica parasites in vitro was found to be restored by the addition of either syngeneic or allogeneic adherent spleen cells as a putative source of macrophages. These results suggested a lack of H-2 restriction in Leishmania-specific T cell responses. However, when T cell blasts generated in vitro in response to the parasite were separated on Percoll density gradients and subsequently maintained for 4 days in the presence of TCGF, their response to L. tropica was found to be strictly dependent on the presence of syngeneic spleen cells. Further studies using congenic recombinant mice demonstrated that proliferation of a parasite-specific blasts required the presence of spleen cells compatible with the responding cells in the I-A region of the MHC. This requirement for I-A compatible adherent cells in the spleen cell populations was further confirmed by a lack of proliferative responses in the presence of spleen cells treated with monoclonal anti-la antibodies and complement. Leishmania-immune F1 blasts responding to the parasite in the context of either parental la-bearing accessory cell could be obtained by positive selection from a F1 hybrid responding cell population. Using flow microfluorometry, the T cell phenotype of the L. tropica-specific blasts was determined to be Thy-1+, lyt-1+, and Lyt-2-.

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

Following the demonstration that hybrids between normal B-lymphocytes and myeloma cell lines continue to secrete antibodies with the same specificity as those produced by the parental B-cells, many groups have tried to use this approach to obtain cell lines expressing T-lymphocyte functions by crossing thymoma lines not expressing any measurable activity with various types of T-cell populations. Although there have been reports that hybrids could be isolated which secrete T-cell products with immunological activity, efforts to produce functionally active hybrids from cytolytic T-cells have all been unsuccessful (refs 6, 7, and M. N. and H. D. Engers, unpublished). We have fused an established, T-cell growth factor (TCGF)-dependent murine cytolytic T-lymphocyte (CTL) line with a mouse thymoma line and have obtained hybrids with cytolytic activity when we selected the hybrids in TCGF-containing medium, while hybrids isolated in the absence of growth factor showed no detectable cytolytic potential.

Conditions for the production of highly effective human T-cell growth factor (TCGF) and its screening in a thymidine incorporation assay are described. Culture medium supplemented with optimum concentrations of TCGF was used in bulk culture systems for the expansion of alloactivated and non-alloactivated T-cell populations. Surface marker studies of cells grown in TCGF-dependent culture showed that the majority were T cells expressing DR antigens. These cells retained their proliferative responsiveness in mixed leucocyte cultures but were unable to stimulate proliferation of normal allogeneic lymphocytes. Cultured cells which derived from mixed leucocyte cultures retained immunological specificity compared with cells not exposed to TCGF both in terms of proliferative responses caused by allogeneic cells in the absence of TCGF, and in terms of cytotoxic activity against 51Cr-labelled target cells. In addition they were capable of suppressing tritiated thymidine incorporation in mixed leucocyte cultures. These experiments show that continuous culture with TCGF can expand a variety of T-cell subpopulations with retention of distinct immunological function.

Large numbers of cells can be recovered from pig intestine with phenotypes suggesting lamina propria rather than intraepithelial origin. Following activation with concanavalin A these cells produced a T-cell growth factor (TCGF) activity which was not inhibited in the presence of a monoclonal antibody recognizing pig interleukin-2 receptors (IL-2R). In contrast, the activity of recombinant human IL-2 and of supernatants from activated spleen cells was almost entirely inhibited by anti-IL-2R. The failure of anti-IL-2R to inhibit the activity of lamina propria-derived TCGF was not apparently owing to interference by soluble receptor with binding of monoclonal to target blast cells as no effect of supernatants on binding was observed. The results suggest that cells derived from the pig intestinal lamina propria fail to produce IL-2 following polyclonal activation in vitro. Consistent with this finding, IL-2 transcripts could be detected by polymerase chain reaction (PCR) following reverse transcription of mRNA derived from spleen and from mesenteric lymph node but not lamina propria lymphocytes, while IL-4 cDNA could be detected from all three sources.

Patients with active Hodgkin's disease (HD) often demonstrate an impaired T-cell proliferative response to phytohemagglutinin (PHA). The present study examined if interleukin regulation of the PHA response was defective in HD. The Hodgkin's PHA response was impaired at all concentrations of PHA utilized. Indomethacin increased the proliferative response but did not bring it to control levels. Stimulation of the cells with both PHA and irradiated Ia+ B cells normalized proliferation despite identical PGE2 concentrations as in the PHA alone cultures. Hodgkin's monocytes produced normal amounts of interleukin 1 (IL-1). Interleukin 2 (IL-2) production by Hodgkin's T cells was decreased in the PHA stimulated cultures, but was normal in the PHA and Ia+ cell stimulated cultures. In response to PHA stimulation alone, Hodgkin's T cells expressed less IL-2 receptor than control cells. The data suggest the diminished PHA response in HD is due to impaired IL-2 production resulting in diminished IL-2 receptor expression. However, when an Ia+ cell source is added to PHA as an additional stimulator, both TCGF production and proliferation are normalized. Monocytes serve to modulate the magnitude of the PHA response through production of both interleukin 1 and PGE2. However, in the presence of sufficient IL-2 production the influence of monocytes is minimized.

A rat monoclonal antibody, obtained after immunization with cells from a cytolytic, TCGF-dependent T cell clone, was found to react with 10 to 20% of murine splenic and lymph node cells and with 65 to 70% of thymocytes. Addition of this antibody to normal spleen cell cultures, or cytotoxic elimination of the cells recognized by the antibody before culture, resulted in the abolishment or profound inhibition of the Con A-induced proliferative responses. The antibody did not react with or inhibit the proliferating, TCGF-reactive T cells, but it inhibited lectin-dependent TCGF production. An analysis of this inhibition, using purified cell populations and the macrophage-derived factor LAF, demonstrated that the cell removed by antibody cytotoxicity was neither the macrophage nor the TCGF-producing T cell, indicating that this antibody eliminates a second helper T cell required for lectin-dependent production of LAF by the accessory cells. These results extend previous observations on the T cell dependence of Con A-induced LAF production and identify a fourth cell type involved in the collaborative processes leading to TCGF-dependent T cell proliferation.

Immunosuppressive acidic protein (IAP, pI 3.0) is a type of alpha 1-acid glycoprotein (alpha 1-AG). The secretion of IAP into the culture fluids of different subpopulations of human peripheral blood leukocytes was examined by a newly devised passive hemagglutination (PHA)-inhibition test. Human peripheral monocytes, an established monoblastoid cell line (THP-1) and peripheral granulocytes produced IAP. However, neither T nor B lymphocytes, nor lymphoblastoid cell lines induced by TCGF or EB virus respectively, produced IAP. The IAP concentration reached a maximum (215 ng/ml) in the culture fluids of peripheral monocytes (1 X 10(6)/ml) when monocytes were stimulated by the addition of either immune complex, carrageenan or endotoxin. The synthesis de novo and shedding of IAP by THP-1 were demonstrated by the immunoprecipitation of radioactive IAP in the culture fluids of [3H]leucine-labeled cells. SDS-polyacrylamide gel electrophoresis of the immunoprecipitates showed two peaks of radioactivity, one comigrated with authentic IAP at 50,000 daltons, and the other at 38,000 daltons, suggesting that two different forms of IAP (and/or alpha 1-AG) are produced from human monocytes.

Cytotoxic T lymphocytes (CTL) against autologous fresh osteosarcoma cells were generated in mixed lymphocyte tumor culture (MLTC), and then were propagated and maintained for up to 19 days with culture medium containing T cell growth factor (TCGF). CTL generated with MLTC and propagated with TCGF (TCGF-CL) lysed fresh autologous osteosarcoma cells, but not autologous phytohemagglutinin-blastoid lymphocytes. More than 90% of the propagated lymphoblastoid cells were E-rosette forming cells. A subpopulation of TCGF-CL was Leu-2a positive and Leu-3a negative CTL. The cytotoxic activity to the autologous tumor detected by 51Cr release assay was not blocked by cold allogeneic tumor cells from 4 out of 5 patients tested. Human leukocyte antigen and chromosomes of TCGF-CL showed no abnormality.

Blood-, lymph node-, and tumour-infiltrating lymphocytes (PBL, LNC, and TIL, respectively) from patients with colonic neoplasms were tested for responsiveness to phytohaemagglutinin (PHA). All populations responded, with LNC and PBL showing comparable reactivities while TIL were less reactive as assessed by incorporation of 3H-thymidine. Increased mitogen responsiveness was observed for T cells enriched by SRBC rosette formation or passage through nylon columns. Mitomycin C-treated LNC and TIL inhibited PHA induced 3H-thymidine incorporation of admixed autologous PBL, suggesting the presence of suppressor cells. Suppressor activity resided primarily in the SRBC rosetting population and was dose-dependent, with increasing numbers of LNC giving greater diminution of PHA response. Suppression by LNC was apparent only when they were added to PBL responders within 6 h of the initiation of stimulation assays, in common with the effects of Concanavalin A (Con A)-induced suppressors on PBL phytomitogen responsiveness. Con A-induced and LNC-suppressor activity could be reversed by addition of lymphocyte-conditioned medium (CM) containing T cell growth factor (TCGF; interleukin IL-2). These data provide further evidence that the suppressor phenomena observed in this system are a function of activated T cells present both in drainage lymph nodes and at the tumour site.

Autologous mixed lymphocyte tumor cell culture (MLTC) induced cytotoxic lymphocytes against autologous tumor cells in 4 out of 14 donors (29%). Further cultivation of MLTC-activated lymphocytes with T cell growth factor (TCGF) resulted not only in the propagation of cytotoxic T lymphocytes (CTL) and an increase of cytotoxicity against autologous tumors, but also in the induction of killer cells against allogeneic tumor cells. Briefly, the results of crisscross tests using fresh tumor cells from 17 donors and lymphocytes from 10 donors indicate that cultivation of MLTC-activated lymphocytes with TCGF generated killer cells against allogeneic tumor cells in most cases, but not against autologous or allogeneic phytohemagglutinin-induced lymphoblasts. These effector cells were mainly OKT3+, OKT8+, OKM1- and Leu7-. Further, the results of cold target inhibition tests undertaken in an autologous tumor killing system suggest that at least 2 different subsets, specifically cytotoxic for autologous tumors and cytotoxic for both autologous and allogeneic tumors, were developed in the CTL induced by autologous MLTC followed by cultivation with TCGF.

The ability of adoptively transferred cytotoxic T cells expanded in T cell growth factor (TCGF) to induce accelerated graft rejection of B10.BR skin grafts from normal B6AF1 recipient mice was tested. In vitro sensitized cells expanded in regular TCGF were capable of accelerating graft rejection when adoptively transferred i.v. 1 day after grafting. Cells expanded in LF-TCGF (lectin-free) were more effective in accelerating graft rejection than cells expanded in lectin containing TCGF. Nonsensitized cells or cells sensitized to irrelevant B10.D2 antigenic determinants in vitro and subsequently expanded in LF-TCGF were unable to induce accelerated rejection of the B10.BR grafts. Expanded cytotoxic effector cells known to be immunologically active in vivo were also unable to accelerate graft rejection if irradiated with 2000 R before adoptive transfer. The ability of cells to lyse the appropriate targets in 4-hr 51Cr release assays correlated poorly with the effectiveness of these cells after in vivo injection. These studies are the first to demonstrate that cytotoxic cells expanded in TCGF are capable of mediating a cytotoxic function in vivo.

Conditions have been defined to measure the in vitro induction of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in human T-lymphocytes by a cell cloning assay. The in vitro growth of mass cultures as well as cell cloning is accomplished by the use of crude T-cell growth factor (TCGF) and irradiated human lymphoblastoid feeder cells. These initial studies employed irradiation of G0 phase peripheral blood mononuclear cells from a single individual. After exposure to gamma-irradiation from a 137Cs source, the cells were stimulated with the mitogen phytohemagglutinin (PHA) and maintained in exponential growth with exogenous TCGF to allow phenotypic expression of the 6-thioguanine-resistant (TGr) mutants. The mutant frequency was determined by measuring cell cloning efficiency in microtiter dishes in the absence and presence of TG, with an optimal selection density of 1 X 10(4) cells/well. The development of this in vitro assay should allow direct study of susceptibility to gamma-irradiation in the human population in terms of both cytotoxicity and mutation induction.

T cells were directly cloned from the cerebrospinal fluid (CSF) of a patient with acute measles encephalitis by limiting dilution in the presence of irradiated feeder cells and T cell growth factor (TCGF). A total of 42 colonies was established. Functional analysis revealed 27 of them to be derived from a cytotoxic T lymphocyte as demonstrated by the ability to exert phytohaemagglutinin (PHA)-dependent cytotoxicity against uninfected allogeneic PHA blasts. Twenty-three of the cytotoxic colonies were specific for measles virus and restricted to self HLA-A or -B antigens. Three clones were also found to give measles virus-specific proliferative responses. The results show that the CSF in measles encephalitis contains a highly enriched population of in vivo sensitized antigen-specific T cells. We propose that the clinical symptoms in measles encephalitis are caused by a T cell-mediated reaction against virus-infected brain cells.

The effect of neonatal thymectomy at various times after birth (Tx-1, Tx-7) on effector and suppressor T cells responsible for cell-mediated cytotoxicity (CMC) for allogenic antigens was determined. Following in-vitro primary mixed lymphocyte cultures, in the absence of T-cell growth factor (TCGF), alloreactive CMC was not detected in spleen cells of Tx-1 mice, but was detected in spleen cells of Tx-7 mice at as high levels as in those of sham-operated mice. However, in the presence of TCGF, as much alloreactive CMC was detected in spleen cells of Tx-1 mice as in those of Tx-7 mice. Furthermore, TCGF production was not detected in spleen cells of Tx-1 mice but was detected in those of Tx-7 mice. In in-vivo experiments, inhibition of allogeneic tumour growth and CMC in spleen cells showed the same pattern as in in-vitro experiments. These results support the concept that the reduction of CMC in Tx-1 mice might be due to a defect in helper function (TCGF-producing capacity) rather than to a defect in cytotoxic T lymphocytes and/or cytotoxic T lymphocyte precursors. Alloreactive suppressor T cells could not be induced in spleen cells of Tx-1 mice but were induced in spleen cells of Tx-7 mice. Therefore, it was suggested that alloreactive suppressor T cells require the presence of the thymus for 7 days after birth in their development.

When the serum content of tissue culture medium is reduced from 10% to 1%, the capacity of T cells to proliferate in response to antigen within that medium is dramatically reduced. Physiological concentrations of platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) are able to partially replace the requirement for serum, in that they are able to increase antigen-driven T-cell proliferation at a serum concentration of 1%. Neither growth factor is mitogenic for T cells in the absence of antigen, and neither is able to act synergistically with T-cell growth factor (TCGF) or IL-2) in the absence of antigen. Antigen-presenting cells (APC) pulsed with antigen in the presence of PDGF or EGF are able to stimulate antigen-specific T-cell proliferation to a greater extent than antigen-presenting cells pulsed in the absence of exogenous PDGF or EGF. Both growth factors increase the expression of MHC Class II antigens on antigen-presenting cells.

The effect of thymus humoral factor (THF) on T cell growth factor (TCGF) production by T cells and the association between splenic macrophages and T cells from young (2-3 months) and old (20-24 months) mice in this respect were studied. Splenocytes were divided into three groups: stimulated with concanavalin A (Con A); preincubated with THF and then stimulated with Con A; or stimulated with Con A and thereafter incubated with THF. These cells were then examined for production of TCGF. Cells treated with Con A and THF as described above were passed on nylon wool to enrich T cell populations and added to mitogen-sensitized (Con A or lipopolysaccharide) adherent splenocytes of old and young mice in the following combinations: young adherent and young T cells; young adherent and old T cells; old adherent and young T cells; and old adherent and old T cells. The results demonstrated that: (A) cells of old mice produced less TCGF than the young; (B) preincubation of splenocytes or nylon-wool enriched T cells with THF increased the production of TCGF consistently in young mice, whereas in the old a significant increase was observed only in some cases; (C) depressed TCGF activity was observed when treatment with THF to splenocytes or nylon-wool enriched T cells from young and old mice was performed after Con A stimulation, and this was also more pronounced in the young; (D) the reduced level of TCGF in the old seemed to be related to a lesion in the T cell compartment, since adherent cells from old and young mouse spleens could support TCGF production by T cells from young mice and not from old.

Adoptive chemoimmunotherapy has cured experimentally induced tumors in animals, but its clinical use has been limited. Six patients were treated with refractory neoplasms in a Phase I study with cyclophosphamide (CPM) and alloactivated haploidentical lymphocytes. Patients received an immunosuppressive dose of CPM (800 mg/m2) followed by haploidentical lymphocytes primed in vitro with alloantigens in mixed lymphocyte culture (MLC). One week later patients received a second infusion of alloactivated lymphocytes expanded in T-cell growth factor (TCGF). The total number of cells given to each patient progressively increased, with a single patient receiving 35.5 X 10(9) cells. Transient febrile responses and delayed-type hypersensitivity reactions at the intravenous sites were the only toxicities noted. A complete clinical response lasting 12 weeks was seen in a single patient with diffuse histiocytic lymphoma. Our experience indicates that adoptive chemoimmunotherapy can be given to patients safely and merits further clinical testing.