Human T-cell leukemia virus type I (HTLV-I) was serially transmitted for 5 passages from rabbit to rabbit by blood transfusion. The virus could be transmitted with 20 ml of whole blood or washed blood cell suspension (fresh or stored for 1-2 weeks at 4 degrees C) but not with cell-free plasma from seroconverted rabbits. Seroconversion occurred 2-4 weeks after blood transfusion and serum anti-HTLV-I titers ranged from 1:20 to 1:640 with the immunofluorescence assay. From transfusion recipients of the 1st to 4th passages, virus-producing cell lines were established by culturing lymphocytes in the presence of T-cell growth factor (TCGF). Three of the 4 cell lines became TCGF-independent after 2-12 months of continuous culture. Blood was transfused between rabbits of opposite sexes and the recipient origin of each cell line was determined by chromosome analysis. We also investigated the effect of X-irradiation (6,000 rad) on blood from seropositive rabbits. Seroconversion likewise occurred in rabbits transfused with blood that had been irradiated immediately before transfusion but not in rabbits transfused with blood that had been irradiated and stored for 1-2 weeks at 4 degrees C. Thus, our rabbit model shows that HTLV-I is serially transmissible by blood transfusion and that this can be prevented by irradiation of blood. The same procedure, therefore, may be useful for the prevention of transfusion-related transmission of HTLV-I in humans.

It has previously been shown that monoclonal antigen-specific mouse CTL lines can be induced to express cytolytic activity with the same specificity as that of splenic natural killer (NK) cells following culture in high concentrations of concanavalin A-induced spleen cell supernatants. In the present experiments, we made use of this in vitro system to explore the regulation of NK activity at the clonal level. Interferon-alpha and interferon-beta and interleukin 2 (IL 2) were potent inducers of NK activity in CTL, demonstrating that these substances can activate NK functions directly without the participation of other cell types. By comparison, IFN-gamma was a poor activator of NK activity in CTL (and also in fresh spleen cells). Three major differences between induction of NK activity by IFN-alpha,beta and IL 2 were noted: IFN induced NK activity selectively without affecting specific cytolysis, whereas IL 2 also enhanced specific killing; IFN acted much more rapidly than IL 2; and IFN did not induce the cells to enter the cell cycle nor were there any obvious morphologic changes. Specific antigen was also a strong inducer of NK activity in CTL, but studies with antisera against the various classes of IFN revealed that this effect was mediated, at least in part, via the release of IFN-beta. By contrast, the same antisera had no effect on NK induction by crude TCGF or by highly purified IL 2, indicating that the regulation of NK activity by IL 2 occurs at the clonal level in an IFN-independent manner. Although, IL 2, IFN, and Ag could apparently act alone to induce NK activity, much greater (synergistic) induction was obtained by various combinations of these regulators, suggesting that the delivery of two (or more) signals to the responder cell was required for full expression of the NK state. As with fresh splenic NK cells, the induced NK state in cloned CTL was intrinsically labile as revealed by its rapid decay in the absence of inducers, but it could nonetheless be maintained indefinitely at very high levels in the continued presence of inducers. This clonal system thus displays a responsiveness to regulatory signals exactly analogous to that of splenic NK cells and provides a unique and exciting opportunity to evaluate the biochemistry of the regulation of NK activity.

Concanavalin A- (Con A) stimulated mouse lymphocytes were separated from unstimulated cell populations by a bovine serum albumin (BSA) density gradient centrifugation 2 days after the stimulation. The stimulated cells divided, but did not initiate a new round of DNA replication when cultured further in the absence of Con A. The divided cells became similar in various properties to unstimulated lymphocytes during a prolonged incubation, suggesting that they entered the resting state. In the present study, the process toward the resting state of lymphocytes from the proliferating state was manifested by: a) a decrease in a cell volume, b) lowering of protein content, c) a loss of responsiveness to T cell growth factor (Interleukin 2) (TCGF(IL2)) probably as a result of disappearance of TCGF(IL2) receptors, d) disappearance of insulin receptors, and e) a duration of time lag between the addition of Con A and the onset of DNA synthesis. In addition, it was found that insulin was not mitogenic for recently divided lymphocytes, but prevented these cells from entering the resting state. This suggests that there may be an in vivo system that keeps stimulated lymphocytes highly responsive to antigens for at least several days without initiating DNA synthesis and cell division.

In general, the human acute T lymphocytic leukemias are composed of malignant expansions of immature T cells lacking membrane receptors for T cell growth factor (TCGF, interleukin 2) and significant immunoregulatory activity. We investigated whether cultured acute lymphocytic leukemic T cell lines can be induced to differentiate and express the Tac antigen, a cell surface protein that contains a TCGF-binding site, after exposure to phorbol 12-myristate 13-acetate (PMA) and/or phytohemagglutinin (PHA). Reactivity of anti-Tac with induced leukemic T cells was studied by three techniques, including: 1) flow microfluorometry; 2) specific binding of [3H]anti-Tac; and 3) receptor immunoprecipitation with anti-Tac and analysis by SDS-PAGE. After exposure to PMA with or without PHA, both JURKAT and HSB-2 acute lymphocytic leukemic T cells displayed Tac antigen within 6 to 8 hr. Induction of receptor expression was blocked by actinomycin D, suggesting a requirement for new mRNA transcription. Induced JURKAT cells contained approximately 7000 Tac molecules per cell, and the binding of anti-Tac to these cells was blocked in a dose-related manner by purified TCGF but not by insulin or purified recombinant interferon-alpha. SDS-PAGE analysis of anti-Tac immunoprecipitates demonstrated that receptors present on induced JURKAT cells were 2000 to 3000 daltons smaller than those present on PHA-activated normal lymphoblasts or induced HSB-2 cells. Induction of JURKAT cells with both PHA and PMA resulted in marked secretion of TCGF as well as the appearance of Tac antigen. After activation of these cells with PMA alone, Tac antigen was similarly expressed, but the level of TCGF synthesis was less than 1% of that obtained after dual induction with PHA and PMA. These data indicate that the signals required for TCGF synthesis and Tac expression are not identical, and furthermore that induction of Tac antigen and TCGF is not obligately linked in these cells.

Activated macrophages are known to release a variety of immunoregulatory substances including the low-molecular-weight substances hydrogen peroxide and lactate. We report here that lactate but not hydrogen peroxide is capable of supporting a substantial production of T-cell growth factor (TCGF) in cultures of accessory cell-depleted splenic T-cell populations after stimulation with concanavalin A. Hydrogen peroxide and its biosynthetic precursor superoxide anion (O2-) mediate, however, a strong augmentation of the TCGF production by accessory cell-depleted T-cell populations in the presence of lactate. Lactate inhibits the incorporation of [3H]thymidine in short-term cultures (18-26 hr) of accessory cell-depleted T cells. This confirms the rule that (optimal) production of T-cell growth factor requires a growth inhibitory signal. Concentrations of hydrogen peroxide which augment TCGF production most effectively (i.e., 1 X 10(-5) M) do not inhibit the incorporation of [3H]thymidine; and higher concentrations (3 X 10(-5)-1 X 10(-4) M) of hydrogen peroxide inhibit both the production of TCGF and the incorporation of [3H]thymidine. In agreement with the augmenting effect of hydrogen peroxide on TCGF production, it was observed that the proliferative response in mixed lymphocyte cultures is suppressed by catalase and augmented by 1 X 10(-5) M H2O2. Proliferative and cytotoxic responses in mixed lymphocyte cultures with an external source of interleukin 2 (IL-2) in contrast, are not augmented by 1 X 10(-5) M H2O2. The relatively high concentration of 1 X 10(-4) M hydrogen peroxide was found to inhibit the proliferative responses in mixed lymphocyte cultures with or without external IL-2 but not the cytotoxic response in the presence of IL-2. This indicates that CTL precursor cells may be relatively resistant against H2O2.

Natural killer (NK) cells are cytotoxic lymphocytes that share numerous cell surface antigens and functional components with T cells. However, in comparison with our knowledge of T cells, little is known about the molecular mechanisms of NK cell activation and function. The following study was initiated as an effort to obtain further information about similarities and differences between NK and T cells at the level of gene expression and also to identify NK-specific cDNA clones for future functional studies of the corresponding gene products. The study used cDNA libraries prepared from an NK clone and from an Epstein-Barr virus transformed B cell lymphoblastoid cell line (LCL). We employed a combination of differential and subtractive hybridization methodologies, which can successfully identify cell-specific cDNA clones representing medium to high abundance transcripts, to identify genes that are expressed in NK cells but not in the LCL. We were particularly interested to ascertain to what extent genes isolated in this manner would be expressed only in NK cells as opposed to being expressed in NK and T cells. Twelve different cross-hybridizing groups were identified that were not expressed in the LCL, and these groups were further characterized: (1) they were used to probe Northern blots prepared from a panel of cells including NK cells, T cells, and B cells: (2) changes in the steady-state level of message following T cell growth factor (TCGF)-induced activation of an NK cell clone were examined for selected isolates; and (3) a partial DNA sequence was determined for each cross-hybridizing group. The DNA sequences of seven groups were identical to previously reported sequences. One group was highly homologous with but not identical to what has been reported as a T cell specific gene, named 519. The DNA sequences of four groups showed no significant homology with the sequences in the GenBank and EMBL databases. The mRNA expression of the newly-identified groups demonstrated several different regulation patterns with respect to cell distribution and level of expression in response to TCGF-activation. Expression of the twelve different genes was examined in three populations of NK cells all of which were CD3- and possessed NK activity. Although these cells differentially expressed the prototype NK markers CD16 and CD56 (the cells were CD16+, CD56-, CD16-, CD56+ and CD16+, CD56+), the expression of all groups of cDNA clones was comparable in the three different types of NK cells despite the phenotypic differences.(ABSTRACT TRUNCATED AT 400 WORDS)

A monoclonal antibody is described which recognises an epitope associated with a receptor for interleukin-2 (IL-2) on pig lymphocytes. The monoclonal antibody inhibits high affinity binding of radiolabelled recombinant human IL-2 (rhIL-2) by pig lymphoblasts and also non-competitively inhibits both pig-TCGF and rhIL-2 maintained proliferation. By flow cytometry the antigen is apparently not present on freshly isolated blood lymphocytes but is detectable on small cells between 6 and 12 h after activation and on large cells by 24-h. These findings are comparable with those obtained using monoclonal antibodies recognising the 55 kDa alpha chain of the human and mouse IL-2 receptor (p55, TAC) expressed on activated cells in vivo and in vitro. However, the molecular weight of the porcine antigen is between 65 and 70 kDa.

In the past 50 years, immunologists have accumulated an amazing amount of information as to how the immune system functions. However, one of the most fundamental aspects of immunity, how the immune system discriminates between self vs. non-self, still remains an enigma. Any attempt to explain this most intriguing and fundamental characteristic must account for this decision at the level of the whole immune system, but as well, at the level of the individual cells making up the immune system. Moreover, it must provide for a molecular explanation as to how and why the cells behave as they do. The "Quantal Theory", proposed herein, is based upon the "Clonal Selection Theory", first proposed by Sir McFarland Burnet in 1955, in which he explained the remarkable specificity as well as diversity of recognition of everything foreign in the environment. The "Quantal Theory" is built upon Burnet's premise that after antigen selection of cell clones, a proliferative expansion of the selected cells ensues. Furthermore, it is derived from experiments which indicate that the proliferation of antigen-selected cell clones is determined by a quantal, "all-or-none", decision promulgated by a critical number of cellular receptors triggered by the T Cell Growth Factor (TCGF), interleukin 2 (IL2). An extraordinary number of experiments reported especially in the past 20 years, and detailed herein, indicate that the T cell Antigen Receptor (TCR) behaves similarly, and also that there are several critical numbers of triggered TCRs that determine different fates of the T cells. Moreover, the fates of the cells appear ultimately to be determined by the TCR triggering of the IL2 and IL2 receptor (IL2R) genes, which are also expressed in a very quantal fashion. The "Quantal Theory" states that the fundamental decisions of the T cell immune system are dependent upon the cells receiving a critical number of triggered TCRs and IL2Rs and that the cells respond in an all-or-none fashion. The "Quantal Theory" accounts fully for the development of T cells in the thymus, and such fundamental cellular fates as both "positive" and "negative" selection, as well as the decision to differentiate into a "Regulatory T cell" (T-Reg). In the periphery, the "Quantal Theory" accounts for the decision to proliferate or not in response to the presence of an antigen, either non-self or self, or to differentiate into a T-Reg. Since the immune system discriminates between self and non-self antigens by the accumulated number of triggered TCRs and IL2Rs, therapeutic manipulation of the determinants of these quantal decisions should permit new approaches to either enhance or dampen antigen-specific immune responses.

Janus kinase 3 (Jak3) is a cytoplasmic tyrosine (Tyr) kinase associated with the interleukin-2 (IL-2) receptor common gamma chain (gamma(c)) that is activated by multiple T-cell growth factors (TCGFs) such as IL-2, -4, and -7. Using human T cells, it was found that a recently discovered variant of the undecylprodigiosin family of antibiotics, PNU156804, previously shown to inhibit IL-2-induced cell proliferation, also blocks IL-2-mediated Jak3 auto-tyrosine phosphorylation, activation of Jak3 substrates signal transducers and activators of transcription (Stat) 5a and Stat5b, and extracellular regulated kinase 1 (Erk1) and Erk2 (p44/p42). Although PNU156804 displayed similar efficacy in blocking Jak3-dependent T-cell proliferation by IL-2, -4, -7, or -15, it was more than 2-fold less effective in blocking Jak2-mediated cell growth, its most homologous Jak family member. A 14-day alternate-day oral gavage with 40 to 120 mg/kg PNU156804 extended the survival of heart allografts in a dose-dependent fashion. In vivo, PNU156804 acted synergistically with the signal 1 inhibitor cyclosporine A (CsA) and additively with the signal 3 inhibitor rapamycin to block allograft rejection. It is concluded that inhibition of signal 3 alone by targeting Jak3 in combination with a signal 1 inhibitor provides a unique strategy to achieve potent immunosuppression.

Mice infected with 5 x 10(3) forms of Trypanosoma cruzi showed a transient, but severe impairment of in vitro spleen cell responses to parasite antigens and to Concanavalin A (Con A). In contrast, inguinal and periaortic lymph node (LN) cells displayed high parasite-specific proliferative responses and only a partial reduction of the Con A-induced proliferation during the acute and chronic phases of infection. Lymphocytes that underwent blastic transformation in T. cruzi-stimulated cell cultures were of the L3T4+ phenotype. Suppression of spleen cell responses occurred in the acute phase whether mice were infected with high (3 x 10(5] or low (5 x 10(3] doses of T. cruzi by intraperitoneal or subcutaneous route. Suppression of the T. cruzi-specific proliferative response of LN cells was only observed in mice infected with high subcutaneous inocula. This suppression, however, was restricted to the LNs draining the site of inoculation without affecting distant LNs. Supernatants from parasite-stimulated proliferating LN cells displayed low or undetectable T cell growth factor (TCGF) activity, in contrast with the high TCGF levels found in supernatants of the same cells stimulated with Con A. Low levels of TCGF were also detected in cultures of LN cells from mice immunized with T. cruzi extracts. Neither the T. cruzi antigen used for in vitro stimulation nor the LN cell supernatants from infected mice inhibited TCGF activity. These findings indicate that (1) parasite-specific responses are present in the LN compartment throughout the acute phase of T. cruzi infection in mice and (2) the proliferative response of L3T4+ LN cells from infected mice to T. cruzi antigens is not associated with a high TCGF secretory response.

We have constructed a general expression vector which allows the synthesis and secretion of processed gene products in Saccharomyces cerevisiae. This vector contains yeast DNA, including the promoter of the mating pheromone (alpha-factor), its downstream leader sequence, and the TRP5 terminator. A cDNA [encoding mature mouse interleukin-2 (IL-2); Yokota et al., Proc. Natl. Acad. Sci. USA 82 (1984) 68-72] was fused immediately downstream to the alpha-factor leader sequence. The resulting recombinant plasmid directed the synthesis of mature mouse IL-2 in S. cerevisiae, with most of the T-cell growth-factor (TCGF) activity secreted into the culture fluid and extracellular space. TCGF activities in the cell extract, as well as in the culture fluid, increased in parallel with cell growth. Production of mature mouse IL-2 was inhibited by tunicamycin (TM), with precursor molecules accumulating in the cell extract. The precursor was processed accurately at the junction between the alpha-factor peptide leader sequence and the coding sequence downstream, yielding mature IL-2. The Mr of the secreted mouse IL-2 determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was 17 kDal, a value expected for the mature mouse IL-2 polypeptide based on the nucleotide (nt) sequence.

We have investigated the mechanisms whereby co-incubation of several types of virus particles with human lymphoid cells in the presence of T cell lectins leads to inhibition of the proliferative response that otherwise ensues. The data indicate that, in the absence of infection, such inhibition can be reversed by the addition to cultures of relatively high concentrations of fluids rich in T cell growth factor (TCGF) activity. The ability of these fluids to achieve such reversal of inhibition is both concentration- and time-dependent. Addition of the factor to virus co-incubated cells more than 26 hr after culture initiation does not restore responsiveness. We have also shown that virus co-incubated cultures are deficient with respect to their ability to synthesize detectable levels of TCGF activity in the presence of phytohemagglutinin. In contrast, the use of relatively dilute virus preparations (less than 10 particles per cell) permits partial responsiveness to lectin as well as the synthesis of moderate levels of TCGF. These finding suggest that viral inhibition of lymphocyte mitogenesis is mediated directly or indirectly by interference with the synthesis of functionally active TCGF activity.

Purified human T cells respond proliferatively to allogenic peripheral blood mononuclear (PBMC) stimulating cells but show no response to murine splenic stimulating cells. Two possible explanations for the lack of xenogeneic response are that human T cells, educated in a human thymus, cannot directly recognize a molecule as disparate as mouse antigen encoded by H-2 and/or that a cytokine(s) produced by the APCs is needed to allow a proliferative response and that the cytokine(s) produced by murine APC do not provide an adequate stimulus to the human T cells under these conditions. We show here that highly purified human T cells can respond directly in an antigen-specific manner to murine stimulating cells if human rIL-1 or rIL-2 or a T cell growth factor (TCGF) preparation are present in the culture. These findings demonstrate that human T cells can recognize murine antigens and that a highly significant response can be obtained if a human cytokine is present to permit that response.

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization (3). Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro (3). Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later and excess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20 degrees C.

T-cell hybridomas have allowed us to define unequivocally a group of 3 distinct molecules, TCGF, T-cell GM-CSF, and PSF, as the products of the activated T-cell. It is becoming increasingly evident that these T-cell-derived molecules, together with a fourth, interferon-gamma, (Wong et al. 1982, 1983), affect a wide range of cell-types. The molecule which we have studied in greatest detail, PSF, probably effects every lineage of non-lymphoid bone-marrow-derived cells. We have evidence that PSF acts in vivo as an important mediator in a pleotropic defence and repair response to antigens that involves all the non-lymphoid elements of the blood. Finally, there is evidence that PSF-dependent cells can become immortal, and that activation and functional expression of the PSF gene can occur in such cells and result in autonomy and tumorigenesis. Clonal sources of T-cell lymphokines and clonal targets for lymphokine assays, formed the basis of recent progress in defining the number and nature of non-antigen-specific T cell products; cloning of the genes coding for these lymphokines should result in a similar impetus to the investigation of the physiology and possible therapeutic role of T-cell lymphokines, and lead to new insights into the control of gene expression and the role of these factors in oncogenesis.

The detailed kinetics of TCGF accumulation in Con A-stimulated spleen cell cultures shows a maximum at 24 hr, with a subsequent decrease in activity. This decrease is not due to the appearance of inhibitory substances "masking" TCGF activity. Pulse experiments show that the rate of TCGF production falls sharply after 18 hr and is completely arrested after 24 hr of Con A stimulation. The arrest in TCGF production is the result neither of culture depletion in medium components nor of limiting accessory cell function or inactivation of the lectin, and it thus seem to be the result of inactivation of TCGF-producing T cells. This regulation is not the result of a TCGF-mediated feedback mechanism but rather of lectin-induced suppressive cells that appear in culture after 24 hr and turn off de novo production of TCGF in fresh cultures.

Interleukin 2 (IL-2), or T-cell growth factor (TCGF), represents the first identified, fully-characterized, purified human interleukin. It is produced mainly by T helper (CD4+) lymphocytes, stimulates cell-mediated immune responses, controls growth and differentiation of B lymphocytes, and intensifies proliferation and activity of all cytotoxic cell clones. IL-2 is a growth factor in vitro and a mediator of self-tolerance in vivo, and therefore interests tumor immunotherapy investigators. The role of IL-2 in the cell cycle of neoplastic cells remains unclear. IL-2 inhibits growth of certain human tumor cells while proliferation of other cells remains intact or is even stimulated. Decreased IL-2 production is often observed in the more advanced clinical stages of human tumors, which provides rational for inclusion of recombinant IL-2 in the immunotherapy for some tumors. On the other hand, tumor cells themselves may produce IL-2 and promote tumor growth. This article summarizes the current physiological role of IL-2 and its role in the pathogenesis of select human diseases. Many papers (including reviews) pertain to the IL-2R receptor. The soluble form of the alpha subunit of the IL-2 receptor (sIL-2Ralpha) is elevated in most proliferative disturbances of the hematopoietic system and in many solid tumors. Special reference to the most important discoveries and our own experience in intracellular detection of IL-2 and IL-2Ralpha are included. IL-2 properties, cellular sources, and targets, including data on its expression in pathological conditions, continue to be supplemented. Attempts to treat tumors are also discussed, using modified varieties of therapy that use IL-2 itself and/or its receptor.

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

T cell growth factors (TCGFs) play a critical role in allograft rejection by promoting the activation and proliferation of alloreactive T cells. To determine whether IL-2 and IL-4 are of quintessential importance in allograft rejection and to identify possible alternative TCGFs, we have bred IL-2(-/-) and IL-4(-/-) double knockout (DKO) mice and studied islet allograft rejection using the DKO mice as allograft recipients. Although mononuclear leukocytes from DKO mice did not mount a proliferative response in vitro in response to anti-CD3 stimulation, crude islet allografts were vigorously rejected by DKO mice (mean survival time 17 +/- 7, n = 8) as compared with wild-type controls (mean survival time 13 +/- 4, n = 7). Treatment of DKO mice with anti-CD3 or rapamycin markedly prolonged the islet allograft survival. An analysis of intragraft cytokine gene transcripts showed robust expression of IL-7 and IL-15. In contrast, intragraft IL-9 gene transcripts were not detected in either wild-type or DKO mice. Provision of exogenous IL-2, IL-4, IL-7, or IL-15, but not IL-9, supports the proliferation of anti-CD3 activated DKO splenic leukocytes in vitro. Blocking the common gamma c of IL-2 receptor, a shared essential signaling component by receptors for IL-2, IL-4, IL-7, IL-9, and IL-15, prolonged the survival of islet allografts in DKO mice. Hence, a T cell dependent allograft rejection enabled by rapamycin-sensitive signals or signals mediated by binding of the gamma c chain occurs in the absence of both IL-2 and IL-4. Non-T cell-derived TCGFs, especially IL-7 and IL-15, may play an active role in supporting allograft rejection.

Lymphokine activities in conditioned medium from activated helper T cell lines are most commonly defined by the proliferation of "specific" lymphokine-dependent cell lines. Various sublines of IL 2-dependent (and ostensibly specific) HT-2 and CTLL cells have now been shown to proliferate in response to BSF-1/IL 4 as well. After activation with antigen or mitogen, D10.G4.1, an antigen-specific cloned T helper cell that has recently been shown to produce IL 4 but not IL 2, secretes two distinct cytokines that induce the growth of HT-2 cells. These "T cell growth factors" (TCGF) can be separated by reversed phase high-performance liquid chromatography (RP-HPLC). The TCGF activity of one of these factors can be blocked by 11B11, an antibody specific for IL 4. The second TCGF activity is not affected by 11B11 or by antibodies specific for IL 2. This TCGF activity can be neutralized by a goat polyclonal antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF), and has a RP-HPLC elution profile identical to that of recombinant GM-CSF. Recombinant GM-CSF induces both proliferation and long-term growth of HT-2 but not CTLL cells, and this activity can be neutralized by the same antibody to GM-CSF. GM-CSF is best known as a factor that induces the maturation and growth of granulocytes and macrophages from bone marrow-derived hematopoietic precursor cells. The ability of GM-CSF to induce the growth of certain T cell lines indicates that this molecule may play a role in T cell-mediated immune responses, either as an autocrine growth factor or a paracrine stimulus from both lymphoid and nonlymphoid tissues that produce this cytokine.

Human T lymphocytes can be maintained in cell culture by the addition of conditioned medium (CM) containing a T cell growth factor (TCGF). This system provides an opportunity to study the presence and modulation of glucocorticoid receptors (GR) by various factors in these cells. GR were present in T cells grown from each of 22 normal individuals; the binding capacity (mean = 3851 +/- 2880 sites/cell) and affinity (Kd = 7.4 X 10(-9)) were similar in rested cultured T cells (CTC) to those reported in peripheral blood T lymphocytes. Treatment of rested CTC with stimuli such as phytohemagglutinin (PHA), CM, or 12-O-tetradecanoylphorbol-13-acetate (TPA) results in a mean increase in GR binding capacity of 3.1-, 3.2- or 2.4-fold respectively without modification of binding affinity. Using an exchange assay to measure occupied and unoccupied GR, we examined the effects of cortisol on its own receptor. Treatment of rested CTC with 10(-7) M cortisol for 24 h decreased GR by more than 50%. Cortisol treatment also blocked the induction of GR by PHA and CM. Since retinoids have been shown to modulate the immune response and to alter the effects of PHA and phorbol esters on lymphocytes, we examined their effects on GR. Retinol decreased GR activity in CTC but only at concentrations which inhibit cell growth. It is concluded that GR activity in human T cells can be modulated by several important factors involved in lymphocyte function.

The antigenic requirements for in vitro proliferation of several cloned continuous lines of H-2-restricted influenza virus-specific cytotoxic T lymphocytes (CTL) has been examined. The cloned CTL lines were established from individual splenic CTL precursors obtained from A/JAPAN/305/57 (H2N2)-immune F1 (C57BL/6 X BALB/c) donors. The lines were isolated (by limiting dilution in liquid culture) and expanded in the presence of A/JAPAN/305/57-infected irradiated syngeneic (F1) spleen cells and T cell growth factor (TCGF) of rat spleen origin. Optimal proliferation (and long-term in vitro cultivation) of these H-2-restricted CTL lines required both specific antigenic stimulation in the form of virus-infected syngeneic spleen cells and an exogenous source of TCGF. In addition, the antigenic requirements for proliferation of these lines directly reflected the pattern of H-2-restricted influenza virus-specific recognition at the level of target cell recognition and lysis.

The cellular mechanisms regulating TCGF production in Con A-stimulated cultures have been investigated. Normal spleen cells, activated by Con A for 24 hr, develop suppressive cells that inhibit de novo production of TCGF by fresh spleen cells. Effector cells mediating suppression are nonadherent, radioresistant, Lyt-2-positive T cells. The induction of suppressor cells is radiosensitive and it requires 18 hr. The kinetics of suppressor cell induction parallels very closely the termination of TCGF production in situ, suggesting the major importance of this mechanism in the control of TCGF production. Reculture of 24-hr Con A-activated cells in the absence of Con A for 24 to 72 hr results in a gradual loss of suppressive activity that can be recalled by readdition of Con A with the same kinetics found in fresh spleen cells. In addition, de novo production of TCGF is readily induced in such cultures upon restimulation with Con A, demonstrating that abrogation of TCGF-production in primary cultures is due to suppression and not to lectin-dependent killing of the TCGF-producing T cells. Measurements of suppressive activity and lectin-dependent cytotoxicity, in various populations of Con A-activated spleen cells, further distinguish these activities. Finally, the reduced suppressive activity of Con A-activated cells after expansion in TCGF excludes that suppressor cells act by absorption or removal of TCGF produced at normal rates. Direct, reversible suppression of TCGF-producing cells by T lymphocytes appears, therefore, to constitute a major mechanism by which cytotoxic T cell responses are regulated.

Supernatants containing T cell growth factor (TCGF) that are produced by incubating human peripheral blood lymphocytes with the mitogenic lectin, phytohemagglutinin (PHA) have previously been shown to allow the successful culture of antigen-reactive T cells. The continued presence of PHA in these preparations, however, complicates the analysis of the direct effects of TCGF. In this report we describe in detail methods used to remove greater than 99% of the exogenous mitogen with a 77% yield of TCGF. TCGF activity precipitated between 50 and 75% saturated ammonium sulfate and was freed of 90% of exogenous PHA. Residual PHA was removed by absorption on affinity columns of Sepharose 4B linked to rabbit anti-PHA antibody. Ninety-nine percent of PHA was removed by these techniques based on assays of the mitogenesis of fresh lymphoid cells and the use of tracer quantities of highly purified radiolabeled PHA. This partially purified TCGF (PP-TCGF) supported the continued growth of mitogen or alloactivated lymphoid cells but not fresh resting cells unless high starting numbers of lymphoid cells are used. An increase of up to 500 times the number of specific lytic units/culture was obtained by expanding alloactivated cells in PP-TCGF when compared to the crude TCGF supernatants.