Generation of activated killer (AK) cells by recombinant interleukin 2 (rIL 2) in collaboration with interferon-gamma (IFN-gamma)

Activation of human peripheral blood mononuclear cells (PBMC) by interleukin 2 (IL 2) and the role of interferon-gamma (IFN-gamma) in the IL 2-induced activation were investigated. Activated killer (AK) cells against NK-resistant tumor cell lines were induced in the medium containing recombinant IL 2 (rIL 2) and autologous serum without any other stimulating agents. AK activity was induced by doses of rIL 2 as low as 3 U/ml, and reached a maximum at 10(3) U/ml. Incubation of PBMC with rIL 2 resulted in IFN-gamma production and augmented NK activity after 1 day of culture, and in induction of AK cells and proliferative response after 2 days of culture. These results suggested that endogenous IFN-gamma was required for rIL 2-induction of AK cells and proliferative response. To prove this, PBMC were cultured with rIL 2 and rIFN-gamma or were pretreated with rIFN-gamma before culture with rIL 2. Both rIFN-gamma treatments of PBMC augmented rIL 2-induced AK activity and proliferative response. rIL 2-induced IFN-gamma production was also enhanced by the rIFN-gamma pretreatment of PBMC. The addition of anti-IFN-gamma antibody to rIL 2 cultures abrogated the rIL 2-induced NK augmentation, AK generation, and proliferative response in proportion to the decreased amounts of endogenous IFN-gamma detectable in culture. rIFN-gamma and/or rIL 2 cultures of PBMC increased Tac antigen expression on cell surfaces as measured by flow cytometry. Enhanced Tac expression by rIL 2 was abrogated by adding anti-IFN-gamma antibody. These data indicate that: 1) AK generation and IFN-gamma production are mediated by IL 2, and 2) IFN-gamma production may be required for IL 2 induction of AK cells and proliferative response. These finding are consistent with the hypothesis that AK generation involves a collaboration between IL 2 and IFN-gamma, in which IL 2 stimulates PBMC to produce IFN-gamma, which in turn acts as a differentiation signal that may be involved in the IL 2-initiated AK generation and proliferative response.     

rIL 2-induced proliferation of human circulating NK cells and T lymphocytes: synergistic effects of IL 1 and IL 2

Cells participating in the rIL 2-induced proliferation of resting PBMC were identified by using different methods of cell purification. NK cells recovered in the light density fraction of Percoll gradients responded, as already known, directly to rIL 2 by strong proliferation. In contrast, large T lymphocytes co-purifying with NK cells, and small T cells sedimenting in the high density area of the Percoll gradients, were virtually unresponsive when cultivated in the sole presence of rIL 2. However, the addition of either irradiated autologous monocytes or highly purified IL 1 allowed both kinds of T cells to undergo cell division. Stringent elimination of possibly contaminating NK cells (NKH-1+) and/or activated T cells (TNKTAR, Tac+, HLA-DR+) from the high density T cells by complement lysis did not impair rIL 2-induced cell proliferation, indicating entire responsiveness of these cells to the synergistic action of IL 1 plus IL 2. Both high density CD4+ and CD8+ participated in this phenomenon, with an apparent advantage for CD4+ cells. All Tac+ cells emerging in a 6-day culture of these cells expressed the WT31 antigen, which indicates that T cells involved in rIL 2-induced proliferation are conventional mature T cells. The relative precursor frequencies of NK cells, large T lymphocytes, and small T lymphocytes that proliferated in response to rIL 2 were analyzed by limiting dilution analysis. The frequencies of clonal growth of NK cells and low density T lymphocytes were approximately the same (1/103 vs 1/185), whereas that of high density T cells was four times lower (1/458). Thus, we clearly demonstrate that resting T cells, defined as such by morphological, density, and phenotypic criteria, are able to proliferate in response to IL 2 in the presence of IL 1 without antigenic or mitogenic triggering.     

Antigenic stimulation regulates the expression of IL 2 receptors in a cytolytic T lymphocyte clone

We have isolated and characterized an H-2Kb-specific CTL clone, B1.8, whose growth is strictly dependent on IL 2 and periodic exposure to stimulator cells. A single, brief encounter with antigen induces on resting B1.8 cells the expression of high-affinity IL 2 receptors, which is maintained for several days, thus allowing cells to undergo at least six rounds of division in the presence of affinity-purified IL 2. We show with the use of monoclonal anti-Lyt-2 or -H-2Kb antibodies, which block specific antigen recognition by B1.8 cells, that continuous antigenic stimulation is not required during this period of growth. After this time, B1.8 cells lose their ability to respond to IL 2 and revert to a resting state, a process that is reversed upon addition of fresh stimulator cells. The decline and subsequent arrest of cell growth correlates with a loss of IL 2 receptors; growth-arrested cells express a 10-fold lower number of receptors than actively growing cells.     

Comparison of the H-2Kb-specific cytolytic T lymphocyte receptor repertoire in Igh recombinant strains

Previous studies in which the H-2Kb-specific cytolytic T lymphocytes (CTL) receptor repertoires were compared for murine strains that express different Igh alleles (C.B-20 and BALB/c) indicated that Igh-linked genes affected the composition of the expressed CTL receptor repertoire. To confirm this genetic localization, and also to individually assess the contributions of Igh-V- and Igh-C-linked genes to CTL repertoire expression, the H-2Kb-specific responses of two Igh recombinant strains, BAB/14 (Igh-Va-Cb) and C.B/R3 (Igh-Vb-Ca), were analyzed. The results indicate that strains which share Igh-V (but not Igh-C) genes demonstrate significant repertoire similarities, whereas strains which share Igh-C (but not Igh-V) are no more similar than strains dissimilar throughout the Igh complex. These results are discussed in the context of the existence of a naturally occurring idiotypic network involving immunoglobulin and CTL receptors.     

Regulation of interleukin 2 receptor expression on a human cytotoxic T lymphocyte clone, synergism between alloantigenic stimulation and interleukin 2

A human T cell clone (termed 40.2.6) established from a rejected human kidney allograft has been studied for its ability to express membrane IL 2 receptors in response to antigen (irradiated cells from the graft's donor) and recombinant IL 2 (rec-IL 2). On antigenic stimulation, the 40.2.6 clone produced low levels (0.15 U/ml) of IL 2 (peak at 24 hr) and incorporated (3H)thymidine (peak at 48 hr). This incorporation was strongly enhanced on addition of rec-IL 2 and was inhibited by the 33B31 antibody, an anti-human IL 2 receptor monoclonal antibody (Mab). The 125I-labeled 33B31 Mab has been used to quantify the density of IL 2 receptors on 40.2.6 cells. Cells not re-exposed to antigen or rec-IL 2 had a level of 33B31-binding sites which declined rapidly (10% of starting value after 2 days). This level remained much more stable when rec-IL 2 (1 U/ml) was present in the medium (80% at day 2). Antigen induced a three- to eightfold increase in the level of 33B31-binding sites which peaked at 24 hr and then declined. When a similar antigenic stimulation was performed in the presence of rec-IL 2 (1 U/ml), the level of 33B31-binding sites peaked at a higher value (eight- to 20-fold increase at day 2), and its subsequent decline was slower. These potentiating effects of rec-IL 2 were dose-dependent and occurred at low concentrations corresponding to the saturation by rec-IL 2 of high affinity IL 2 receptor sites. Finally, high affinity IL 2 receptors, as measured by the binding of 35S-labeled rec-IL 2, were found to be similarly up-regulated by antigen and rec-IL 2. Together, our results obtained on a monoclonal human T cell population with highly purified rec-IL 2 demonstrate that rec-IL 2 and antigen act in synergy to induce the expression of both high and low affinity membrane IL 2 receptors.     

Species-specificity of T cell stimulating activities of IL 2 and BSF-1 (IL 4): comparison of normal and recombinant, mouse and human IL 2 and BSF-1 (IL 4)

Mouse and human interleukin 2 (IL 2) both cause proliferation of T cells of the homologous species at high efficiency. Human IL 2 also stimulates proliferation of mouse T cells at similar concentrations, whereas mouse IL 2 stimulates human T cells at a lower (sixfold to 170-fold) efficiency. In contrast, the T cell stimulating activities of mouse and human B cell stimulatory factor 1 (interleukin 4; IL 4) appear to be species specific over the range of concentrations tested; we detected no activity of mouse IL 4 on human T cells, or human IL 4 on mouse T cells.     

Leu-11+ lymphocytes with natural killer (NK) activity are precursors of recombinant interleukin 2 (rIL 2)-induced activated killer (AK) cells

Precursors of activated killer (AK) cells cytotoxic for human noncultured metastatic melanoma and colon carcinoma were characterized. These cells required 3 days incubation with recombinant interleukin 2 (rIL 2) and DNA synthesis for the induction of AK activity. Both negative and positive cell purification methods were used to identify the subpopulation of cells containing AK precursors. By complement-mediated cell depletion studies, AK precursors were largely present in the Leu-11+ fraction, and to a much lesser extent in the Leu-7+ and Leu-2a+ fractions; they were absent in Leu-3a+ and Leu-4+ cells. Lymphocyte subpopulations were then purified with a cell sorter to positively select for the subset containing AK precursors. Leu-11+ cells had the highest level of AK activity and proliferative response when cultured for 3 days with rIL 2 as well as the highest level of NK activity before culture. Leu-7+ cells had neither AK activity nor a proliferative response when cultured with rIL 2, although they still possessed high NK activity. The same levels of AK and NK activity were found in Leu-2a+ and Leu-2a- fractions, but both activities were absent among Leu-4+ and Leu-3a+ cells. Further fractionation with a two-step sorting technique showed that the highest AK activity resided in the Leu-7-Leu-11+ cell fraction. Morphologically, this subfraction was granular lymphocytes. Titration experiments or rIL 2-responsive cells showed that the number of cells required to achieve a comparable level of rIL 2 proliferative response were as follows: 35 X 10(3) cells from unseparated PBL, 10 X 10(3) cells from Leu-11+ cells, 3.3 X 10(3) from Leu-7-Leu-11+ cells, and 640 X 10(3) cells from Leu-7+ cells. These results indicate that the lymphocyte subpopulation that proliferates in the presence of rIL 2 and then develops AK activity was a subpopulation of Leu-11+ granular lymphocytes, which also possessed the highest NK activity. These Leu-11+ cells lacked the antigens defined by the Leu-7, Leu-3a, or Leu-4 antibodies. Although Leu-7+ cells did not respond to rIL 2 by themselves, they may play a role in the induction of AK activity.     

Monoclonal antibody OKT11A inhibits and recombinant interleukin 2 (IL 2) augments expression of IL 2 receptors at a pretranslational level

The expression of receptors for interleukin 2 (IL 2) represents a critical event regulating the growth of normal T lymphocytes. We investigated the effects of the inhibitory monoclonal antibody OKT11A (anti-sheep erythrocyte receptor) and of purified recombinant IL 2 (rIL 2) on the expression of IL 2 receptors by activated T cells at both the protein and the mRNA levels. Adding OKT11A antibody (0.5 microgram/ml) to phytohemagglutinin (PHA)-stimulated cultures of human peripheral blood mononuclear cells (PBMC) markedly suppressed cellular proliferation (assessed by [3H]thymidine incorporation) and IL 2 receptor expression (determined by immunofluorescence assay by using the anti-IL 2-receptor antibody, anti-Tac). Northern blot analysis performed with the use of a cDNA probe specific for the human IL 2 receptor gene demonstrated that OKT11A antibody also decreased the accumulation of IL 2 receptor mRNA induced by PHA in PBMC. Purified rIL 2 (10 U/ml) alone had little effect on the expression of IL 2 receptors in unstimulated PBMC cultures. In combination with PHA or with PHA plus OKT11A, however, rIL 2 augmented both the expression of IL 2 receptor protein on PBMC and the accumulation of IL 2 receptor mRNA in PBMC. Adding anti-Tac antibody to PBMC cultures to block the interaction of IL 2 with its receptor diminished the accumulation of IL 2 receptor mRNA induced by PHA. Taken together, these data demonstrate that OKT11A antibody inhibits and IL 2 augments expression of IL 2 receptors on PHA-stimulated T cells, at least in part, at a pretranslational level.     

LPS and specific T cell responses: interleukin 1 (IL 1)-independent amplification of antigen-specific T helper (TH) cell proliferation

Lipopolysaccharide (LPS) fraction of endotoxin induces a significant potentiation of the antigen-specific proliferative response of T helper (TH) cell lines. This effect was obtained with LPS from different bacterial sources and reproduced with the lipid A moiety of endotoxin. Purified adherent spleen cells used as antigen-presenting cells (APC) support this LPS-enhanced TH cell proliferation. In addition, the effect of endotoxin on specific TH cell responses was found to be absolutely dependent on the interaction between TH lymphocytes and APC through antigen-specific recognition. Thus, it was not observed in the absence of specific antigen or when monoclonal antibodies against class II MHC products or against L3T4 antigens were used to inhibit the T cell-APC interaction. Similarly, it was found that APC from the B6.CH-2bm12 mutant do not support the LPS-mediated enhancing effect. Furthermore, interleukin 1 (IL 1) appears not to be involved in LPS-mediated enhancement, and this effect is not reproduced by muramyl dipeptide (MDP)-mediated activation of APC.     

Absolute requirement for adherent cells in the production of human interleukin 2 (IL2)

Activated T lymphocytes appear to require a growth factor, interleukin 2 (IL2), for continued proliferation. It has been hypothesized that T lymphocytes serving an amplifier function produce IL2 in response to an activating signal such as antigen or mitogen; in addition, the producer lymphocytes receive a second signal from macrophages. The present experiments demonstrated that human IL2 production in culture is totally depleted by exhaustive removal of adherent cells and can be completely restored by replacement of the adherent cells.     

Monoclonal antibodies to human recombinant interleukin 1 (IL 1)beta: quantitation of IL 1 beta and inhibition of biological activity

Monoclonal antibodies (McAb) were developed to the Mr 17,500 form of human recombinant interleukin 1, IL 1 beta. Four McAb have been identified that inhibit the biological activity of IL 1 beta. McAb H34 and H67, at 1 microgram/ml (6 X 10(-9) M), completely inhibit the capacity of 1 ng/ml (6 X 10(-11) M) recombinant IL 1 beta to stimulate the proliferation of murine thymocytes or human fibroblasts in vitro. McAb H6 and H21 are approximately 10-fold less potent, and completely inhibit IL 1 beta activity at 10 micrograms/ml (6 X 10(-8) M) in both assays. The McAb do not have a significant effect on the biological activity of human recombinant IL 1 alpha in either assay. These McAb block the binding of recombinant [125I]IL 1 beta to IL 1 receptors on mouse 3T3 fibroblasts and have affinity constants for IL 1 beta in the range of 10(9) to 10(10) liters/mol. Competition studies suggest that two nonoverlapping epitopes on the IL 1 beta molecule are recognized by the McAb. H6 and H34 recognize one epitope, and H21 and H67 another. McAb H6 and H67 have been used together in a two-site ELISA to detect IL 1 beta. The sensitivity of the ELISA, which is 15 pg/ml (0.86 pM), approaches the limit of sensitivity of the thymocyte proliferation assay. The ELISA and thymocyte proliferation assay were used to quantitate IL 1 beta in E. coli LPS-stimulated human monocyte culture supernatants (HMCS). The level of IL 1 beta detected by ELISA in culture supernatants from eight donors ranged from 1.7 to 5.6 ng/ml, with a mean value of approximately 3 ng/ml. By comparison, the thymocyte proliferation assay gave levels of IL 1 in HMCS that were eight fold higher when quantitated by using recombinant IL 1 beta as a standard. This discrepancy with the bioassay used was reflected by the three fold higher maximum stimulation of thymocyte proliferation by HMCS as compared with recombinant IL 1 alpha or IL 1 beta, and only 45% inhibition of HMCS IL 1 activity by McAb. Thus, factors other than IL 1 beta account for the IL 1-like activity in monocyte culture supernatant as measured by the bioassay. The ILB1 McAb and ELISA allow for the first time-sensitive, accurate, and convenient quantitation of IL 1 beta levels in biological fluids or specimens.     

Role of interleukin 1 in early T cell development: Lyt-2-L3T4- thymocytes bind and respond in vitro to recombinant IL 1

Thymocytes that bear neither Lyt-2 nor L3T4 differentiation antigens (2-4- thymocytes) contain the precursors for mature L3T4+Lyt-2- and Lyt-2-L3T4+ T cells. In the present study we determined the capacity of 2-4- cells to respond to recombinant interleukin 1 (rIL 1) in vitro. The presence of rIL 1 enhanced IL 2-dependent proliferation to the lectins Con A and PHA by threefold to eightfold. In a second assay, rIL 1 enhanced proliferation and IL 2 production by 2-4- cells in response to phorbol myristate acetate (PMA) and the calcium ionophore, ionomycin. Using a direct IL 1 binding assay, we were able to detect both high-affinity (Kd approximately 5 pM) and low-affinity (Kd approximately 200 pM) classes of IL 1 receptors on freshly isolated 2-4- cells. Bound IL 1 was rapidly internalized, suggesting that such receptors were functional. These results are compatible with a role for interleukin 1 during thymocyte maturation.     

Characteristics of mouse lymphoid cells proliferating in vitro by recombinant human interleukin 2 (TGP-3): comparison between normal and nude mice

Various lymphoid cells obtained from BALB/c and BALB/c nu/nu mice were cultured in vitro with recombinant human interleukin 2 (rIL 2), and the characteristics of responder cells to rIL 2 were analyzed. Spleen cells, lymph node cells, and thymocytes except for bone marrow cells obtained from BALB/c mice remarkably proliferated in response to rIL 2. On the other hand, among lymphoid cells obtained from BALB/c nu/nu mice, only lymph node cells showed significant proliferation by rIL 2. Flow cytometric analysis revealed that mainly two types of lymphoid cells were proliferating in response to rIL 2 in BALB/c mice, i.e., Thy 1+, Lyt 1-, Lyt 2- and Thy 1+, Lyt 1-, Lyt 2+ cells. On the other hand, most of the proliferating cells were Thy 1+, Lyt 1-, Lyt 2- cells in BALB/c nu/nu mice. Treatment with various antibodies plus complement revealed that the majority of IL 2-responsive cells in BALB/c mice were Thy 1+, Lyt 1+, and Lyt 2+, although a minor part of them were Thy 1-, Lyt 1-, and Lyt 2-. On the other hand, a predominant type of the IL 2-responsive cells in BALB/c nu/nu mice were Thy 1-, Lyt 1-, and Lyt 2-, though some were Thy 1+. Nonspecific killer activity against tumor cells increased to variable extents in all of the lymphoid cells of both strains after culture with rIL 2. Our results indicate that mouse responder cells to rIL 2 have the following characteristics. First, the responder cells exist abundantly among spleen, lymph nodes, and thymus in normal mice, though their cell lineages are heterogeneous; one is of T cell lineage and the other of natural killer (NK) cell lineage. Second, nude mice are defective in the responder cells of T cell lineage but not of NK cell lineage. Moreover, the responder cells in nude mice predominantly accumulate in the lymph nodes but not other lymphoid organs.     

Heterologous antibody responses in mice with chronic T. cruzi infection: depressed T helper function restored with supernatants containing interleukin 2

Antibody production to sheep erythrocytes (SRBC) or hapten-conjugated SRBC (TNP-SRBC) was studied in mice with chronic Trypanosoma cruzi infections. Studies in vivo demonstrated that both IgM and IgG anti-SRBC responses were suppressed during chronic infection. Secondary IgG responses were suppressed regardless of whether the primary immunization was given before or after infection. The ability of cells from infected mice to provide help for antibody production was examined in vitro. Anti-SRBC responses were restored to cultures of whole spleen cells from infected mice by the addition of interleukin 2 (IL 2)-rich supernatants, indicating that these cells were capable of antibody production when sufficient help was provided. T cells from SRBC-primed infected mice were unable to provide significant help to normal B cell/M phi cultures for in vitro anti-TNP or anti-SRBC responses. The percentages of Thy-1+, Lyt-1+, and Lyt-2+ spleen cells were not significantly different between normal and infected mice. Anti-TNP and anti-SRBC responses were restored to cultures that contained T cells from infected mice and normal B cell/M phi by the addition of IL 2-rich spleen cell supernatants. The suppression of in vitro antibody responses in mice with chronic T. cruzi infections was associated with a lack of T cell help, which was provided by exogenous spleen cell supernatant.     

Monoclonal antibodies to interferon-gamma inhibit interleukin 2-dependent induction of growth and maturation in lectin/antigen-reactive cytolytic T lymphocyte precursors

In this study we tested the effect of monoclonal antibodies (moAb) AN-18 to murine IFN-gamma on the generation of cytolytic T cells (CTL) from a homogeneous population of precursor cells (CTL-P). As responder cells, highly purified Lyt-2+ C57BL/6 lymph node T cells were used that had been positively selected by flow cytofluorometry on a cell sorter. Lyt-2+ cells were set up in bulk culture or in limiting dilution (LD) either with Con A or with P815 tumor cells as antigen and recombinant human interleukin 2 (rec.hIL 2) in the presence or absence of moAb AN-18 and tested for growth and development of CTL. The results show that moAb AN-18 but not the unrelated moAb AN-37 diminished or abrogated proliferative and cytolytic responses of Lyt-2+ lymphocytes to lectin and rec.hIL 2 in a dose-dependent manner. The inhibitory activity of the antibodies could be abolished by neutralizing moAb AN-18 with recombinant murine IFN-gamma (rec.mIFN-gamma) before their addition to culture. Kinetic analysis shows that the inhibitory effect of moAb AN-18 is only optimal when added at the beginning of culture or up to 48 hr after initiation. The frequencies of CTL-P responding either to Con A or to P815 tumor cells and rec.hIL 2 were reduced up to 10-fold in the presence of moAb AN-18. The inhibitory capacity of moAb AN-18 was also operative in cultures containing on the average one antigen-specific CTL-P. Together with the finding that activated CTL-P secrete IFN-gamma in response to rec.hIL 2 in a dose-dependent manner, the data suggest that endogenous IFN-gamma collaborates with exogenous IL 2 in the induction of CTL-P. The generation of CTL may therefore represent a case of autocrine growth regulation of normal lymphocytes, in which the same cell synthesizes and responds to its own factor.     

Cytotoxic and proliferative lymphocyte responses to allogeneic and xenogeneic antigens in vitro.

In vitro lymphoproliferative responses to foreign histocompatibility antigens are phylogenetically restricted. Responses occur most readily to allogeneic or closely related xenogeneic leucocytes, but not to unrelated xenogeneic cells. Specific cytotoxic T cell responses to foreign histocompatibility antigens show the same phylogenetic restriction. This lack of xenoreactivity is not due to a lack of precursor cells for the xenoantigens; guinea-pig lymphocytes, although normally unresponsive to mouse antigens, have a similar precursor frequency for these antigens as do lymphocytes of allogeneic mouse strains. Specific cytotoxic responses of guinea-pig lymphocytes to mouse antigens can be generated if a factor released from con A stimulated guinea-pig spleen cells is added to the culture medium. The factor produced by con A-activated spleen cells (CS) is also phylogenetically restricted in its action; CS must be obtained from animals homologous with the donor of the responding lymphocytes.     

Down-regulation of interleukin 1 (IL 1) receptor expression by IL 1 and fate of internalized 125I-labeled IL 1 beta in a human large granular lymphocyte cell line

The regulation of interleukin 1 (IL 1) receptor expression on a human large granular lymphocyte cell line, YT, and fate of internalized 125I-labeled IL 1 beta (125I-IL 1 beta) were studied. YT cells were selected for this study, because this cell line expresses a large number of specific high-affinity receptor for IL 1, responds biologically to exogenously added IL 1 by expressing high-affinity IL 2 receptors, and does not produce IL 1. YT cells constitutively express approximately 7 X 10(3) IL 1 receptors/cell with a Kd approximately 10(-10) M. Neither IL 2, phorbol myristic acid, nor lipopolysaccharide affected the total binding of 125I-IL 1 beta by YT cells. In contrast, the capacity of YT cells to bind 125I-IL 1 beta when incubated at 37 degrees C for 3 to 16 hr with a low dose of purified IL 1 beta (approximately 6 U/ml) was reduced by greater than 80%. The loss of binding capability gradually recovered by 16 hr after removal of IL 1 beta from cultured YT cells. The apparent loss of IL 1 receptor expression was accompanied by the internalization of 125I-IL 1 beta into cells. Acid treatment of YT cells to remove bound 125I-IL 1 beta at 4 degrees C showed that 50% of the 125I-IL 1 beta bound to cells could no longer be recovered after 30 min at 37 degrees C, and this increased to 80% after 3 hr at 37 degrees C. Fractionation of cell extracts on Percoll gradient additionally showed 125I-IL 1 beta to appear intracellularly after receptor binding on plasma membranes, and to be successively transferred to some membranous organelles (d approximately equal to 1.037) through an intermediate density organelle (d approximately equal to 1.050), and to finally end up in lysosomal cell fractions (d approximately equal to 1.05 to 1.08) after approximately 3 hr at 37 degrees C. Only approximately 5% of internalized 125I-IL 1 beta was released into culture media by 6 hr of incubation at 37 degrees C. However, the radioactivity in the TCA soluble fraction of the culture media increased gradually by 6 hr and a lysosomotropic enzyme, ethylamine, significantly inhibited both the transfer of internalized 125I-IL 1 beta to the lysosomal fraction and the degradation of 125I-IL 1 beta. This study represents the first evidence of autoregulation of IL 1 receptors by IL 1 and internalization of IL 1 molecules after binding to receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

A mitogenic factor, released by stimulated human mononuclear cells and distinct from interleukin 2 (IL 2), B cell growth factor (BCGF), and interleukin 1 (IL 1)

Two lymphocyte mitogenic factors, interleukin 2 (IL 2) and blastogenic factor (BF), are generated concomitantly in human mixed lymphocyte cultures (MLC). The latter mitogenic factor is directly mitogenic for unstimulated lymphocytes, whereas the former mitogenic factor acts only on previously activated lymphocytes. Both factors had a m.w. range, as determined by gel filtration, of 18,000 to 30,000. Thus, these two factors were inseparable on the basis of m.w. size. However, BF and IL 2 were separable during ion exchange chromatography on the DEAE cellulose and phenyl-Sepharose chromatography. In addition, BF activity in the supernatants of MLC reached a maximum after day 5, whereas IL 2 activity peaked at day 3, thus distinguishing BF from IL 2 kinetically. These results clearly indicate that BF activity is mediated by molecules distinct from IL 2. The biochemical relationship between B cell growth factor (BCGF) and BF was also examined. Because BF was readily separable from BCGF by Con A-Sepharose chromatography, BF is distinguishable from BCGF. No augmentation of PHA-stimulated C3H mouse thymocyte proliferation was associated with the preparation of partially purified BF, demonstrating that BF and IL 1 are distinct molecules. Taken together, these results indicate that BF is clearly distinct from IL 2, BCGF, and IL 1. BF-containing MLC supernatants have direct mitogenic activity on both T and B cells. Both T and B cell blastogenic activities copurified during ammonium sulfate precipitation, gel filtration, DEAE cellulose ion exchange chromatography, and hydrophobic chromatography. Thus, these two activities appear to be biochemically inseparable. Monoclonal anti-Tac, that has been suggested to recognize the receptor for human IL 2, was highly inhibitory to the T cell response to the phenyl-Sepharose preparations of BF (IL 2-free). In contrast, this antibody had minimal or no effect on BF-induced B cell proliferation. However, when MLC supernatants were absorbed with a cloned IL 2-dependent T cell line, only IL 2 activity, but not BF activity, was removed, demonstrating that BF and IL 2 have different binding specificities. The precise mechanism(s) by which anti-Tac inhibits BF-induced proliferation of T cells is unknown at present. Additionally, during the course of these experiments, we observed that Con A-Sepharose chromatography could be used as a simple one-step method of separating BCGF from IL 2.