In vivo administration of purified human interleukin 2. II. Half life, immunologic effects, and expansion of peripheral lymphoid cells in vivo with recombinant IL 2

Purified recombinant human interleukin 2 (RIL 2) derived from E. coli containing the inserted gene encoding for IL 2 was administered to 20 patients with a variety of malignancies. Toxicity was dose related and included fever, chills, malaise, arthralgias, myalgias, and unexpectedly, weight gain related to marked fluid retention. All patients receiving more than 10(5) U/kg total cumulative dose developed evidence of fluid retention, and all patients requiring discontinuance of RIL 2 (11/20) received total doses of between 2.54 X 10(5) U/kg to 15.4 X 10(5) U/kg. The limiting dose with this preparation was 3000 U/kg/hr by continuous administration or 10(6) U/kg by bolus administration. IL 2 was rapidly cleared from the plasma, with a half life of 6.9 min, and a later delayed clearance was consistent with a two-compartment model, with slower release from the extravascular space back into the plasma compartment. A marked change in lymphoid cells in the periphery was noted with an early depletion of all lymphoid cells, followed by an expansion of such cells with continuous IL 2 administration. A twofold to 16-fold expansion of total lymphoid cells in the peripheral blood could be demonstrated. TAC+ cells representing up to 25% of the circulating peripheral blood mononuclear cells could be demonstrated with 3 wk of continuous RIL 2 administration. Interferon-gamma levels increased in patients treated with IL 2. Precursors of lymphokine-activated killer cells generated under standard conditions were depleted within 2 to 3 min after IL 2 administration, but repopulated the peripheral blood after 7 to 10 days of continuous IL 2 administration. No tumor regression was seen in any of the cancer patients treated with IL 2 alone.     

Cellular source of soluble interleukin 2 receptors in serum of mice after recombinant interleukin 2 administration.

Previous studies have shown that peripheral blood mononuclear cells activated in vitro not only express cell-associated interleukin 2 receptors (IL2R) but also release a soluble form of this receptor. In this study, we demonstrate that administration of human recombinant IL 2 (rIL 2) to mice results in increased spleen weights, splenic natural killer (NK) cell cytolytic activity, and serum levels of soluble IL2R. However, compared with rIL 2-treated heterozygote controls, beige mice treated with rIL 2 displayed similar elevations in serum soluble IL2R but significantly less splenic NK activity. Likewise, administration of anti-asialo GM1 antiserum to rIL 2-treated mice resulted in a dramatic reduction in splenic NK cytolytic activity, but no reduction in serum soluble IL2R. Conversely, while rIL 2 treatment of BALB/c mice produced increased splenic NK activity and serum soluble IL2R, similar treatment of BALB/c nude mice resulted in elevation of only splenic NK activity. These studies demonstrate that administration of rIL 2 to normal mice can elevate both serum IL2R levels and splenic NK cytolytic activity. However, the results suggest that T cells are likely to be the source of elevated serum IL2R after rIL 2 administration.     

Human recombinant interleukin 2-activated sheep lymphocytes lyse sheep pulmonary microvascular endothelial cells

Administration of lymphokine-activated killer (LAK) cells in combination with interleukin 2 (IL-2) has been effective in reducing tumor mass in humans, but has been accompanied by significant toxicity. We used a chronic awake sheep model to investigate the cause of the vascular leak syndrome associated with IL-2 administration. Sheep repeatedly infused with human recombinant IL-2 (hrIL-2) developed mild pulmonary hypertension, systemic hypotension, acidemia, hypoxemia, and increased flow of protein rich lung lymph. We hypothesized that LAK cells may damage lung endothelium in vivo and cause increased lung vascular permeability. Sheep peripheral blood and lung lymph lymphocytes incubated in vitro with hrIL-2 generated cytotoxic activity for human K-562 cells and sheep pulmonary microvascular endothelial cells. In addition, cytotoxic effector cells were isolated from the peripheral blood of a sheep which had received hrIL-2. These observations suggest that LAK cells possess the ability to damage endothelial cells and may contribute to an increased pulmonary vascular permeability observed following hrIL-2 infusion in sheep.     

Prerequisite for the induction of lymphokine-activated killer cells from T lymphocytes

Human blood mononuclear cells were separated into Leu-11+7-NK, Leu-11-7+, and Leu-11-7-T cells by means of a combination of the Percoll gradient method and C-mediated cytolysis using mAb. When purified Leu-11+7-NK, Leu-11-7+, and Leu-11-7-T cells were cultured with rIL 2 (500 U/ml) for 6 days in a medium supplemented with 10% FCS, Leu-11+7-NK cells responded at the maximum level and Leu-11-7+ cells responded moderately as shown by both cell-proliferation response and cytotoxic activity generated. On the other hand, Leu-11-7-T cells did not respond at all to rIL-2. However, when Leu-11-7-T cells were cultured with rIL-2 in a medium supplemented with 10% autologous serum, they showed considerable responsiveness to rIL-2. In addition, much greater response to Leu-11-7-T cells were produced by the addition of monocytes. Monocyte cytokines, neither IL 1, IFN-gamma, TNF, nor their combination were able to substitute for monocytes in the induction culture. In contrast, the response level of Leu-11+7- NK cells remained unchanged irrespective of supplementation with autologous serum to medium or the addition of monocytes to the culture. These results indicated that culture conditions in the experiments significantly affected the results as to determination of lymphokine-activated killer cell precursors, especially the result pertaining to the conversion of T lymphocytes to lymphokine-activated killer cells. Under appropriate conditions, not only NK cells but also T cells are important precursors of lymphokine-activated killer cells.     

Recombinant interleukin-2 and lymphokine-activated killer cells in renal cancer patients: I. phenotypic and functional analysis of the peripheral blood mononuclear cells

Summary The efficacy of recombinant interleukin-2 (rIL-2) or rIL-2 plus lymphokine-activated killer (LAK) cells in cancer therapy has been demonstrated by several groups both in experimental models in animals and clinical trials in humans, but their effects in vivo have yet to be clarified.Starting February 1988, we have treated 12 patients affected by advanced renal cancer with rIL-2 + LAK cells according to an open, non-randomized, phase II trial. Immediately before each rIL-2 infusion and during the last day of infusion, immunological tests were performed on the patients' peripheral blood mononuclear cells. During rIL-2 infusion we have observed a slight increase of the spontaneous cell proliferation and of natural killer (NK) and LAK activity; phenotypic analysis showed a significant decrease in the CD4⁺ T-lymphocyte subset, both in percentage and in absolute number. Conversely, before each cycle CD4⁺ cells increased when compared to basal values. No significant variations were observed in the CD8⁺ T-lymphocyte subset. Furthermore, a significant increase of the NK cells (CD3^– CD56⁺ CD16⁺) was evident during rIL-2 infusion.     

Extravasation of intravascular fluid mediated by the systemic administration of recombinant interleukin 2

Adoptive immunotherapy with lymphokine-activated killer cells and recombinant interleukin 2 (IL 2) can produce significant reduction of visceral metastases in tumor-bearing mice and, as shown recently, in humans with disseminated cancer. Because further dose escalations of IL 2 have been prevented by the development of a vascular leak syndrome (VLS) in both mice and humans, we investigated this VLS in mice undergoing the systemic administration of high-dose IL 2. A model for quantitating capillary permeability was used in which 125I-bovine serum albumin was injected i.v., and 2 hr later, tissues were counted in a gamma analyzer. A permeability index (PI) was calculated by dividing the mean counts per minute (cpm) of tissues from IL 2-treated mice by those from control animals. The injection of IL 2 produced increases in vascular permeability that were most pronounced in the thymus, spleen, lungs, liver, and kidneys (PI = 18.0, 10.0, 9.7, 6.7, and 6.3, respectively, on day 6). The development of the VLS was highly dependent on the number of days of IL 2 treatment (for example, the lungs contained 638, 1382, 3350, and 6187 cpm after 0, 1, 3, and 6 days of IL 2, respectively). Moreover, the degree of the VLS was directly related to the dose of IL 2 administered. Measurement of the wet and dry weights of lungs from IL 2-treated mice demonstrated that IL 2 produced a dramatic increase in their water weight (from 0.10 g at base line to 0.22 g after 200,000 U of IL 2 for 6 days). The injection of the IL 2 excipient failed to induce capillary leakage in tissues. Immunosuppression of mice by pretreatment irradiation (500 rad) or by injection of cyclophosphamide or by concurrent use of cortisone acetate markedly reduced or eliminated the development of the VLS. Similarly, the VLS was not observed in nude mice receiving IL 2. Thus, the administration of IL 2 produces a dose-limiting VLS that may be mediated, directly or indirectly, by host lymphoid elements.     

Release of lymphokines after infection with Epstein Barr virus in vitro. II. A monocyte-dependent inhibitor of interleukin 1 downregulates the production of interleukin 2 and interferon-gamma in rheumatoid arthritis

Epstein Barr virus (EBV)-infection of normal peripheral blood mononuclear cells (PBMC) in vitro induces IFN-alpha secretion from B cell and natural killer (NK) cell populations, and IFN-gamma secretion from T cells. IFN-gamma depends on prior elaboration of IL 2 and IL 1 that originates from monocytes and NK cells. PBMC from rheumatoid arthritis (RA) patients released moderately elevated levels of IFN-alpha (236 +/- 62 U/ml vs 168 +/- 34 in normals). In contrast, IFN-gamma was significantly lower in RA (88 +/- 34 U/ml vs 209 +/- 32) with an associated deficit in IL 2. A monocyte-dependent factor was shown to be responsible for this deficit, since monocyte depletion of RA cultures normalized the levels of IL 2 and IFN-gamma. Significantly lower levels of IL 1 activity were present in the supernatants of RA PBMC cultures as compared with normal cultures, and this was shown to be associated with presence of a nondialyzable IL 1 inhibitor. This inhibitor was capable of preventing the IL 1-dependent synthesis of IL 2 and IFN-gamma by normal PBMC. Exogenous IL 1 or IL 2 restored the deficient IFN-gamma secretion in RA PBMC. Thus, the deficient ability of RA lymphocytes to control EBV infection may be secondary to impairment of a monocyte-T cell interaction at the level of IL 1.     

Clinical trials of intrasplenic arterial infusion of interleukin-2 (IS-IL-2) to patients with advanced cancer

We tried a infusion of interleukin-2 (IL-2) of a relatively low dose via an intrasplenic arterial catheter connected to a chronometric infusion (IS-IL-2). Eighteen patients of colorectal cancer with metastases to the liver or lung or of unresectable hepatoma received a 24 hour continuous infusion with low dose recombinant of IL-2 (mainly 8 × 10⁵ JRU/day) for 25–40 days. All patients tolerated this protocol of the therapy and the main toxic effects were fever and general fatigue. Such serious toxicity as previously reported by high dose IL-2 therapy was not observed. Data of hepatic and renal functions were normal. IS-IL-2 therapy induced a high incidence of eosinophilia (12/18) and thrombocythemia (12/18). Peripheral natural killer (NK) and LAK activities were augmented in all patients and total white blood cell counts were increased during IS-IL-2 therapy. An increase in IL-2 receptor expression of peripheral blood mononuclear cells and significant rises in numbers of Leull (CD16)+, OKMl(CD11)+ and OKIal(HLA-DR)+ were observed. Of 18 patients 12 were evaluable for their response to therapy. Partial response (PR) was observed in one unresectable hepatoma and 11 demonstrated no change (NC) or progressive disease (PD). Six patients were not evaluable because of additional therapy (3 cases) or decreasing tumor cell markers having no measurable lesions (3 cases). Three patients of colorectal cancer from an unresectable group were presumed to have micrometastases to the liver as suggested by an elevated serum CEA level. After receiving IS-IL-2 therapy they demonstrated a decrease in the serum CEA level for more than 3 years after treatment. We conclude that continuous IS-IL-2 administration can result in an increase of their therapeutic efficacy of IL-2 administration and in a decrease its toxicity.     

Augmentation of cell number and LAK activity in peripheral blood mononuclear cells activated with anti-CD3 and interleukin-2

Summary A wide variety of human cancers currently have no effective treatment and are potential targets for lymphokine-activated killer (LAK) cellular immunotherapy. Relapsed acute lymphocytic leukemia (ALL) and neuroblastoma are two of the major therapeutic challenges in pediatric oncology today. However, one problem which makes LAK immunotherapy in children particularly difficult is obtaining the large numbers of cells required. Present adult therapeutic LAK protocols have utilized short-term (5 day) cultures of interleukin-2 (IL2)-activated cells which are initially obtained from leukophersis. Since routine use of this procedure in small children is not practical, we have investigated a different approach to obtain increased cell numbers by activation of peripheral blood mononuclear cells with OKT3, a mitogenic anti-CD3 monoclonal antibody, and IL2. Cell growth and LAK activity in OKT3+IL2-activated cultures were compared to cultures activated with IL2 alone in 2 children with relapsed ALL and 2 children with stage IV neuroblastoma. OKT3+IL2-activated cultures had marked increases in cell number: after 14 days the OKT3+IL2-activated cultures yielded an approximately 500-fold increase in cell number compared to a 7-fold increase for cultures activated with IL2 alone. In vitro ⁵¹Cr release assays were used to estimate LAK activity of the cultures at 7 and 14 days. When tested against HL60, a natural killer (NK)-resistant tumor cell line, not only were total cytolytic units greatly increased in OKT3+IL2-stimulated cultures but lytic activity on a per cell basis (lytic units/1×10⁶ cells) had also markedly increased on day 14 of culture. Phenotypic analysis demonstrated that 80% to 90% of cells in OKT3+IL2-stimulated cultures were CD3+ T cells. Variable low percentages of CD16+ NK cells were seen in these cultures. In summary, OKT3+IL2 activation resulted in a large increase in cell yield and the development of high level LAK activity using peripheral blood mononuclear cells from children with cancer. This approach may facilitate the utilization of increased cell numbers in future adoptive immunotherapy protocols, especially in pediatric patients.Supported by the Children's Cancer Research Fund, and the USPHS Training Grant T32CA09445Supported by NIH AI17687, AI18326, AI19007, and AI72626     

Defect of CD2- and CD3-mediated activation pathways in T cells of atopic patients: role of interleukin 2.

In the present work we analyzed the proliferative response of T lymphocytes from 11 atopic patients stimulated in vitro via either the CD2 or the CD3 pathway of cell activation. In both cases we found a significant decrease of thymidine incorporation in cell DNA in comparison with T cells from normal donors. The mechanism of this impaired proliferative response was analyzed. Atopic patients' T cells were found to secrete low quantities of interleukin 2 (IL2) and to express low amounts of Tac antigen, measured as both a percentage of Tac-positive cells and a mean fluorescence intensity of Tac antigen per cell. Addition of recombinant IL2 to cultures completely restored both cell proliferative response and Tac antigen expression. This effect was specific of IL2 since addition of IL1 or IL4 did not significantly affect T cell proliferative response. We conclude that atopic patients' T lymphocytes have a defect in both CD2 and CD3 pathways of cell activation relying on impairment of IL2 production, without involving IL2 responsiveness or other lymphokine defects.     

The synergistic effects of interleukin 2 and interleukin 7 on the proliferation and autologous tumor cell lysis of tumor-associated lymphocytes

Interleukin-7 (IL-7) has an ability to stimulate the proliferation of pre-B cells. It has been shown that IL-7 can also activate T lymphocytes. We here demonstrate that IL-7 in combination with interleukin-2 (IL-2) can drive cell proliferation and enhance the autologous tumor cell lysis by peripheral blood mononuclear cells (PBMC) and autologous mixed lymphocyte tumor cell culture (MLTC)-derived effector cells (MLTC cells). These synergistic effects of IL-2 and IL-7 on the proliferation and the augmentation of autologous tumor cell lysis were found for both effector cells. These effects were inhibited by neutralizing antibodies to IL-2 or IL-7, and by a combination of both antibodies, significantly. In terms of phenotypical expression, CD3 positive cells comprised the vast majority of MLTC cells after culture in medium containing IL-2 and IL-7 with an increase of IL-2 receptor positive cells.Abbreviations CD cluster differentiation - IFN interferon - IL interleukin - JRU Japanese Reference Unit - LAK lymphokine activated killer - mAb monoclonal antibody - MLTC mixed lymphocyte tumor cell culture - PBMC peripheral blood mononuclear cells - TILs tumor infiltrating lymphocytes     

Immunomodulatory effects of systemic low-dose recombinant interleukin-2 and lymphokine-activated killer cells in humans

Summary The adoptive immunotherapy of human cancer using lymphokine-activated killer (LAK) cells in combination with high-dose systemic recombinant interleukin-2 (rIL-2) has been associated with global changes in several hematological and immunological parameters while imposing profound toxicity on patients. We have evaluated an alternative LAK cell therapy utilizing low-dose systemic rIL-2 in 27 consecutive patients with metastatic cancer. We report that the administration of systemic low-dose rIL-2 is also characterized by significant changes in immunological and hematological parameters, which are qualitatively similar to those induced by high-dose rIL-2. Low-dose systemic rIL-2, given by i.v. bolus, is cleared to baseline levels within 240 min of administration. The induction of lymphocytosis and eosinophilia, which has characterized other protocols, is also a feature of this protocol. In addition, low-dose systemic rIL-2/LAK cell immunotherapy results in increased peripheral blood mononuclear cell (PBMC) expression of T-cell activation markers such as OKIa, OKT10 and IL-2 receptor. PBMC sampled approximately 100 h after the final infusion of LAK cells demonstrated a statistically significant increase in their ability to kill natural killer (NK)-sensitive and NK-resistent cell lines such as K562 and Daudi compared to baseline values (P <.05). These data suggest that rIL-2-based immunotherapy using low-dose rIL-2 is capable of inducing quantitative hematological and immunological changes while (in combination with LAK cells) retaining the ability to mediate tumor regressionin vivo. Dr. Eberlein was a recipient of an American Cancer Society Career Development Award. This work is supported in part by NIH Grant CA-40555 and the Clinical Research Center Grant 20-9299     

Augmentation of human natural cell-mediated cytotoxicity by recombinant human interleukin 2

Human peripheral blood mononuclear cells (PBMC) demonstrated increased natural cell-mediated cytotoxicity (NCMC) activity after only 5 min of exposure to purified recombinant human IL 2 or interferon (IFN)-gamma. The mechanism of NCMC augmentation by treatment with IL 2 is not entirely dependent on IFN-gamma production because: a) IL 2 was found to augment NCMC activity at levels which did not induce detectable IFN-gamma; b) IL 2 required only 5 min of exposure to PBMC to augment NCMC activity, whereas 3 hr of contact were required to demonstrate detectable IFN-gamma levels; c) the levels of NCMC enhancement by treatment with IL 2 exceeded the amount of NCMC enhancement that could be due to IFN alone; d) anti-recombinant IFN-gamma, which totally eliminated the augmentation of NCMC enhancement by IFN-gamma, only partially reduced the augmentation of NCMC activity by IL 2; and e) combination treatment of PBMC with IL 2 and IFN-gamma resulted in a synergistic enhancement of NCMC. The results strongly support the conclusion that augmentation of NCMC by IL 2 and IFN-gamma involve overlapping mechanisms.     

Epinephrine-induced changes in the distribution of lymphocyte subsets in peripheral blood of humans

We have previously demonstrated that mitogen responsiveness of mononuclear cells (MNC) from peripheral blood is reduced after a single injection of epinephrine to human subjects. The purpose of the present study was to characterize the relative distributions of MNC subsets after epinephrine administration using monoclonal antibodies and conventional cell markers. The absolute number of circulating MNC increased 64% within 30 min after injection of epinephrine, and returned to baseline by 2 hr. Analysis of MNC subsets revealed that there were no changes in the relative percentages of total T lymphocytes [T3+ cells, or neuraminidase-treated sheep red blood cell rosettes (EN-rosettes)], B lymphocytes (B1+, or cells with surface-bound immunoglobulin), or monocytes (by morphologic criteria) after epinephrine administration. The percentage of inducer T cells (T4+) declined at 30 and 60 min postinjection. Overall, the percentage of suppressor/cytotoxic T cells (T8+) did not change after injection of epinephrine; however, analysis of individual subjects revealed opposing responses of this subset. The T4:T8 ratio was 2.19 before injection, declined to 1.56 at 60 min, then increased to 3.10 2 hr postinjection. The percentage of natural killer/killer cells (HNK-1+) increased from a baseline of 15.5% before epinephrine injection to 29.6% at 30 min postinjection, then declined to 11.4% at 2 hr. Therefore, the administration of physiologic doses of epinephrine results in changes in the relative proportions of lymphocyte subsets in peripheral blood, in addition to reduced mitogen responsiveness as reported previously.     

Identification of cellular mechanisms operational in vivo during the regression of established pulmonary metastases by the systemic administration of high-dose recombinant interleukin 2

The systemic administration of high-dose recombinant IL 2 mediated significant reductions of established 3-day pulmonary micrometastases from both weakly immunogenic and nonimmunogenic sarcomas. However, when treatment with IL 2 was delayed for 10 days after the injection of tumor cells in an attempt to treat grossly visible pulmonary macrometastases, only those established from weakly immunogenic sarcomas remained susceptible. Established 10-day pulmonary nodules from the nonimmunogenic sarcomas became refractory to IL 2 therapy. We utilized selective depletion of lymphocyte subsets in vivo by the systemic administration of specific monoclonal antibodies to cells bearing either the L3T4 or Lyt-2 marker or a heteroantiserum to cells bearing the ASGM-1 glycosphingolipid to identify lymphocytes involved in IL 2-induced tumor regression. Cells with potent lymphokine-activated killer (LAK) activity against fresh tumor targets in vitro were identified in the lungs of IL 2-treated mice. By flow cytometry analysis, the majority of these effector cells were Thy-1+, L3T4-, Lyt-2-, ASGM-1+. Depletion in vivo of ASGM-1+ cells before the onset of IL 2 administration eliminated the successful therapy of 3-day pulmonary metastases from nonimmunogenic sarcomas, with concurrent elimination of LAK cell activity in the lungs. In mice with 3-day pulmonary metastases from weakly immunogenic sarcomas, both Lyt-2+ cells and ASGM-1+ cells were involved in IL 2-mediated tumor regression, but Lyt-2+ cells appeared to be the more potent mediator in the response. Lyt-2+ cells were also involved in the elimination of grossly visible 10-day macrometastases from these weakly immunogenic tumors. Depletion of L3T4+ cells had no effect on tumor regression. Thus, although LAK effectors derived from ASGM-1+ precursors can eliminate pulmonary micrometastases regardless of tumor immunogenicity, Lyt-2+ cells are predominant effectors in the elimination of both pulmonary micro- and macrometastases from weakly immunogenic sarcomas.     

Destruction of autologous human lymphocytes by interleukin 2-activated cytotoxic cells

Human and murine lymphocyte populations differentiate into lymphokine activated killer (LAK) cells after in vitro or in vivo exposure to interleukin 2 (IL 2). LAK cells mediate destruction of neoplastic tissue in vitro and have been reported to spare normal tissue. However, systemic toxicity is observed in mice and patients receiving IL 2 infusions. Some aspects of this toxicity are similar to that seen in graft-vs-host disease, suggesting that IL 2 may cause an immune-mediated destruction of normal tissues. We have evaluated this issue by examining the destructive potential of fresh human lymphocytes cultured in media containing highly purified recombinant human IL 2. In the absence of any exogenous antigen or allogeneic stimulating cells, strong proliferative responses were induced after 6 days of exposure to IL 2. Lymphocytes harvested from these 6-day cultures were highly cytotoxic to K562 and Daudi target cells. These IL 2-activated cells were also cytotoxic against autologous and allogeneic normal lymphocyte target cells. This autologous lymphocyte destruction was detected in media containing autologous serum and was directly dependent on the concentration of IL 2 added to the cultures. These studies demonstrate that populations of IL 2-activated lymphocytes, containing LAK activity, can mediate low-level but significant destruction of normal lymphocytes in vitro.     

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.     

Cell numbers and in vitro responses of leucocytes and lymphocyte subpopulations following maximal exercise and interval training sessions of different intensities.

In vitro lymphocyte function and the mobilisation of peripheral blood leucocytes was examined in eight trained subjects who undertook an incremental exercise test to exhaustion and a series of interval training sessions. Venous blood samples were obtained before the incremental test, immediately after, and 30, 60, and 120 min after the test. Interval training sessions were undertaken on separate days and the exercise intensities for each of the different sessions were 30%, 60%, 90% and 120% of their maximal work capacity respectively, as determined from the incremental exercise test. There were 15 exercise periods of 1-min duration separated by recovery intervals of 2 min in each session. Venous blood samples were obtained immediately after each training session. Significant increases in lymphocyte subpopulations (CD3+, CD4+, CD8+, CD20+, and CD56+) occurred following both maximal and supramaximal exercise. This was accompanied by a significant decrease in the response of cultures of peripheral blood lymphocytes to Concanavalin A (ConA), a T-cell mitogen. The state of lymphocyte activation in vivo as measured by CD25+ surface antigen was not, however, affected by acute exercise. The total number of lymphocytes, distribution of lymphocyte subpopulations and in vitro lymphocyte response to ConA had returned to pre-exercise levels within half an hour of termination of exercise but serum cortisol concentrations had not begun to fall at this time. There was a significant decrease in the CD4+:CD8+ cell ratio following exercise; this was more the result of increases in CD3-CD8+ cells (CD8+ natural killer cells) than to CD3+CD8+ cells (CD8+ T-lymphocytes). Decreased responsiveness of T-cells to T-cell mitogens, postexercise, may have been the result of decreases in the percentage of T-cells in postexercise mixed lymphocyte cultures rather than depressed cell function. The cause of this was an increase in the percentage of natural killer cells which did not respond to the T-cell mitogen. The results indicated that while a substantial immediate in vitro "immunomodulation" occurred with acute exercise, this did not reflect an immunosuppression but was rather the result of changes in the proportions of reactive cells in mononuclear cell cultures. We have also demonstrated that the degree of the change in distribution of lymphocyte subpopulation numbers and responsiveness of peripheral blood mononuclear cells in in vitro mitogen reactions increased with increasing exercise intensity. Plasma volume changes may have contributed to some of the changes seen in leucocyte population and subpopulation numbers during and following exercise.     

Synergy between recombinant interleukin 2 (rIL 2) and IL 2-depleted lymphokine-containing supernatants in facilitating allogeneic human cytolytic T lymphocyte responses in vitro

Supernatants from human mixed leukocyte cultures or lectin-depleted supernatants from cultures of PHA-activated human peripheral blood leukocytes were depleted of IL 2 by passage over an anti-human rIL2 immunoadsorbent column. The column eluates were concentrated, dialyzed, and tested for their ability to synergize with human rIL 2 in facilitating human cytolytic T lymphocyte (CTL) responses to allogeneic, uv-irradiated HT144 melanoma cells in vitro. CTL were generated in the presence of 1 X 10(-4) M hydrocortisone sodium succinate in order to minimize the generation of nonspecific lymphokine-activated killer (LAK) cells. IL 2-depleted lymphokine-containing supernatant (LKS), alone or in the presence of less than or equal to U/ml rIL 2 did not stimulate significant CTL responses. Recombinant IL 2 at greater than 2 U/ml stimulated weak CTL responses in the absence of LKS. However, strong synergistic CTL responses were observed when both IL 2-depleted LKS and greater than 2 U/ml rIL 2 were added to the cultures. CTL generated in these cultures could be distinguished from nonspecific LAK cells on the basis of their i) specificity, ii) T3 phenotype, and iii) kinetics of generation. Nevertheless, rIL 2 and IL 2-depleted LKS were sometimes observed to synergize in facilitating the generation of nonspecific LAK cells as well as the generation of specific CTL. When the times at which rIL 2 and IL 2-depleted LKS were added to the cultures were varied, IL 2 was found to be required early in CTL responses, whereas the synergistic factor(s) in LKS seemed to act later. Recombinant human interferon-gamma was unable to replace LKS in synergizing with rIL 2 to elicit CTL responses. In summary, these experiments suggest that LKS contains a late-acting factor(s), antigenically distinct from IL 2, which synergizes with IL 2 in facilitating human CTL responses.     

Regulation of hepatic acute phase protein synthesis by products of interleukin 2 (IL 2)-stimulated human peripheral blood mononuclear cells

Cancer patients injected with recombinant human IL 2 develop marked changes in serum concentrations of hepatic acute-phase proteins. To determine if this acute-phase response involves a change in the rate of hepatic protein synthesis and if it is due to a direct effect of IL 2 on hepatocytes, human hepatoma-derived hepatocytes (Hep-3B cells) were incubated in medium containing IL 2 or in culture supernatants from IL 2-activated human peripheral blood mononuclear cells (PBMNC). The rate of synthesis of two acute-phase proteins, complement protein factor B and albumin, was determined by the incorporation of a radiolabeled amino acid precursor into newly synthesized protein as measured by analytical gel electrophoresis of immunoprecipitates. IL 2 in concentrations from 1 to 1000 U/ml had no effect on the synthesis of factor B or albumin; conversely, there was a dose-dependent increase in the rate of synthesis of factor B and decrease in albumin synthesis mediated by culture supernatants of IL 2-activated PBMNC. The magnitude of the effect of acute-phase protein synthesis was dependent on the IL 2 concentration used for the activation of PBMNC. The rate of factor B synthesis increased approximately 4.0-fold in the presence of culture supernatants of PBMNC activated with either opsonized heat-killed Staphylococcus albus or with 1000 U/ml IL 2. Preincubation of the IL 2-activated PBMNC culture supernatants with an antiserum specific for recombinant IL 1-beta completely neutralized the capacity of the supernatants to stimulate factor B synthesis, whereas antisera specific for human IL 1-alpha or for tumor necrosis factor had no effect. These results indicate that the indirect effect of IL 2 on hepatic acute phase protein synthesis is mediated by IL 1-beta.