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.     

Systemic administration of recombinant interleukin 2 stimulates in vivo lymphoid cell proliferation in tissues

Recent work in our laboratory has demonstrated that the repeated injections of high doses of recombinant interleukin 2 (IL 2) can dramatically reduce the number of established pulmonary and hepatic metastases and the growth of intradermal tumors in a variety of murine tumor models. We have thus undertaken studies to define the mechanisms underlying these in vivo effects of IL 2. Using an in vivo DNA-labeling technique in which we employed 5-[125I]iodo-2'-deoxyuridine (125IUdR), we examined the in vivo cell proliferation in the tissues of mice treated with IL 2. A proliferation index (PI) was calculated by dividing the raw counts per minute (cpm) of tissues in IL 2-treated mice by the cpm in corresponding tissues of control animals. At an IL 2 dose of 6000 U given i.p. three times a day, the highest 125IUdR incorporation was seen in the lungs, liver, spleen, kidneys, and mesenteric lymph nodes (PI = 6.9, 6.9, 5.1, 7.1, 24.6, respectively, at 5 days). The amount of lymphoid proliferation in these organs was a direct function of the dose of IL 2 administered. Other tissues including thymus, intestines, skin, and hind limb showed no significant increase in 125IUdR uptake even after host treatment with the highest doses of IL 2. Blood and brain demonstrated intermediate incorporation of the radiolabel. Preirradiation of the host largely eliminated the proliferative response to IL 2. Histologic studies of normal and irradiated mice receiving IL 2 corroborated the result of the 125IUdR findings. In normal IL 2-treated mice, large collections of activated lymphoid cells were seen, most prominently in the lungs, liver, and kidneys, whereas markedly decreased lymphoid proliferation was evident histologically in preirradiated mice. A fluorescein-labeled monoclonal antibody directed against the Thy-1.2 surface determinant was used to identify these dividing cells in frozen tissue sections as T lymphoid cells. Activated lymphocytes isolated from the lungs, liver, spleen, and mesenteric lymph nodes of IL 2-treated mice demonstrated significant lysis of a fresh murine sarcoma target in short-term 51Cr-release assays. These studies demonstrate that the systemic administration of recombinant IL 2 causes in vivo activation and proliferation of host lymphoid cells and has important implications for the adoptive immunotherapy of tumors.     

Augmentation of the therapeutic efficacy of adoptive tumor immunotherapy by in vivo administration of slowly released recombinant interleukin 2

Summary Immunization of C57BL/6 mice with MMC-treated syngeneic lymphoma cells, MBL-2, caused the generation of antitumor effector cells in vivo and the immunized mice permanently rejected viable MBL-2 lymphoma cells. Both plastic nonadherent T cells and plastic adherent MØ obtained from MBL-2 immunized mouse peritoneal exudate cells revealed strong cytotoxic activity against MBL-2 lymphoma cells, whereas immune spleen cells were not highly active against MBL-2 lymphoma cells in vitro. However, systemic adoptive transfer of immune spleen cells into the MBL-2-bearing mice by i.v. infusion in conjunction with i.p. cyclophosphamide (100 mg/kg) treatment cured the mice of tumor. This therapeutic efficacy of immune spleen cells was reflected by the number of transferred effector cells and over 5×10⁷ immune spleen cells were required to cure the mice completely. The cells mediating in vivo rejection of MBL-2 lymphoma cells were Thy 1.2⁺ T cells. This ACIT was specific against MBL-2 lymphoma cells and had no effect on the growth of other syngeneic tumors, B16 melanoma or BMC6A fibrosarcoma. In vivo administration of recombinant interleukin 2 (r-IL 2) combined with ACIT greatly modulated the cure rate of tumor-bearing mice. In addition, we found that slowly released r-IL 2 administratered from an ALZET miniosmotic pump was more effective in augmenting the therapeutic efficacy of immune spleen cells in ACIT than a single injection of the same total dose of r-IL 2.     

Antibody-dependent cellular cytotoxicity mediated by murine lymphocytes activated in recombinant interleukin 2

The incubation of murine splenocytes in recombinant interleukin 2 (RIL 2) gives rise to lymphokine-activated killer (LAK) cells that can lyse fresh, NK-resistant tumor cells but not normal cells in 4-hr 51Cr-release assays. Lysis by this IL 2-activated cell population was enhanced up to 100-fold by prior reaction of target cells with specific antisera reactive with antigens on the target cells. This antibody-dependent cellular cytotoxicity (ADCC) also resulted in lysis of fresh normal target cells, which are not usually susceptible to LAK lysis. The ADCC was evident after 24 hr of incubation of splenocytes in RIL 2, but peak lytic activity was reached after 3 to 4 days of incubation. The concentrations of RIL 2 needed for the in vitro activation of the effectors in order to attain maximal ADCC was between 100 and 3000 U/ml and parallel the IL 2 concentrations required to generate LAK cells. ADCC mediated by IL 2-activated splenocytes was completely blocked by anti-FcR monoclonal antibodies. Although antisera directed against MHC antigens were used in most experiments, anti-B16 monoclonal antibodies have also shown the ability to induce ADCC mediated by RIL 2-activated syngeneic and allogeneic cells. Treatment of the precursor splenocyte populations with anti-asialo GM1 and complement eliminated the direct LAK activity and the antibody-dependent cytotoxicity, suggesting that both direct and indirect tumor cell lysis may be caused by the same effector cell. ADCC mediated by LAK cell populations represents another possible mechanism for the in vivo therapeutic effects of these cells.     

The anti-tumor efficacy of lymphokine-activated killer cells and recombinant interleukin 2 in vivo: direct correlation between reduction of established metastases and cytolytic activity of lymphokine-activated killer cells

Our previous studies demonstrated that the incubation of human peripheral blood lymphocytes or murine splenocytes in recombinant interleukin 2 (RIL 2) resulted in the generation of lymphokine-activated killer (LAK) cells capable of lysing a broad spectrum of fresh tumors in short-term chromium-release assays. Moreover, injections of LAK cells plus RIL 2 were highly effective in eliminating established 3 day metastases in the lung and liver (1-3). We have examined several parameters to define whether or not the cytolytic activity of LAK cells as measured in vitro correlated directly with the in vivo anti-tumor efficacy of adoptively transferred LAK cells. LAK cells plus RIL 2 could mediate marked reductions of established pulmonary metastases in mice rendered T cell deficient by adult thymectomy and lethal, total body irradiation followed by reconstitution with T cell-depleted bone marrow and spleen cells. Thus there was no requirement for additional T lymphocytes of host origin for successful therapy with adoptively transferred LAK cells plus RIL 2. Fresh splenocytes depleted of T cells by anti-Thy-1.2 monoclonal antibody plus complement generated LAK cells that were as highly lytic to fresh tumor in vitro and were as effective in reducing established pulmonary metastases as those generated from untreated or complement-treated splenocytes. Thus the precursor to LAK cells with anti-tumor activity in vitro and in vivo did not express the Thy-1 antigenic marker. In contrast, treatment of LAK effector cells (those generated from a 3-day incubation of fresh, normal splenocytes in RIL 2) with anti-Thy-1.2 antibody plus complement reduced or abolished their in vitro cytolytic activity. However, when combined with the systemic administration of RIL 2, these T cell-depleted, non-lytic LAK cells remained as effective in reducing the number of established pulmonary metastases upon adoptive transfer as untreated or complement-treated lytic LAK cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of systemic administration of mouse recombinant interferon-gamma on the lung tumor metastases in mice

The purpose of this study was to examine the effective anti-metastatic activity by multiple i.v. administrations of mouse recombinant interferon-gamma (IFN-gamma) against pulmonary metastases of 3LL or B16-BL6 melanoma cells after surgical excision of primary tumors. Multiple treatments with IFN-gamma reduced effectively the incidence of pulmonary tumor metastases. Repeated 4 consecutive treatment modalities with IFN-gamma showed remarkable reduction of lung tumor colonies, and also rendered alveolar macrophages (AM) cytotoxic against B16-BL6 cells. In contrast, 14 consecutive administrations of IFN-gamma at any doses (10(2) and 10(3) U/mouse) could not activate macrophages to become cytotoxic, but were effective in regressing metastases. Thus, antimetastatic activity of IFN-gamma may be due to the stimulation of host immune defense systems such as induction of tumoricidal macrophages, presumably the direct antiproliferative action to tumor cells, or both actions under the appropriate administration conditions. We found that the systemic administration of IFN-gamma under appropriate multiple treatment modalities results in the reduction of the lung metastases and can activate AM to become tumor cytotoxic at relatively low doses (10(2) U). High-dose IFN-gamma in the multiple administration schedule was also effective for the reduction of lung tumor colonies, but strongly suppressed the nonspecific immune function and could not activate tumoricidal properties of AM.     

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.     

Augmentation of immunity to herpes simplex virus by in vivo administration of interleukin 2

The immune mechanisms responsible for recovery from herpesvirus infections are multiple and include a principle role for aspects of T cell immunity. Our investigations add further support for this notion. We show that the ability of immune lymphocytes from animals infected i.p. 6 wk previously with herpes simplex virus type one (HSV-1) clear virus more effectively when interleukin 2 (IL 2) is injected into recipients of the adoptive transfers. Mice were treated on two consecutive days with cyclophosphamide and infected in the pinnae with 4 X 10(6) plaque-forming units of HSV-1. Three hours post-infection lymphocyte populations were injected i.v., and after a further 3 days the pinnae were removed, homogenized, and the content of infectious virus assayed. Purified IL 2 obtained from EL-4 cells either was given i.v. 2 hr before and 24 and 48 hr after cell injection or was given subcutaneously 2 hr before and 3, 24, and 48 hr after cell injection. The latter three injections were given i.p. and suspended in 15% gelatin. The immune lymphocyte cell populations were splenocytes and were either injected immediately after preparation of cell suspensions or after 5 days in vitro secondary stimulation with HSV-1. This latter cell population showed greater viral clearance activity, a function shown previously to be a property of Lyt-2+ cells. The clearance activity of cells was markedly enhanced in animals given IL 2 but only with a regimen that included injections in gelatin, a procedure that enhances in vivo circulation time of IL 2. The cell involved in clearance was a T cell and principally the Lyt-2+ subset. Treatment of recipient mice with anti-asialo GM-1 did not affect the clearance efficiency, indicating that NK cells were not responsible for the observed effect. Our experiments indicate that IL 2 may provide an important regulator of immune function in vivo and may warrant its investigation as a therapeutic agent to enhance antiviral immunity in certain circumstances.     

Modulation of the expression of interleukin 2 receptors of human thymocytes by recombinant interleukin 2

The effect of purified recombinant interleukin 2 on the expression of the receptors for interleukin 2 by human thymocytes was examined. Interleukin 2 augmented the expression of interleukin 2 receptors and interferon-gamma synthesis by thymocytes activated with concanavalin A, and it was required to maintain the growth of thymocytes in vitro and the expression of interleukin 2 receptors. The increase observed in the number of receptor bearing thymocytes and in the density of receptors due to interleukin 2 occurred within the first 2 days of culture. Dexamethasone inhibited the expression of interleukin 2 receptors, the synthesis of interferon-gamma, and the early proliferation and protein synthesis of lectin-activated thymocytes during the first 2 days of culture. The inhibitory effect of dexamethasone on the expression of interleukin 2 receptors and on the synthesis of interferon-gamma was reversed by interleukin 2, whereas its effect on proliferation and on protein synthesis during the first two days of culture was not reversed by interleukin 2. Interleukin 2 induced the proliferation of thymocytes in vitro, even in the absence of activation by lectin; however, the number of cells displaying receptors which could be detected with anti-Tac remained low throughout the first week of culture and interferon-gamma synthesis was not observed. Nonetheless, interleukin 2-induced proliferation was inhibited by anti-Tac on a dose dependent manner. The results of the study document that recombinant interleukin 2, like purified natural interleukin 2, is required for the expression of interleukin 2 receptors, for interferon-gamma synthesis, and for the growth of thymocytes in vitro.     

The anti-tumor efficacy of lymphokine-activated killer cells and recombinant interleukin 2 in vivo

We showed previously that adoptive immunotherapy with the combination of LAK cells and recombinant IL 2 (RIL 2) can markedly reduce pulmonary micrometastases from multiple sarcomas established 3 days after the i.v. injection of syngeneic tumor cells in C57BL/6 mice. In this report, we analyzed the factors required for successful therapy. Titration analysis in vivo revealed an inverse relationship between the number of pulmonary metastases remaining after treatment and both the number of LAK cells and the amount of RIL 2 administered. Fresh or unstimulated splenocytes had no anti-tumor effect; a 2- to 3-day incubation of splenocytes in RIL 2 was required. LAK cells generated from allogeneic DBA (H-2d) splenocytes were as effective in vivo as syngeneic, C57BL/6 (H-2b) LAK cells. The anti-metastatic capacity of LAK cells was significantly reduced or eliminated when irradiated with 3000 rad before adoptive transfer. The combined therapy of LAK cells plus RIL 2 was shown to be highly effective in mice immunosuppressed by 500 rad total body irradiation and in treating macrometastases established in the lung 10 days after the i.v. injection of sarcoma cells. Further, reduction of both micrometastases and macrometastases could also be achieved by RIL 2 alone when administered at higher levels than were required with LAK cells. The value of LAK cell transfer and of IL 2 administration for the treatment of tumors established at other sites is currently under investigation.     

Systemic administration of LPD prepared with CpG oligonucleotides inhibits the growth of established pulmonary metastases by stimulating innate and acquired antitumor immune responses

Intravenous (i.v.) administration of a lipopolyplex consisting of a ternary complex of DOTAP:cholesterol cationic liposomes, protamine sulfate, and noncoding plasmid DNA (LPD-pDNA) is capable of stimulating a potent Th-1 cytokine response and inhibiting the growth of established tumors in mice. Both activities are mainly elicited by unmethylated CpG motifs in the plasmid DNA (pDNA) component, which are bacterial in origin. Since oligodeoxynucleotides (ODN) that possess a consensus immunostimulatory CpG motif of RRCpGYY (R is purine and Y is pyrimidine) can mimic the immunostimulatory actions of bacterial DNA, we hypothesized that i.v. administration of LPD prepared with GpG-ODN would mimic the ability of LPD-pDNA to stimulate Th-1 cytokines and antitumor activity and provide an improved vector for probing the immune mechanisms underlying the observed antitumor effects. These hypotheses were tested for the treatment of established 24JK experimental pulmonary metastases that are syngeneic in C57BL/6 mice. Mice treated with LPD containing 25 microg of the prototypical phosphodiester (PO) CpG-ODN 1668 (tccatGACGTTcctgatgct, motif capitalized) demonstrated a dramatic reduction in lung tumor burden (>80% inhibition, P<0.01) compared to dextrose-treated controls. The antitumor effect was dependent on the CpG dinulceotide and correlated with the ability to stimulate serum Th-1 cytokines (TNF-alpha, IL-12, and IFN-gamma). Both activities required assembly of CpG-ODN in a cationic liposome/DNA complex (lipoplex) or the LPD lipopolyplex. LPD delivery of both PO-1668 and phosphorothioated (PS)-1668 stimulated a greater cytokine response compared to delivery of free ODN. Furthermore, within the LPD complex, both PO- and PS-1668 had similar ability to stimulate Th-1 cytokines with respect to potency and duration of response, thus eliminating the need for the PS modification. In tumor cell lysis assays, LPD-CpG DNA stimulated development of an acquired, tumor-specific CD8+ cytotoxic T-lymphocyte (CTL) activity that was dependent on CpG DNA. LPD was also capable of stimulating NK activity; however, this was not dependent on CpG DNA. Only formulations that concomitantly stimulated NK activity and CpG-specific, Th-1 cytokine were capable of stimulating the development of tumor-specific CTL activity and significant inhibition of tumor growth. Thus, we propose a model where CpG DNA in complex with cationic liposome-based lipoplexes or lipopolyplexes stimulates antitumor NK activity and CpG-stimulated Th-1 cytokine production. The combination of these two activities of the innate immune system subsequently direct the development of an acquired, tumor-specific CTL response that in total are effective for inhibiting the growth of established tumors in mice.     

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.     

In vivo administration of purified human interleukin 2. I. Half-life and immunologic effects of the Jurkat cell line-derived interleukin 2

A total of 12 patients with cancer or the acquired immunodeficiency syndrome have been treated with Jurkat-derived purified human interleukin 2 (IL 2). The toxicity was dose-related and consisted primarily of fever, chills, malaise, and mild reversible hepatic dysfunction. No evidence of clinical efficacy was seen when IL 2 was administered at doses of up to 2000 micrograms by bolus or continuous infusion once a week for 4 wk. No significant chronic immunologic effects (changes in mitogen responsiveness of induction of cytotoxic cells) were demonstrated. IL 2 was measured in the serum of patients, and a half-life of approximately 5 to 7 min was demonstrated with a second component of clearance of 30 to 120 min. Heating the serum at 56 degrees C for 30 min allowed for detection of smaller quantities of IL 2 by removing a serum inhibitor whose effect was seen at dilutions of up to 1/80 in our biologic assay. Sustained levels of IL 2 could be maintained by continuous infusion. Acute effects of IL 2 administration included a rapid decrease in peripheral mononuclear cells with a shift to cells of macrophage lineage and a rapid decrease in total T lymphocytes and T lymphocyte subsets. IL 2 responsiveness of peripheral mononuclear cells decreased within 15 min of IL 2 administration, with a concurrent decrease in the ability to generate lymphokine-activated killer cells. These changes did not recover until 48 hr after IL 2 administration. A rise in serum ACTH and cortisol levels was seen after the administration of 1 to 2 mg of IL 2. Future studies will evaluate the role of larger quantities of recombinant IL 2 given alone or in conjunction with in vitro-generated lymphokine-activated killer cells.     

The biological activity in vivo of recombinant murine interleukin 1 in the rat

The present study summarizes the biological response of rats to infusion with recombinant murine IL-1 (rIL-1) cloned in Escherichia coli. Thirty-seven male rats (135-180 g) were infused over a 6-hr period with either 0.008 M guanidine hydrochloride (the vehicle) or E. coli product (both groups are controls) or 1000, 3750, 7500, 15,000, or 37,500 LAF units/hr of rIL-1. The controls and the group receiving 1000 LAF units/hr of rIL-1 did not exhibit a change in body temperature during the experiment. A mild fever was noted with 3750 LAF units/hr which became significantly elevated with 7500 and 15,000 LAF units/hr. At a dose of 37,500 LAF units/hr of rIL-1 (in 0.08 M guanidine hydrochloride) the rats became hypothermic and died. An equivalent dose of guanidine hydrochloride alone (0.08 M) was not fatally toxic although the rats did become hypothermic. Plasma zinc levels were significantly depressed and white blood cell count elevated at 6 hr postinfusion onset. Resting energy expenditure (REE) was significantly depressed during an infusion of 7500 and 15,000 LAF units/hr of rIL-1 despite a concurrent elevation in body temperature. Whole-body leucine kinetics were unchanged by infusion with rIL-1. Plasma fibrinogen and serum haptoglobin and copper levels were not altered by rIL-1. In conclusion, murine rIL-1 is similar to monocytic-derived IL-1 in that it produces a fever, hypozincemia, and leukocytosis; however, rIL-1 does not induce changes in protein metabolism.     

Effect of recombinant interleukin 2 (R-IL2) on in vivo growth of murine myeloma X5563

Summary The present study deals with the effect of recombinant interleukin 2 (R-IL2) on in vivo growth of murine myeloma X5563. Administration of R-IL2 (5×10⁴ J.U./mouse per day) s.c. starting 1 day after X5563 inoculation i.d. had a marginal effect on the growth of X5563, and all the mice repeatedly given R-IL2 from day 1 to day 17 died. However, daily administration of R-IL2 starting 7 days after the tumor inoculation was highly effective and significantly lengthened survival time compared with the control mice injected with vehicle alone. About 50% of the treated mice were completely cured, and survived for more than a month after the therapy ceased. In a representative experiment, where the growth of X5563 was slow because of the small number of inoculated tumor cells, all the mice (n=6) given R-IL2 from day 11 to day 23 showed complete cure of the established X5563 solid tumor. These mice showed in vivo protective immunity and in vitro cytotoxic T cell responses to X5563 tumor antigens. Histologically, a large number of macrophages and lymphocytes had infiltrated the area around the necrotic X5563 tumor mass in the mice which had received R-IL2 therapy. These results suggest that repeated injections of R-IL2 at the local site after tumor development can augment antitumor immunological responses and subsequently induce tumor regression.     

Induction of colony stimulating factor in vivo by recombinant interleukin 1 alpha and recombinant tumor necrosis factor alpha 1

In response to a potent inflammatory challenge, such as Gram-negative endotoxin, a number of cytokines are induced that, in turn, mediate many of the pathophysiologic alterations associated with endotoxicity. In this study, we have observed two endotoxin-associated monokines, recombinant interleukin-1 alpha (rIL 1 alpha) and recombinant tumor necrosis factor alpha (rTNF alpha), to induce colony stimulating factor (CSF) in vivo. The CSF activities produced in response to rIL 1 alpha or rTNF alpha gave rise to a mixture of granulocyte-macrophage colonies and were induced in a dose- and time-dependent fashion, peaking within 3 hr of cytokine injection (preceding peak CSF induction by endotoxin by several hours). Combined injection of suboptimal concentrations of rIL 1 alpha and rTNF alpha were additive, and simultaneous injection of optimal concentrations of each failed to increase CSF levels over that observed with either cytokine alone. Unlike endotoxin, neither cytokine induced interferon in vivo. These findings extend our understanding of the cytokine cascade that is operative in an inflammatory response and may account for many of the observed hematopoietic alterations that accompany inflammation.     

Identification of the viral and cellular microRNA interactomes during SARS-CoV-2 infection

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Induction of antibody-dependent cellular cytotoxicity in vivo by IFN-alpha and its antitumor efficacy against established B16 melanoma liver metastases when combined with specific anti-B16 monoclonal antibody

We have previously shown the ability of different cytokines to induce antibody-dependent cellular cytotoxicity (ADCC) in murine cells in vitro. In addition we found that the administration to mice of IL-2-induced cells which mediated ADCC and that these cells were phenotypically similar to the cells induced in vitro. In the present study we tested the ability of various cytokines, including IL-1, TNF, IFN-alpha, and IFN-gamma to induce ADCC in vivo. We found that both IFN-alpha and IFN-gamma induced ADCC in the livers and spleens of C3H/Hen-treated mice and that these cytokines together with TNF enhanced the IL-2-induced ADCC in vivo. In C57BL/6 mice which, as previously shown, exhibit relatively low ADCC activity, IFN-alpha and IFN-gamma increased the IL-2-induced ADCC only when 100,000 U of IL-2 were used for priming. The effect of IFN-alpha on ADCC was dose dependent and was optimal after the administration of 200,000 U of the cytokine given three times a day for 3 days. Similar to the cells induced in vivo by IL-2, the precursors of the cells mediating ADCC were asialo GM1+ whereas the effectors were mainly nonadherent, Thy-1+ cells. IFN-alpha-generated cells mediating ADCC in the liver and spleen and, when combined with IL-2, ADCC was induced in the thymus as well. This effect of IFN-alpha on the induction of ADCC was exploited in an immunotherapy model in which we found that IFN-alpha significantly enhanced the antibody-mediated antitumor effect on established B16 melanoma liver micrometastases. Furthermore, when IL-2 and IFN-alpha administration was combined with the administration of mAb, a significantly reduced number of established 6- to 8-day B16 melanoma liver macrometastases and prolonged survival of tumor-bearing mice were seen. These studies imply that the administration of appropriate cytokine combinations may be a useful adjunct to the administration of mAb for the treatment of cancer in humans.     

Therapeutic effects of tumor-reactive type 1 and type 2 CD8+ T cell subpopulations in established pulmonary metastases.

Cytolytic CD8+ T cells fall into two subpopulations based on cytokine-secretion. Type 1 CD8+ cells (Tc1) characteristically secrete IFN-gamma, whereas type 2 CD8+ cells (Tc2) secrete IL-4 and IL-5. We assessed the relative therapeutic effects of adoptively transferred OVA-specific Tc1 and Tc2 CD8+ cells in mice bearing established OVA-transfected B16 melanoma lung metastases. Both Tc1 and Tc2 subpopulations mediated a reduction in lung tumor growth that subsequently prolonged survival times in mice with both early (day 7) and more advanced (day 14) levels of tumor development. CD8+ T cell populations recovered from spleens of tumor-bearing mice receiving Tc1 or Tc2 cells showed markedly enhanced tumor Ag-specific cytolytic and cytokine-releasing activities that correlated with delays in tumor cell growth and progression. Initially, both tumor-reactive Tc1 and Tc2 effector cells accumulated at the tumor site with nearly equal frequency. Tc1 cells persisted, whereas Tc2 cell numbers progressively diminished over time. Titration of Tc1 and Tc2 effector cells showed that protection was dose dependent with the former being 5-fold more effective. Tc2 cells achieved a comparable reduction in lung tumor cell growth at higher concentrations of cell transfer. Tc1 effectors from IFN-gamma-deficient mice were less therapeutically effective than wild-type mice, but there was no significant reduction in activity between corresponding Tc2 populations. We speculate that the effectiveness of Tc1 and Tc2 cells may depend on different mechanisms. These studies suggest a potential role for Tc1 and Tc2 CD8+ subpopulations in tumor regression and immunotherapy.