The fate of interleukin-2 after in vivo administration

Interleukin 2 (IL 2) activity was measured in the serum of mice, using a bioassay, following various methods of IL 2 administration. IL 2 has a serum half-life of 3.7 min +/- 0.8 min (mean +/- SD) in mice after i.v. injection, and a serum IL 2 titer of 2 units/ml could be sustained for less than 30 min after injection of highly concentrated IL 2 solutions. Intraperitoneal (i.p.) and subcutaneous (s.c.) injection of similar amounts of IL 2 prolonged the duration of serum IL 2 activity at greater than 2 units/ml for 2 and 6 hr, respectively. Administration of IL 2 in a gelatin base was capable of sustaining serum IL 2 levels at greater than 2 units/ml for up to 16 hr. The reason for the short in vivo half-life of i.v. injected IL 2 was studied. No inhibitors of IL 2 could be detected in our cell growth assay of IL 2 activity. Exposure of IL 2 to mouse blood or serum had no impact on IL 2 titers. To evaluate the possibility that IL 2 was binding to lymphoid cells in vivo, the fate of IL 2 was studied in nude mice, in mice 4 days after 1000 rads total body irradiation, and in splenectomized mice. The serum half-life in these modified mice was identical to that in normal mice. The kidney appeared to be the main site of IL 2 clearance. The rates of IL 2 disappearance in mice rose from a control T 1/2 of 2.5 to 3.5 min in sham-operated animals to up to 84 min in mice with ligated renal pedicles. IL 2 was not excreted in an active form in the urine and ureteral ligation had only a small effect on the serum half-life of IL 2, implying probable renal tubular catabolism of filtered IL 2.     

Adoptive immunotherapy of a syngeneic murine leukemia with a tumor-specific cytotoxic T cell clone and recombinant human interleukin 2: correlation with clonal IL 2 receptor expression

The successful adoptive immunotherapy of the syngeneic Friend virus-induced murine leukemia FBL-3 was mediated by a proliferative MHC-restricted, tumor-specific CTL clone in combination with recombinant human IL 2. This clone was previously shown to express the L3T4-, Lyt-1+, Lyt-2+ surface phenotype. Activation of the clone for 48 hr in vitro with irradiated tumor cells induced the expression of IL 2 receptors and markedly increased clonal proliferation in response to recombinant IL 2. Intravenous injection of 2 X 10(7) 48 hr in vitro-activated cloned cells, followed by 6 days of systemic (i.p.) administration of IL 2 resulted in the complete regression of tumors and the cure of 50% of the treated mice. IL 2 alone had no effect on tumor growth, whereas the injection of nonactivated (resting) clone plus IL 2 or activated clone without IL 2 had small but insignificant effects on tumor growth and survival. These results indicated that the in vivo effector functions of cloned T cells may be markedly enhanced by the concurrent systemic administration of recombinant IL 2 and by the induction of optimal IL 2 receptor expression on the cloned T cells at the time of cell 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.     

Interleukin 2 administered in vivo induces the growth of cultured T cells in vivo

The capacity of exogenous IL 2 to induce the growth of antigen-activated T lymphocytes in vivo was evaluated. The in vivo growth of adoptively transferred T lymphocytes that had been previously cultured long-term with IL 2 was initially examined, because in vitro such T cells are exquisitely dependent upon exogenous IL 2 for proliferation and survival. Daily administration of IL 2 in vivo, beginning on the day of cell transfer, induced these IL 2-dependent long-term cultured T lymphocytes to proliferate in vivo, and the magnitude of in vivo growth was proportional to the dose of IL 2 administered. The capacity of IL 2 to induce the in vivo growth of antigen-activated T cells not previously exposed in vitro to exogenous IL 2 was similarly studied. T lymphocytes from the spleens of immune mice, activated by 5-day culture with tumor antigen before transfer, survived poorly in vivo when injected with antigen alone, but demonstrated marked proliferation in vivo in response to antigen and exogenous IL 2. By contrast, immune spleen cells transferred with antigen, but without prior culture, proliferated without supplementary exogenous IL 2. Moreover, the growth of noncultured donor T cells was not augmented by the administration of exogenous IL 2, implying that noncultured spleen cells immune to tumor antigens can produce sufficient amounts of endogenous IL 2 in vivo to sustain maximal T cell growth over the time period examined. Importantly, the ability of exogenous IL 2 to induce donor T cell growth in vivo correlated with its ability to function in vivo to augment the anti-tumor efficacy of specifically immune donor T cells in models for the adoptive therapy of disseminated antigenic murine leukemia. Thus, the current studies highlight the potential of exogenous IL 2 to induce T cell growth in vivo and suggest that the administration of IL 2 in vivo may be useful for augmenting T cell responses that are relatively deficient in the production of endogenous IL 2.     

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.     

Reconstitution of nude mouse T cell function in vivo: IL 2-independent effect of human T cells

The i.p. injection of 1 to 5 X 10(6) heavily irradiated human T lymphocytes resulted in the lasting reconstitution of T cell functions in young mice bearing the nu/nu mutation. IgM and IgG responses to immunization with sheep red cells or ovalbumin, splenic lectin responses, and the expression of easily detectable Thy-1 determinants on up to 20% of spleen cells could be documented for several months after the injection of human cells. Only a narrow cell dose range was effective. Injection of larger cell numbers not only failed to induce immune reconstitution but also resulted in the development of resistance to subsequent treatments. Only mature T cells, but not thymocytes, could induce nude mouse T cell development. Lymphoblasts from one patient with acute T cell leukemia consistently immune-reconstituted nude recipients. These cells were completely unable to produce IL 2 in vitro. In contrast, the IL 2-producing T cell line Jurkat was ineffective, indicating that the abilities to produce IL 2 and to induce nude mouse T cell development are independent. In an extension of earlier models of the nude mouse immune defect, two distinct T precursor cell pools are proposed as the major components of an extrathymic differentiation pathway. As an adequate trigger of differentiation, interaction with thymus-processed T cells guides the development of precursors in the first cell pool towards populating the second IL 2-responsive pool of T precursor cells.     

Dendritic cells injected via different routes induce immunity in cancer patients

Dendritic cells (DC) represent potent APCs that are capable of generating tumor-specific immunity. We performed a pilot clinical trial using Ag-pulsed DC as a tumor vaccine. Twenty-one patients with metastatic prostate cancer received two monthly injections of DC enriched and activated from their PBMC. DC were cocultured ex vivo with recombinant mouse prostatic acid phosphatase as the target neoantigen. Following enrichment, DC developed an activated phenotype with up-regulation of CD80, CD86, and CD83 expression. During culture, the DC maintained their levels of various adhesion molecules, including CD44, LFA-1, cutaneous lymphocyte-associated Ag, and CD49d, up-regulated CCR7, but lost CD62 ligand and CCR5. In the absence of CD62 ligand, such cells would not be expected to prime T cells efficiently if administered i.v. due to their inability to access lymphoid tissue via high endothelial venules. To assess this possibility, three patient cohorts were immunized with Ag-pulsed DC by i.v., intradermal (i.d.), or intralymphatic (i.l.) injection. All patients developed Ag-specific T cell immune responses following immunization, regardless of route. Induction of IFN-gamma production, however, was seen only with i.d. and i.l. routes of administration, and no IL-4 responses were seen regardless of route, consistent with the induction of Th1-type immunity. Five of nine patients who were immunized by the i.v. route developed Ag-specific Abs compared with one of six for i.d. and two of six for i.l. routes. These results suggest that while activated DC can prime T cell immunity regardless of route, the quality of this response and induction of Ag-specific Abs may be affected by the route of administration.     

In vivo development of cytolytic T lymphocytes (CTL) to hapten-altered self: MIs-disparate cells facilitate the response by neutralizing IL 2 inhibitor

Inability to develop CTL in vivo to hapten-altered self can be attributed in part to an inhibitor of interleukin 2 (IL 2) that is present in the serum of normal mice. We have shown earlier that hapten-specific CTL can be generated in C3H mice (H-2k, MIsc) provided CBA/J (H-2k MIsd) spleen cells are injected simultaneously with hapten-modified syngeneic spleen cells into the hind foot paws. In efforts to determine whether serum levels of the inhibitor of IL 2 are altered as a consequence of this successful immunization method, we have compared the activity of the inhibitor in serum at intervals after the injection of syngeneic spleen cells, CBA spleen cells, or TNP-C3H spleen cells alone or together with CBA spleen cells, by using a murine IL 2-dependent, cloned cytotoxic T cell line, CT-6. The results indicate that inhibitor was neutralized optimally 48 to 72 hr after injection of TNP-C3H spleen cells mixed with CBA/J spleen cells. The order in which neutralization occurred was as follows: TNP-C3H cells + CBA/J cells greater than CBA cells greater than TNP-C3H cells greater than normal C3H spleen cells. Furthermore, supernatants from cultures of C3H lymph node cells stimulated in vivo with CBA/J cells also contained IL 2 activity. Thus, injection of CBA/J cells with TNP-modified syngeneic spleen cells produces IL 2 in vivo in sufficient quantity to neutralize the activity of the inhibitor as well as to facilitate the maturation of pre-CTL into hapten-altered self-specific CTL.     

Recombinant interleukin 2 stimulates in vivo proliferation of adoptively transferred lymphokine-activated killer (LAK) cells

We previously reported that the adoptive transfer of lymphokine-activated killer (LAK) cells plus repetitive injections of recombinant interleukin 2 (IL 2) produced a marked reduction in established pulmonary metastases from a variety of murine sarcomas. The requirement for the exogenous administration of IL 2 prompted a subsequent examination of the role of IL 2 in the in vivo function of transferred LAK cells. The in vivo proliferation and migration patterns of lymphoid cells in C57BL/6 mice were examined after i.v. transfer of LAK cells alone, i.p. injection of IL 2 alone, or the combination of LAK cells and IL 2. A model for in vivo labeling of the DNA of dividing cells was used in which mice were injected with 5-[125I]-iodo-2'-deoxyuridine (125IUdR) and, 20 hr later, their tissues were removed and were counted in a gamma analyzer. A proliferation index (PI) was calculated by dividing the mean cpm of organs of experimentally treated mice by the mean cpm of organs of control mice. In animals given LAK cells alone, the lungs and liver demonstrated little if any uptake of 125IUdR above saline-treated controls (PI = 2.5 and 0.8, respectively, on day 5), whereas the same organs of mice receiving 6000 U of IL 2 alone displayed higher radiolabel incorporation (PI = 7.1 and 5.9, respectively). When mice were given LAK cells plus 6000 U of IL 2, their tissues showed an additional increase in 125IUdR uptake. In the spleen, kidneys, and mesenteric lymph nodes, IL 2 treatment alone (6000 U) produced elevated PI values that were not, however, additionally increased if LAK cells were also administered. To separate the stimulatory effects of IL 2 on host lymphocyte proliferation from similar IL 2 effects on injected LAK cells, these studies were repeated in mice immunosuppressed by 500 rad total body irradiation. Pre-irradiation of the host sufficiently reduced endogenous lymphoid expansion stimulated by IL 2 so as to allow the demonstration that IL 2 also induced the proliferation of the transferred LAK cells. A variety of studies confirmed that the injected LAK cells were actively proliferating in tissues in vivo under the influence of IL 2. Substitution of "normal" LAK cells with fresh and cultured (without IL 2) splenocytes, or irradiated LAK cells did not result in increased 125IUdR uptake in tissues. Histologic studies corroborated the findings of the 125IUdR incorporation assays and revealed extensive lymphoid proliferation in irradiated mice receiving LAK cells plus IL 2.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vivo and in vitro expression of an interleukin 2 receptor by murine B and T lymphocytes

Interleukin 2 (IL 2), which is well established to be a T cell growth factor, has more recently been shown to stimulate B lymphocyte growth and differentiation in vitro. Responsiveness of B and T cells to IL 2 has been associated with expression of a cell membrane IL 2 receptor (IL 2R). To investigate the role of IL 2 in B cell growth and differentiation in vivo, a system was used in which the injection of mice with a goat antibody to mouse IgD (GaM delta) induces polyclonal T-independent B cell proliferation first, and later induces polyclonal T-dependent B cell proliferation and IgG secretion. IL 2R expression by splenic B and T lymphocytes from GaM delta injected mice was studied by a dual label immunofluorescence technique. Although GaM delta was found to be a strong inducer of B cell IL 2R expression in vitro, even in serum-free medium, and stimulated up to 50% of splenic T cells to express considerable quantities of IL 2R in vivo, it failed to induce more than minimal B cell IL 2R expression in vivo. Concanavalin A and bacterial lipid A also induced B cells to express IL 2R to a much greater extent in vitro than in vivo. Although these agents and GaM delta acted synergistically to stimulate B cell IL 2R expression both in vitro and in vivo, a single agent induced B cell IL 2R expression to a considerably greater extent in vitro than did all three agents acting together in vivo. In vitro GaM delta-induced B cell IL 2R expression was not suppressed by inclusion of IL 2 in the culture medium but was suppressed by the presence of 10% normal mouse serum or plasma. These observations suggest that polyclonal T-dependent B cell proliferation and antibody secretion may not require an interaction between B cells and IL 2; the in vivo environment may downregulate IL 2R expression by B cells: and in vivo B cell IL 2R expression and consequently, induction of B cell responsiveness to IL 2, may require stimuli beyond those sufficient to induce B cell IL 2R expression and IL 2 responsiveness in vitro.     

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.     

Generation of alloreactive cytotoxic T lymphocytes: production of T cell and macrophage helper factors in addition to IL 1 and IL 2 by peritoneal cells from mice immunized to Listeria monocytogenes

We investigate the production and biological activity of soluble helper factors produced by peritoneal T cells and macrophage derived from mice primed in vivo with Listeria monocytogenes. Supernatant fluids from co-cultures of these immune T cells and activated macrophages contained Interleukin 1 (IL 1) and Interleukin 2 (IL 2), and had the ability to assist the generation of cytotoxic T lymphocytes (CTL) from a population of nylon wool nonadherent spleen cells sensitized to allogeneic heat-treated thymocytes. The ability to assist CTL development involved T cell and macrophage factors in addition to IL 1 and IL 2. Immune T cells cultured alone produced a factor, devoid of significant IL 2 activity, that assisted CTL development only if adherent cells were present in the responding population. Activated macrophage produced a 38,000 dalton component, distinct from IL 1 on the basis of m.w., that assisted the development of CTL from nylon wool nonadherent splenic cells. Supernatants fluids from co-cultures of immune T cells and allogeneic, nonactivated macrophage contained a CTL helper factor but did not contain IL 1 or IL 2 activities. In contrast, supernatant fluids from co-cultures of immune T cells and syngeneic, nonactivated macrophage contained all 3 activities. This suggests a genetic restriction for the production of IL 1 and IL 2 that does not restrict the production of a CTL helper factor. These results demonstrate that T cell- and macrophage-derived helper factors distinct from IL 1 and IL 2 participate in the development of CTL.     

Differential effect of anti-beta 2-microglobulin on IL 2 production and IL 2 receptor expression in the primary mixed lymphocyte culture reaction

The mechanism of inhibition of the proliferative response in primary mixed lymphocyte culture (1 degree MLC) by antibodies to beta 2-microglobulin (beta 2m) was investigated. It is demonstrated that anti-beta 2m antibodies inhibit the production of interleukin 2 (IL 2). In contrast, the expression of IL 2 receptor is not affected by anti-beta 2m. The addition of purified exogenous IL 2 to the antibody-treated 1 degree MLC can completely restore the proliferative response, indicating that anti-beta 2m does not interfere with IL 2 binding to its receptor. Similarly, anti-beta 2m does not interfere with the capacity of IL 2-dependent T cell lines or T cell clones to respond to exogenous IL 2. The inhibition of cell proliferation and IL 2 production by anti-beta 2m is maximal when the antibody is added at the beginning of 1 degree MLC culture, and no effect of anti-beta 2m is seen when added after 3 days of culture. Anti-beta 2m has no effect on mitogen-induced cell proliferation and IL 2 production. Anti-beta 2m acts on the responder cell population, as demonstrated in experiments in which responder cells or stimulator cells are treated separately with the antibody. The expression of HLA-class II antigens (i.e., HLA-DR and DQ (DC) on the T cells activated on 1 degree MLC is not affected by anti-beta 2m. These studies indicate that the HLA-beta 2m class I antigen complex plays a role in T lymphocyte activation via release of IL 2, and suggest the existence of different mechanisms for activation of IL 2 producers and IL 2 responders in 1 degree MLC.     

Role of interleukin 2 (IL 2) and hemopoietin-1 (H-1) in the generation of mouse natural killer (NK) cells from primitive bone marrow precursors

The development of natural killer (NK) cells from bone marrow (BM) precursors was studied. Recombinant interleukin 2 (IL 2) was able to induce the in vitro development of NK cells when added to cultures of mouse BM cells. Treatment of donor mice with 5-fluorouracil (150 mg/kg i.v.), which eliminates more differentiated cells but spares less differentiated cells, appears to augment NK cell development. The "NK stem cell" was found to be asialo GM1-, Thy-1+, Lyt-2-, and Lyt-1-. The cells generated in vitro had a typical phenotype of NK cells, being asialo GM1+, Lyt-5+, Thy-1+, Lyt-2-, and Lyt-1-. These effector cells also had specificity characteristics of NK cells lysing the NK-susceptible YAC-1 and K562 targets, but not the NK-resistant EL/4 or allogeneic and syngeneic blasts. Hemopoietin-1 (H-1), a factor which acts on very primitive multipotent BM cells, was able to cooperate with IL 2, increasing the development of NK cells. In contrast, other factors such as interleukin 3 or colony-stimulating factor did not cause induction of NK activity when added to cultures of BM cells, indicating that this effect, i.e., induction of NK cell development, is peculiar to IL 2. These results indicate that IL 2 can act as a differentiation as well as growth factor for NK cells, and that H-1 can promote the development of functional activity in a lymphocyte subpopulation as well as affect the differentiation of myelomonocytic and other cell lineages. This experimental system appears quite useful for characterization of BM precursors for NK cells, and should help to better understand the relationship of the NK cell lineage to the T cell or other lineages.     

2'Deoxycoformycin and deoxyadenosine affect IL 2 production and IL 2 receptor expression of human T cells

Congenital deficiency of the enzyme adenosine deaminase (ADA) leads to severe combined immunodeficiency. 2'Deoxycoformycin (dCF), a tightly binding inhibitor of ADA, can induce the metabolic state of ADA deficiency. In vivo, the drug causes specific impairment of lymphocyte function and shows strong immunosuppressive properties. However, to decide whether inhibition of the enzyme ADA offers an attractive approach for immunosuppressive therapy, more information is needed about the immunologic mechanisms affected. In human T cells, we investigated the effect of dCF and deoxyadenosine (AdR) on cell activation, interleukin 2 (IL 2) production, and IL 2 receptor induction after allogeneic and lectin-induced stimulation. After allogeneic stimulation, dCF and AdR affected several events in T cellular immune response. Early events in T cell activation showed to be most sensitive to the drugs. Primary MLC was completely inhibited by concentrations as low as 1 microM dCF and 1 microM AdR. The addition of human recombinant IL 2 (rIL 2) could not abrogate the inhibitory effect of the drugs. Apart from activation of T cells, the drugs interfered with proliferation of activated T cells. Two events in activated T cells were affected: IL 2 production and IL 2 receptor expression. In secondary MLC, IL 2 production was markedly reduced in the presence of 9 microM dCF and 60 microM AdR. These concentrations appeared also to affect IL 2 receptor expression in 12-day primary MLC cells stimulated with rIL 2. Lectin stimulation was also affected by the drugs. In phytohemagglutinin (PHA)-stimulated cultures, 9 microM dCF and 60 microM AdR resulted in inhibition of proliferation and IL 2 receptor expression, whereas IL 2 production was normal. It is concluded that dCF and AdR interfere with several events in T cellular immune response such as cell activation, IL 2 production, and IL 2 receptor expression. According to these results, inhibition of the enzyme ADA seems an attractive approach to immunosuppressive therapy.     

Activation and route of administration both determine the ability of bone marrow-derived dendritic cells to accumulate in secondary lymphoid organs and prime CD8<Superscript>+</Superscript> T cells against tumors

Aims

To examine the effects of route of administration and activation status on the ability of dendritic cells (DC) to accumulate in secondary lymphoid organs, and induce expansion of CD8⁺ T cells and anti-tumor activity.

Methods

DC from bone marrow (BM) cultures were labeled with fluorochromes and injected s.c. or i.v. into naïve mice to monitor their survival and accumulation in vivo. Percentages of specific CD8⁺ T cells in blood and delayed tumor growth were used as readouts of the immune response induced by DC immunization.

Results

The route of DC administration was critical in determining the site of DC accumulation and time of DC persistence in vivo. DC injected s.c. accumulated in the draining lymph node, and DC injected i.v. in the spleen. DC appeared in the lymph node by 24 h after s.c. injection, their numbers peaked at 48 h and declined at 96 h. DC that had spontaneously matured in vitro were better able to migrate compared to immature DC. DC were found in the spleen at 3 h and 24 h after i.v. injection, but their numbers were low and declined by 48 h. Depending on the tumor cell line used, DC injected s.c. were as effective or more effective than DC injected i.v. at inducing anti-tumor responses. Pre-treatment with LPS increased DC accumulation in lymph nodes, but had no detectable effect on accumulation in the spleen. Pre-treatment with LPS also improved the ability of DC to induce CD8⁺ T cell expansion and anti-tumor responses, regardless of the route of DC administration.

Conclusions

Injection route and activation by LPS independently determine the ability of DC to activate tumor-specific CD8⁺ T cells in vivo.

     

Mechanisms of human T cell response to mitogens: IL 2 induces IL 2 receptor expression and proliferation but not IL 2 synthesis in PHA-stimulated T cells

Human peripheral blood T cells were purified by a four-step procedure which included depletion of plastic-adherent cells, rosetting with sheep red blood cells, nylon wool passage, and treatment with mouse monoclonal antibodies to human Ia antigens plus complement. The purified T cells completely failed to proliferate to phytohemagglutinin (PHA). Bacterially derived recombinant human interleukin 2 (IL 2) reconstituted the proliferative response of resting T cells to PHA. The optimal concentration of IL 2 required was 100 to 200 U/ml. IL 2 alone caused no T cell proliferation. Both PHA and IL 2 needed to be present together for the proliferation of T cells to occur. Incubation of T cells with either PHA or IL 2 alone for up to 18 hr, followed by washing, then by the addition of the reciprocal reagent, resulted in no T cell proliferation. Expression of IL 2 receptors and of Ia antigens, as assessed by indirect immunofluorescent staining, revealed that both PHA and IL 2 needed to be present for Tac and Ia antigen expression by T cells. T cells incubated with PHA and IL 2 for 18 to 42 hr acquired responsiveness to IL 2. These T cells remained absolutely dependent on IL 2 for proliferation to occur. In contrast to T cells stimulated with PHA in the presence of monocytes, T cells stimulated with PHA and IL 2 released no detectable IL 2. The failure of IL 2 secretion was not caused by down-regulation of IL 2 production by IL 2 itself, because the addition of IL 2 to cultures of T cells stimulated with PHA in the presence of monocytes did not interfere with IL 2 production. These results indicate that IL 2 is a sufficient signal to induce the expression of its receptor in PHA-stimulated T cells and subsequent proliferation but is not sufficient to cause endogenous IL 2 release.     

Pharmacokinetics of an antiangiogenic ribozyme (ANGIOZYME) in the mouse.

Vascular endothelial growth factor (VEGF) is a growth factor that contributes to the angiogenesis of developing tumors. To interfere with the action of VEGF, a nuclease-stabilized ribozyme, ANGIOZYME, has been developed against VEGF receptor subtype Flt-1 mRNA. To determine which routes of administration would be useful for systemic delivery of this ribozyme, a dose of 30 mg/kg [32P]ANGIOZYME was administered as an i.v., i.p., or s.c. bolus. Concentrations of ANGIOZYME in plasma, femur, kidney, liver, and lung were examined. ANGIOZYME was well absorbed after i.p. (90%) or s.c. administration (77%), with peak plasma concentrations occurring 30 minutes after dosing. Total body clearance after a single dose of 30 mg/kg ANGIOZYME was 20 ml/min/kg, and the elimination half-life was 33 minutes. The apparent volume of distribution at steady-state ranged from 0.5 to 1.3 L/kg. ANGIOZYME was detected in the four tissues examined through the 3 hour sampling period after i.v. or i.p. administration. After s.c. administration, ANGIOZYME was detected in femur, kidney, and lung but not in the liver. The highest concentrations of ANGIOZYME were found in kidney and femur with all three routes. Because of the rapid and extensive absorption after extravascular injections, either i.p. or s.c. administration could be considered for use in pharmacodynamic studies examining the effects of ANGIOZYME or other ribozymes with similar chemical modifications.     

Generation of tumor-specific cytotoxic T lymphocytesin vivo by combined treatment with inactivated tumor cells and recombinant interleukin-2

In order to search for a new therapy that would maximize the effect of interleukin-2 (IL-2) in evoking antitumor immunity in vivo, the therapeutic effect of a combination of mitomycin-C(MMC)-treated tumor cells and recombinant IL-2 was examined for its induction of antitumor activity against established melanoma metastasis. In C57BL/6 mice intravenously (i. v.) injected with B16 melanoma cells on day 0, the combined treatment with an intraperitoneal (i. p.) injection of MMC-treated melanoma cells on day 6 and 2500 U rIL-2 (twice daily) on days 7 and 8 markedly reduced the number of pulmonary metastases. This antitumor activity was more effective than that in untreated controls and mice that were injected with MMC-treated melanoma cells alone or rIL-2 alone. When the i. p. injection of MMC-treated tumor cells was replaced by other syngeneic tumor cells, antitumor activity against metastatic melanoma was not induced. The antitumor activity induced by this treatment increased in parallel with an increase in the dose of rIL-2 injected. In contrast, an i. p. injection of soluble tumor-specific antigens alone could induce only a marginal level of antitumor activity, and this activity was not augmented by subsequent i. p. injections of rIL-2. In vivo treatment with anti-CD8 monoclonal antibody (mAb), but not with anti-CD4 mAb or anti-asialo-GM1 antibody, abrogated the antitumor activity induced by this combined therapy. This suggests that the antitumor effect was dependent on CD8⁺ T cells. Lung-infiltrating lymphocytes from mice that had been i. v. injected with melanoma cells 11 days before and were treated with this combined therapy, showed melanoma-specific cytolytic activity. This combined therapy also showed significant antitumor activity against subcutaneously inoculated melanoma cells. These results demonstrate that the combined therapy of an i. p. injection of MMC-treated tumor cells and subsequent and consecutive i. p. administration of rIL-2 increases antitumor activity against established metastatic melanoma by generating tumor-specific CD8⁺ CTL in vivo.     

Possibility of reducing the luteolytic dose of cloprostenol in cyclic dairy cows

The administration of cloprostenol by intravulvosubmucous (i.v.s.m.) injection at 1 2 and 1 4 of the dose usually given by intramuscular (i.m.) injection, was tested in dairy cows for luteolysis and estrus synchronization. The i.m. injection was used in ten adult cows at the usual dose of 500 mug/animal. Eleven adult cows and 11 heifers were treated i.v.s.m. with a dose equivalent to 250 mug/animal and 125 mug/animal, respectively. Two injections of cloprostenol were administered 11 days apart to the cows not detected in oestrus after a single injection. Forty-three out of the total 46 animals were detected to be in dioestrus at the time of at least one of the injections, as reflected by the plasma progesterone concentrations at the time of treatments. Three out of the 43 animals injected during dioestrus were refractory to the luteolytic effect of cloprostenol; this appeared to be independent of the dosage and the route of administration (refractory cows were: one adult cow treated i.m. and two treated i.v.s.m. with 125 mug of cloprostenol). The mean time interval from injection to the onset of heat was 82.8 hours with a confidence limit for 95% of probability between 67.9 hours and 92.7 hours. The difference between treatments is not significant. The results suggest that in heifers and adult cows cloprostenol can be given i.v.s.m. route at a reduced dose of 1 4 of the usual 500 mug i.m. dosage without affecting the luteolytic effect of the drug or fertility.