Combination therapy with adriamycin and interleukin 2 augments immunity against murine renal cell carcinoma.

The mechanism(s) through which combination treatment with Adriamycin and recombinant interleukin 2 (rIL2) affects murine renal cell carcinoma was investigated. A single dose of Adriamycin (ADM) administered 1 day after implantation of tumor cells s.c. delayed the formation of tumor and significantly extended the median survival time. About 40% of the treated mice remained tumor free for more than 6 months. Treating mice with a similar dose of ADM 12 to 16 days after implantation of tumor caused complete regression of established tumors within 10 to 12 days in more than 90% of the mice. However, after a transient tumor free period of 15 to 20 days recurrence of tumor was observed in all treated mice. In contrast, a regimen that included treating tumor bearing mice with a single dose of ADM followed by a daily i.p. injection of rIL2 5 x 10(4) units for 10 days delayed the recurrence of tumors and significantly prolonged the survival time compared to the median survival time of mice treated with ADM alone. About 20 to 30% tumor bearing mice remained tumor free for 4 months following treatment with ADM and rIL2. Treatment with rIL2 alone produced no antitumor response. In addition, rIL2 itself was not inhibitory for tumor cell growth nor did it modulate the cytotoxic response of renal cell carcinoma cells to ADM in vitro. Mice that were cured following treatment with ADM and rIL2 were resistant to a rechallenge with viable tumor cells, and cured mice also expressed a tumor specific T-cell mediated delayed hypersensitivity reaction. The immune cells that mediate tumor rejection were identified as Thy-1.2+ T-cells. Taken together, these results indicate that antitumor activity of combined Adriamycin/rIL2 treatment is at least partly attributable to the production of tumor specific immunity.

Interactive effects of alpha-interferon A/D and interleukin 2 on murine lymphokine-activated killer activity: analysis at the effector and precursor level.

Studies from our laboratory and others have demonstrated that alpha-interferon (IFN alpha) can regulate natural killer cells and lymphokine-activated killer (LAK) cell activation. In vitro experiments have shown that IFN alpha has differential effects on both natural killer cells and LAK activity when combined with interleukin 2 (IL2); IFN alpha synergized with IL2 to augment natural killer cells activity while it suppressed the IL2-induced LAK response. Here we demonstrated that IFN alpha A/D can also regulate IL2-induced LAK activity in vivo with enhanced or suppressed activity depending on the IFN alpha A/D dose. The enhanced response was observed with the combination when 80,000 units/day of IFN alpha A/D were used and was detectable in the spleen, lung, and peritoneum. When a high dose of IFN alpha A/D was combined with IL2, a moderate reduction in LAK activity was noted in the spleen and peritoneum. In contrast, a high dose IFN alpha A/D augmented IL2-induced LAK activity in the lung even though it reduced the level of cellular infiltration. We have also evaluated the effect that IL2, IFN alpha A/D, and IL2 plus IFN alpha A/D have on the frequency of LAK precursors in the spleen and lung using limiting dilution analysis. Treatment of normal mice with IL2 alone increased the frequency of LAK precursor (LAKp) in the lung. This increase was associated with an infiltration of Thy-1+, asialo-GM1+, Lyt-2- lymphocytes into the lungs. Moreover, treatment with IL2 plus IFN alpha A/D enhanced the frequency of LAKp over that observed with IL2 alone. Treatment with the combination did not change the phenotype of LAKp in the lung from that seen with IL2. The increase in LAKp frequency induced by the combined treatment may not be a direct effect of IFN alpha A/D on precursor cells since IFN alpha A/D alone did not increase the frequency of LAKp in vivo or in vitro when added to limiting dilution analysis cultures. In contrast to what occurred in the lung, a consistent increase in LAKp was not seen in the spleen after treatment with IL2 or with the combination, although LAK activity was observed. These results demonstrated that in addition to inducing lytic activity from LAK effectors in vivo, IL2 treatment increased the number of precursor cells within the lung. Moreover, IFN alpha A/D in combination with IL2 influenced the level of LAKp in situ.

In vitro and in vivo responses to interleukin 12 are maintained until the late SIV infection stage but lost during AIDS.

The in vitro proliferative responses of macaque peripheral blood mononuclear cells (PBMCs) to IL-12 appeared similar before and early after SIV infection, whereas macaque PBMCs sampled during symptomatic stages of SIV infection showed markedly decreased responses. IL-12 was administered to SIVmac239-infected rhesus macaques either during the asymptomatic or the AIDS stage of infection in efforts to evaluate the effect of this cytokine on immune responses, viral loads, and hematopoietic functions in vivo. IFN-gamma secretion levels induced during the asymptomatic or early symptomatic phase were similar to preinfection induced levels, whereas in later AIDS stages this response was lost. The constitutive levels of other measured cytokines were not affected by IL-12 administration in vivo. The frequency and activity of circulating NK cells were markedly enhanced at early stages but not at symptomatic stages of SIV infection. pCTL frequencies were enhanced at early symptomatic stages but not at late AIDS stages. Despite its immunomodulatory effect, IL-12 did not seem to exacerbate or inhibit the replication of SIV in vivo, or the frequency of circulating infected lymphocytes. IL-12 administration was associated with a significant yet subclinical and transient decrease in hematocrit and hemoglobin levels without evidence of hemolysis, hemodilution, or reduction in the frequency of colony-forming unit potential of bone marrow CD34+ cells. This phenomenon may be explained by a functional inhibition of differentiation rather than an altered generation of bone marrow precursors. Thus, these results suggest that IL-12 may benefit HIV-1-infected patients only as long as their immune system retains its capability to respond to cytokine stimulation.

Therapeutic efficacy of interleukin-2 activated killer cells against adriamycin resistant mouse B16-BL6 melanoma.

Development of multidrug-resistance (MDR) remains a major cause of failure in the treatment of cancer with chemotherapeutic agents. In our efforts to explore alternative treatment regimens for multidrug-resistant tumors we have examined the sensitivity of MDR tumor cell lines to lymphokine activated killer (LAK) cells. Adriamycin (ADM) resistant B16-BL6 melanoma, L1210 and P388 leukemic cell lines were tested for sensitivity to lysis by LAK cells in vitro. While ADM-resistant B16-BL6 and L1210 sublines were found to exhibit at least 2-fold greater susceptibility to lysis by LAK cells, sensitivity of ADM-resistant P388 cell was similar to that of parental cells. Since ADM-resistant B16-BL6 cells were efficiently lysed by LAK cells in vitro, the efficacy of therapy with LAK cells against the ADM-resistant B16-BL6 subline in vivo was evaluated. Compared to mice bearing parental B16-BL6 tumor cells, the adoptive transfer of LAK cells and rIL2 significantly reduced formation of experimental metastases (P less than 0.009) and extended median survival time (P less than 0.001) of mice bearing ADM-resistant B16-BL6 tumor cells. Results suggest that immunotherapy with LAK cells and rIL2 may be a useful modality in the treatment of cancers with the MDR phenotype.

Differences in murine gut-associated lymphoid tissues in generating broadly nonspecific cytotoxic cells in response to interferon alpha A/D and interleukin 2.

We examined the response of cells of murine gut-associated lymphoid tissues to agents that augment the activity of natural killer (NK) cells. Specifically, we studied the effect of polyinosinic: polycytidylic acid (Poly I:C) in vivo, and recombinant interferon alpha A/D (rIFN alpha A/D) and recombinant interleukin 2 (rIL2) in vitro on lymphoid cells of mesenteric lymph nodes (MLN) and Peyer's patches (PP) in generating cytotoxicity against NK-sensitive (YAC-1) and NK-insensitive (B16BL6) tumor targets. The effect of these agents on spleen cells was examined for comparison with their effect on MLN and PP cells and as a positive control. MLN and PP cells lacked spontaneous NK activity: however, NK activity could be augmented to different levels by the three agents. The treatment of mice in vivo with Poly I:C induced considerable cytotoxicity in the spleen and MLN but only a weak cytotoxic response in PP. The in vitro enhancement of NK activity by rIFN alpha A/D was strong in the spleen, intermediate in MLN, and consistently poor in PP. The weak NK augmentation by rIFN alpha A/D in PP was not restricted to a single mouse strain. PP cells from five strains of mice responded poorly to rIFN alpha A/D. Furthermore, NK augmentation by rIFN alpha A/D in PP cells did not improve after passing the responder cells through nylon wool, indicating that the lack of augmentation of NK activity was not the result of a preponderance of B cells or the masking of NK cells by adherent lymphoid populations in PP. In contrast to weak augmentation of NK activity by rIFN alpha A/D, considerable IL2-induced lymphocyte-activated killer (LAK) activity against NK-insensitive B16BL6 tumor cells was induced in PP. Limiting-dilution analysis showed that the frequency of LAK precursors in the MLN and PP was not markedly different from that of the spleen. The differences among spleen, MLN, and PP lymphoid populations in generating the broadly nonspecific cytotoxic effector cells in response to rIFN alpha A/D or rIL2 may result from differences in the pools of different pre-NK cells or to differential sensitivity of the same pool of pre-NK cells to rIFN alpha A/D and rIL2 in different anatomical locations.

Anti-inflammatory action of IL-4. Negative regulation of contact sensitivity to trinitrochlorobenzene.

We examined the effects of IL-4, a cytokine produced by Th2 cells, on the development of an Ag-specific, T cell-mediated inflammatory response in a hapten-induced model of contact sensitivity (CS). Intravenous administration of IL-4 was ineffective in modulating the development of CS when administered on days 0, 1, and 2 after sensitization with the hapten trinitrochlorobenzene. In contrast, such treatment significantly reduced the response when given on the day of challenge. Conversely, treatment with anti-IL-4 mAb on day 4 markedly increased the magnitude of CS but was without effect when administered on days 0, 1, and 2. These results suggest that IL-4 interferes with CS at the efferent but not the afferent limb of the response. IL-4 had no inhibitory effect on the ability of immune lymph node cells to transfer adoptively CS or their proliferation upon restimulation with hapten. However, the expression of CS by immune cells was severely curtailed in mice treated with IL-4 prior to immune cell transfer. Furthermore, IL-4 inhibited monokine (gamma-IFN inducible protein [IP-10] and TNF-alpha) expression in macrophages induced by treatment with culture supernatants from the Ag-stimulated immune lymph node cells. These results indicate that suppression of Ag-specific inflammatory CS response by IL-4 may be mediated at least in part through inhibition of cytokine production by mononuclear phagocytes infiltrating the site.

Oral administration of the contact sensitizer trinitrochlorobenzene: initial sensitization and subsequent appearance of a suppressor population.

Our earlier studies have demonstrated that intragastric administration of the hapten trinitrochlorobenzene (TNCB) 2 to 3 weeks prior to attempting sensitization with epidermally applied hapten can abrogate development of systemic contact sensitivity (CS). In this paper, we have examined whether onset of tolerance following intragastric administration of the hapten is preceded by development of hapten-specific CS. Indeed, CS was found to be present 5 days after feeding TNCB and in most experiments the response decreased significantly by Days 10 to 12. The kinetics of development of CS by the oral and epidermal routes were strikingly similar except that the magnitude of reactivity (up to 5 days) in orally sensitized mice was somewhat less than that of epidermally sensitized mice. With the exception of Peyer's patches (PP), effector cells of CS were recovered from such gut-associated lymphoid tissues as mesenteric lymph nodes (MLN), lamina propria, and lymphocytes that are present in the intraepithelial compartment of the intestinal wall. These cells as well as spleen cells of TNCB-fed mice were able to adoptively transfer CS to naive mice. The capacity of MLN and spleen cells of TNCB-fed mice to confer CS adoptively was abrogated after treating cells with anti-Thy 1.2 and anti-Lyt 1.1 antibodies plus complement thereby identifying them as T lymphocytes. Although CS decreased by 10-12 days after feeding TNCB, the decline was reversed by pretreating mice with cyclophosphamide (CY) 2 days before giving the hapten. Whereas spleen cells from animals fed hapten 5 days earlier transferred CS readily, those from mice fed hapten 12 days earlier did not. However, when 12-day spleen cells were depleted of Lyt 2+ cells their ability to adoptively transfer CS was restored. These observations indicate that feeding TNCB to mice initially produces CS, mediated by Thy 1.2+, Lyt 1.1+ lymphocytes. CS is subsequently down-regulated by activation of Lyt 2+ suppressor cells, precursors of which are sensitive to CY.

Restricted replication of vesicular stomatitis virus in T lymphocytes is coincident with a deficiency in a cellular protein kinase required for viral transcription.

Vesicular stomatitis virus (VSV) fails to replicate in mouse T lymphocytes unless the cells have been mitogenically stimulated with concanavalin A (Con A). We have examined the possibility that the failure of VSV to replicate in unstimulated T lymphocytes can be attributed to a deficiency in a host protein kinase which activates the viral P protein by phosphorylation, thus rendering it transcriptionally competent. Soluble extracts were prepared from purified mouse T lymphocytes, with or without prior treatment with Con A. The ability of these extracts to phosphorylate bacterially synthesized P protein of two VSV serotypes was measured in vitro. Activity of the protein kinase on the P proteins of the Indiana or New Jersey serotypes of VSV increased, on average 2.4- and 2.1-fold respectively, after treatment of the cells with 3 micrograms/ml Con A. Higher concentrations of Con A induced proportional increases (up to 10-fold) in the activity of the host protein kinase. Activities of the kinase phosphorylating the P protein in separate populations of CD4- and CD8-containing murine T lymphocytes increased similarly on mitogenic activation. No biochemical or immunological differences were observed between the T cell protein kinase and the previously characterized protein kinase (casein kinase II) from BHK-21 cells. The activity of the kinase that phosphorylates the P protein did not vary in CV-1 cells on treatment with alpha- or gamma-interferon, both of which inhibited VSV replication. Similarly, casein kinase II activities in Raji and SIRC cells, which do not normally support VSV growth, were the same as in BHK-21 cells. Thus restriction of VSV replication in these cells, in contrast to T lymphocytes, was not associated with a deficiency in the host casein kinase II activity.

L3T4 (CD4+) cells that mediate contact sensitivity to trinitrochlorobenzene express I-A determinants.

The present study has further characterized the T cell-mediated inflammatory response of contact sensitivity (CS) to the hapten trinitrochlorobenzene (TNCB) in mice. A discernible CS response was found to be induced as early as 2 days after epicutaneous application of TNCB. The response peaked on Days 4 to 5 and it then declined to a nearly undetectable level by Days 10 to 11. Examination of the draining lymph nodes demonstrated that development of CS coincided with an increase in cellular proliferation and in the total number of cells present. Despite a severalfold increase in the cellular contents of the draining lymph nodes of sensitized mice, the relative percentages of most subsets of T cells remained unchanged. Flow cytometric studies revealed that the subpopulation of T cells characterized as Thy 1.2+ L3T4+ I-A+ increased substantially in comparison to its presence in unsensitized mice. Whether the Thy 1.2+ L3T4+ I-A+ cells that increased following sensitization represented the effector population that mediates CS was then examined. Four-day immune lymph node T cells or L3T4 cells positively selected from them were capable of adoptively transferring CS to normal mice. However, these cells, after treatment with anti-Ia antibody or anti-I-A monoclonal antibody and complement, were unable to transfer CS. These findings imply that expression of I-A determinants may indicate antigen-induced T cell activation in vivo and that L3T4 cells that mediate CS are I-A positive.

Immune and hematopoietic parameters in HIV-1-infected chimpanzees during clinical progression toward AIDS.

Until recently, chimpanzees were considered susceptible to human immunodeficiency virus type 1 (HIV-1) infection, but refractory to disease induction based on the asymptomatic status of all experimentally infected chimpanzees after over 10 years postinfection (PI). However, a decline in peripheral CD4+ T cells was noted in one chimpanzee (C499) of the Yerkes cohort of HIV-1 infected apes, after 11 years PI concurrent with increasing plasma viral load. These clinical signs were followed by the occurrence of opportunistic infections, thrombocytopenia, and progressive anemia leading to euthanasia. A second chimpanzee (C455) was transfused with blood from C499 collected during the symptomatic stage. Shortly thereafter, this second animal showed a rapid decline in peripheral CD4+ T-cell levels and sustained high viral load. Hematological analyses showed a 50% decrease in CFU-GM for both apes during the symptomatic phase and a reduction of 40% and 73% of the total CFU despite normal levels of CD34+ cells in the bone marrow. Cryopreserved sequential PBMC samples from these two chimpanzees were analyzed for constitutive and PHA-P induced levels of cytokines and chemokines. Data show that whereas there were no detectable constitutive levels of mRNA coding for IL-2, 4, and 10, there appears to be a transient increase in IFN-gamma message level coincident with increased viremia and this IFN-gamma synthesis decreased with disease progression. PHA-induced cytokine mRNA analysis showed low or undetectable levels of IL-4 and IL-10 mRNA in all samples and a marked decrease in the levels of IL-2 shortly after HIV infection. In addition, there was also a gradual decrease in IFN-gamma mRNA with progression of disease. Of interest were the findings of high to normal levels of PHA-induced synthesis of the chemokines MIP-1alpha, MIP-1beta, and RANTES in samples during the asymptomatic and early symptomatic period, which also dramatically decreased at late stages of the disease. These data suggest important roles for IL-2, IFN-gamma, and the chemokines in the regulation of immune responses in HIV-1-infected chimpanzees.

Expression and in vitro evaluation of rhesus macaque wild type (wt) and modified CC chemokines.

Several human CC chemokines have been shown to inhibit HIV/ SIV infection in vitro, providing the rationale for their potential use in vivo. However, because of their inherent physiological effect, such chemokines are reasoned to be of limited therapeutic value due to potential side effects. The knowledge that amino terminus modified or deleted human RANTES retains its receptor binding properties but loses its signaling properties has provided a means to use such modified chemokines in vivo for possible therapeutic benefits. In efforts to test the efficacy of such modified chemokines, our laboratory has cloned, sequenced, and prepared recombinant forms of wild-type (wt) and amino-terminus modified rhesus macaque chemokines MIP-1alpha, MIP-1beta, and RANTES. These sets of chemokines were tested for their potential to inhibit SIV infection and induce signaling. The data showed that whereas wt chemokines retained both virus inhibitory and signaling functions, corresponding amino-terminus modified chemokines only showed virus inhibitory effects without detectable signaling effects. Such reagents will be valuable for evaluation of their therapeutic potential in vivo, either alone or as adjuncts to other chemotherapeutic drugs.

Markedly elevated levels of interferon (IFN)-gamma, IFN-alpha, interleukin (IL)-2, IL-10, and tumor necrosis factor-alpha associated with fatal Ebola virus infection.

The role of immune mechanisms in the pathogenesis of Ebola hemorrhagic fever (EHF) remains to be elucidated. In this report, the serum cytokine levels of patients who died of EHF were compared with those of patients who recovered and those of control patients. A marked elevation of interferon (IFN)-gamma levels (>100 pg/mL) was observed in sequential serum samples from all fatal EHF cases compared with patients who recovered or controls. Markedly elevated serum levels of interleukin (IL)-2, IL-10, tumor necrosis factor (TNF)-alpha, and IFN-alpha were also noted in fatal EHF cases; however, they had a greater degree of variability. No differences were noted in serum levels of IL-4 and IL-6. mRNA quantitation from blood clots of the same patients showed relatively elevated levels of TNF-alpha and IFN-alpha in samples from EHF patients. Taken together, these results suggest that a high degree of immune activation accompanies and potentially contributes to a fatal outcome in EHF patients.

Induction of long-term protective effects against heterologous challenge in SIVhu-infected macaques.

A group of three rhesus macaques were inoculated with SIV isolated from a human (SIVhu) accidentally exposed and infected with SIVsm. Extensive sequence analyses of SIVhu obtained from the human and macaques following infection indicated the presence of truncated nef. Not only did nef fail to repair itself in vivo postinfection (p.i.), but instead, further mutations added additional stop codons with increasing time p.i. Infection of these animals was associated with minimal acute viral replication, followed by undetectable plasma viral loads and only intermittent PCR detection up to 5 years p.i. The three SIVhu infected and three control monkeys were then challenged with the heterologous highly pathogenic SHIV89.6p. All three controls became infected and showed rapid declines in peripheral CD4(+) lymphocytes, disease, and death at 10 and 32 weeks p.i., respectively. In contrast, all three animals previously infected with SIVhu are healthy and exhibit stable CD4(+) lymphocyte levels and undetectable plasma viral loads at >20 months post-SHIV89. 6p challenge. Only transient, low levels of SHIV replication were noted in these animals. Whereas responses to SIVgag/pol were noted, no evidence for SIV/SHIV envelope cross-reactivity was detected by antibody or CTL analyses, suggesting that the protective immune mechanisms to the heterologous challenge isolate were most likely not directed to envelope but rather to other viral determinants.