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Microsatellite stable colorectal cancers (MSS-CRC) are resistant to anti-PD-1/PD-L1 therapy but the combination of immune checkpoints inhibitors (ICI) could be a clue to reverse resistance. Our aim was to evaluate ex vivo the capacity of the combination of atezolizumab (anti-PD-L1) and tiragolumab (anti-TIGIT) to reactivate the immune response of tumor infiltrating lymphocytes (TILs) in MSS-CRC. We analysed CRC tumor tissue and the associated blood sample in parallel. For each patient sample, extensive immunomonitoring and cytokine production were tested. We generated an ex vivo assay to study immune reactivity following immune stimulation with checkpoint inhibitors of tumor cell suspensions. Three microsatellite instable (MSI) and 13 MSS-CRC tumors were analysed. To generalize our observations, bioinformatics analyses were performed on public data of single cell RNA sequencing of CRC TILs and RNA sequencing data of TCGA. Atezolizumab alone could only reactivate T cells from MSI tumors. Atezolizumab and tiragolumab reactivated T cells in 46% of MSS-CRC samples. Reactivation by ICK was observed in patients with higher baseline frequency of Th1 and Tc1 cells, and was also associated with higher baseline T cell polyfunctionality and higher CD96 expression. We showed that a high frequency of CD96 expression on T cells could be a surrogate marker of atezolizumab and tiragolumab efficacy. Together these data suggest that the association of atezolizumab and tiragolumab could restore function of CD4 and CD8 TILs in MSS-CRC and could be tested in a clinical trial in colorectal cancer patients with MSS status. [ABSTRACT FROM AUTHOR]

The C–C chemokines, macrophage inflammatory protein (MIP)1α and MIP1β are potent chemoattractants for the monocytes, which form an important component of the stroma of tumor tissue and may regulate tumor growth and associated inflammation. We examined the role of MIP1α and MIP1β in inducing the release of inflammatory cytokines and the generation of tumoricidal monocytes from the peripheral blood monocytes (PBM) of healthy women and patients with carcinoma of breast (CaBr). Interleukin-1 (IL-1) and tumor necrosis factor (TNF) α release by the PBM was markedly stimulated by MIP1α in CaBr patients, but only marginally so in healthy women. In contrast, MIP1β stimulated the release of these cytokines by the PBM of healthy women, but failed to do so in CaBr patients. MIP1α, but not MIP1β, synergized with LPS in inducing the release of IL-1 from the PBM of both healthy women and CaBr patients. Both MIP1α and MIP1β augmented respiratory bursts in PBM and generated tumoricidal PBM that killed T24 cells, MIP1α being more effective in CaBr patients and MIP1β in healthy women. IFN- γ co-stimulated and IL-4 suppressed MIP1α and β-induced cytotoxicity in PBM. The synergy of IFN- γ was more marked with MIP1α than with MIP1β. The differential effects of MIP1α and MIP1β on the PBM of healthy women and CaBr patients co-related with the levels of expression of CCR1 and CCR5 in these monocytes. The expression of CCR5 was higher than that of CCR1 in the PBM of healthy women and the PBM of the CaBr patients showed overexpression of CCR1 and downregulation of CCR5. [ABSTRACT FROM AUTHOR]

We evaluated the safety and immunogencity of a novel vaccine directed against autologous TNFα in a Phase I fixed dose escalation trial. The vaccine consisted of two recombinant TNFα proteins, with specific peptides replaced by foreign immunodominant T cell epitopes from tetanus toxoid. The main objectives were to establish a safe dose and evaluate the vaccines ability to raise neutralising TNFα antibodies. Secondary objectives were improvements in body weight and tumour response. Thirty-three patients were vaccinated with three doses (20, 100, or 400 μg) of TNFα vaccine at 2-weekly intervals adjuvanted with aluminium hydroxide. Anti-TNFα antibody titres were measured by both a RIA, using soluble native TNFα as the antigen, and by an ELISA using immobilized partly denatured TNFα. Eleven patients (33%) had mild grade1/2 injection site reactions at the higher doses. In 10 of 20 patients, serum antibodies recognize denatured TNFα in the ELISA, whereas, antibody titres against native TNFα in the RIA were undetectable. This suggests that the production process had partly denatured the vaccine preventing the formation of cross-reacting antibodies to native TNFα. In conclusion, TNFα vaccine was able to elicit vaccine specific antibodies. However, since the antibodies were only able to cross-react with partly denatured TNFα, evaluation of safety and tumour responses to the TNFα vaccine was compromised. [ABSTRACT FROM AUTHOR]

To investigate cytokine regulation in cells of freshly excised lymphoid tissues, rigorous quantitative reverse transcription/polymerase chain reaction (QRT-PCR) assays were developed to measure attomole (10−18 mol) amounts of the mRNA for seven cytokines: interleukin-1α (IL-1α), IL-1β, tumor necrosis factor α (TNFα), interferon γ (IFNγ), transforming growth factor β (TGFβ), IL-2 and IL-6. RNA was purified from single-cell suspensions of immune tissues (spleen, thymus and resident peritoneal cells). Data are presented demonstrating the utility of these assays for quantifying basal levels of all seven cytokine mRNAs in the freshly isolated splenocytes and thymocytes. Studies to establish the usefulness of these assays for measuring changes in the levels of cytokine mRNA focused on IL-1α, IL-1β, TNFα and IL-2 in splenocytes, thymocytes and resident peritoneal cells. Using the QRT-PCR assays developed, levels of cytokine mRNA could be quantified in RNA samples obtained both from freshly isolated cells and from cells following short-term (≤26 h) culture. These measurements established basal in vivo levels of specific cytokine mRNAs and demonstrated specific modulation of their levels by cell culture and by the inclusion of immune stimulants (bacterial lipopolysaccharide or the plant lectin concanavalin A) in the culture. These data provide new information on both basal and stimulated cytokine levels that is required for valid interpretations of the roles of cytokine expression in immune regulation. [ABSTRACT FROM AUTHOR]

We have previously illustrated the importance of B7-2 expression for the enhanced generation of cytotoxic T lymphocyte (CTL) activity by stimulation cultures of tumor bearer splenic cells to which tumor necrosis factor α (TNFα) has been added. Here we show that the B7-1 molecule is also important for CTL generation by such stimulation cultures, although to a much lesser extent than the B7-2 molecule. In addition, we show the importance of CD40/CD40L interaction for the expression of the B7-2 molecule, but not the B7-1 molecule, by tumor bearer splenic cells stimulated in vitro in the presence of TNF. The CD40/CD40L interaction is also shown to be important for the generation of CTL activity by tumor bearer splenic cells stimulated in vitro in the presence of exogenous TNF. However, the CD40/CD40L interaction is less important for the generation of enhanced CTL activity than for the expression of an elevated level of B7-2. Specifically, blockade of CD40/CD40L interaction, which reduced the level of B7-2 expressed by tumor bearer splenic cells stimulated in vitro in the presence of TNF to the level of B7-2 expressed by tumor bearer splenic cells stimulated in vitro in the absence of exogenous TNF, failed to reduce the level of CTL generated to the level generated by tumor bearer splenic cells stimulated in the absence of exogenous TNF. Finally, blockade of CD40/CD40L interaction was inferior to blockade of B7-2/CD28 interaction in inhibiting the generation of CTL activity by tumor bearer splenic cells stimulated in the presence of exogenous TNF. Thus, although CD40/CD40L interaction is important for the generation of enhanced CTL activity by stimulation cultures of tumor bearer splenic cells to which TNF has been added, TNF also mediates its potentiating effect for CTL generation by such stimulation cultures via other mechanisms that are independent of CD40/CD40L interaction but dependent on B7-2 expression. [ABSTRACT FROM AUTHOR]

There is strong evidence that antitumor activity of interleukin-12 (IL-12) in vivo is mediated, in part, through interferon (IFNγ) produced by IL-12-stimulated natural killer and T cells. Since IFNγ and tumor necrosis factor α (TNFα) have been reported to synergize in antitumor effects in a number of models, we decided to examine whether the combined treatment with recombinant mouse IL-12 and recombinant human TNFα would produce similar effects. The efficacy of the combined IL-12/TNFα immunotherapy was evaluated in three tumor models in mice: B16F10 melanoma, Lewis lung (LL/2) carcinoma and L1 sarcoma. Intratumoral daily injections of 1 μg IL-12 in combination with 5 μg TNFα into B16F10-melanoma-bearing mice resulted in a significant retardation of the tumor growth as compared with that in controls and in mice treated with either cytokine alone. Similar effects were obtained using 0.1 μg IL-12 and 5 μg TNFα in LL/2 carcinoma and L1 sarcoma models. Antitumor activity against L1 sarcoma was still preserved when TNFα at a low dose (1 μg) was combined with 0.1 μg IL-12 and applied for a prolonged time. Potentiation of antitumor effects, which was observed in IL-12/TNFα-based immunotherapy, could result from at least three different mechanisms, partly related to stimulation of IFNγ and TNFα production in treated mice: (a) direct cytostatic/cytotoxic effects on tumor cells, (b) induction of antitumor activity of macrophages, and (c) inhibition of blood vessel formation in the tumor. Our studies demonstrate that combination tumor immunotherapy with IL-12 and TNFα may be more effective than single-cytokine treatment, and suggest possible mechanisms by which IL-12 and TNFα may exert potentiated therapeutic effects against locally growing tumors. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor α (TNFα) is a cytokine, produced by lymphocytes and monocytes, with cytotoxic activity against some but not all tumor cell lines. Resistance to the cytolytic effects of TNFα has been reported in cell lines with autocrine TNFα production. The purpose of this study was to investigate whether human primary malignant melanoma and tumor infiltrating lymphocytes produce TNFα in vivo. Optimal conditions for in situ hybridization for TNFα mRNA in paraffin-embedded tissue were established. Analysis of 13 primary malignant melanomas and 3 metastatic lesions with different degrees of immunohistochemical TNFα positivity demonstrated that, in some tumors, both melanoma cells and leukocytes contained TNFα mRNA and protein. These findings demonstrate variable production of TNFα in primary and metastatic melanoma in vivo.The previously described resistance to TNFα cytolytic activity may, therefore, be clinically important. [ABSTRACT FROM AUTHOR]

We have reported that patients with metastatic melanoma treated with an autologous, dinitrophenol-modified vaccine develop inflammatory responses at tumor sites. Histologically, these inflamed lesions are characterized by T cell infiltration, which is sometimes associated with tumor cell destruction. We tested biopsy specimens of eight subcutaneous metastases that had developed inflammation following vaccine treatment for expression of mRNA for interferon γ(IFNγ), interleukin-4 (IL-4), tumor necrosis factor α (TNFα), and IL-10. Post-vaccine, inflamed biopsies contained mRNA for IFNγ (5/8), IL-4 (4/8) or both (3/8), and for TNFα (4/7). In contrast, IFNγ mRNA was detected in only 1/17 and TNFα mRNA in 2/16 control specimens (pre-treatment lymph node metastases or non-inflamed subcutaneous metastases). mRNA for IL-10, a cytokine with anti-inflammatory properties, was detected in 24/25 melanoma metastases and was independent of lymphoid content; in situ the reverse transcriptase/polymerase chain reaction confirmed that melanoma cells were the major source. These findings may provide a new parameter by which to measure the effects of cancer immunotherapy. [ABSTRACT FROM AUTHOR]

A combination treatment protocol initiated 12 days after tumor injection, when the tumor was large, by administering cyclophosphamide (CY, 150 or 250 mg/kg) intraperitoneally followed by intravenous tumor necrosis factor α (TNFα, 1000 units/injection) on days 13, 16, 18, 21, and 23, resulted in about 60% longterm survival (i.e., survival for at least 60 days) in the syngeneic C57BL/6 mouse/EL4 lymphoma model system. The establishment of a specific antitumor immune memory and its possible therapeutic relevance was verified by reinjecting 60-day survivors with EL4 cells; all 60-day survivors that had received the combination treatments rejected the implants and survived for a further 60 days. Thymic cellularity was reduced during treatment and its recovery appeared to correlate with long-term survival and immunity. Thymocytes from mice treated with the combination were found to express significant levels of specific anti-EL4 cytolytic activity following a 4-day stimulation culture with X-irradiated EL4 cells and low concentrations of interleukin-2. This response could not be generated with thymocytes from naive animals. In each case the effect seen with the combination of a moderate CY dose (150 mg/kg) with TNFα was better than that seen with either dose of CY alone and equal to or better than that seen with the higher dose of CY combined with TNFα. These results indicate that treatment with a single moderate dose of CY in combination with TNFα is effective against a large, established tumor in this murine model. Furthermore, all the long-term survivors induced by this treatment developed protective immunity against reimplanted tumor and demonstrated a long-term specific immune memory in the thymus. [ABSTRACT FROM AUTHOR]

Hyperthermic isolated limb perfusion is an established method of treatment for regionally advanced melanoma. Recent studies suggest that exogenously administered cytokines potentiate tumor response in patients with in-transit melanoma. We hypothesized that isolated limb perfusion induces an immunogenic response characterized by increased circulating levels of cytokines in the pump circuit, potentially contributing to the antitumor effect. We obtained blood samples from the perfusion circuit and systemic circulation at various intervals from patients undergoing isolated chemotherapeutic perfusion for melanoma. Samples were analyzed for serum cytokine profiles by enzyme-linked immunosorbent assay. When compared with baseline values, significant increases in serum levels of interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor (TNF) occurred within the perfusion circuit during isolated limb perfusion ( P<0.05). In addition, there was a corresponding increase in IL-8 within the systemic circulation at the 60-min interval ( P<0.05), suggesting some degree of leakage from the isolated circuit due to the extremely high levels of IL-8 in the perfusion circuit. A transient but insignificant decrease in circulating levels of neutrophils was also observed during the perfusion process, which may be attributed to margination. Increased levels of cytokines IL-6, IL-8, and TNF occurred within the isolated circuit during hyperthermic limb perfusion and may contribute to tumor response seen in patients treated with isolated limb perfusion. [ABSTRACT FROM AUTHOR]

Intravesical immunotherapy for bladder cancer is the most effective form of tumour immunotherapy. Following repeated instillations of bacillus Calmette-Guerin (BCG) organisms into the bladder large quantities of several cytokines are detected in the urine. These cytokines include interleukins IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, tumour necrosis factor α(TNFα), interferon γ(IFNγ) and also soluble intercellular adhesion molecule ICAM-1. In the work reported here we simultaneously quantified urinary levels of TNFα, TNFβ, TNF receptor I and TNF receptor II by enzyme-linked immunosorbent assay (ELISA) techniques and compared this with bioactive levels of TNF. This was undertaken with a limited number of patients throughout a course of six instillations of immunotherapy. Sequential instillations of BCG induced secretion of TNFα and TNFβ into urine. These cytokines were not always secreted simultaneously, perhaps suggesting differential regulation of their synthesis. Maximal concentrations of TNFα were 675 pg/ml and TNFβ 47 pg/ml. High levels of both species of soluble TNF receptor were readily identified in urine. Maximal levels of sTNF-RI were 6200 pg/ml (range from 0) and for sTNF-RII 7800 pg/ml (range from 0). Contrasting with earlier published observations concerning cytokine levels, the concentration of soluble receptor did not increase with repeated instillation. In apparent contrast with the ELISA data, very low levels of bioactive TNF were identified by the L929 bioassay (maximum concentration 1 U/ml) despite the elevated concentration of immunoreactive TNF. The large concentrations of soluble TNF receptor in patients' urine samples could account for the apparently low bioactivity as determined by the L929 cytotoxicity assay. The precise nature of the role of TNF in BCG immunotherapy remains undetermined; however, it is thought that proinflammatory cytokines are in part responsible for the clinical efficacy of this therapeutic approach. Whether other cytokines are antogonised by soluble binding proteins remains to be determined. Furthermore, whether TNF is bioactive in the bladder wall and only neutralised in the urine also requires investigation. [ABSTRACT FROM AUTHOR]

During the past decade, particular attention has been focused on treatment of bladder cancer patients with the bacterial agent bacillus Calmette-Guérin (BCG). In these studies, bladder cancer patients were instilled with BCG (75mg/50ml) once per week for 6 weeks, 1-2 weeks following trans-urethreal resection of the bladder. Cystoscopy was performed after 6 weeks and, unless tumor progression was present, monthly treatments were given for 1 year. Blood was drawn 2 h after the last instillation, and monocytes were isolated (5×10 cells/ml) and treated, or not, with lipopolysaccharide (LPS) (20 μg/ml) for tumor necrosis factor α (TNFα), interleukin-1α (IL-1α) and interleukin-6 (IL-6) release. The levels of monokines were determined by a monokine-specific enzyme-linked immunosorbent assay. Out results clearly show that, after 18 h incubation, macrophages from BCG-treated bladder cancer patients produced from 2.8- to 1.9-fold and from 2.0- to 1.3-fold greater amounts of TNFα and IL-1α respectively, compared to macrophages from healthy controls, 5-fold higher than bladder cancer patients not treated with BCG. IL-6 was not affected. In another set of experiments macrophages (5×10 cells/ml) from healthy subjects were pretreated, or not, with BCG (100 μg/ml) overnight and treated, or not, with LPS 20 μg/ml alone and in combination with interleukin-1 receptor antagonist (IL-1ra) 250 ng/ml. Macrophages treated with BCG had a strong stimulatory effect on IL-1α release (9.45 ng/ml) while LPS was less effective (3.59 ng/ml). The combination of BCG plus LPS produced an additive effect on IL-1α release (13.71 ng/ml) compared to the effect of the compound alone. The addition of IL-1ra (250 ng/ml) to BCG was not effective, while when IL-1ra was added to BCG plus LPS only a partial inhibition of IL-1α release was found (9.83 ng/ml), compared to BCG plus LPS without IL-1ra (13.71 ng/ml). These effects seem to be related to the inhibition of IL-1α stimulated with LPS, but not BCG. The priming effect of BCG exerted on LPS-stimulated monocyte production of TNFα and IL-1α from bladder cancer patients led us to study the possible modulation of fibrinogen and C-reactive protein in the serum of BCG-treated cancer patients. The plasma levels of fibrinogen and C-reactive protein were higher (approximately twice) in BCG-treated patients compared to values obtained in untreated patients or healthy controls. We conclude that the beneficial immunotherapeutic effects of BCG in bladder cancer patients are related to its capacity to prime macrophages to enhance the release of TNFα and IL-1α, but not IL-6 in response to physiological secondary stimuli, or through the direct stimulation of BCG on IL-1α or TNFα, which are directly involved in the killing of cancer cells. Moreover, the increase of IL-1α or TNFα in BCG bladder cancer patients may lead to high plasma levels of fibrinogen and C-reactive protein, two proteins responsible for the acute-phase response. [ABSTRACT FROM AUTHOR]

We have recently demonstrated that prothymosin α (ProTα) when administered intraperitoneally (i.p.) protects DBA/2 mice against the growth of syngeneic leukemic L1210 cells through the induction of tumoricidal peritoneal cells producing high levels of tumor necrosis factor α (TNFα) [Papanastasiou et al. (1992) Cancer Immunol Immunother 35: 145]. In this report we tested further immunological alterations that may be caused by the administration of ProTα in vivo. We demonstrate that i.p. injections of ProTα enhance natural killer (NK) cell activity and induce lymphokine-activated (LAK) activity in vivo. Thus, splenocytes from ProTα-treated DBA/2 animals exhibited significantly higher cytotoxic activity (up to threefold) against the NK-sensitive YAC cell line and the NK-resistant P815 and L1210 syngeneic tumor cells, as compared to splenocytes from syngeneic control mice. The enhancement of the cytotoxic profile of DBA/2 splenocytes was associated with increased percentages of CD8 cells, NK cells and activated CD3 cells. The ProTα-induced effect persisted for 30 days after the end of the ProTα treatment period and returned to normal levels 20 days later. SPlenocytes from non-treated DBA/2 animals generated high NK and LAK activities in response to ProTα in vitro. The ProTα-induced NK an LAK activities reached 84% and 75% respectively of what was obtained with interleukin-2 (IL-2). High concentrations of TNFα and IL-2 were generated in response to ProTα in LAK cultures. These findings suggest that ProTα may provide an overall protective effect against tumor growth in vivo through induction of NK and LAK activities possibly indirectly via the production of IL-2 and TNFα in the spleen, peritoneal cavity and probably other lymphoid organs. [ABSTRACT FROM AUTHOR]

Eleven metastatic cancer patients were studied during three different regimens of immunotherapy with interleukin-2 (IL-2) and/or interferon α (IFNα): group A received 4 days of IL-2 i.a. infusion ( n=3), group B IFNα s.c. during 5 days ( n=4), followed on day 3 by 5 days of a continuous IL-2 i.v. infusion, and group C had 4 days of IL-2 i.v. infusion together with s.c. IFNα on days 1 and 4 ( n=4). Soluble tumor necrosis factor receptors (sTNFR) p55 and p75 and TNFα concentrations in serum were analyzed before therapy and daily during 8 days of the first therapy cycle. sTNFR was measured by radioimmunoassay. sTNFR p55 increased in all patient groups from a baseline value of 5.2±0.9 ng/ml to a maximum of 13.6±1.2 ng/ml by days 3-4 ( P=0.003). sTNFR p75 increased from 7.6±1.1 ng/ml to peak values of 30.1±2.6 ng/ml in groups A and B ( P=0.02). In group C the sTNFR p75 response was weak (NS). In group B, the increase of both p55 and p75 occurred only after addition of IL-2 to IFNα. TNFα increased weakly during treatment with IFNα alone (group B); it rose strongly during IL-2 and the combined treatment (groups A-C) from 8±2 pg/ml to 115±13 pg/ml ( P=0.003). In group B, it reached the maximum 24 h after addition of IL-2 to IFNα and decreased thereafter. there was a significant relationship between TNFα and sTNFR p55 or sTNFR p75 in groups A and C, ( P=0.001), but not in group B. Group C was also investigated during the third therapy cycle. The increase of sTNFR p75 was stronger ( P=0.01) and that of TNFα weaker than in the first cycle; the sTNFR p55 response was similar in both cycles. In conclusion sTNFR p55 and p75 are rapidly induced during IL-2 and IL-2+IFNα treatment, the increase of sTNF receptors parallels or exceeds that of TNFα and may influence the immunomodulatory effects of TNFα during cytokine therapy. [ABSTRACT FROM AUTHOR]

The possibility of in vivo removal of metastatic tumour cells from lymph nodes by local intradermal administration of an anti-CD3 monoclonal antibody (mAb) was examined. Murine tumour cells in the lymph nodes were completely eradicated by intradermal injections of the mAb. This treatment was effective for removal of Lewis lung cancer cells from lymph nodes, but not for removal of subcutaneous tumours of this cell line. This treatment induced in vivo cytotoxicity in the regional lymph nodes against the syngeneic tumour cells. The following in vitro studies suggested that the cytotoxicity was probably mediated mainly by CD4 T cells, with slight participation of CD8 T cells. Normal lymph node and spleen cells showed cytotoxicity after in vitro incubation with the mAb for 2 days. Cell sorting with a fluoresceinactivated cell sorter showed that CD4 T cells developed during the incubation to lyse syngeneic tumor cells directly by themselves, macrophages not being involved in this tumour cell lysis. The lytic activity was detected in the cellular fractions, but not in the culture supernatants of these T cells. Furthermore, it was completely blocked by specific antiserum for tumour necrosis factor-α (TNFα). An immunoprecipitation study revealed that these T cells expressed TNFα molecules of 26 kDa, but not of 17 kDa, suggesting that tumour cell lysis was caused by membraneintegrated TNFα molecules. These results strongly suggest that local administration of anti-CD3 antibody is a very effective and appropriate procedure for eradication of metastatic tumour cells from regional lymph nodes. [ABSTRACT FROM AUTHOR]

Transfection of tumor cells with a vector containing the entire coding sequence of human interleukin-2 (hIL-2) was previously shown to convert the tumorigenic murine fibrosarcoma line CMS-5 into a non-tumorigenic line. The failure of the IL-2-secreting tumor to grow in conventional (immunocompetent) mice was attributed to the activation of CD8 T cells that exhibited tumor specificity and memory. In order to determine whether or not the IL-2 produced by the tumor may be activating tumor cytotoxic effector cells other than B or T cells we have repeated this study using immunodeficient SCID and SCID-beige mice as syngeneic tumor recipients. In contrast to the rapid growth of the wild-type tumor, the hIL-2-transfected cells (N2A/IL2/CMS5) did not grow, or grew more slowly and regressed, in the mice that lack functional B and T cells. The inhibition of tumor growth associated with the local release of IL-2 was reversed in mice treated with antiasialo-GM1 antibodies specific for natural killer (NK) lineage cells. In contrast to the studies with conventional mice, the IL-2-dependent effector cells in the immunodeficient mice exhibited no evidence of memory. In vitro analysis of spleen cells from tumor-bearing mice revealed the presence of effector cells able to lyse YAC-1 target cells as well as the wild-type CMS-5 and the IL-2-transfected variant tumor lines but unable to lyse P815 cells. The pattern of selective target cell killing and the kinetics of killing were indistinguishable from those observed using tumor necrosis factor α (TNFα) the mediator associated with natural cytotoxicity cell killing of tumor cells. Histopathology of the IL-2-secreting tumors in SCID mice reveals the presence of infiltrating lymphoid cells and macrophages that were not observed in the CMS-5 tumors. Consistent with the notion that the tumor killing in the SCID mice was mediated by TNFα, mice bearing IL-2-secreting tumors had elevated levels of serum TNFα and little or no effector cell activity, or TNFα was found in tumor-bearing mice treated with anti-asialo-GM1 antibody. The results indicate that the cytokine-induced tumor regression observed in the IL-2-transfected tumors is a more complex phenomenon than previously recognized and one that is mediated by effector cells of the NK cell and/or monocyte/macrophage lineages, in addition to CD8 T cells. [ABSTRACT FROM AUTHOR]

We have recently reported that autologous tumor-specific cytotoxic T lymphocyte (CTL) lines and clones can be developed from lymphocytes infiltrating ovarian malignant ascites (TAL). In this study, we investigated the biological effects of tumor necrosis factor α (TNFα) in the induction, expansion, long-term proliferation and lytic function of CD8 TAL. TNFα up-regulated the IL-2 receptor (IL-2R) α chain (Tac antigen) on the surface of CD3 CD8 CD4 TAL, enhanced the proliferation of autologous tumor-specific CTL, and potentiated their lytic function in long-term cultures. Furthermore, in the induction and expansion phase of CD8 TAL, the presence of TNFα was associated with a selective increase in CD8 IL-2R (Tac) cells, and subsequent decrease in CD4 IL-2R (Tac) cells. These results suggest that the observed facilitation of the outgrowth of CD8 cells in TAL cultures may be due, at least in part, to the up-regulation of IL-2R, and indicate the usefulness of TNFα in the analysis of signalling in autologous tumor-reactive CTL. [ABSTRACT FROM AUTHOR]

The effect of prothymosin α (ProTα) on the survival of DBA/2 mice inoculated with syngeneic tumour cells was studied. DBA/2 mice inoculated intraperitoneally (i.p.) with 2×10 syngeneic leukaemic L1210 cells developed ascites within 8-12 days and died 10-14 days later. Treatment with ProTα consistently inhibited the development of ascites in 20% of the treated animals and prolonged the survival of 40%-60% of the animals up to 70 days. The most effective treatment schedule of ProTα was 300 ng/mouse given i.p. at 2-day intervals for 3 weeks followed by a rest period of 7 days, prior to tumour cell inoculation. Peritoneal exudate (PE) cells collected from mice treated with the optimal dose of ProTα produced, in the absence of exogenous stimulus, six- to eightfold higher levels of tumour necrosis factor α (TNFα) than PE cells from control mice. Furthermore these cells exhibited cytotoxic activity against several tumour cell lines including the syngeneic L1210, the TNF-insensitive P815 mastocytoma, the human MOLT-4 lymphoblastic leukaemia, as well as the murine TNF-sensitive L929 fibroblast cell line. Kinetic studies revealed that both production of TNFα and tumoricidal activity peaked 7 days after the last injection of ProTα and were maintained at high levels over a period of 1 month. Injections with 150 ng ProTα slightly improved the survival of mice whereas higher (500 ng and 1000 ng) doses of ProTα and a wide range of thymosin α1 doses remained without any effect. PE cells collected from these mice produced extremely low levels of TNFα and exhibited negligible tumoricidal activity. Our data demonstrate that ProTα has a protective effect in vivo against the growth of adoptively transfered tumour cells and suggest that this effect is, at least in part, mediated by ProTα-activated PE cells. These cells were demonstrated to produce high levels of TNFα in vitro and to exhibit activity against both TNF-sensitive and TNF-resistant cell lines. [ABSTRACT FROM AUTHOR]

In view of cellular adoptive immunotherapy we have studied monocyte-mediated cytostasis and cytotoxicity against U 937 cells, a human histiocytic lymphoma cell line. Highly purified human monocytes and monocytederived macrophages were activated with interferon γ (IFN) or tumour necrosis factor α (TNF) to antileukemic immune effector cells. Antileukemic activity of human monocytes was dependent on monocyte differentiation into macrophages and on a dose- and time-dependent activation with IFN or TNF. Maximum cytostasis of 97.0±0.7% (mean ± SEM) (conventional [H]dT uptake assay) and 81.9±5.3% cytotoxicity (modified MTT assay) of U 937 cells was obtained by monocytes activated with 100 U/ml IFN for at least 24 h at an effector-to-target-cell ratio of 10. U 937 cells premodified with IFN showed an increase in susceptibility to monocyte-mediated cytotoxicity. U 937 cells premodified with TNF were almost resistant to monocyte-mediated cytotoxicity while activated monocytes maintained their cytotoxic potential. These data show that IFN and TNF are potent activators of monocyte-mediated cytotoxicity. Furthermore, IFN and TNF might be involved in the regulation of the susceptibility of leukemic cells to lysis by interactions with monocytes or macrophages. [ABSTRACT FROM AUTHOR]

Few clinical responses have occurred in preliminary studies using the cytokines tumor necrosis factor α (TNFα) or interferon γ(IFNγ) in cancer patients. This may be related to the observation that many malignant cell lines are resistant to lysis by these cytokines in vitro. Resistance to lysis by TNFα or IFNγ in many cells is controlled by a protein-synthesis-dependent mechanism, such that when protein synthesis is inhibited cells become sensitive to lysis by these cytokines. Because there is some evidence that TNFα and IFNγ act through different lytic mechanisms and are opposed by different resistance mechanisms, we treated a panel of eight cell lines, five derived from human cervical carcinomas (ME-180, MS751, SiHa, HT-3, and C-33A) and three derived from ovarian carcinomas (Caov-3, SK-OV-3, and NIH: OVCAR-3) with both TNFα and IFNγ to determine whether such combination treatment might maximize in vitro cell lysis. Our results showed that pretreatment with IFNγ followed by exposure to TNFα in the presence of protein synthesis inhibitors increased lysis of seven of the eight cell lines above that seen with either TNFα or IFNγ and inhibitors of protein synthesis. Only the cell line C-33A was resistant to lysis by TNFα and IFNγ, when exposed to these agents both alone and in combination with protein synthesis inhibitors. Clinically, combining the cytokines TNFα and IFNγ with protein synthesis inhibitors may maximize the in vivo lytic effects of these cytokines. [ABSTRACT FROM AUTHOR]