Combinations of the cytokines IL-12, IL-2 and IFN-alpha significantly augment whereas the cytokine IL-4 suppresses the cytokine-induced antibody-dependent cellular cytotoxicity of monoclonal antibodies 17-1A and BR55-2

Since some cytokines effectively enhance the cytotoxicity of monoclonal antibodies, we investigated whether a combination of cytokines can augment the antibody-dependent cellular cytotoxicity (ADCC) of monoclonal antibodies 17-1A and BR55-2 against the colorectal carcinoma cell line HT29. Since monocytes/macrophages are important effector cells for ADCC, we used a new flow cytometric cytotoxicity assay, which allows the analysis of long-term-ADCC exerted by these cells. In our previous studies with peripheral blood mononuclear cells from normal donors, we found that IL-2, IL-12 and IFN-alpha increase ADCC. Therefore, we examined whether combination of these three cytokines with IL-2, IL-4, IL-6, IL-10, IL-12, IFN-alpha, IFN-gamma, GM-CSF, M-CSF and TNF-alpha may yield higher ADCC than obtained by the application of single cytokines. Indeed, we found that the combinations IL-2/IFN-alpha, IL-2/IL-12 and IL-12/IFN-alpha potentiated ADCC. Interestingly, the ineffective single cytokines TNF-alpha and GM-CSF in the combinations IL-2/TNF-alpha, IFN-alpha/TNF-alpha and IFN-alpha/GM-CSF also proved to enhance ADCC. In contrast, IL-4 significantly suppressed the IL-2, IL-12 and IFN-alpha-induced ADCC. In addition, the immunosuppressive cytokine IL-10 in higher concentrations significantly suppressed the IL-12-induced-ADCC. Our results may be useful to find combinations of cytokines and mAb for the treatment of cancer.     

Clinical effects of monoclonal antibody 17-1A combined with granulocyte/macrophage-colony-stimulating factor and interleukin-2 for treatment of patients with advanced colorectal carcinoma

Granulocyte/macrophage-colony-stimulating factor (GM-CSF) has previously been indicated to enhance the therapeutic effect of the anti-colorectal carcinoma mAb17-1A as well as to augment in vivo immune effector functions. In vitro interleukin-2 (IL-2) augmented GM-CSF-induced antibody-dependent cellular cytotoxicity, a mechanism considered to be of significance for the therapeutic effect of mAb. A treatment regimen was elaborated that combined mAb17-1A (400 mg at day 3 of a 10-day treatment cycle) with the simultaneous administration of GM-CSF (250 μmg/m² once daily) and IL-2 (2.4 × 10⁶ U/m² twice daily) for 10 days. The treatment cycle was repeated once a month. Twenty patients with advanced colorectal carcinoma were included in the study. One patient obtained a partial remission and 2 patients stable disease for 7 and 4 months respectively. The median survival time from the start of mAb therapy was 8 months. Owing to allergic reactions, the planned mAb17-1A dose had to be reduced by repeated infusions. At the fourth treatment cycle only 25% received the planned mAb dose. In 3 patients the GM-CSF and IL-2 dose was reduced because of side-effects. The subjective tolerability of the treatment was considered good or acceptable in more than 80% of the patients. The increment in white blood cell subsets induced by the cytokines decreased by increasing number of courses. This particular regimen did not augment the therapeutic effect of mAb17-1A anticipated from in vitro data but rather hampered the clinical effect of the antibody. The reason for this is not clear but a possibility might be the induction of immune suppression in vivo resulting from an impaired human anti-(mouse Ab) and anti-idiotypic antibody response as well as antibody-dependent cellular cytotoxicity, on the basis of a comparison of mAb17-1A/GM-CSF/IL-2- and mAb17-1A/GM-CSF-treated patients. Received: 25 February 1999 / Accepted: 15 July 1999     

Cytotoxic functions of blood mononuclear cells in patients with colorectal carcinoma treated with mAb 17-1A and granulocyte/macrophage-colony-stimulating factor

Summary Unconjugated monoclonal antibodies (mAb) may induce tumour regression in patients. The mechanisms of action are complex. Antibody-dependent cellular cytotoxicity (ADCC) is considered one of the effector functions. Augmentation of the killing capacity of cytotoxic cells may thus be a way to increase the therapeutic potential of mAb. Granulocyte/macrophage-colony-stimulating factor (GM-CSF) has been shown to enhance this function in vitro. Eighteen patients with metastatic colorectal carcinoma received GM-CSF (250 µg m^–2 day^–1 s.c.) for 10 days and a single infusion of the anti-(colon carcinoma) mAb 17-1A (mouse IgG2A) (400 mg) on day 3 of the cycle. The cycles were repeated once a month four times. Neutrophils, eosinophils, monocytes and lymphocytes increased significantly in a biphasic way. However, at the fourth cycle the rise in white blood cells was significantly lower compared to the preceding courses. ADCC (SW948, a human CRC cell line, + mAb 17-1A) or peripheral blood mononuclear cells (PBMC) was significantly (P <0.05) augmented by day 6 of a cycle and then declined gradually and, at the end of a cycle, the ADCC activity had returned to the pretreatment level. The spontaneous cytotoxicity of PBMC against the natural-killer-resistant cell line, SW948, varied in a similar way. During GM-CSF treatment there was also a significant increase in FcRI⁺ (CD64), FcRII⁺ (CD32), FcRIII⁺ (CD16) and CD14⁺ cells but not of CD56⁺ cells.     

Antitumor response of regional lymph node lymphocytes in human lung cancer

 Monoclonal antibodies (mAb) are promising substances for the treatment of colorectal carcinoma, but the efficiency of this therapy still needs further improvement. We used a flow-cytometric cytotoxicity test to determine the efficacy of the cytokines interferon α (IFNα) and γ (IFNγ), interleukin-2 (IL-2), macrophage-colony-stimulating factor (M-CSF), granulocyte/macrophage-CSF (GM-CSF) and tumor necrosis factor α (TNFα) in enhancing the antibody-dependent cellular cytoxicity (ADCC) of the mAb 17-1A and the mAb BR55-2 against the colorectal carcinoma cell line HT29. In experiments performed at an effector to target ratio of 9:1, with peripheral blood mononuclear cells from five healthy volunteers as effector cells, we found that IFNα, IFNγ and IL-2 significantly augmented the ADCC of both mAb at concentrations between 3 ng/ml and 30 ng/ml. The other three cytokines were not effective. In further experiments we examined combinations of the three effective cytokines in different concentrations. The combination of IFNα and IL-2 proved to be optimal in enhancing ADCC of both mAb. Thus, the examination of ADCC by flow cytometry may reveal potentially useful combinations of cytokines and mAb for the treatment of colorectal carcinoma. Received: 11 September 1997 / Accepted: 19 February 1998     

Granulocyte-monocyte colony-stimulating-factor augments the interleukin-2-induced cytotoxic activity of human lymphocytes in the absence and presence of mouse or chimeric monoclonal antibodies (mAb 17-1A)

Summary Blood lymphocytes stimulated for 96 h with interleukin-2 (IL-2; 100 BRMP U/ml) (lymphokine-activated killer, LAK, cells) or granulocyte-monocyte colonystimulating-factor (GM-CSF) (10 ng/ml) became cytotoxic for Daudi cells. IL-2 was significantly more effective than GM-CSF. Only IL-2-activated cells killed SW948 (a human colorectal carcinoma cell line) while GM-CSF-stimulated cell did not. GM-CSF and IL-2 acted synergistically in a dose-dependent fashion for induction of a highly effective cytotoxic cell population (IL-2/GM-CSF cells). Il-2/GM-CSF cells were statistically significantly more effective than LAK cells in lysing Daudi cells and SW948 (P <0.05). The enhancing effect was most pronounced during the first 48–96 h of activation. Incubation periods longer than 192 h did not contribute to augmented cytotoxicity. The combination of IL-2 and GM-CSF significantly increased the number of CD25⁺ cells compared to IL-2 and GM-CSF alone. Furthermore, IL-2/GM-CSF cells were significantly more effective in antibody-dependent cellular cytotoxicity assays (SW948 + mAb 17-1A) than LAK cells. The chimeric mAb 17-1A was significantly more effective in tumor cell lysis than the mouse mAb. Thus, combination of various biological therapeutics might be a way to enhance their antitumoral effects.     

Granulocyte/macrophage-colony-stimulating factor augments the induction of antibodies,especially anti-idiotypic antibodies,to therapeutic monoclonal antibodies

A group of 86 patients with advanced colorectal carcinoma were treated with the mouse (m) (IgG2A) or chimeric (c) monoclonal antibody (mAb) 17-1A. Prior to therapy, no patient had detectable levels of antibodies to mAb17-1A. All mmAb17-1A-treated patients (n=76) developed antibodies against both idiotypic and isotypic determinants. Addition of granulocyte/macrophage-colony-stimulating factor (GM-CSF) to mmAb17-1A significantly enhanced the induction of anti-idiotypic (ab₂) as well as anti-isotypic antibodies. Of the mmAb17-1A-treated patients, 16 developed type I allergic reactions. These patients had significantly higher concentrations of anti-(mouse Ig) antibodies than patients without type I reactions. Of these 16 patients, 5 had received mmAb17-1A alone; they constituted 9% of this group (5/56). The remaining 11 patients had been given mmAb17-1A together with GM-CSF, and represented 55% of this treatment group (11/20). The difference was statistically significant (P<0.001). Of 10 patients, 9 (90%) treated with cmAb17-1A and GM-CSF developed ab₂. The ab₂ concentration in this patient group was significantly lower compared to those treated with mmAb-17A. Anti-(mouse Ig) antibodies caused clinical symptoms requiring therapeutic intervention in fewer than 10% of the patients treated with mmAb17-1A alone. With the addition of GM-CSF, the antibody concentration as well as the frequency of allergic side-effects calling for medical action increased significantly. Significantly more patients with a high ab₂ concentration (at least 15g/ml) 1 month after completion of mAb therapy responded to mAb treatment as compared to those with a low ab₂ concentration (P<0.05). Moreover, patients with a high ab₂ concentration (at least 15 g/ml) had a median survival time of 15 months while those with a lower concentration survived for a median time of 9 months (P=0.01).     

Immunogenic regions of the GA733-2 tumour-associated antigen recognised by autoantibodies of patients with colorectal carcinoma

The tumour-associated antigen (TAA) GA733-2 is overexpressed by >90% of human colorectal carcinomas (CRC). The antigen has previously been shown to be recognised by B and T cells. The aim of the present study was to define B cell epitopes of GA733-2. Fifteen percent of CRC patients with no previous immunotherapy have recently been shown to elicit an anti-GA733-2 IgG antibody response. Sera of these patients ( n=136) were analysed by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies against 23 partly overlapping synthetic peptides (18 amino acids: aa) derived from the extracellular domain of GA733-2. An 18-aa long sequence at the N-terminal region of the antigen (peptide 2) was found to be an immunodominant B cell epitope. Fifty percent of the patients had antibodies against peptide 2, while 8% to 9% had antibodies against peptides 1, 4, 7, 8 or 20. In healthy donors ( n=30) antibodies against peptides 2 and 8 were also detected in 13% and 3% of cases respectively, while no antibodies were found against the other peptides and the complete protein. Thirteen percent of CRC patients ( n=30) with no IgG antibodies against the GA733-2 antigen elicited antibodies against peptide 2. The specificity of peptide-reactive sera was verified by inhibition ELISA. The binding of sera to GA733-2 was significantly inhibited by peptides to which CRC sera bound, but not by control peptides. Binding to peptide 2 of sera showing both peptide 2 and GA733-2 reactivity was specifically inhibited by the complete GA733-2 antigen, while binding of peptide 2-reactive sera showing no GA733-2 reactivity was not inhibited. CRC sera interfered with the binding of monoclonal antibody (mAb) 17-1A and mAb C215 that recognise distinct epitopes of GA733-2. No significant correlation was found between the presence of anti-peptide antibodies in CRC patients and clinical stage or overall survival. The results provide additional evidence for immune recognition of CRC by the host.     

Humoral anti-idiotypic and anti-anti-idiotypic immune response in cancer patients treated with monoclonal antibody 17-1A

 A group of 96 patients with advanced colorectal carcinoma were treated with the mouse (m) or chimeric (c) (mouse variable regions × human IgG1 constant regions) monoclonal antibody (mAb) 17-1A recognizing the tumour-associated antigen GA733-2. Eighty-two of the 83 patients treated with mmAb17-1A and 69% of the patients given cmAb17-1A (n = 13) developed anti-idiotypic antibodies (ab₂). Auto-antibodies binding to tumour cells expressing GA733-2 were found in 7% of the patients. In a further 38 patients (40%) antitumour-cell antibodies, i.e. anti-anti-idiotypic antibodies (ab₃), were induced by the mAb17-1A therapy. Patients with detectable ab₃ after treatment had significantly higher ab₂ levels than those not developing ab₃. Addition of granulocyte/macrophage-colony-stimulating factor (GM-CSF) to mmAb17-1A significantly enhanced the induction of ab₂ as well as induction of anti-anti-idiotypic antibodies (ab₃), compared to mmAb17-1A alone. Patients with a high increase in antitumour-cell antibodies (ab₃) induced by the therapy lived significantly longer than patients with no or a low level of induction of ab₃ (P = 0.016). The results indicate that induction of an idiotypic network response might be an important effector mechanism in mAb therapy. Received: 20 October 1995 / Accepted: 18 December 1995     

Granulocyte-monocyte-colony-stimulating factor augments the cytotoxic capacity of lymphocytes and monocytes in antibody-dependent cellular cytotoxicity

Summary Human peripheral blood mononuclear cells (lymphocytes and monocytes) were preincubated for 0–24 h with human recombinant granulocyte-monocyte-colony-stimulating factor (GM-CSF) and used as effector cells in an 18 h antibody-dependent cellular cytotoxicity (ADCC) assay with SW948 (a human colorectal carcinoma cell line) as target cells and mAb 17-1A. A significant increase in the lytic capability was noted after 0.5–2 h of preactivation while longer preincubation times did not significantly increase the lytic potential. GM-CSF at 0.01 g/ml induced the best tumor cell lysis while higher concentrations were inhibitory. GM-CSF pretreatment induced a statistically significant increase in the lytic capacity of both monocytes and lymphocytes in ADCC as well as in the spontaneous cytotoxicity.     

The role of monocytes and natural killer cells in mediating antibody-dependent lysis of colorectal tumour cells

Monocytes and natural killer (NK) cells are known to be important effector cell populations in mediating antibody-dependent cell-mediated cytotoxicity (ADCC). Purified monocyte and NK effector cell populations, from normal and colorectal cancer (CRC) patients, together with a number of murine (17-1A and 323/A3) and their chimaeric (c17-1A) or humanised (3622W94) equivalents, and chimaeric (c) SF25 were compared for their ability to mediate ADCC of colorectal tumour cells. The chimaeric and humanised antibodies were significantly better at mediating tumour lysis than their murine equivalents with all-effector populations. When effector cells from CRC patients were used the cSF25 antibody was significantly better than 3622W94 (P < 0.02) which, in turn, was significantly better than c17-1A (P < 0.03). Depletion of NK cells produced a decrease in specific tumour lysis with all antibodies. In addition a higher rate of NK cell death was observed in CRC patients during the assay than in normal controls. The chimaeric and humanised antibodies stained a similar percentage of the HT-29 target cells (>80%), but 3622W94 bound to significantly more cells from primary tumour biopsies than cSF-25 (P = 0.001). Together, the results suggest that NK cells are the most important effector cell type mediating ADCC in vitro, that there is some impairment of NK function in CRC patients, and that cSF25 is the most potent antibody. For use in vivo the anti-Ep-CAM antibody 3622W94 would appear to be the most suitable reagent for further study. Received: 3 June 1999 / Accepted: 22 July 1999     

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

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

Mechanisms of macrophage cytotoxicity in IL-2 and IL-12 mediated tumour regression

IL-2 and IL-12 are promising anti-tumour agents. However, little attention has been paid to the role of macrophages during IL-2/IL-12 mediated tumour rejection. We studied the role of macrophages during IL-2/IL-12 mediated tumour rejection in DBA/2 mice bearing syngeneic SL2 lymphoma. Local treatment with IL-2 and IL-12 cured 85% of mice with severe metastasised tumour load. In vivo depletion studies showed that macrophages were required for the anti-tumour effect of IL-2 and IL-12. Macrophages could kill tumour cells both non-specifically and by antibody-dependent cellular cytotoxicity (ADCC). Treatment with IL-2, IL-12 or IL-2/IL-12 enhanced production of specific IgG1 immunoglobulins, while treatment with IL-12 and IL-2/IL-12 additionally induced IgG2a production. FcgammaRII and/or III were essential for ADCC expression after treatment with IL-2 and IL-12. These data show for the first time the essential role of macrophages during IL-2/IL-12 mediated tumour rejection and also suggest that IL-2 and IL-12 act via different mechanisms.     

Defucosylated anti-CCR4 monoclonal antibody exercises potent ADCC-mediated antitumor effect in the novel tumor-bearing humanized NOD/Shi-scid, IL-2Rγnull mouse model

Purpose  There are no suitable small animal models to evaluate human antibody-dependent cellular cytotoxicity (ADCC) in vivo, due to species incompatibilities. Thus, the first aim of this study was to establish a human tumor-bearing mouse model in which human immune cells can engraft and mediate ADCC, but where the endogenous mouse immune cells cannot mediate ADCC. The second aim was to evaluate ADCC mediated in these humanized mice by the defucosylated anti-CC chemokine receptor 4 (CCR4) monoclonal antibody (mAb) which we have developed and which is now in phase I clinical trials. Experimental design  NOD/Shi-scid, IL-2Rγ^null (NOG) mice were the recipients of human immune cells, and CCR4-expressing Hodgkin lymphoma (HL) and cutaneous T-cell lymphoma (CTCL) cell lines were used as target tumors. Results  Humanized mice have been established using NOG mice. The chimeric defucosylated anti-CCR4 mAb KM2760 showed potent antitumor activity mediated by robust ADCC in these humanized mice bearing the HL or CTCL cell lines. KM2760 significantly increased the number of tumor-infiltrating CD56-positive NK cells which mediate ADCC, and reduced the number of tumor-infiltrating FOXP3-positive regulatory T (Treg) cells in HL-bearing humanized mice. Conclusions  Anti-CCR4 mAb could be an ideal treatment modality for many different cancers, not only to directly kill CCR4-expressing tumor cells, but also to overcome the suppressive effect of Treg cells on the host immune response to tumor cells. In addition, using our humanized mice, we can perform the appropriate preclinical evaluation of many types of antibody based immunotherapy.     

Effects of oral deoxynivalenol exposure on immune-related parameters in lymphoid organs and serum of mice vaccinated with porcine parvovirus vaccine

Mice were exposed to deoxynivalenol (DON) via drinking water at a concentration of 2 mg/L for 36 days. On day 8 of treatment, inactivated porcine parvovirus vaccine (PPV) was injected intraperitoneally. The relative and absolute weight of the spleen was significantly decreased in the DON-treated group (DON). Antibody titers to parvovirus in serum were 47.9?±?2.4 in the vaccination group (Vac), but 15.2?±?6.5 in the group treated with DON and vaccine (DON?+?Vac). The IgA and IgG was not different in the DON, Vac an,d DON?+?Vac groups. IgM was significantly lower only in the DON?+?Vac group. However IgE was significantly increased in the Vac and DON?+?Vac group, but no change was observed between the Vac and DON?+?Vac groups. The concentrations of IL-2, IL-4, GM-CSF, MCP-1 and Rantes in serum, and IL-1α in mesenteric lymph node and MIP-1β in spleen were significantly increased by DON treatment compared to control. The concentrations of IL-2, IL-5, IL-6, IL-9, IL-12, IL-13 and Rantes in thymus, of IL-2 in spleen, and of IL-1α, IL-1β, IL-3, IL-5, IL-10, IL-17, G-CSF, GM-CSF and MCP-1 in mesenteric lymph nodes were significantly decreased in mice compared to those in the Vac group, while concentrations of IL-1α, IL-2, IL-9, IL-13,G-CSF, GM-CSF, IFN-γ, MCP-1, MIP-1α and TNF-α were significantly increased in serum compared to the Vac group. In conclusion, the results presented here indicate that exposure to DON at 2.0 mg/L via drinking water can disrupt the immune response in vaccinated mice by modulating cytokines and chemokines involved in their immune response to infectious disease.     

Protection against gram-negative bacteremia in neutropenic mice with recombinant granulocyte-macrophage colony-stimulating factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates production of neutrophils in bone marrow and may decrease the incidence of infection during neutropenia. We evaluated the protective role of recombinant GM-CSF against Pseudomonas aeruginosa challenge in neutropenic mice. CD-1 mice treated with cyclophosphamide on days 1 and 2 of the experiment were given GM-CSF (1, 2, or 4 micrograms/day) starting at day 4 of the experiment according to the following protocol: 1) 1 microgram of GM-CSF 2 hr and 24 hr after challenge; 2) 1 microgram 24 hr before challenge, 2 hr and 24 hr after challenge; 3) 2 micrograms injected 24 hr before and 2 hr after challenge; 4) 2 micrograms given 24 hr before and 2 micrograms given 2 hr and 24 hr after challenge; 5) 4 micrograms administered 2 hr and 24 hr after challenge; and 6) saline and bovine albumin controls. The number of blood neutrophils by days 4 and 5 was similar for GM-CSF-treated and untreated animals. Survival was significantly greater in animals given 2 micrograms of GM-CSF at 24 hr before and at 2 hr and 24 hr after challenge with Pseudomonas. Neutrophils and splenic macrophages obtained from GM-CSF-treated mice (2 micrograms/animal) produced significantly greater amounts of O2- (204 +/- 36 nmoles/10(5) cells) than controls (21 +/- 10 nmoles/10(5) cells). Additionally, neutrophils and macrophages from GM-CSF-treated mice killed significantly more bacteria (P. aeruginosa) in vitro and had a greater number of C3b and Fc receptors (78 +/- 12% and 89 +/- 8%) than did cells obtained from control animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

IL-6单克隆抗体对自身免疫性心肌炎的保护作用及其机制

目的：观察白介素-6（interleukin,IL-6）单克隆抗体（IL-6 mAb）治疗Lewis大鼠自身免疫心肌炎（EAM）的疗效。探讨IL-6与辅助性T细胞17（Th17）、调节性T细胞（Treg）在EAM发病中的机制。方法：将34只8-10周龄Lewis大鼠随机分为正常对照组（n=6）,EAM组（n=12）,IL-6mAb干预组（n=16）。对EAM组和干预组注射心肌肌凝蛋白,干预组于免疫注射后第1、7至第20天腹腔注射IL-6 mAb1nlg,分别于急性峰值期（第21天）、慢性持续期（第84天）取材,观察心肌炎症浸润、纤维化、细胞凋亡以判断IL-6mAb疗效。检测脾脏TH17、Treg细胞数量和功能,比较各组血清中IL-6、IL-10、IL-17和转化生长因子．β（TGF-β）的浓度,实时定量PCR测定外周血STAT3、RORγt、Foxp3mRNA水平,对EAM源性脾细胞进行体外IL-6mAb刺激,并用ELISA法测定IL-10、IL-17和TGF-β的浓度。结果：炎症积分、纤维化积分、凋亡指数IL-6mAb干预组较EAM组明显下降（P〈0．01）。急性峰值期（21d组）EAM组TH17和Treg细胞数量上调,干预组则受明显抑制（P〈0．01）;21d干预组血清IL-6、IL-10、IL-17和TGF-β的浓度较EAM组明显下降（P〈0．01）;21d干预组外周血STAT3、RORγt、Foxp3mRNA水平下降（P〈0．01）;体外IL-6mAb刺激EAM源性脾细胞,IL-10、IL-17和TGF-β表达明显增加。结论：IL6mAb对EAM有明显的保护作用,IL6mAb通过抑制Th17、Treg细胞的数量和功能,实现对EAM的保护作用。     

Anti-idotype and recombinant antigen in immunotherapy of colorectal cancer

The CO17-1A/GA733 antigen (Ag), bound by monoclonal antibodies (MAb) CO17-1A and GA733 that define two different epitopes on the Ag, has proven a useful target in passive and active immunotherapy of colorectal carcinoma (CRC). Previous studies suggest that the antitumor effects demonstrated in MAb-treated patients may be mediated by idiotypic cascades. In approaches to active immunotherapy against the Ag, polyclonal goat and monoclonal rat anti-idiotypic antibodies (Ab2) directed against MAb CO17-1A or GA733 (Ab1) were administered as alum precipitates to 54 patients with CRC (stage Dukes' B, C, and D). The majority of the patients treated with the various Ab2 preparations developed anti-anti-idiotypic antibodies (Ab3) that specifically bound to the CO17-1A or GA733 epitope and shared idiotopes with the corresponding Ab1. Approximately 30% of the patients tested developed specific cellular immunity, i.e., Ag-specific T-cells mediating delayed-type hypersensitivity (DTH) reaction in vivo or proliferating on stimulation with the Ag in vitro. The humoral and cellular immune responses may underlie the clinical responses observed in some of the treated patients. Recently, the CO17-1A/GA733 Ag has been molecularly cloned and expressed in baculo-, adeno-, and vaccinia viruses. In preclinical studies, these recombinant Ag preparations elicited specific humoral immunity (cytotoxic antibodies) and cellular immunity (DTH-reactive and proliferative T-cells), similar to the native Ag. Antibody titers elicited in experimental animals by recombinant Ag were significantly higher than those elicited by Ab2, presumably because Ag expresses numerous epitopes, whereas Ab2 mimics a single epitope. Recombinant CO17-1A/GA733 Ag has potential as a vaccine for CRC patients.     

Flow cytometric analysis on reactivity of human T lymphocyte-specific and cytokine-receptor-specific antibodies with peripheral blood mononuclear cells of chimpanzee (Pan troglodytes), rhesus macaque (Macaca mulatta), and squirrel monkey (Saimiri sciureus)

Abstract: There are relatively few monoclonal antibodies (mAb) that have been characterized for their applicability in studies on the immune system of various nonhuman primates. In the present study, we identified a large number of mAb that can be used in future immunological studies in three different nonhuman primates, i.e., chimpanzees, rhesus macaques, and squirrel monkeys. The reactivity of 161 anti-human mAb to T-cell antigens and cytokine receptors were tested on peripheral blood mononuclear cells (PBMC) from the three primate species by flow cytometric analysis. A total of 105 (65%), 73 (45%), and 68 (42%) antibodies reacted with PBMC from chimpanzees, rhesus macaques, and squirrel monkeys, respectively. Out of the 161 mAb, 38 reacted with all three species and 112 reacted with one or two of the species. No specific reaction was observed with mAb to receptors to GM-CSF, 4–1BB, FLT3, FLX2, common β-chain, IL-1 (type I receptor), and IL-8.     

Granulocyte/macrophage colony-stimulating factor inhibits IL-12 production of mouse Langerhans cells

We investigated the capacity of mouse Langerhans cells (LC) to produce IL-12, a central cytokine in a Th1 type of immune responses. We prepared purified LC (>95%) from BALB/c mouse skin by the panning method using anti-I-Ad mAb. An ELISA showed that purified LC spontaneously produced IL-12 p40, and that its production was up-regulated following simultaneous stimulation with anti-CD40 mAb and IFN-gamma. Surprisingly, GM-CSF strikingly inhibited IL-12 p40 production by anti-CD40/IFN-gamma-stimulated LC (% inhibition = 97.0 +/- 0.9% at 1 ng/ml GM-CSF). Supernatants of 48-h cultured keratinocytes (KC) also caused the inhibition of LC IL-12 p40 secretion, and this effect was neutralized by anti-GM-CSF mAb. IL-1alpha (1 ng/ml)-stimulated KC produced much more GM-CSF than unstimulated KC (60.9 +/- 0.2 pg/ml vs 20.9 +/- 1.7 pg/ml), and IL-1alpha-stimulated KC supernatants strongly inhibited IL-12 p40 production by anti-CD40/IFN-gamma-stimulated LC (% inhibition = 89.4 +/- 1.4%). A bioassay using an IL-12-dependent T cell line demonstrated the correlation of the level of IL-12 p40 with the bioactivity of IL-12. These results provide important implications for the pathogenesis of atopic dermatitis, which involves the participation of LC and KC with the capacity to produce IL-12 and GM-CSF, respectively.     

Cytotoxicity of white blood cells activated by granulocyte-colony-stimulating factor,granulocyte/macrophage-colony-stimulating factor and macrophage-colony-stimulating factor against tumor cells in the presence of various monoclonal antibodies

Unconjugated monoclonal antibodies (mAb) kill tumor cells in vivo by activating immune functions. One of these is ADCC (antibody-dependent cellular cytotoxicity). The efficacy of mAbs might be augmented if the cytotoxic capacity of the effector cells could be increased. In this study the augmenting effect of granulocyte-colony-stimulating factor (G-CSF), granulocyte/macrophage(GM)-CSF and macrophage(M)-CSF was analyzed. Effector cells [peripheral blood mononuclear cells (PBMC) or granulocytes] were activated for 4–6 h by the respective CSF and assayed in an 18-h Cr⁵¹-release assay. Human colorectal, lymphoma, glioma and melanoma cell lines were target cells. Mouse mAbs of different isotypes, as well as chimeric and humanized mAbs, were used. mAbs having the human Fc part of the IgG molecule were the most effective. The killing capacity of PBMC as well as of granulocytes was statistically significantly enhanced when mAbs were added. M-CSF and GM-CSF were the best CSF for augmenting the lytic capacity of PBMC in ADCC. G-CSF had no significant effect on PBMC. Spontaneous cytolysis of PBMC was significantly augmented only by M-CSF. Granulocytes were, in general, significantly less effective than PBMC but may be equally effective killer cells together with mouse or human mAbs of the IgG1 isotype, particularly against melanoma cells. Granulocytes may also be significantly stimulated to increased lytic capacity when activated with G-CSF or GM-CSF. On the basis of the present evaluation, clinical trials in tumor patients are warranted, combining mAbs with GM-CSF or M-CSF. Preference might be given to GM-CSF as this cytokine activates both PBMC and granulocytes.