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. 2011 Aug 23;105(5):687-93.
doi: 10.1038/bjc.2011.306. Epub 2011 Aug 9.

Supernatants from lymphocytes stimulated with Bacillus Calmette-Guerin can modify the antigenicity of tumours and stimulate allogeneic T-cell responses

W M Liu  1 , D W Fowler, A M Gravett, P Smith, A G Dalgleish
Affiliations

Supernatants from lymphocytes stimulated with Bacillus Calmette-Guerin can modify the antigenicity of tumours and stimulate allogeneic T-cell responses

W M Liu et al. Br J Cancer. .

Abstract

Background: Reduced expression of class 1 human leucocyte antigens (HLA1) is often a mechanism by which tumours evade surveillance by the host immune system. This is often associated with an immune function that is unable to mount appropriate responses against disease, which can result in a state that favours carcinogenesis.

Methods: In the current study, we have explored the effects of Bacillus Calmette-Guerin (BCG) on the cytokine output of leucocytes, which is a key determinant in generating antitumour action, and have also assessed the effect of these cytokine cocktails on HLA1 expression in solid tumour cell lines.

Results: BCG potently activated a broad range of leucocytes, and also enhanced the production of cytokines that were Th(1)-predominant. Supernatants from BCG-treated leucocytes significantly increased the expression of HLA1 on the surface of cancer cell lines, which correlated with increased cytolytic T-cell activity. We also showed that the increased HLA1 expression was associated with activation of intracellular signalling pathways, which was triggered by the increases in the Th(1)-cytokines interferon-γ and tumour necrosis factor-α, as counteracting their effects negated the enhancement.

Conclusion: These studies reaffirm the role of BCG as a putative immunotherapy through their cytokine-modifying effects on leucocytes and their capacity to enhance tumour visibility.

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Figures

Figure 1
Figure 1
Effect of BCG on the active status of immune cells and cytokine production. Peripheral-blood mononuclear cells harvested from pathologically normal subjects were exposed to BCG for up to 72 h before assessing the percentage of CD69-expressing cells by nested flow cytometric analyses. The gating profile (A) allowed for identification of five immune subsets, and the percentage of CD69 positive cells assessed (B). The amounts of six cytokines produced by PBMCs in response to BCG were also assessed (C). Each data column represents the mean and s.d. of five separate individuals at the 72-h time-point, except NKT, where n=2 as only 2/5 donors had detectable levels of NKT cells. P-values from paired t-tests.
Figure 2
Figure 2
Effect of supernatants on HLA1 expression in tumour cells. Five tumour cell lines were cultured for 24 h with the supernatants derived from PBMCs treated with BCG (BCG-supernatant), before assessing HLA1 expression on the tumour cells. The basal levels of HLA1 expression expressed as MFI relative to the isotype control are presented in the text. The magnitudes of these expressions were compared with those on tumours after stimulation with supernatants from untreated PBMCs (CONT-supernatant). Data were presented as raw MFI (A) or relative to the respective controls (B). The relationship between HLA1 expression and cytotoxic effect of allogeneic CD8+ T-cells were assessed by measuring LDH activity (C). Effector:target ratios were at 20 : 1 and each data column represented the mean and s.d. of at least three separate experiments. P-values were from paired tests.
Figure 3
Figure 3
Effect of supernatants on intracellular signalling proteins. Cells were cultured with BCG supernatants for 24 h before western blotting for the proteins indicated. AKT, ERK, JNK and p38 MAPK were the proteins assessed as they represented a broad range of signalling elements indicating receptor activation and intracellular functioning. Samples were designated ‘BCG’ if treated with BCG supernatants, and expressions were compared with those from tumours cultured with CONT supernatants (‘un’) and basal expressions (‘B’: where tumours were cultured in standard medium). Both the phosphorylated (P) and total (T) levels were assessed, and densitometry were performed showing the effect of supernatants on the phospho:total ratio. Cell lines that were unaffected by BCG supernatants with regard to HLA1 expression are shown in (A), while those exhibiting increases in HLA1 expression following treatment in BCG supernatant are shown in (B). *P<0.05 when compared with the untreated control as determined by paired t-tests.
Figure 4
Figure 4
Effect of JNK inhibition on HLA1 expression. HCT116 and MCF7 cells were treated with BCG supernatant +/− the JNK inhibitor SP600125, and the effect of reduced JNK action on HLA1 expression assessed. The increase in phosphorylated JNK induced by BCG supernatant was negated by SP600125 (A), and the ability of BCG supernatant to enhance HLA1 expression compromised by JNK inhibition (B and C). Representative histograms from MCF7 experiments are shown (B), and each data column shows means and s.d.'s of a minimum of four separate experiments.
Figure 5
Figure 5
Effect of anti-IFN-γ and anti-TNF-α on HLA1 expression. A549, HCT116 and MCF7 cells were cultured with BCG supernatant in the presence or absence of neutralising antibodies against IFN-γ and TNF-α. Antagonising these cytokines caused reversal of the increases in HLA1 seen as a consequence of BCG-supernatant culture (A). Furthermore, culturing with the neutralising antibodies disrupted the extent of cytotoxicity seen with admixing with CD8+ T-cells (B). Each data column represents the mean and s.d. of at least three separate experiments.
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