Dendritic cells stimulated by MUC1 antigen could induce lymphocytes' anergy through PD-L2 but not through PD-L1 patway
Authors: K. Wojas-Krawczyk, I. Homa-Mlak, P. Krawczyk, T. Kucharczyk, J. Milanowski
Journal: Annals of Oncology (2016)

Clinical trials with antigen-specific immunotherapy have failed in NSCLC patients. We have read again with great interest the article by Mitchell et al. published in Annals of Oncology concerning antigen-specific immunotherapy with tecemotide (L-BLP25) given as maintenance therapy in stage III unresectable non-small-cell lung cancer (NSCLC) patients, who underwent chemoradiotherapy [1.Mitchell P. Thatcher N. Socinski M.A. et al.Tecemotide in unresectable stage III non-small-cell lung cancer in the phase III START study: updated overall survival and biomarker analyses.Ann Oncol. 2015; 26: 1134-1142Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar]. L-BLP25 liposomal vaccine is based on MUC1 antigen—a glycoprotein widely over-expressed in common cancers such as lung cancer. The mentioned START trial showed that immunotherapy with tecemotide was safe although the study failed to meet the primary end point for overall survival in the whole group of treated patients [2.Butts C. Socinski M.A. Mitchell P.L. et al.Tecemotide (L-BLP25) versus placebo after chemoradiotherapy for stage III non-small-cell lung cancer (START): a randomised, double-blind, phase 3 trial.Lancet Oncol. 2014; 15: 59-68Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar]. Similarly, the MAGRIT clinical study was also unsuccessful. In this study, MAGE-A3-based immunotherapy was used for adjuvant treatment of stage II or IIIA NSCLC patients who underwent surgical resection [3.Vansteenkiste J.F. Cho B. Vanakesa T. et al.MAGRIT, a double-blind, randomized, placebo-controlled Phase III study to assess the efficacy of the recMAGE-A3 + AS15 cancer immunotherapeutic as adjuvant therapy in patients with resected MAGE-A3-positive non-small cell lung cancer (NSCLC).ESMO. 2014; (Congress: abstract no 11730)Google Scholar]. Isolated groups of patients benefit from mentioned antigen-specific immunotherapy. A certain type of tumor cells gene signature (MAGRIT study) or high concentration of soluble MUC1 antigen and antinuclear antibodies in serum (START trial) positively correlated with survival prolongation in patients receiving immunotherapy. Moreover, concurrent chemoradiotherapy prior to immunotherapy, in contrast to sequential one, was a positive predictive factor for tecemotide [1.Mitchell P. Thatcher N. Socinski M.A. et al.Tecemotide in unresectable stage III non-small-cell lung cancer in the phase III START study: updated overall survival and biomarker analyses.Ann Oncol. 2015; 26: 1134-1142Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 2.Butts C. Socinski M.A. Mitchell P.L. et al.Tecemotide (L-BLP25) versus placebo after chemoradiotherapy for stage III non-small-cell lung cancer (START): a randomised, double-blind, phase 3 trial.Lancet Oncol. 2014; 15: 59-68Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 3.Vansteenkiste J.F. Cho B. Vanakesa T. et al.MAGRIT, a double-blind, randomized, placebo-controlled Phase III study to assess the efficacy of the recMAGE-A3 + AS15 cancer immunotherapeutic as adjuvant therapy in patients with resected MAGE-A3-positive non-small cell lung cancer (NSCLC).ESMO. 2014; (Congress: abstract no 11730)Google Scholar]. Nowadays, there are effective methods of anticancer immunotherapy targeting immune checkpoint molecules (PD-1, PD-L1, PD-L2, CTLA-4 etc.). Upregulation of inhibition molecules on tumor cells or on antigen-presenting cells (APCs) induces anergy of lymphocytes in tumor tissues and in lymph nodes. Blockade of inhibitory immune checkpoints reactivates lymphocytes to kill tumor cells [4.Swaika A. Hammond W.A. Joseph R.W. Current state of anti-PD-L1 and anti-PD-1 agents in cancer therapy.Mol Immunol. 2015; 67: 4-17Crossref PubMed Scopus (171) Google Scholar, 5.He J. Hu Y. Hu M. Li B. Development of PD-1/PD-L1 pathway in tumor immune microenvironment and treatment for non-small cell lung cancer.Nature Rev Immunol. 2015; 5 (:): 13110Google Scholar]. We came up with an idea to check whether active immunotherapy with tumor-specific antigens might upregulate expression of immunological checkpoint molecules on APCs, which could be responsible for providing negative signal for lymphocyte activity. Until now, there are no reports concerning PD-L1 or PD-L2 expression changes on APC during antigen-specific immunotherapy. Therefore, our study aimed to clarify some reasons of failure of large clinical trials using active antigen-specific immunotherapy in NSCLC patients. In our study, autologous dendritic cells (DCs) were generated from CD14-positive, magnetically sorted monocytes, taken from 20 unresectable and treatment-naïve lung adenocarcinoma patients. The cells were cultured in CellDCGrow medium supplemented with interleukin-4 (IL-4) and granulocyte-macrophage colony stimulating factor. After tumor necrosis factor-α stimulation, mature DCs were incubated with tumor-specific antigens: MUC1, MAGE-A3, epidermal growth factor receptor and cytomeglovirus (as positive stimulator) or only with CellDCGrow medium (as negative control). We used flow cytometry and specific monoclonal antibodies to analyze immunophenotype of the generated DCs, especially in terms of costimulatory molecules from B7 family (CD80 and CD86) including PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273). Autologous DCs were well generated and after cytokine stimulation showed phenotype specific for fully mature DCs (CD14-/CD1a-/CD11c+/CD83+/CD209+), with high expression of chemokine receptors (CCR7+/CXCR3+/CD54+). Expression of CD80 costimulatory molecule was significantly (P = 0.01) higher after MUC1 antigen stimulation compared with unstimulated cells (Figure 1). We observed insignificant increase in the expression of PD-L1 and CD86 after antigen stimulation. But most importantly, the expression of PD-L2 antigen was significantly (P = 0.01) higher after MUC1 stimulation when compared with unstimulated DCs (Figure 1). Programmed cell death protein 1 (PD-1) is expressed mainly on lymphocytes. Its two ligands, PD-L1 and PD-L2 (both coinhibitory) are expressed on different tissues [4.Swaika A. Hammond W.A. Joseph R.W. Current state of anti-PD-L1 and anti-PD-1 agents in cancer therapy.Mol Immunol. 2015; 67: 4-17Crossref PubMed Scopus (171) Google Scholar, 5.He J. Hu Y. Hu M. Li B. Development of PD-1/PD-L1 pathway in tumor immune microenvironment and treatment for non-small cell lung cancer.Nature Rev Immunol. 2015; 5 (:): 13110Google Scholar]. The restricted expression of PD-L2, largely to APCs, is consistent with a role in regulating T-cell priming or polarization, whereas the broad distribution of PD-L1 suggests a more general role in protecting peripheral tissues from excessive inflammation [6.Rozali E.N. Hato S.V. Robinson B.W. Lake R.A. Lesterhuis W.J. Programmed death ligand 2 in cancer-induced immune suppression.Clin Dev Immunol. 2012; (2012: ID: 656340)Crossref PubMed Scopus (232) Google Scholar]. We postulate that antigen-specific immunotherapy is effective due to the induction of antigen presentation and costimulatory molecule expression, but it can fail because of the induction of coinhibitory molecule expression on APCs. It is possible that the inhibition of lymphocytes' function takes place in lymph nodes via interaction of PD-1 on lymphocyte surface with its ligand PD-L2 on APCs stimulated by antigenic vaccine. It is also possible that PD-L2 blockade could enhance the effectiveness of anticancer antigen-specific immunotherapy. The authors have declared no conflicts of interest.