Nat Rev Malignancy. Importantly, a dense stromal CD8+ T cell infiltration was strongly associated with improved OS only in HLA-E unfavorable tumors Peretinoin (p=0.005) and its prognostic effect was completely abolished when tumors highly expressed HLA-E (p=0.989). CONCLUSIONS CD8+ T cell infiltration strongly contributes to a better prognosis in NSCLC when the tumor cells retain the expression of classical HLA Peretinoin class I and do not express HLA-E. Therefore, analysis of HLA-A, -B/C and HLA-E expression should be included as biomarkers to predict the response to immunotherapy. [43, 44] and this NFIL3 has resulted in a currently ongoing phase I/II trial in which patients with advanced head and neck malignancy are treated with an anti-NKG2A monoclonocal antibody (ClinicalTrials.gov, Identifier: “type”:”clinical-trial”,”attrs”:”text”:”NCT02331875″,”term_id”:”NCT02331875″NCT02331875). Potentially, a combination of antibodies to these two different checkpoints may even have a synergistic effect. In conclusion, our results confirm the pivotal protective role of tumor infiltrating CD8+ T cells in NSCLC and in addition show that their effect is particularly apparent when the tumor cells retain the expression of classical HLA class I Peretinoin and do not express the non-classical molecule HLA-E. These results warrant the inclusion of HLA-A, -B/C and HLA-E as biomarkers to predict the response to immunotherapy and the use of HLA-E or NKG2A blocking antibodies for the treatment of NSCLC. MATERIALS AND METHODS Study populace We retrospectively identified 197 patients diagnosed with non-small cell lung cancer (NSCLC), subtype adenocarcinoma, in the Leiden University Medical Center (LUMC) between 2000 and 2013. All patients underwent preoperative staging and were classified as stage I/II NSCLC and subsequently underwent surgical resection of the primary tumor with systematic lymph node dissection. After surgical removal of the tumor and its draining lymph nodes, patients were considered disease free. Tumor tissue, clinical data and follow-up data were collected from all patients. Staging of NSCLC was decided according to the TNM (Tumor, Node, Metastasis) classification using Peretinoin the updated guidelines of the International Association for the Study of Lung Cancer (IASLC) [45]. The use of archival tumor blocks was in accordance with guidelines from the Dutch Federation of Medical Research Association. Since this retrospective study does not fall under the scope of the Medical Research Involving Human Subjects Act (WMO), it was not subject to a prior review by a Medical Ethical Committee and written informed consent was not obtained. However, patient data were anonymized. Antibodies Mouse monoclonal antibodies HCA-2 (anti HLA-A, 1:1000) and HC-10 (anti HLA B/C, 1:500) were used to detect expression of the free heavy chain of the HLA class I molecule. Rabbit anti-human 2-microglobulin (anti-2M; clone A-072, DAKO, 1:2000) and mouse anti-human HLA-E (clone MEM-E/02; Serotec, Germany [1:200]) antibodies were used in order to detect the light chain and non-classical HLA-E heavy chain respectively. Mouse monoclonal CD8 antibody (clone IA5, Leica Biosystems, Germany [1:500]) was used for the detection of the CD8+ T-cells. Immunochemistry Formalin-fixed, paraffin embedded tumor blocks were cut in 4 m sections using a microtome and deparaffinized in xylene. The endogenous peroxidase activity was blocked for 20 minutes using 0.3% hydrogen peroxide/methanol. The samples were subsequently rehydrated in 70% and 50% ethanol and antigen retrieval was performed by heating the samples to 97C for 10 minutes in citrate buffer (either pH 9.0 or pH 6.0, DAKO, Glostrup, Denmark). Antibodies were diluted in phosphate buffered saline (PBS, Fresenius Kabi Bad Homburg, Germany) with 1% bovine serum albumin (BSA) and incubated overnight at room heat. The slides were stained immunohistochemically with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (DAKO envision) for 30 minutes at room heat. NovaRed (Vector, Burlingame, USA) was applied as a chromagen followed by counterstaining with Mayer’s hematoxylin (Klinipath). All washing steps were done with PBS. All slides were mounted with Pertex mounting medium (HistoLab, Sweden). The microscopic evaluation and analysis of the HCA2, HC10, 2M and HLA-E staining was performed by two impartial observers without prior knowledge of clinical or histopathological parameters (observer one 100% of the cohort, observer two 20% of the cohort). The inter-observer agreement was assessed by calculating Cohen’s kappa coefficient resulting in a coefficient of 0.70 for all those stainings which indicates a substantial inter-observer agreement. The grade of tumor differentiation was decided and classified as either poorly differentiated, moderately differentiated or well differentiated based on the.