Evaluation of T lymphocyte subpopulations in Actinic Keratosis, In Situ and Invasive Squamous Cell Carcinoma of the Skin Aristea Stravodimou 1 , Vassiliki Tzelepi 1 , Helen Papadaki 2 , Athanasia Mouzaki 3 , Sophia Georgiou 4 , Maria Melachrinou 1 and Eleni P. Kourea 1 1 Department of Pathology, School of Medicine, University of Patras, Patras, Greece 2 Department of Anatomy, School of Medicine, University of Patras, Patras, Greece 3 Division of Hematology, Department of Internal Medicine, School of Medicine, University of Patras, Patras, Greece 4 Department of Dermatology, School of Medicine, University of Patras, Patras, Greece Corresponding Author: Eleni Kourea Department of Pathology, School of Medicine, University of Patras, Patras, Greece Complete address: Meganitou 7, 26442, Patras, Greece Tel. Number: +30 2610 969191 +30 6977276407 Fax number +30 2610 991810 Εmail address: hkourea@yahoo.com Abbreviated title: T-lymphocytes in squamous carcinogenesis Key Words: Invasive Squamous Cell Carcinoma, Actinic Keratosis, In Situ Squamous Cell Carcinoma, Tumor Infiltrating Lymphocytes (TILs), regulatory T-cells (Tregs). Abstract Background: Tumor infiltrating lymphocytes (TILs) represent important regulators of carcin- ogenesis. Cutaneous invasive squamous cell carcinoma (inSCC) develops through precursor lesions, namely in situ squamous cell carcinoma (isSCC) and actinic keratosis (AK), represent- ing a natural model of carcinogenesis. The study evaluates TIL subpopulations in inSCC and its precursors by comparing two semi-quantitative scoring systems, and assesses the pres- ence of regulatory T-cells (Tregs) in these lesions. Methods: Paraffin sections from 33 cases of AK, 19 isSCCs and 34 inSCCs with adjacent pre- cursor lesions or normal skin were immunostained for CD3, CD4, CD8 and Foxp3. TIL sub- groups were evaluated by the semi-quantitative Klintrup-Mäkinen score, and by a more de- tailed modification of this system. Treg counts were assessed by image analysis quantifica- tion. Results: An increase of all TIL subpolulations from precursor lesions towards inSCC was shown by both scoring systems. Treg counts progressively increased from normal skin to AK and isSCC, but decreased in inSCC. Tregs were more numerous in pT2 and around indolent inSCCs compared to T1 and aggressive subtypes. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/cup.13123 This article is protected by copyright. All rights reserved. Accepted Article