INTRODUCTION Regulatory mechanisms used by CD4 + T lymphocytes place (T helper lymphocytes) these cells in the central position of a specific immune response. During pregnancy, these cells play a pivotal role in the maintenance of pregnancy. In mouse, an expansion of T reg lymphocytes of CD4 + CD25 + FOXP3 + phenotype in the uterine draining lymph nodes is clearly visible. Additionally, their depletion after allogenic matings is known to cause pregnancy failure (1). Likewise, the ratio of cytokines produced by Th1/Th2 lymphocytes at the feto-maternal interface is an indicator of successful or failing pregnancy (2). On the other hand, the precise role of peripheral activity of CD4 + lymphocytes in the context of pregnancy has not yet been determined. Recent papers have demonstrated that in humans not only production of Th1 type cytokines is decreased (3, 4), but also the overall adaptive immune response is weakened in favor of the innate immune mechanism protecting the mother from infectious antigens (5). In mice, the serum cytokine expression pattern during pregnancy is not clear (6), but an enlarged group of activated CD4 + CD25 + spleen lymphocytes observed at early and more advanced stages of gestation suggests pregnancy-related regulation of peripheral immunity in this species (7, 8). Our previous work indicated differential expression of costimulatory molecules on splenic antigen-presenting cells (APCs) within the first three days after mating in pregnant females in comparison to pseudopregnant mice. This data support the hypothesis of an early awareness of the immune system of the presence of paternal antigens (9). Exact effects of the mentioned phenomenon in the context of lymphocyte function are not clear. However, it may be assumed that APCs' specific phenotype during preimplantation pregnancy influences the activity of their counterparts - CD4 + lymphocytes. The proteomic approach has been successfully applied in studies aimed at examining the lymphocyte function in both physiological and pathological conditions (10, 11). The first gel- based proteomic reference map of T CD4 + lymphocytes from non- JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2014, 65, 5, 719-731 www.jpp.krakow.pl A. CHELMONSKA-SOYTA 1,2 , M. OZGO 3 , A. LEPCZYNSKI 3 , A. HEROSIMCZYK 3 , K. BUSKA-PISAREK 1 , A. KEDZIERSKA 1 , D. NOWAK 4 , A.J. MAZUR 4 PROTEOME OF SPLEEN CD4 LYMPHOCYTES IN MOUSE PREIMPLANTATION PREGNANCY 1 Laboratory of Reproductive Immunology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland; 2 Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland; 3 Department of Physiology, Cytobiology and Proteomics, Faculty of Biotechnology and Animal Husbandry, West Pomeranian University of Technology, Szczecin, Poland; 4 Department of Cell Pathology, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland Pregnancy exerts profound impact on female immune system. The first signs of pregnancy recognition by immune system are observed even before implantation. The most visible effects are present in the local compartment, i.e. in uterine draining lymph nodes and the decidua, while peripheral changes are less obvious. In our recent paper we indicated that costimulation phenotype of APCs in spleens of female mice during the preimplantation period of pregnancy differs from mice in pseudopregnancy. However, the effect of differential costimulation in the context of the T lymphocyte function at periphery in early pregnancy is still unknown. For that reason, we decided to investigate global protein expression in splenic CD4 + lymphocytes in order to identify and validate the most important biomarkers characteristic for the preimplantation period of pregnancy at periphery. Two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) were utilized to analyze the protein expression pattern of magnetically sorted CD4 + lymphocytes from spleens of pregnant and pseudopregnant females at 3.5 days after mating. The first goal of this study was to create a 2-DE map of the splenic CD4 + T cells of pregnant mice. As a result, 106 protein spots from 373 were identified using MS. The comparison of lymphocyte protein patterns between pregnant and pseudopregnant mice depicted differential expression of 11 identified proteins belonging to the group of proteins involved in cytoskeletal structure, cell motility and metabolism. Profoundly diminished expression of cofilin-1, F-actin capping protein subunit alpha and malate dehydrogenase proteins in lymphocytes of pregnant mice indicates that preimplantation pregnancy could change the activation state of peripheral CD4 + lymphocytes. Key words: CD4 + lymphocytes, early pregnancy, proteome, spleen, mouse, antigen-presenting cells, cofilin-1