Endosomal trafficking of open Major Histocompatibility Class I conformers - Implications for presentation of endocytosed antigens

Major Histocompatibility Class I (MHC-I) molecules are present at the cell surface either as fully conformed trimolecular complexes composed of heavy chain, beta-2-microglobulin ( 2m) and antigenic peptide or as various open forms, devoid of the peptide and/or 2m. While the role of fully conformed MHC-I is well studied, the physiological role of open conformers is neglected. We have shown that fully conformed MHC-I and open MHC-I conformers segregate at the PM and during endosomal trafficking resulting in the exclusion of open MHC-I from the early endosomal/juxtanuclear recycling route. As a result, open MHC-I conformers are internalized with a higher rate than fully conformed counterparts. Although the majority of internalized open MHC-I is directed into the acidic late endosomal (LE) compartments, only a fraction of them is degraded. Namely, a significant fraction of open MHC-I is present in a subset of LEs with the capacity of recycling and/or exocytosis. Therefore, it should be examined whether exogenous peptide loading may occur during traveling of MHC-I proteins through LE compartments, especially in a subset of less acidic LEs that detach from the core of perinuclear acidic LEs and migrate toward the cell periphery. Given that the acidic LE environment is not favorable for Peptide loading, an endosomal compartment with the recycling capacity and less acidic environment that allows stabilization of newly formed trimolecular complexes is proper site for exogenous peptide loading. We propose that a LE compartment which collect and retain open MHC-I conformers should be taken into consideration as a site of exogenous peptide loading

Expression of cytolytic protein–perforin in peripheral blood lymphocytes in severe traumatic brain injured patients

Purpose: The purpose of this study was to investigate the changes of cytotoxic protein-perforin in peripheral blood lymphocytes in severe TBI patients and possible correlation between severity of TBI and perforin expression. Methods: Flow cytometry was used for simultaneous detection of intracellular perforin and cell surface antigens of peripheral blood lymphocytes of 20 severe TBI patients on day 1, 4 and 7 after the onset of injury. Peripheral blood mononuclear cells from 20 healthy volunteers were used as control. Clinical and laboratory parameters were also recorded. Results: There was a statistically significant decrease of perforin-positive lymphocytes including T, natural killer (NK) and NKT cells on day 4 as compared with day 1 after the brain injury or healthy controls. On day 7, perforin expression was restored in lymphocyte of cytotoxic phenotype (CD8 + T lymphocytes, NK cells, and NKT cells) compared with day 1. High positive correlation was found between the severity of TBI and frequency of perforin-positive cells on day 4 when the occurrence of the intra-hospital infections was the highest. Conclusion: Severe TBI significantly decreases perforin expression in T lymphocytes, NK and NKT cells, which indicate a possible mechanism underlying the high susceptibility to infections. © 2010 Elsevier Ltd. All rights reserved

Early Changes in Frequency of Peripheral Blood Lymphocyte Subpopulations in Severe Traumatic Brain‐Injured Patients

Abstract Infections are leading causes of increased morbidity and mortality of severe traumatic brain‐injured (STBI) patients. The mechanism underlying the susceptibility to the infections is still unexplained. The purpose of the study was to investigate changes in frequency of leucocytes subpopulations in peripheral blood of patients with STBI during the course of intensive care treatment. Twenty patients with STBI were included in the study. Healthy age‐ and sex‐ volunteers served as control. Peripheral blood samples were taken from these patients at day 1, 4 and 7, and peripheral blood mononuclear cells (PBMC) were isolated. The percentage of T, B lymphocyte, NK and NKT cells as well as monocytes was analysed by simultaneous detection of surface antigens using fluorochrome‐conjugated monoclonal antibodies. The two major subsets of T lymphocytes (CD3 CD56 CD4 and CD3 CD56 CD8 ) and NK cells (CD3 CD56 and CD3 CD56 ) were also analysed by flow cytometry. Extracranial infections were presented in 55% patients with STBI. At day 4, the percentage of T lymphocytes with cytotoxic phenotype significantly diminished and their numbers restored at day 7. The frequency of NKT cells showed the identical time‐dependent pattern, whereas the percentage of NK cells diminished on day 4 but did not restore after 7 days. The frequency of B lymphocytes did not change significantly during the time investigated, whereas the percentage of monocytes increased immediately after the injury and gradually diminished. The decrease in cells with cytotoxic phenotype might explain high incidence of susceptibility to infection of patients with STBI

Granulysin expression and the interplay of granulysin and perforin at the maternal–fetal interface

Granulysin (GNLY) is a cytolytic/apoptotic molecule highly expressed in immune cells, particularly NK cells, at the maternal-fetal interface. The primary function of GNLY is to carry out lysis or apoptosis induction in target cells, tumor cells or cells infected by intracellular pathogens. To exert some of its functions GNLY needs to collaborate with perforin. The purpose of this study was to determine: (a) the expression of GNLY at the gene and protein levels at the maternal-fetal interface, (b) the relationship(s) between GNLY and perforin, and (c) GNLY secretion by NK cells stimulated by the NK-sensitive K562 cell line and its HLA-C and HLA-G transfectants. GNLY and perforin genes were found to be highly activated at the interface. GNLY mRNA was present at significantly higher levels compared with other cytolytic/apoptotic molecules. Confocal microscopy analysis showed that most first trimester pregnancy decidual lymphocytes simultaneously contained both GNLY and perforin protein in their cytoplasm, with a punctuate pattern consistent with granule localization. In contrast to peripheral blood, in unstimulated decidual lymphocytes GNLY and perforin rarely co-localized (10% of GNLY-positive cells and 20% of perforin-positive cells were positive for both proteins). Contact between decidual lymphocytes and K562 cells caused GNLY and perforin to be expressed in the same granules (approximately 50% co-localization), i.e., to attain the pattern seen in peripheral blood lymphocytes. The abundant GNLY secretion by decidual NK cells compared with peripheral blood NK cells after 2. h of contact with the NK-sensitive K562 cells and K562 transfectants was striking