Skin transplantation in a mouse model of naïve T-cell deficiency

Coronin 1 is one of seven mammalian coronin family members that is preferentially expressed in hematopoietic cells and brain tissue (Ferrari et al. 1999; Jayachandran et al. 2014). It was initially discovered in macrophages where it is involved in Mycobacterium tuberculosis pathogenesis and survival (Ferrari et al. 1999). Interestingly, analysis of coronin 1-deficient mice showed a drastic reduction in the numbers of T cells in the periphery (Föger et al. 2006; Shiow et al. 2008; Haraldsson et al. 2008; Mueller et al. 2008). However, segregation and development of T cells in the thymus showed no significant changes when compared with wild type mice (Mueller et al. 2008). Recent data demonstrate that despite the paucity in naïve T cells, coronin 1-deficient mice are resistant to infections but fail to induce autoimmune diseases (Tchang et al. 2013; Haraldsson et al. 2008; Shiow et al. 2008; Siegmund et al. 2011). The physiological relevance of coronin 1 deficiency in allorejection is yet to be discovered, however, the importance of T cells in this process suggests a critical role for coronin 1. When organs are transplanted between members of the same species, an immune response to such a graft develops alloreactivity. It consists of the same mechanism as an immune response for defense against pathogens but it is mediated against differences in major histocompatibility complexes (MHC) class I and II molecules between the host and the donor (Goldsby et al. 2002). The goal of my project was to dissect the ability of coronin 1-deficient T cells to recognize foreign MHC complexes and to reject the allograft. I focused on finding the mechanisms that allow coronin 1-deficent mice to keep the graft while at the same time drive the immunity against infection. To this end, I combined skin transplantation experiments with in-depth in vitro analysis. Interestingly, we found prolonged survival of Balb/c skin transplanted onto coronin 1-deficient BL/6 recipients. Further investigations showed tolerance induction in a minor mismatch setting where BL/6 skin presenting minor histocompatibility antigens from Balb/c were transplanted onto coronin 1-deficient mice. This could be the outcome of reduced frequency and/or impaired activation of T cells in the absence of coronin 1. However, despite increasing coronin 1-deficient CD4+ T cells numbers, we could not induce rejection in Rag2-/- mice transplanted with bm12Rag2-/- skin. Although the retained lower counts of CD4+ T cells could indicate a defect in proliferation or antigen recognition after skin transplantation, further in vitro experiments suggested that coronin 1-deficient T cells were able to recognize antigen through the T cell receptor (TCR) and respond, could become activated and could proliferate. Furthermore, cytokines and cell markers analysis suggest that coronin 1-deficient T cells were under the stress of high activation (IFNγhi, CD5hi, IL-7Rlo) and intense proliferation (Ki-67hi) that could cause exhaustion (PD-1hi) and consequently lead to their death (FasL). As the same markers were not changed in the thymus of coronin 1-deficient mice and the mice could produce recent thymic emigrants this can provide insight into the time point at which coronin 1 is essential for T cells’ survival. In-depth analysis of T cells in vitro and in vivo confirmed the different impact of coronin 1-deficiency in CD8+ and CD4+ T cells, as the prolonged graft survival resembled the depletion of CD4+ T cells in skin transplantation model (Goldsby et al. 2002). In a CD4+ T cell-dependent model, where bm12Rag2-/- skin was transplanted onto coronin 1-deficient mice, we observed acceptance of the graft. What is more, the bm1 (CD8-dependent) skin was rejected in coronin 1-deficient recipients upon transplantation. This absence or delay in rejection in coronin 1-deficient mice caused by induction of tolerance may be due to elevated rate of regulatory T cells (Tregs), the suppressive abilities of conventional CD4+ T cells or combination of these. Finding increased frequencies of Tregs in the coronin 1-deficient mice, compared to wild type mice, supported this statement. In-depth analysis of regulatory T cells in coronin 1-deficient mice showed no difference in suppressive abilities compared with wild type cells. Moreover, we demonstrated that the cells did not generate or proliferate better in coronin 1-deficient mice. Nevertheless, their superior survival over conventional coronin 1-deficient CD4+ T cells in vitro could explain their higher frequencies and could be linked to enhanced expression of PD-1. This, in turn, promotes apoptosis in antigen-specific T cells while increasing survival of Tregs (Francisco, Sage, and Sharpe 2010). Overall, the data suggests a critical role of coronin 1 in T cell-mediated allograft rejection. However, its precise function and mechanistic details are subject for further study

Disruption of Coronin 1 Signaling in T Cells Promotes Allograft Tolerance while Maintaining Anti-Pathogen Immunity

The ability of the immune system to discriminate self from non-self is essential for eradicating microbial pathogens but is also responsible for allograft rejection. Whether it is possible to selectively suppress alloresponses while maintaining anti-pathogen immunity remains unknown. We found that mice deficient in coronin 1, a regulator of naive T cell homeostasis, fully retained allografts while maintaining T cell-specific responses against microbial pathogens. Mechanistically, coronin 1-deficiency increased cyclic adenosine monophosphate (cAMP) concentrations to suppress allo-specific T cell responses. Costimulation induced on microbe-infected antigen presenting cells was able to overcome cAMP-mediated immunosuppression to maintain anti-pathogen immunity. In vivo pharmacological modulation of this pathway or a prior transfer of coronin 1-deficient T cells actively suppressed allograft rejection. These results define a coronin 1-dependent regulatory axis in T cells important for allograft rejection and suggest that modulation of this pathway may be a promising approach to achieve long-term acceptance of mismatched allografts

Allele-Independent Turnover of Human Leukocyte Antigen (HLA) Class Ia Molecules.

Major histocompatibility complex class I (MHCI) glycoproteins present cytosolic peptides to CD8+ T cells and regulate NK cell activity. Their heavy chains (HC) are expressed from up to three MHC gene loci (human leukocyte antigen [HLA]-A, -B, and -C in humans), whose extensive polymorphism maps predominantly to the antigen-binding groove, diversifying the bound peptide repertoire. Codominant expression of MHCI alleles is thus functionally critical, but how it is regulated is not fully understood. Here, we have examined the effect of polymorphism on the turnover rates of MHCI molecules in cell lines with functional MHCI peptide loading pathways and in monocyte-derived dendritic cells (MoDCs). Proteins were labeled biosynthetically with heavy water (2H2O), folded MHCI molecules immunoprecipitated, and tryptic digests analysed by mass spectrometry. MHCI-derived peptides were assigned to specific alleles and isotypes, and turnover rates quantified by 2H incorporation, after correcting for cell growth. MHCI turnover half-lives ranged from undetectable to a few hours, depending on cell type, activation state, donor, and MHCI isotype. However, in all settings, the turnover half-lives of alleles of the same isotype were similar. Thus, MHCI protein turnover rates appear to be allele-independent in normal human cells. We propose that this is an important feature enabling the normal function and codominant expression of MHCI alleles

Branch as a way of conducting business activity in Poland by foreign entrepreneur

Kwestia działalności podmiotów zagranicznych na terenie Polski uregulowana została w ustawie z dnia 2 lipca 2004 roku o swobodzie działalności gospodarczej. Przepisy ustawy realizują unijną zasadę swobody przedsiębiorczości, wyrażoną w Traktacie o Funkcjonowaniu Unii Europejskiej. Równocześnie w świetle Ustawy przedsiębiorcy zagraniczni mogą prowadzić działalność gospodarczą na terenie Polski zgodnie z zasadą wzajemności bądź narodowego traktowania lub w świetle umów międzynarodowych, w zależności od miejsca siedziby lub zamieszkania przedsiębiorcy.Przedsiębiorca zagraniczny w celu prowadzenia działalności gospodarczej na rynku polskim może założyć spółkę zależną (lub filię), oddział bądź przedstawicielstwo. Spółka zależna (inaczej spółka córka) różni się od filii ilością posiadanych przez przedsiębiorcę zagranicznego udziałów (przedsiębiorca zagraniczny posiada całość udziałów filii i większość spółki-córki). Są to jednak formy niezależne pod względem prawnym, działające według prawa kraju rejestracji. Przedstawicielstwo lub oddział to podmioty zależne prawnie od przedsiębiorcy zagranicznego (gdyż nie posiadają osobowości prawnej), które podlegają prawu kraju pochodzenia przedsiębiorcy zagranicznego. Przedstawicielstwo może prowadzić działalność wyłącznie w zakresie reklamy i promocji przedsiębiorstwa macierzystego. Oddział natomiast może prowadzić działalność gospodarczą wyłącznie w zakresie przedmiotu działalności przedsiębiorcy zagranicznego. Przedsiębiorca zagraniczny prowadzący działalność gospodarczą na terenie Polski w postaci oddziału obowiązany jest ustanowić osobę upoważnioną w oddziale do reprezentowania przedsiębiorcy zagranicznego, a także uzyskać wpis oddziału do rejestru przedsiębiorców, zgodnie z przepisami Ustawy o Krajowym Rejestrze Sądowym. Oddział przedsiębiorcy zagranicznego obowiązany jest używać w celu oznaczenia oryginalnej nazwy przedsiębiorstwa macierzystego wraz z przetłumaczoną na język polski formą prawną tego przedsiębiorstwa oraz członem „oddział w Polsce”. Ponadto, oddział zobligowany jest do prowadzenia oddzielnej rachunkowości w języku polski. Nadzór nad oddziałem przedsiębiorcy zagranicznego sprawuje minister właściwy do spraw gospodarki, a nad oddziałami banków zagranicznych, oddziałami zagranicznych zakładów ubezpieczeń, oddziałem zagranicznej instytucji kredytowej oraz oddziałem zagranicznej firmy inwestycyjnej, która prowadzi na terenie Polski działalność maklerską nadzór sprawuje Komisja Nadzoru Finansowego. Działalność oddziału może zakończyć się jedynie poprzez likwidację, co wynika z faktu, iż nie posiada on zdolności upadłościowej i układowej. Likwidacja może być skutkiem decyzji przedsiębiorcy zagranicznego bądź decyzji ministra właściwego do spraw gospodarki zakazującej wykonywania działalności gospodarczej przez przedsiębiorcę zagranicznego w postaci oddziału.According to the Act concerning freedom of economic activity dated 2nd July 2004 year, foreign entrepreneurs may conduct business activity in Poland. Polish act differentiates foreign entrepreneurs – as a consequence of the company seat or man’s residence they may establish business in Poland as it is regulated by the rule of mutuality, rule of national treatment or accordingly to international agreement. The rule of freedom of establishment is expressed in the Treaty on the functioning of the European Union. In order to conduct business activity in Poland foreign entrepreneur may establish subsidiary company (however company may possess all shares in subsidiary company or majority of shares), branch or agency (representation). Subsidiary company is legally independent and is regulated by the law of the country of establishment (Polish law). Contrary, branches and agencies are depended on foreign entrepreneur (because they do not have legal personality) and they are functioning accordingly to the law of the company seat or residence of foreign entrepreneur. Representation may conduct activity limited to advertisement and promotion of foreign entrepreneur. On the other hand branch may conduct business activity limited to the scope of economic activity conducted by home company. Foreign entrepreneur is obliged to indicated authorized person who will represent branch in country of registration and to fill the registration in Entrepreneurs’ Register of the National Court Register. Branch has to use original name of foreign entrepreneur with the indication of entrepreneur’s legal form translated into Polish and added words "oddział w Polsce". Moreover, branch is obliged to keep separate account books in Polish. Minister in charge of economy is supervising branches in Poland, however it is Polish Financial Supervision Authority who supervises branches of foreign banks, branches of foreign insurances companies, branches of foreign loan institutions and branches of foreign investment companies, which are conducting in Poland stockbroking activity. Business activity of branch may be terminated only by liquidation, what is consequence of the fact that branch has no bankruptcy or arrangement capacity. Liquidation may be consequence of the decision of foreign entrepreneur or decision of minister in charge of economy, forbidding conducting business activity in Poland by foreign entrepreneur in the form of branch

MHCI turnover in LCL721 cells.

<p>LCL721 cells were labeled with ≈ 5% ²H₂O in media and folded MHCI molecules immunoprecipitated with W6/32. ²H incorporation into tryptic peptides was quantified by LC-MS. (A-C) Fractional synthesis was calculated for different peptides derived from HLA-A (A), HLA-B (B), and HLA-C molecules (C) (mean ± SD of different mass isotopomers) and plotted against labeling time. Full sequences and analytical metrics for all informative peptides (identified by four amino acids in single-letter code or by charge) are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161011#pone.0161011.s004" target="_blank">S2 Table</a>. In (A) and (B), allele- and isotype-specific peptides exhibited no significant differences in fractional protein synthesis (p = 0.32 and p = 0.29, respectively, by F test); in (C), only C1-specific peptides were identified. Single-exponential curve fits (with 95% confidence intervals) are based on a pooled analysis of all peptides from each isotype. (D) Exponential growth of LCL721 cells during ²H₂O labeling. The corresponding time course of the fraction of new cells is shown in panels (A-C), for comparison with protein synthesis. Panels (A-D) were from the same experiment. (E) Fractional synthesis rates (per hour, mean ± SEM) of MHCI isotypes (from (A-C)), compared to fractional cell growth rates (from (D)). Two independent experiments are shown. Fractional synthesis rates for individual peptides are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161011#pone.0161011.s004" target="_blank">S2 Table</a>. (F) Turnover rates of different MHCI isotypes, calculated from the excess of mean fractional protein synthesis rates over the cell growth rate.</p

Effect of ²H₂O labeling on peptide mass isotopomer distributions.

<p>(A) Mass isotopomer distributions of a MHCI-derived, B isotype-specific tryptic peptide from KG-1 cells after labeling with ²H₂O for various times. Lines connect data at each time point. Within error, each mass isotopomer changed from its initial (unlabeled) to final plateau (fully-labeled) value at the same rate, which is identical to the rate of protein fractional synthesis. (B) For the same peptide, MIDA models for the unlabeled and fully-labeled mass isotopomer distributions (dashed and solid lines, respectively) were compared with experimental data (symbols). RMSD values were 0.20% and 0.25%, respectively, for unlabeled and fully-labeled samples).</p

HLA-B fractional synthesis in unstimulated MoDCs.

<p>Analysis as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161011#pone.0161011.g006" target="_blank">Fig 6</a>, except that separate curve fits are shown for HD2 (p = 0.04, F test). The significance of this result is doubtful, as explained in the text. B*27+ donors are identified; note that B27 allele-specific peptides proved suitable for analysis in HD6 and 7, but not in HD4 and 5.</p

HLA-C fractional synthesis in unstimulated MoDCs.

<p>Analysis as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161011#pone.0161011.g006" target="_blank">Fig 6</a>.</p