Transcriptional signatures of Itk-deficient CD3+, CD4+ and CD8+ T-cells

<p>Abstract</p> <p>Background</p> <p>The Tec-family kinase Itk plays an important role during T-cell activation and function, and controls also conventional versus innate-like T-cell development. We have characterized the transcriptome of Itk-deficient CD3⁺T-cells, including CD4⁺and CD8⁺subsets, using Affymetrix microarrays.</p> <p>Results</p> <p>The largest difference between Itk^-/-and Wt CD3⁺T-cells was found in unstimulated cells, e.g. for killer cell lectin-like receptors. Compared to anti-CD3-stimulation, anti-CD3/CD28 significantly decreased the number of transcripts suggesting that the CD28 co-stimulatory pathway is mainly independent of Itk. The signatures of CD4⁺and CD8⁺T-cell subsets identified a greater differential expression than in total CD3⁺cells. Cyclosporin A (CsA)-treatment had a stronger effect on transcriptional regulation than Itk-deficiency, suggesting that only a fraction of TCR-mediated calcineurin/NFAT-activation is dependent on Itk. Bioinformatic analysis of NFAT-sites of the group of transcripts similarly regulated by Itk-deficiency and CsA-treatment, followed by chromatin-immunoprecipitation, revealed NFATc1-binding to the <it>Bub1</it>, <it>IL7R, Ctla2a</it>, <it>Ctla2b</it>, and <it>Schlafen1 </it>genes. Finally, to identify transcripts that are regulated by Tec-family kinases in general, we compared the expression profile of Itk-deficient T-cells with that of Btk-deficient B-cells and a common set of transcripts was found.</p> <p>Conclusion</p> <p>Taken together, our study provides a general overview about the global transcriptional changes in the absence of Itk.</p

Models for infections in immunodeficiency

Increased susceptibility to infections in immunodeficient patients is a well-known phenomenon. The spectrum of infections reflects the underlying defect. Experimental infections in mouse strains with different types of defined immunodeficiencies allow studying the course of infectious diseases in immunocompromised hosts. In such models it is possible to investigate the relationship between immunity and infection and the role of the immune system both in the protection against microorganisms and in the pathogenesis. However, analogous mutations in man and mice do not always result in similar phenotype. Another obstacle is that there is strong host specificity for some microorganisms, and establishment of relevant mouse models for human infections can be a problem. Within this project we have been working with three pathogens: human pathogen Campylobacter jejuni and mouse pathogens polyomavirus and Mycoplasma pulmonis. Several rodent models have been developed in order to mimic human campylobacteriosis but none of them has gained acceptance as universal model of the infection, since it was impossible to produce illness similar to that seen in humans. The aim of our study (paper I) was to examine the possibility of developing a relevant animal model of campylobacteriosis by infecting scid mice, lacking both B and T cells, with enterotoxin producing C. jejuni strain. In addition, the mice underwent antibiotic treatment prior to infection in order to disrupt the intestinal flora, and enhance gut colonisation. Both immunocompetent controls and scid mice became colonised by C. jejuni for 7 weeks. Any clinical and histological signs of the disease were, however, not observed. This suggests that other factors than normal intestinal flora and acquired immunity must be involved in the resistance of mice to this bacterium. Mycoplasma infections were performed with M. pulmonis which is a causative agent of murine respiratory disease (papers III, IV, V). M. pulmonis infection in mice resembles in many aspects human disease caused by M. pneumoniae, thus providing a relevant model of the infection. Several mouse strains with severe combined immunodeficiencies (scid/beige, scid), B cell deficiencies (xid, Btk-/-, muMT, lambda5-/- ) and immunocompetent control strains of respective backgrounds were infected. Bacteriological, histological and immunological analyses were performed. The results can be summarised to: 1) the course of mycoplasmal disease is highly dependent on the immune status of the host 2) antibodies have a protective role regarding systemic disease, 3) humoral immunity is involved in the pathogenesis of pulmonary lesions, 4) proinflammatory cytokines, locally produced in the chondrocytes, may contribute to the aetiology of Mycoplasma-associated arthritis, 5) different genetic backgrounds of mouse strains influence the severity of the mycoplasmosis. The importance of antibodies for elimination of polyomavirus infection was also studied (paper II). B cell deficient xid and muMT (referred in the paper as IgM-/-) mice, CD8-/-/muMT double k.o. mice, and normal control mice were infected with polyomavirus as adults. The mice were tested for presence of polyoma DNA with a polyoma specific polymerase chain reaction during 6 weeks post infection. A protective role of antibodies with regard to prevention against persistent polyomavirus infection was shown

"Bråkar, skriker fula ord om jag var mobbad skulle jag bli rädd"

Studien avser att belysa frekvensen av mobbning, typ av mobbning, kunskap om var den sker och, om det finns någon skillnad i pojkars respektive flickors beteende i avseende till om någon mobbar eller blir mobbad. En skola vidtalades. Ett frågeformulär med 22 frågor med fasta svarsskalor och en öppen fråga användes. Tendensen är att det är fler flickor än pojkar som har blivit mobbade. Det är en signifikant skillnad mellan pojkar och flickor på den öppna frågan. De senare som definierar mobbning som verbal eller att retas medan pojkarna framför allt nämner att slåss. Få av eleverna har svarat att de varit med om att mobba andra elever någon gång. När det har hänt är det flera pojkar/en enskild pojke, än flickor som mobbat både pojkar och flickor. Sist kommer en enskild flicka som mobbar. Platsen är skolgården

"Bråkar, skriker fula ord om jag var mobbad skulle jag bli rädd"

Studien avser att belysa frekvensen av mobbning, typ av mobbning, kunskap om var den sker och, om det finns någon skillnad i pojkars respektive flickors beteende i avseende till om någon mobbar eller blir mobbad. En skola vidtalades. Ett frågeformulär med 22 frågor med fasta svarsskalor och en öppen fråga användes. Tendensen är att det är fler flickor än pojkar som har blivit mobbade. Det är en signifikant skillnad mellan pojkar och flickor på den öppna frågan. De senare som definierar mobbning som verbal eller att retas medan pojkarna framför allt nämner att slåss. Få av eleverna har svarat att de varit med om att mobba andra elever någon gång. När det har hänt är det flera pojkar/en enskild pojke, än flickor som mobbat både pojkar och flickor. Sist kommer en enskild flicka som mobbar. Platsen är skolgården

Ibrutinib Has Time-dependent On- and Off-target Effects on Plasma Biomarkers and Immune Cells in Chronic Lymphocytic Leukemia

Ibrutinib is a covalently binding inhibitor of the B-cell receptor signaling-mediator Bruton’s tyrosine kinase (BTK) with great efficacy in chronic lymphocytic leukemia (CLL). Common side effects like atrial fibrillation (AF), bleeding and infections might be caused by ibrutinib’s inhibition of other kinases in non-B cells. Five-year follow-up of plasma biomarkers by proximity extension assay and immune cell numbers by flow cytometry during ibrutinib treatment revealed that 86 of the 265 investigated plasma biomarkers significantly changed during treatment, 74 of which decreased. Among the 12 markers that increased, 6 are associated with cardiovascular diseases and therefore potentially involved in ibrutinib-induced AF. Comparison between healthy donors and X-linked agammaglobulinemia (XLA) patients, who have nonfunctional BTK and essentially lack B cells, showed indicative changes in 53 of the 265 biomarkers while none differed significantly. Hence, neither B cells nor BTK-dependent pathways in other cells seem to influence the levels of the studied plasma biomarkers in healthy donors. Regarding immune cells, the absolute number of T cells, including subsets, decreased, paralleling the decreasing tumor burden. T helper 1 (Th1) cell numbers dropped strongly, while Th2 cells remained relatively stable, causing Th2-skewing. Thus, long-term ibrutinib treatment has a profound impact on the plasma proteome and immune cells in patients with CLL

BTK gatekeeper residue variation combined with cysteine 481 substitution causes super-resistance to irreversible inhibitors acalabrutinib, ibrutinib and zanubrutinib

Irreversible inhibitors of Bruton tyrosine kinase (BTK), pioneered by ibrutinib, have become breakthrough drugs in the treatment of leukemias and lymphomas. Resistance variants (mutations) occur, but in contrast to those identified for many other tyrosine kinase inhibitors, they affect less frequently the “gatekeeper” residue in the catalytic domain. In this study we carried out variation scanning by creating 11 substitutions at the gatekeeper amino acid, threonine 474 (T474). These variants were subsequently combined with replacement of the cysteine 481 residue to which irreversible inhibitors, such as ibrutinib, acalabrutinib and zanubrutinib, bind. We found that certain double mutants, such as threonine 474 to isoleucine (T474I) or methionine (T474M) combined with catalytically active cysteine 481 to serine (C481S), are insensitive to ≥16-fold the pharmacological serum concentration, and therefore defined as super-resistant to irreversible inhibitors. Conversely, reversible inhibitors showed a variable pattern, from resistance to no resistance, collectively demonstrating the structural constraints for different classes of inhibitors, which may affect their clinical application

The transcriptional regulator PLZF induces the development of CD44 high memory phenotype T cells

Transcriptional pathways controlling the development of CD44hi memory phenotype (MP) T cells with “innate-like” functions are not well understood. Here we show that the BTB (bric-a-brac, tramtrack, broad complex) domain-containing protein promyelocytic leukemia zinc finger (PLZF) is expressed in CD44hi, but not in CD44lo, CD4+ T cells. Transgenic expression of PLZF during T cell development and in CD4+ and CD8+ T cells induced a T cell intrinsic program leading to an increase in peripheral CD44hi MP CD4+ and CD8+ T cells and a corresponding decrease of naïve CD44lo T cells. The MP CD4+ and CD8+ T cells produced IFNγ upon PMA/ionomycin stimulation, thus showing innate-like function. Changes in the naïve versus memory-like subset distribution were already evident in single-positive thymocytes, indicating PLZF-induced T cell developmental alterations. In addition, CD1d-restricted natural killer T cells in PLZF transgenic mice showed impaired development and were severely reduced in the periphery. Finally, after anti-CD3/CD28 stimulation, CD4+ transgenic T cells showed reduced IL-2 and IFNγ production but increased IL-4 secretion as a result of enhanced IL-4 production of the CD44hiCD62L+ subset. Our data indicate that PLZF is a novel regulator of the development of CD44hi MP T cells with a characteristic partial innate-like phenotype