Characterization of CD94/NKG2 receptors on cytotoxic lymphocytes

Lektin-ähnliche Natürliche Killer (NK) -Zell Rezeptoren werden von NK und einem Teil der T Zellen exprimiert und vermitteln Schutz vor Pathogenen und Tumoren. Dazu gehört die Familie der heterodimeren CD94/NKG2 Rezeptoren. Diese besitzen entweder inhibierende oder aktivierende Funktion, erkennen das “selbst-Antigen” HLA-E und spielen eine wichtige Rolle bei der Regulierung der Zellaktivität. Ein erstes Ziel dieser Arbeit war es, die Verteilung von CD94/NKG2A und CD94/NKG2C Rezeptoren innerhalb der Lymphozyten des periphären Blutes (PBL) zu untersuchen und dadurch Subpopulationen von NK- und T- Zellen zu identifizieren. Die durchflusszytometrische Analyse ergab Subpopulationen von variierender Größe in Abhängigkeit vom individuellen Spender. Die größte Subpopulation wurde über den inhibierenden Rezeptor CD94/NKG2A definiert, wohingegen CD94/NKG2C stets von einer kleineren Population exprimiert wurde. Ein sehr geringer Prozentsatz an Zellen koexprimierte beide Rezeptoren. Dies gilt sowohl für NK- als auch T- Zellen. Des Weiteren haben wir untersucht, inwieweit die Expression von NKG2 Isoformen eine Auswirkung auf die Funktionen der Subpopulationen hat. Dafür wurde die Expansion dieser Subpopulationen in Kokulturen von mononukleären Zellen des periphären Blutes (PBMC) mit einer HLA Klasse Ia negativen, aber HLA-E transfektierten Zelllinie analysiert. Weiters wurde die Degranulierungs- Aktivität über CD107a gemessen. In der Kokultur zeigten NKG2C+ Zellen verstärkte Expansion im Vergleich zu NKG2A+ Zellen. Die wichtigsten funktionellen Unterschiede zwischen NKG2A und NKG2C positiven Subpopulationen waren eine Inhibition der Degranulation von frisch isolierten NKG2A+ Zellen in Abhängigkeit von HLA-E und ein HLA-E-vermittelte Aktivierung von zuvor kokultivierten, NKG2C+ Zellen. Dies deutet darauf hin, dass der “selbst-Ligand” HLA-E voraktivierte Zellen über NKG2C induzieren kann. Schließlich wurden Methoden etabliert, um Zellexpansion im Zusammenhang mit Zytomegalovirus-infizierten Fibroblasten und Endothelzellen zu untersuchen, da NKG2 Rezeptoren für die Kontrolle von CMV durch das Immunsystem eine wichtige Rolle spielen dürften. Eine vom Spender abhängige, CMV assoziierte Amplifizierung von NKG2 exprimierenden NK und T-Zell Subpopulationen wurden festgestellt. Zusammenfassend konnten durch die in dieser Arbeit durchgeführten Experimente Subpopulationen auf NK- und T- Zellen anhand der Expression von NKG2 Rezeptoren definiert und funktionale Unterschiede im Zusammenhang mit HLA-E und CMV festgestellt werden.Lectin-like NK cell receptors are expressed on NK cells and a fraction of T lymphocytes and are involved in providing protection against invading pathogens and cancer. Among these, the heterodimeric NKG2/CD94 receptors, comprising activating and inhibitory family members, recognize the “self” marker HLA-E and have been implicated as receptors of major importance. The goal of the first part of this work was to investigate the distribution of the CD94/NKG2A and CD94/NKG2C receptors within peripheral blood lymphocytes (PBL) to define subpopulations of NK and T lymphocytes expressing these receptors. Flow cytometry analysis displayed subpopulations of variable sizes between different individuals. Usually expression of the inhibitory CD94/NKG2A defined the largest NK subpopulation, whereas NK cells with the activating CD94/NKG2C constituted a smaller subpopulation. Cells with expression of both receptors were also found at very low percentage. The receptors were further detected on T lymphocytes with similar distribution of NKG2A and NKG2C subpopulations. In the next part, we asked the question wether the NKG2 isoforms would provide different functions to the subpopulations. The expansion of these subpopulations in co-cultures of peripheral blood mononuclear cells (PBMC) with an HLA class Ia-negative, but HLA-E transfected cell line, was investigated as well as the degranulation upon target cell encounter as measured by CD107a surface expression. In co-culture with the HLA-E cells, the NKG2C+ subset displayed preferential expansion as compared to NKG2A+ cells. Major functional differences of NKG2A and NKG2C subpopulations were in freshly isolated cells an inhibition of degranulation of the NKG2A cells by HLA-E, whereas after co-culture the NKG2C cells acquired increased killing capacity for the HLA-E cells suggesting that the self-ligand HLA-E can function as triggering ligand after preactivation of the cells. Finally, the tools to investigate cellular expansion in the context of cytomegalovirus (CMV) infections of fibroblasts and endothelial cells were prepared and initially tested as NKG2 receptors have been implicated in CMV immunosurveillance. Corresponding amplifications of NK and T cell subpopulations with NKG2 receptors were observed, however were to a large degree dependent on the individual donors. In summary, this work has defined subpopulations of NK as well as T cells by expression of NKG2 receptors for which differences in function could be shown in the context of cells with expression of the cognate ligand HLA-E and CMV infection

Fc Glycan-Modulated Immunoglobulin G Effector Functions

Immunoglobulin G (IgG) molecules are glycoproteins and residues in the sugar moiety attached to the IgG constant fragment (Fc) are essential for IgG functionality such as binding to cellular Fc receptors and complement activation. The core of this sugar moiety consists of a bi-antennary heptameric structure of mannose and N-acetylglucosamine (GlcNAc), further decorated with terminal and branching residues including galactose, sialic acid, fucose, and GlcNAc. Presence or absence of distinct residues such as fucose and sialic acid can dramatically alter pro- and anti-inflammatory IgG activities which could be harnessed for immunotherapeutic purposes. Here we review recent advances in understanding the role of the IgG-Fc glycan during immune responses and for immunotherapy with a focus on sialic acid and intravenous immunoglobulin (IVIG) treatment

Fc-Galactosylation of Human Immunoglobulin Gamma Isotypes Improves C1q Binding and Enhances Complement-Dependent Cytotoxicity

Binding of the complement component C1q to the CH2 domain of antigen-bound immunoglobulin gamma (IgG) activates the classical complement pathway and depends on its close proximity to Fc fragments of neighboring antibodies. IgG subclasses contain a highly conserved asparagine 297 (N)-linked biantennary glycan within their CH2 domains, the core structure of which can be extended with terminal galactose and sialic acid residues. To investigate whether Fc-glycosylation regulates effector functions of human IgG subclasses, we cloned the antigen-binding region of the CD20-specific monoclonal antibody rituximab into IgG isotype expression vectors. We found that Fc-galactosylation enhances the efficacy of CD20-targeting complement-fixing antibodies for C1q binding and complement-mediated tumor cell lysis. Increased efficacies were restricted to IgG1 and IgG3 subclasses indicating that Fc-galactosylation alone is not sufficient for IgG2 and IgG4 to acquire complement-fixing properties. Addition of terminal galactose to the N-glycan specifically improved binding of C1q without changing antigen- and FcγRIIIa-binding affinities of IgG isotypes. These data indicate that Fc galactosylation can be harnessed to enhance the complement-activating properties of IgG1 and IgG3 antibodies

Terminal complement activation is increased and associated with disease severity in CIDP

Chronic inflammatory demyelinating polyneuropathy (CIDP) is the most common chronic autoimmune neuropathy. While both cell-mediated and humoral mechanisms contribute to its pathogenesis, the rapid clinical response to plasmapheresis implicates a circulating factor responsible for peripheral nerve injury. We report that treatment-naïve patients with CIDP show increased serum and CSF levels of the anaphylatoxin C5a and the soluble terminal complement complex (sTCC). Systemic terminal complement activation correlates with clinical disease severity as determined by the Inflammatory Neuropathy Cause and Treatment (INCAT) disability scale. These data indicate that complement activation contributes to peripheral nerve injury and suggest that complement inhibition should be explored for its potential therapeutic merit in CIDP

Fc-Galactosylation of Human Immunoglobulin Gamma Isotypes Improves C1q Binding and Enhances Complement-Dependent Cytotoxicity

Binding of the complement component C1q to the CH2 domain of antigen-bound immunoglobulin gamma (IgG) activates the classical complement pathway and depends on its close proximity to Fc fragments of neighboring antibodies. IgG subclasses contain a highly conserved asparagine 297 (N)-linked biantennary glycan within their CH2 domains, the core structure of which can be extended with terminal galactose and sialic acid residues. To investigate whether Fc-glycosylation regulates effector functions of human IgG subclasses, we cloned the antigen-binding region of the CD20-specific monoclonal antibody rituximab into IgG isotype expression vectors. We found that Fc-galactosylation enhances the efficacy of CD20-targeting complement-fixing antibodies for C1q binding and complement-mediated tumor cell lysis. Increased efficacies were restricted to IgG1 and IgG3 subclasses indicating that Fc-galactosylation alone is not sufficient for IgG2 and IgG4 to acquire complement-fixing properties. Addition of terminal galactose to the N-glycan specifically improved binding of C1q without changing antigen- and FcγRIIIa-binding affinities of IgG isotypes. These data indicate that Fc galactosylation can be harnessed to enhance the complement-activating properties of IgG1 and IgG3 antibodies

Efficacy of Intravenous Immunoglobulin in Neurological Diseases

Owing to its anti-inflammatory efficacy in various autoimmune disease conditions, intravenous immunoglobulin (IVIG)-pooled IgG obtained from the plasma of several thousands individuals-has been used for nearly three decades and is proving to be efficient in a growing number of neurological diseases. IVIG therapy has been firmly established for the treatment of Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, and multifocal motor neuropathy, either as first-line therapy or adjunctive treatment. IVIG is also recommended as rescue therapy in patients with worsening myasthenia gravis and is beneficial as a second-line therapy for dermatomyositis and stiff-person syndrome. Subcutaneous rather than intravenous administration of IgG is gaining momentum because of its effectiveness in patients with primary immunodeficiency and the ease with which it can be administered independently from hospital-based infusions. The demand for IVIG therapy is growing, resulting in rising costs and supply shortages. Strategies to replace IVIG with recombinant products have been developed based on proposed mechanisms that confer the anti-inflammatory activity of IVIG, but their efficacy has not been tested in clinical trials. This review covers new developments in the immunobiology and clinical applications of IVIG in neurological diseases

Interleukins 12 and 15 induce cytotoxicity and early NK-cell differentiation in type 3 innate lymphoid cells

Type 3 innate lymphoid cells (ILC3s) fulfill protective functions at mucosal surfaces via cytokine production. Although their plasticity to become ILC1s, the innate counterparts of type 1 helper T cells, has been described previously, we report that they can differentiate into cytotoxic lymphocytes with many characteristics of early differentiated natural killer (NK) cells. This transition is promoted by the proinflammatory cytokines interleukin 12 (IL-12) and IL-15, and correlates with expression of the master transcription factor of cytotoxicity, eomesodermin (Eomes). As revealed by transcriptome analysis and flow cytometric profiling, differentiated ILC3s express CD94, NKG2A, NKG2C, CD56, and CD16 among other NK-cell receptors, and possess all components of the cytotoxic machinery. These characteristics allow them to recognize and kill leukemic cells with perforin and granzymes. Therefore, ILC3s can be harnessed for cytotoxic responses via differentiation under the influence of proinflammatory cytokines

Protection from experimental autoimmune encephalomyelitis by polyclonal IgG requires adjuvant-induced inflammation

BACKGROUND Intravenous immunoglobulin (IVIG) proved to be an efficient anti-inflammatory treatment for a growing number of neuroinflammatory diseases and protects against the development of experimental autoimmune encephalomyelitis (EAE), a widely used animal model for multiple sclerosis (MS). METHODS The clinical efficacy of IVIG and IVIG-derived F(ab')2 fragments, generated using the streptococcal cysteine proteinase Ide-S, was evaluated in EAE induced by active immunization and by adoptive transfer of myelin-specific T cells. Frequency, phenotype, and functional characteristics of T cell subsets and myeloid cells were determined by flow cytometry. Antibody binding to microbial antigen and cytokine production by innate immune cells was assessed by ELISA. RESULTS We report that the protective effect of IVIG is lost in the adoptive transfer model of EAE and requires prophylactic administration during disease induction. IVIG-derived Fc fragments are not required for protection against EAE, since administration of F(ab')2 fragments fully recapitulated the clinical efficacy of IVIG. F(ab')2-treated mice showed a substantial decrease in splenic effector T cell expansion and cytokine production (GM-CSF, IFN-γ, IL-17A) 9 days after immunization. Inhibition of effector T cell responses was not associated with an increase in total numbers of Tregs but with decreased activation of innate myeloid cells such as neutrophils, monocytes, and dendritic cells. Therapeutically effective IVIG-derived F(ab')2 fragments inhibited adjuvant-induced innate immune cell activation as determined by IL-12/23 p40 production and recognized mycobacterial antigens contained in Freund's complete adjuvant which is required for induction of active EAE. CONCLUSIONS Our data indicate that F(ab')2-mediated neutralization of adjuvant contributes to the therapeutic efficacy of anti-inflammatory IgG. These findings might partly explain the discrepancy of IVIG efficacy in EAE and MS

The Amount of BCL6 in B Cells Shortly after Antigen Engagement Determines Their Representation in Subsequent Germinal Centers

It is unknown whether the incremental increases in BCL6 amounts in antigen-activated B cells influence the unfolding differentiation before germinal center (GC) formation. By comparing shortly after immunization the distribution of conventional B cells to those enforced to express BCL6 at the upper quartile of normal and those lacking BCL6 altogether, we determined that B cell representation in the stages before the GC compartment was related to BCL6 amounts. This was not by increased proliferation or suppression of early plasmablast differentiation, but rather by preferential recruitment and progression through these early stages of B cell activation, culminating in preferential transition into GC. Once established, this bias was stable in GC over several weeks; other BCL6-regulated GC B cell behaviors were unaffected. We propose that setting BCL6 amounts very early in activated B cells will be central in determining clonal representation in the GC and thus memory populations