Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or ...

Saarland University

Lytic granule (LG)-mediated apoptosis is the main mechanism by which CTL kill virus-infected and tumorigenic target cells. CTL form a tight junction with the target cells, which is called the immunological synapse (IS). To avoid unwanted killing of neighboring cells, exocytosis of lytic granules (LG) is tightly controlled and restricted to the IS. In this study, we show that in activated human primary CD8(+) T cells, docking of LG at the IS requires tethering LG with CD3-containing endosomes (CD3-endo). Combining total internal reflection fluorescence microscopy and fast deconvolution microscopy (both in living cells) with confocal microscopy (in fixed cells), we found that LG and CD3-endo tether and are cotransported to the IS. Paired but not single LG are accumulated at the IS. The dwell time of LG at the IS is substantially enhanced by tethering with CD3-endo, resulting in a preferential release of paired LG over single LG. The SNARE protein Vti1b is required for tethering of LG and CD3-endo. Downregulation of Vti1b reduces tethering of LG with CD3-endo. This leads to an impaired accumulation and docking of LG at the IS and a reduction of target cell killing. Therefore, Vti1b-dependent tethering of LG and CD3-endo determines accumulation, docking, and efficient lytic granule secretion at the IS.

Docking of lytic granules at the immunological synapse in human CTL requires Vti1b-dependent pairing with CD3 endosomes.

Store-operated Ca(2+) entry is a major Ca(2+) entry mechanism that is present in most cell types. In immune cells, store-operated Ca(2+) entry is almost exclusively mediated by Ca(2+) release-activated Ca(2+) (CRAC) channels. Ca(2+) entry through these channels and the corresponding cytosolic Ca(2+) signals are required for many immune cell functions, including all aspects of T-cell activation. ORAI proteins are the molecular correlates for the CRAC channels. The three human members, ORAI1, ORAI2 and ORAI3, are activated through the stromal interaction molecules (STIM)1 and 2 following depletion of endoplasmic reticulum Ca(2+) stores. Different combinations of STIM and ORAI can form different CRAC channels with distinct biophysical properties. In this article, we review and discuss mechanistic and functional implications of two important CRAC/ORAI inhibitors, 2-APB and BTP2, and the antibiotic G418 that has also been reported to interfere with ORAI channel function. The use of pharmacological tools should help to assign distinct physiological and pathophysiological functions to different STIM-ORAI protein complexes.

Pharmacology of ORAI channels as a tool to understand their physiological functions.

&#x7EC6;&#x80DE;&#x6BD2;&#x6027; T &#x6DCB;&#x5DF4;&#x7EC6;&#x80DE; (Ctl) &#x7684;&#x4E3B;&#x8981;&#x529F;&#x80FD;&#x662F;&#x6D88;&#x9664;&#x75C5;&#x539F;&#x611F;&#x67D3;&#x548C;&#x81F4;&#x7624;&#x7EC6;&#x80DE;&#x3002;&#x8FD9;&#x662F;&#x4E3B;&#x8981;&#x662F;&#x901A;&#x8FC7;&#x80DE;&#x5410;&#x6EB6;&#x9897;&#x7C92; (LGs) &#x542B;&#x6709;&#x7EC6;&#x80DE;&#x6BD2;&#x7684;&#x7EC4;&#x4EF6;&#x5982;&#x7A7F;&#x5B54;&#x7D20;&#x548C;&#x514D;&#x75AB;&#x7A81;&#x89E6; (IS) &#x5728; granzymes &#x4ECB;&#x5BFC;&#x7684;&#x3002;&#x53EF;&#x6EB6;&#x6027; NSF &#x9644;&#x4EF6; &#xFF08;&#x9677;&#x9631;&#xFF09; &#x53D7;&#x4F53;&#x86CB;&#x767D;&#x5F02;&#x6784;&#x4F53;&#x662F;&#x4F17;&#x6240;&#x5468;&#x77E5;&#x9700;&#x7684;&#x56CA;&#x6CE1;&#x80DE;&#x4E2D;&#x795E;&#x7ECF;&#x5143;&#x7A81;&#x89E6;&#xFF0C;&#x4F46;&#x53EA;&#x662F;&#x90E8;&#x5206;&#x7684;&#x7406;&#x89E3;&#x662F;&#x5728; Ctl &#x4E2D;&#x7684;&#x6F5C;&#x5728;&#x529F;&#x80FD;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x5BA1;&#x67E5;&#x4E86;&#x7F51;&#x7F57;&#x86CB;&#x767D;&#x8868;&#x8FBE;&#x7684;&#x4E4B;&#x524D;&#x548C;&#x4E4B;&#x540E;&#x6FC0;&#x6D3B;&#x4E3B;&#x4EBA;&#x7C7B; CD8(+) T &#x7EC6;&#x80DE;&#x4E0E;&#x786E;&#x5B9A;&#x5176;&#x4EA4;&#x611F;&#x795E;&#x7ECF;&#x4E0E; LGs &#x548C; CD3 &#x540E;&#x662F;&#x5F62;&#x6210;&#x4E0E;&#x76EE;&#x6807;&#x5355;&#x5143;&#x683C;&#x3002;&#x6211;&#x4EEC;&#x53D1;&#x73B0;&#x5728;&#x795E;&#x7ECF;&#x7EC6;&#x80DE;&#x4E2D;&#x7684;&#x51E0;&#x4E2A;&#x5173;&#x952E;&#x7F51;&#x7F57;&#x86CB;&#x767D;&#x8D28;&#x4E0D;&#x8868;&#x793A;&#x5728; Ctl&#xFF0C;&#x5982; syntaxin1B2 &#x548C;&#x7BA1;&#x7406;&#x5355;&#x5143; 25&#x3002;Vti1b&#x3001; Stx8 &#x548C; Stx16 &#x4E86; LGs&#x3001; Stx3&#x3001; Stx4&#x3001; Stx6&#x3001; Stx7&#x3001; Stx8&#x3001; Stx13&#x3001; Vti1b&#x3001; VAMP3 &#x548C; VAMP4 &#x4E0E; CD3 &#x7EF4;&#x540C;&#x65F6;&#x4E0E;&#x4EA4;&#x611F;&#x795E;&#x7ECF;&#x7684;&#x6700;&#x9AD8;&#x5B66;&#x4F4D;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x6570;&#x636E;&#x63D0;&#x4F9B;&#x4E86;&#x5B8C;&#x6574;&#x7684;&#x8868;&#x8FBE;&#x5F0F;&#x7684;&#x7B2C;&#x4E00;&#x4E2A;&#x914D;&#x7F6E;&#x6587;&#x4EF6;&#x548C;&#x672C;&#x5730;&#x5316;&#x7684;&#x5708;&#x5957;&#x5C0F;&#x5B66; CD8(+) T &#x7EC6;&#x80DE;&#xFF0C;&#x4E3A;&#x8FDB;&#x4E00;&#x6B65;&#x4E86;&#x89E3;&#x4ED6;&#x4EEC;&#x7684;&#x6F5C;&#x5728;&#x4F5C;&#x7528;&#x7684; T &#x7EC6;&#x80DE;&#x529F;&#x80FD;&#x5960;&#x5B9A;&#x57FA;&#x7840;&#x3002;

&#x7F51;&#x7F57;&#x86CB;&#x767D;&#x8868;&#x8FBE;&#x548C;&#x7EC6;&#x80DE;&#x6BD2; T &#x6DCB;&#x5DF4;&#x7EC6;&#x80DE;&#x7684;&#x672C;&#x5730;&#x5316;&#x3002;

The major function of cytotoxic T lymphocytes (CTLs) is to eliminate pathogen-infected and tumorigenic cells. This is mediated mainly through the exocytosis of lytic granules (LGs) containing cytotoxic components, such as perforin and granzymes at the immunological synapse (IS). The soluble NSF attachment receptor (SNARE) protein isoforms are well known to be required for vesicle exocytosis in neuronal synapses, but their potential function in CTLs is only partly understood. Here, we examined the expression of SNARE proteins before and after the activation of primary human CD8(+) T cells and determined their co-localization with LGs and CD3 after IS formation with target cells. We found that several key SNARE proteins in neuronal cells were not expressed in CTLs, such as syntaxin1B2 and SNAP-25. Vti1b, Stx8 and Stx16 had the highest degrees of co-localization with LGs while Stx3, Stx4, Stx6, Stx7, Stx8, Stx13, Vti1b, VAMP3 and VAMP4 co-localized with CD3. Our data provide the first complete expression profile and localization of SNAREs in primary human CD8(+) T cells, laying the groundwork for further understanding their potential role in T-cell function.

SNARE protein expression and localization in human cytotoxic T lymphocytes.

La fonction principale des lymphocytes T cytotoxiques (CTL) est d&#39;&eacute;liminer les cellules infect&eacute;es par le pathog&egrave;ne et tumorig&egrave;nes. C&#39;est v&eacute;hicul&eacute;e principalement par le biais de l&#39;exocytose des granules lytiques (LGs) comportant des composantes cytotoxiques, perforine et granzymes &agrave; la synapse immunologique (IS). Les solubles NSF fixation du r&eacute;cepteur (SNARE) isoformes prot&eacute;iques sont bien connus pour &ecirc;tre requis pour l&#39;exocytose des v&eacute;sicules dans les synapses neuronales, mais leur fonction potentielle en CTL n&#39;est que partiellement comprise. Ici, nous avons &eacute;tudi&eacute; l&#39;expression des prot&eacute;ines SNARE avant et apr&egrave;s que l&#39;activation du primaire humain CD8 T cells et d&eacute;termin&eacute; leur co-localisation avec LGs et CD3 apr&egrave;s formation IS avec les cellules cibles. Nous avons constat&eacute; que plusieurs prot&eacute;ines SNARE cl&eacute;s dans les cellules neuronales &eacute;taient pas exprim&eacute;es en CTL, tels que syntaxin1B2 et SNAP-25. Vti1b, Stx8 et Stx16 ont les plus hauts degr&eacute;s de co-localisation avec LGs tandis que Stx3, Stx4, Stx6, Stx7, Stx8, Stx13, Vti1b, VAMP3 et VAMP4 co-localisaient avec CD3. Nos donn&eacute;es fournissent le premier profil d&#39;expression complet et la localisation des pi&egrave;ges dans des cellules humaines primaires de T CD8, pr&eacute;parer le terrain pour mieux comprendre leur r&ocirc;le potentiel dans la fonction des cellules T.

Expression des prot&eacute;ines SNARE et la localisation dans les lymphocytes T cytotoxiques humains.

Die Hauptfunktion von zytotoxischen T-Lymphozyten (CTLs) soll Erreger infiziert und tumorigenic Zellen eliminiert. Dies ist vor allem durch die Exozytose lytischer Granulat (LGs) mit zytotoxischen Komponenten wie Perforin und Granzymes bei der immunologischen Synapse (IS) vermittelt. Die l&ouml;slichen NSF Anlage Rezeptor (SNARE) Protein-Isoformen sind bekannt f&uuml;r die Vesikel-Exozytose in neuronalen Synapsen erforderlich sein, aber ihre potenziellen Funktion in CTLs ist nur teilweise verstanden. Hier haben wir den Ausdruck der SNARE-Proteine, vor und nach die Aktivierung der prim&auml;ren menschlichen CD8(+) T Zellen und ihre Doppelhybridisierung mit LGs und CD3 nach IS Bildung mit Zielzellen bestimmt gepr&uuml;ft. Wir fanden, dass mehrere wichtige SNARE-Proteine in neuronalen Zellen nicht in CTLs, wie syntaxin1B2 und SNAP-25 ausgedr&uuml;ckt wurden. Vti1b, Stx8 und Stx16 hatten die h&ouml;chsten Grade der Doppelhybridisierung mit LGs w&auml;hrend Stx3, Stx4, Stx6, Stx7, Stx8, Stx13, Vti1b, VAMP3 und VAMP4 co-localized mit CD3. Unsere Daten liefern die erste vollst&auml;ndige Ausdruck Profil und Lokalisierung von SNAREs in den prim&auml;ren menschlichen CD8(+) T-Zellen, den Grundstein f&uuml;r das weitere Verst&auml;ndnis ihre m&ouml;gliche Rolle in der T-Zell-Funktion.

Schlinge Proteinexpression und Lokalisierung in menschlichen cytotoxische T-Lymphozyten.

La principale funzione dei linfociti T citotossici (CTL) &egrave; di eliminare le cellule infettate da patogeni e tumorigeno. Questo &egrave; mediato principalmente attraverso l&#39;esocitosi dei granuli litici (LGs) contenenti componenti citotossici, quali perforina e granzymes presso la sinapsi immunologica (IS). I solubile NSF attachment recettore (rullante) proteina isoforme sono ben noti per essere richiesto per esocitosi della vescicola nelle sinapsi neuronali, ma la loro funzione potenziale in CTL &egrave; solo parzialmente compreso. Qui, abbiamo esaminato l&#39;espressione delle proteine SNARE prima e dopo l&#39;attivazione del primario umano cellule T cellule e determinato la loro collocazione con LGs e CD3 dopo formazione &egrave; con le cellule bersaglio. Abbiamo trovato che parecchie proteine SNARE chiave nelle cellule neuronali non sono stati espressi in CTL, come syntaxin1B2 e SNAP-25. Vti1b, Stx8 e Stx16 erano i pi&ugrave; alti gradi di collocazione con LGs mentre Stx3, Stx4, Stx6, Stx7, Stx8, Stx13, Vti1b, VAMP3 e VAMP4 co-ha localizzato con CD3. I nostri dati forniscono la primo espressione completa il profilo e la localizzazione delle insidie nelle cellule umane primarie di cellule T, ponendo le basi per comprendere ulteriormente il loro ruolo potenziale nella funzione delle cellule T.

Espressione della proteina SNARE e localizzazione in linfociti T citotossici umani.

&#x7D30;&#x80DE;&#x50B7;&#x5BB3;&#x6027; T &#x30EA;&#x30F3;&#x30D1;&#x7403; (Ctl) &#x306E;&#x4E3B;&#x8981;&#x306A;&#x6A5F;&#x80FD;&#x306F;&#x3001;&#x75C5;&#x539F;&#x4F53;&#x611F;&#x67D3;&#x3068;&#x5E79;&#x7D30;&#x80DE;&#x3092;&#x6392;&#x9664;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x306F;&#x30A8;&#x30AD;&#x30BD;&#x30B5;&#x30A4;&#x30C8;&#x30FC;&#x30B7;&#x30B9; &#x30D1;&#x30FC;&#x30D5;&#x30A9;&#x30EA;&#x30F3;&#x3084;&#x514D;&#x75AB;&#x5B66;&#x306E;&#x30B7;&#x30CA;&#x30D7;&#x30B9; (IS) &#x3067; granzymes &#x306A;&#x3069;&#x306E;&#x7D30;&#x80DE;&#x6BD2;&#x6027;&#x6210;&#x5206;&#x3092;&#x542B;&#x3080;&#x6EB6;&#x83CC;&#x9846;&#x7C92; (LGs) &#x306E;&#x4E3B;&#x306B;&#x3092;&#x901A;&#x3058;&#x3066;&#x4EF2;&#x4ECB;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x53EF;&#x6EB6;&#x6027; NSF &#x6DFB;&#x4ED8;&#x30D5;&#x30A1;&#x30A4;&#x30EB; (&#x30B9;&#x30CD;&#x30A2;&#x30C9;&#x30E9;&#x30E0;) &#x53D7;&#x5BB9;&#x4F53;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x30A2;&#x30A4;&#x30BD; &#x30D5;&#x30A9;&#x30FC;&#x30E0;&#x304C;&#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x306B;&#x304A;&#x3051;&#x308B;&#x5C0F;&#x80DE;&#x306E;&#x30A8;&#x30AD;&#x30BD;&#x30B5;&#x30A4;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x304C;&#x5FC5;&#x8981;&#x306B;&#x306A;&#x308B;&#x3053;&#x3068;&#x306F;&#x3088;&#x304F;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x304C;&#x3001;Ctl &#x306B;&#x304A;&#x3051;&#x308B;&#x305D;&#x306E;&#x6F5C;&#x5728;&#x7684;&#x306A;&#x6A5F;&#x80FD;&#x306F;&#x90E8;&#x5206;&#x7684;&#x306B;&#x3057;&#x304B;&#x7406;&#x89E3;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x524D;&#x306B;&#x3001;&#x3068;&#x5F8C;&#x306E;&#x30D7;&#x30E9;&#x30A4;&#x30DE;&#x30EA;&#x4EBA;&#x9593;&#x3060;&#x308D;&#x3046;&#x3053;&#x306E; T &#x306E;&#x6D3B;&#x6027;&#x5316;&#x7D30;&#x80DE;&#x3057;&#x3001;&#x305D;&#x306E; LGs &#x3068; CD3 &#x5927;&#x8178;&#x83CC;&#x6A19;&#x7684;&#x7D30;&#x80DE;&#x3068; IS &#x5F62;&#x6210;&#x5F8C;&#x6C7A;&#x5B9A; SNARE &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x767A;&#x73FE;&#x3092;&#x8ABF;&#x3079;&#x305F;&#x3002;&#x6211;&#x3005; &#x306F;&#x3001;&#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x306E;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x30AD;&#x30FC; SNARE &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x304C; syntaxin1B2 &#x3068;&#x30B9;&#x30CA;&#x30C3;&#x30D7; 25 &#x306A;&#x3069; Ctl &#x3067;&#x8868;&#x73FE;&#x3055;&#x308C;&#x3066;&#x3044;&#x306A;&#x3044;&#x3053;&#x3068;&#x3092;&#x767A;&#x898B;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;Vti1b&#x3001;Stx8&#x3001;Stx16 Stx3&#x3001;Stx4&#x3001;Stx6&#x3001;Stx7&#x3001;Stx8&#x3001;Stx13&#x3001;Vti1b&#x3001;VAMP3 &#x3068; VAMP4 &#x3068; CD3 co-localized &#x306A;&#x304C;&#x3089; LGs &#x5185;&#x306E;&#x6700;&#x9AD8;&#x5EA6;&#x3092;&#x6301;&#x3063;&#x3066;&#x3044;&#x305F;&#x3002;&#x6211;&#x3005; &#x306E;&#x30C7;&#x30FC;&#x30BF;&#x306F;&#x3001;&#x6700;&#x521D;&#x306E;&#x5B8C;&#x5168;&#x306A;&#x8868;&#x73FE;&#x306E;&#x30D7;&#x30ED;&#x30D5;&#x30A3;&#x30FC;&#x30EB;&#x3068;&#x3055;&#x3089;&#x306B; T &#x7D30;&#x80DE;&#x306E;&#x6A5F;&#x80FD;&#x5185;&#x3067;&#x306E;&#x6F5C;&#x5728;&#x7684;&#x306A;&#x5F79;&#x5272;&#x3092;&#x7406;&#x89E3;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x57FA;&#x790E;&#x3092;&#x6577;&#x8A2D;&#x30B9;&#x30CD;&#x30A2; &#x30D7;&#x30E9;&#x30A4;&#x30DE;&#x30EA;&#x306E;&#x3072;&#x3068;&#x3060;&#x308D;&#x3046;&#x3053;&#x306E; T &#x7D30;&#x80DE;&#x306E;&#x5C40;&#x5728;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;

SNARE &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x767A;&#x73FE;&#x3068;&#x7D30;&#x80DE;&#x50B7;&#x5BB3;&#x6027; T &#x30EA;&#x30F3;&#x30D1;&#x7403;&#x306B;&#x304A;&#x3051;&#x308B;&#x30ED;&#x30FC;&#x30AB;&#x30EA;&#x30BC;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3002;

Cytotoxic T &#xC138;&#xD3EC; (Ctl)&#xC758; &#xC8FC;&#xC694; &#xAE30;&#xB2A5;&#xC740; &#xBCD1;&#xC6D0; &#xCCB4;&#xC5D0; &#xAC10;&#xC5FC; &#xBC0F; tumorigenic &#xC140;&#xC744; &#xC81C;&#xAC70; &#xD558;&#xB294;. &#xC774;&#xAC83;&#xC740; &#xC8FC;&#xB85C; &#xC6A9;&#xADE0;&#xC131;&#xACFC; &#xB9BD; (LGs) perforin &#xB4F1; &#xBA74;&#xC5ED; &#xB0C5; (IS)&#xC5D0; granzymes cytotoxic &#xAD6C;&#xC131; &#xC694;&#xC18C; &#xD3EC;&#xD568; &#xB41C; exocytosis&#xB97C; &#xD1B5;&#xD574; &#xC911;&#xC7AC;. &#xC218;&#xC6A9; &#xC131; NSF &#xCCA8;&#xBD80; &#xD30C;&#xC77C; (&#xC62C; &#xBB34;) &#xC218;&#xC6A9; &#xCCB4; &#xB2E8;&#xBC31;&#xC9C8; isoforms &#xC798; &#xC2E0;&#xACBD; &#xC2DC; &#xB0C5; &#xC2A4;&#xC758; &#xC18C;&#xD3EC; exocytosis&#xC5D0; &#xD544;&#xC694;&#xD55C; &#xAC83;&#xC73C;&#xB85C; &#xC54C;&#xB824;&#xC838; &#xC788;&#xC9C0;&#xB9CC; Ctl&#xC5D0; &#xADF8;&#xB4E4;&#xC758; &#xC7A0;&#xC7AC;&#xC801;&#xC778; &#xAE30;&#xB2A5;&#xC740; &#xBD80;&#xBD84;&#xC801;&#xC73C;&#xB85C; &#xC774;&#xD574;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC; &#xC778;&#xAC04;&#xC758; &#xAE30;&#xBCF8; CD8(+) T &#xD65C;&#xC131;&#xD654; &#xC138;&#xD3EC; &#xBC0F; &#xB300;&#xC0C1; &#xC140;&#xACFC; IS &#xD615;&#xC131; &#xD6C4; LGs &#xBC0F; CD3 &#xADF8;&#xB4E4;&#xC758; co-localization &#xACB0;&#xC815; &#xD6C4; SNARE &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xC2DD;&#xC744; &#xC870;&#xC0AC; &#xD588;&#xB2E4;. &#xC6B0;&#xB9AC; syntaxin1B2, &#xC2A4;&#xB0C5;-25 &#xB4F1; &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC5D0; &#xC5EC;&#xB7EC; &#xAC00;&#xC9C0; &#xC8FC;&#xC694; &#xC62C; &#xBB34; &#xB2E8;&#xBC31;&#xC9C8; Ctl&#xC5D0; &#xD45C;&#xD604; &#xD558;&#xC9C0; &#xD588;&#xB2E4; &#xBC1C;&#xACAC;. Vti1b, Stx8, Stx16 Stx3, Stx4, Stx6, Stx7, Stx8, Stx13, Vti1b, VAMP3 &#xBC0F; VAMP4 co-localized c d 3&#xC640; &#xD568;&#xAED8; &#xD558;&#xB294; &#xB3D9;&#xC548; Lgs&#xC640; co-localization&#xC758; &#xAC00;&#xC7A5; &#xB192;&#xC740; &#xD559;&#xC704;&#xB97C; &#xD588;&#xB2E4;. &#xC6B0;&#xB9AC;&#xC758; &#xB370;&#xC774;&#xD130; &#xCCAB; &#xBC88;&#xC9F8; &#xC644;&#xC804; &#xD55C; &#xC2DD; &#xD504;&#xB85C;&#xD544;&#xACFC; SNAREs &#xC778;&#xAC04;&#xC758; &#xAE30;&#xBCF8; CD8(+) T &#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xB354; T-&#xC138;&#xD3EC; &#xAE30;&#xB2A5;&#xC5D0; &#xADF8;&#xB4E4;&#xC758; &#xC7A0;&#xC7AC;&#xC801;&#xC778; &#xC5ED;&#xD560;&#xC744; &#xC774;&#xD574; &#xD558;&#xAE30; &#xC704;&#xD55C; &#xAE30;&#xCD08;&#xB97C; &#xB204;&#xC6CC;&#xC758; &#xC9C0;&#xC5ED;&#xD654;&#xB97C; &#xC81C;&#xACF5; &#xD569;&#xB2C8;&#xB2E4;.

SNARE &#xB2E8;&#xBC31;&#xC9C8; &#xC2DD; &#xBC0F; &#xC9C0;&#xC5ED;&#xD654; &#xC778;&#xAC04;&#xC758; cytotoxic T &#xC138;&#xD3EC;.

La principal funci&oacute;n de los linfocitos T citot&oacute;xicos (CTL) es eliminar las c&eacute;lulas infectadas por el pat&oacute;geno y tumor&iacute;gena. Esto es mediado principalmente a trav&eacute;s de la exocitosis de gr&aacute;nulos l&iacute;ticos (LGs) que contengan componentes citot&oacute;xicos, como perforina y granzymes en la sinapsis inmunol&oacute;gica (IS). Las isoformas de prote&iacute;na solubles de NSF accesorio del receptor (trampa) son muy conocidas por ser necesario para la exocitosis de la ves&iacute;cula en la sinapsis neuronales, pero s&oacute;lo en parte se entiende su funci&oacute;n potencial en CTLs. Aqu&iacute;, se analiz&oacute; la expresi&oacute;n de prote&iacute;nas SNARE antes y despu&eacute;s de la activaci&oacute;n de primaria T CD8 humanos c&eacute;lulas y determin&oacute; su colocalizaci&oacute;n con LGs y CD3 despu&eacute;s de la formaci&oacute;n es con las c&eacute;lulas diana. Encontramos que varias prote&iacute;nas clave de la trampa en c&eacute;lulas neuronales no fueron expresadas en CTLs, tales como syntaxin1B2 y SNAP-25. Vti1b, Stx8 y Stx16 tuvieron los m&aacute;s altos grados de co-localizaci&oacute;n con LGs mientras Stx3, Stx4, Stx6, Stx7, Stx8, Stx13, Vti1b, VAMP3 y VAMP4 co-localized con CD3. Nuestros datos proporcionan el primer perfil de la expresi&oacute;n completa y la localizaci&oacute;n de trampas en las c&eacute;lulas CD8 T humanas primarias, sentando las bases para la comprensi&oacute;n adicional su papel potencial en funci&oacute;n de las c&eacute;lulas T.

Expresi&oacute;n de la prote&iacute;na de trampa y localizaci&oacute;n en los linfocitos T citot&oacute;xicos humanos.

Calcium (Ca(2+)) influx is required for the activation and function of all cells in the immune system. It is mediated mainly by store-operated Ca(2+) entry (SOCE) through Ca(2+) release-activated Ca(2+) (CRAC) channels located in the plasma membrane. CRAC channels are composed of ORAI proteins that form the channel pore and are activated by stromal interaction molecules (STIM) 1 and 2. Located in the membrane of the endoplasmic reticulum, STIM1 and STIM2 have the dual function of sensing the intraluminal Ca(2+) concentration in the ER and to activate CRAC channels. A decrease in the ER's Ca(2+) concentration induces STIM multimerization and translocation into puncta close to the plasma membrane where they bind to and activate ORAI channels. Since the identification of ORAI and STIM genes as the principal mediators of CRAC channel function, substantial advances have been achieved in understanding the molecular regulation and physiological role of CRAC channels in cells of the immune system and other organs. In this review, we discuss the mechanisms that regulate CRAC channel function and SOCE, the role of recently identified proteins and mechanisms that modulate the activation of ORAI/STIM proteins and the consequences of CRAC channel dysregulation for lymphocyte function and immunity. 

Molecular regulation of CRAC channels and their role in lymphocyte function.

Killing cancer cells by cytotoxic T lymphocytes (CTL) and by natural killer (NK) cells is of vital importance. Cancer cell proliferation and apoptosis depend on the intracellular Ca(2+) concentration, and the expression of numerous ion channels with the ability to control intracellular Ca(2+) concentrations has been correlated with cancer. A rise of intracellular Ca(2+) concentrations is also required for efficient CTL and NK cell function and thus for killing their targets, in this case cancer cells. Here, we review the data on Ca(2+)-dependent killing of cancer cells by CTL and NK cells. In addition, we discuss emerging ideas and present a model how Ca(2+) may be used by CTL and NK cells to optimize their cancer cell killing efficiency. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Calcium, cancer and killing: the role of calcium in killing cancer cells by cytotoxic T lymphocytes and natural killer cells.

A simple, economic, time-resolved killing assay.

Cytotoxic T lymphocytes (CTLs) play an important role in the immune system by recognizing and eliminating pathogen-infected and tumorigenic cells. In order to achieve their function, T cells have to migrate throughout the whole body and identify the respective targets. In conventional immunology studies, interactions between CTLs and targets are usually investigated using tedious and time-consuming immunofluorescence imaging. However, there is currently no straightforward measurement tool available to examine the interaction strengths. In the present study, adhesion strengths and migration of single human CD8(+) T cells on pre-coated field-effect transistor (FET) devices (i.e. fibronectin, anti-CD3 antibody, and anti-LFA-1 antibody) were measured using impedance spectroscopy. Adhesion strengths to different protein and antibody coatings were compared. By fitting the data to an electronically equivalent circuit model, cell-related parameters (cell membrane capacitance referring to cell morphology and seal resistance referring to adhesion strength) were obtained. This electronically-assessed adhesion strength provides a novel, fast, and important index describing the interaction efficiency. Furthermore, the size of our detection transistor gates as well as their sensitivity reaches down to single cell resolution. Real-time motions of individually migrating T cells can be traced using our FET devices. The in-house fabricated FETs used in the present study are providing a novel and very efficient insight to individual cell interactions. 

Human T cells monitored by impedance spectrometry using field-effect transistor arrays: a novel tool for single-cell adhesion and migration studies.

Cytotoxic T lymphocytes (CTL) eliminate pathogen-infected and cancerous cells mainly by polarized secretion of lytic granules (LG, containing cytotoxic molecules like perforin and granzymes) at the immunological synapse (IS). Members of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family are involved in trafficking (generation, transport and fusion) of vesicles at the IS. Syntaxin 8 (Stx8) is expressed in LG and colocalizes with the T cell receptor (TCR) upon IS formation. Here, we report the significance of Stx8 for human CTL cytotoxicity. We found that Stx8 mostly localized in late, recycling endosomal and lysosomal compartments with little expression in early endosomal compartments. Down-regulation of Stx8 by siRNA resulted in reduced cytotoxicity. We found that following perforin release of the pre-existing pool upon target cell contact, Stx8 down-regulated CTL regenerate perforin pools less efficiently and thus release less perforin compared to control CTL. CD107a degranulation, real-time and end-point population cytotoxicity assays, and high resolution microscopy support our conclusion that Stx8 is required for proper and timely sorting and trafficking of cytotoxic molecules to functional LG through the endosomal pathway in human CTL. 

Syntaxin 8 is required for efficient lytic granule trafficking in cytotoxic T lymphocytes.

Ca microdomains and spatially resolved Ca signals are highly relevant for cell function. In T cells, local Ca signaling at the immunological synapse (IS) is required for downstream effector functions. We present experimental evidence that the relocation of the MTOC towards the IS during polarization drags mitochondria along with the microtubule network. From time-lapse fluorescence microscopy we conclude that mitochondria rotate together with the cytoskeleton towards the IS. We hypothesize that this movement of mitochondria towards the IS together with their functionality of absorption and spatial redistribution of Ca is sufficient to significantly increase the cytosolic Ca concentration. To test this hypothesis we developed a whole cell model for Ca homoeostasis involving specific geometries for mitochondria and use the model to calculate the spatial distribution of Ca concentrations within the cell body as a function of the rotation angle and the distance from the IS. We find that an inhomogeneous distribution of PMCA pumps on the cell membrane, in particular an accumulation of PMCA at the IS, increases the global Ca concentration and decreases the local Ca concentration at the IS with decreasing distance of the MTOC from the IS. Unexpectedly, a change of CRAC/Orai activity is not required to explain the observed Ca changes. We conclude that rotation-driven relocation of the MTOC towards the IS together with an accumulation of PMCA pumps at the IS are sufficient to control the observed Ca dynamics in T-cells during polarization.

Cytoskeleton rotation relocates mitochondria to the immunological synapse and increases calcium signals.

We consider random search processes alternating stochastically between diffusion and ballistic motion, in which the distribution function of ballistic motion directions varies from point to point in space. The specific space dependence of the directional distribution together with the switching rates between the two modes of motion establishes a spatially inhomogeneous search strategy. We show that the mean first passage times for several standard search problems-narrow escape, reaction partner finding, reaction escape-can be minimized with a directional distribution that is reminiscent of the spatial organization of the cytoskeleton filaments of cells with a centrosome: radial ballistic transport from the center to the periphery and back, and ballistic transport in random directions within a concentric shell of thickness Δ_{opt} along the domain boundary. The results suggest that living cells realize efficient search strategies for various intracellular transport problems economically through a spatial cytoskeleton organization that involves radial microtubules in the central region and only a narrow actin cortex rather than a cell body filled with randomly oriented actin filaments. 

Optimality of Spatially Inhomogeneous Search Strategies.

DNA methylation changes in peripheral blood DNA have been shown to be associated with solid tumors. We sought to identify methylation alterations in whole blood DNA that are associated with breast cancer (BC). Epigenome-wide DNA methylation profiling on blood DNA from BC cases and healthy controls was performed by applying Infinium HumanMethylation450K BeadChips. Promising CpG sites were selected and validated in three independent larger sample cohorts via MassARRAY EpiTyper assays. CpG sites located in three genes (cg06418238 in RPTOR, cg00736299 in MGRN1 and cg27466532 in RAPSN), which showed significant hypomethylation in BC patients compared to healthy controls in the discovery cohort (p < 1.00 x 10-6) were selected and successfully validated in three independent cohorts (validation I, n =211; validation II, n=378; validation III, n=520). The observed methylation differences are likely not cell-type specific, as the differences were only seen in whole blood, but not in specific sub cell-types of leucocytes. Moreover, we observed in quartile analysis that women in the lower methylation quartiles of these three loci had higher ORs than women in the higher quartiles. The combined AUC of three loci was 0.79 (95%CI 0.73-0.85) in validation cohort I, and was 0.60 (95%CI 0.54-0.66) and 0.62 (95%CI 0.57-0.67) in validation cohort II and III, respectively. Our study suggests that hypomethylation of CpG sites in RPTOR, MGRN1 and RAPSN in blood is associated with BC and might serve as blood-based marker supplements for BC if these could be verified in prospective studies.

DNA methylation array analysis identifies breast cancer associated RPTOR, MGRN1 and RAPSN hypomethylation in peripheral blood DNA.

Natural killer (NK) cells play a central role during innate immune responses by eliminating pathogen-infected or tumorigenic cells. In the microenvironment, NK cells encounter not only target cells but also other cell types including non-target bystander cells. The impact of bystander cells on NK killing efficiency is, however, still elusive. In this study we show that the presence of bystander cells, such as P815, monocytes or HUVEC, enhances NK killing efficiency. With bystander cells present, the velocity and persistence of NK cells were increased, whereas the degranulation of lytic granules remained unchanged. Bystander cell-derived HO was found to mediate the acceleration of NK cell migration. Using mathematical diffusion models, we confirm that local acceleration of NK cells in the vicinity of bystander cells reduces their search time to locate target cells. In addition, we found that integrin β chains (β1, β2 and β7) on NK cells are required for bystander-enhanced NK migration persistence. In conclusion, we show that acceleration of NK cell migration in the vicinity of HO-producing bystander cells reduces target cell search time and enhances NK killing efficiency.

Bystander cells enhance NK cytotoxic efficiency by reducing search time.

Breast cancer (BC) is the leading cancer in women worldwide. Changes in DNA methylation in peripheral blood could be associated with malignant diseases. Making use of screening results by llumina 27K Methylation Assay, we validated demethylation of five CpG sites of S100P gene in blood cell DNA of BC patients by three independent retrospective studies with subjects from different centers (Validation I: 235 familial BC case and 206 controls, odds ratio per -1% methylation > 1.03, and P < 6.00 × 10-8 for all five CpG sites; Validation II: 189 sporadic BC case and 189 controls, odds ratio per -1% methylation > 1.03, P < 8.0 × 10-5 for four CpG sites; Validation III: 156 sporadic BC case and 151 controls, odds ratio per -1% methylation > 1.03, P < 6.0 × 10-4 for four CpG sites). In addition, the blood-based S100P methylation pattern was similar among BC patients with differential clinical characteristics regardless of stage, receptor status and menopause status. The observed BC-associated decreased S100P methylation in blood mainly originates from the leucocytes subpopulations but not B cells. The methylation levels of most S100P CpG sites were inversely correlated with the expression of S100P in leucocytes (P < 1.2 × 10-4) and in tissue (P < 1.1 × 10-4). This study reveals significant association between blood-based decreased S100P methylation and BC, and provides another proof for the application of altered DNA methylation signatures from blood cells as potential markers for the detection of BC, especially for the early stage.

The association between breast cancer and S100P methylation in peripheral blood by multicenter case-control studies.

The actin-binding protein profilin1 (PFN1) plays a central role in actin dynamics, which is essential for cytotoxic T lymphocyte (CTL) functions. The functional role of PFN1 in CTLs, however still remains elusive. Here, we identify PFN1 as the only member of the profilin family expressed in primary human CD8 T cells. Using in vitro assays, we find that PFN1 is a negative regulator of CTL-mediated elimination of target cells. Furthermore, PFN1 is involved in activation-induced lytic granule (LG) release, CTL migration and modulation of actin structures at the immunological synapse (IS). During CTL migration, PFN1 modulates the velocity, protrusion formation patterns and protrusion sustainability. In contrast, PFN1 does not significantly affect migration persistence and the rates of protrusion emergence and retraction. Under in vitro conditions mimicking a tumor microenvironment, we show that PFN1 downregulation promotes CTL invasion into a 3D matrix, without affecting the viability of CTLs in a hydrogen peroxide-enriched microenvironment. Highlighting its potential relevance in cancer, we find that in pancreatic cancer patients, PFN1 expression is substantially decreased in peripheral CD8 T cells. Taken together, we conclude that PFN1 is a negative regulator for CTL-mediated cytotoxicity and may have an impact on CTL functionality in a tumor-related context.

Human profilin 1 is a negative regulator of CTL mediated cell-killing and migration.

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are required to eliminate cancer cells. We analysed the Ca dependence of CTL and NK cell cytotoxicity and found that in particular CTLs have a very low optimum of [Ca ] (between 122 and 334 nm) and [Ca ] (between 23 and 625 μm) for efficient cancer cell elimination, well below blood plasma Ca levels. As predicted from these results, partial down-regulation of the Ca channel Orai1 in CTLs paradoxically increases perforin-dependent cancer cell killing. Lytic granule release at the immune synapse between CTLs and cancer cells has a Ca optimum compatible with this low Ca optimum for efficient cancer cell killing, whereas the Ca optimum for CTL migration is slightly higher and proliferation increases monotonously with increasing [Ca ] . We propose that a partial inhibition of Ca signals by specific Orai1 blockers at submaximal concentrations could contribute to tumour elimination.

Bin Qu

Department of Biophysics,
Center for Integrative Physiology and Molecular Medicine (CIPMM),
School of Medicine,
Department of Biophysics, Center for Integrative Physiology and Molecular Medicine (CIPMM), School of Medicine

Saarland University

Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

Bin Qu

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biophysics,Center for Integrative Physiology and Molecular Medicine (CIPMM),School of Medicine,Department of Biophysics, Center for Integrative Physiology and Molecular Medicine (CIPMM), School of Medicine

Saarland University

Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

Department of Biophysics,
Center for Integrative Physiology and Molecular Medicine (CIPMM),
School of Medicine,
Department of Biophysics, Center for Integrative Physiology and Molecular Medicine (CIPMM), School of Medicine