Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer‐reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state‐of‐the‐art handbook for basic and clinical researchers.DFG, 389687267, Kompartimentalisierung, Aufrechterhaltung und Reaktivierung humaner Gedächtnis-T-Lymphozyten aus Knochenmark und peripherem BlutDFG, 80750187, SFB 841: Leberentzündungen: Infektion, Immunregulation und KonsequenzenEC/H2020/800924/EU/International Cancer Research Fellowships - 2/iCARE-2DFG, 252623821, Die Rolle von follikulären T-Helferzellen in T-Helferzell-Differenzierung, Funktion und PlastizitätDFG, 390873048, EXC 2151: ImmunoSensation2 - the immune sensory syste

Fraktalkinrezeptor (CX3CR1) positive Makrophagen fördern eine IL-22 Produktion während einer C. rodentium Infektion

Citrobacter rodentium ist ein gram-negatives Bakterium, welches ein Mauspathogen darstellt. C. rodentium verursacht eine mukosale Inflammation. Dabei hat das Bakterium besonders engen Kontakt mit Fraktalkinrezeptor/CX3CR1 Makrophagen (MP). Die CX3CR1 MP haben über Interleukine (IL) einen Einfluss auf eine kleine Lymphozyten-Population des angeborenen Immunsystems. Dabei handelt es sich "Innate lymphoid cells" (ILC), welche eine entscheidende Rolle bei der Klärung der Citrobacter Infektion spielen. Die ILC schütten als Reaktion auf die Infektion IL-22 aus. Das IL-22 wirkt auf die Epithelzellen, die mit der verstärkten Expression antimikrobieller Peptide beginnen. Der Infektionsverlauf mit C. rodentium wurde in der C57/Bl6, in der CX3CR1-GFP, in der CD11c.DOG und der CX3CR1-GFP x RAG-/- Maus untersucht. Die Lymphozytenpopulationen wurden dabei mittels Durchflusszytometrie ermittelt. Der Infektionsgrad mit C. rodentium wurde mit Nalidixinplatten aus Stuhl und Organen ermittelt. Wir konnten anhand einer Infektionskurve aus Stuhlproben feststellen, dass CX3CR1-GFP Mäuse längere Zeit benötigen, um C. rodentium nach einer Infektion erfolgreich zu beseitigen. Des Weiteren war der Anteil an C. rodentium, der in verschiedenen Organen wie Leber, Milz und den mesenterischen Lymphknoten gefunden wurde, in CX3CR1-GFP Tieren deutlich erhöht. Nach der Untersuchung unterschiedlicher Zytokine konnte das IL-22 ermittelt werden, dass in CX3CR1-GFP Mäusen im Vergleich zu C57/Bl6 signifikant reduziert ist. Die weitere Untersuchung der CX3CR1 Knockout Mäuse hat ergeben, dass durch die verminderte Ausschüttung von IL-22 auch der Anteil an antimikrobiellen Peptiden gesenkt ist. Diese Ergebnisse bringen uns zu dem Schluss, dass Fraktalkin über die ILC die Expression von IL-22 kontrolliert. Das IL-22 wiederum wirkt auf die Ephitelzellen des Kolons, welche antimikrobielle Peptide produzieren und somit zur Beseitigung des Bakteriums beitragen

The Early Activation Marker CD69 Regulates the Expression of Chemokines and CD4 T Cell Accumulation in Intestine

Migration of naïve and activated lymphocytes is regulated by the expression of various molecules such as chemokine receptors and ligands. CD69, the early activation marker of C-type lectin domain family, is also shown to regulate the lymphocyte migration by affecting their egress from the thymus and secondary lymphoid organs. Here, we aimed to investigate the role of CD69 in accumulation of CD4 T cells in intestine using murine models of inflammatory bowel disease. We found that genetic deletion of CD69 in mice increases the expression of the chemokines CCL-1, CXCL-10 and CCL-19 in CD4(+) T cells and/or CD4(-) cells. Efficient in vitro migration of CD69-deficient CD4 T cells toward the chemokine stimuli was the result of increased expression and/or affinity of chemokine receptors. In vivo CD69(-/-) CD4 T cells accumulate in the intestine in higher numbers than B6 CD4 T cells as observed in competitive homing assay, dextran sodium sulphate (DSS)-induced colitis and antigen-specific transfer colitis. In DSS colitis CD69(-/-) CD4 T cell accumulation in colonic lamina propria (cLP) was associated with increased expression of CCL-1, CXCL-10 and CCL-19 genes. Furthermore, treatment of DSS-administrated CD69(-/-) mice with the mixture of CCL-1, CXCL-10 and CCL-19 neutralizing Abs significantly decreased the histopathological signs of colitis. Transfer of OT-II×CD69(-/-) CD45RB(high) CD4 T cells into RAG(-/-) hosts induced CD4 T cell accumulation in cLP. This study showed CD69 as negative regulator of inflammatory responses in intestine as it decreases the expression of chemotactic receptors and ligands and reduces the accumulation of CD4 T cells in cLP during colitis

Absence of CD69 leads to the increased expression of <i>CCL-1</i>, <i>CXCL-10</i> and <i>CCL-19</i>.

<p>RNA was isolated from the frozen mesenteric lymph nodes (MLN), small intestinal (si) and colonic (co) tissue samples or from sorted CD4⁺ and CD4⁻ spleen cells of non-treated B6 or CD69^−/− animals and reverse transcribed to complementary DNA. Relative expression of <i>CCL-1</i> (<b>A</b> and <b>D</b>), <i>CXCL-10</i> (<b>B</b> and <b>E</b>) and <i>CCL-19</i> (<b>C</b> and <b>F</b>) genes compared to <i>β-actin</i> gene is analyzed by qRT-PCR. Mean (± SEM) for at least six mice per each strain is presented. *p≤0.05; **p≤0.005; ***p≤0.0005; N.S. – not significant.</p

Increased migratory potential of CD69-deficient CD4 T cells to the mucosal intestinal tissues <i>in vivo</i>.

<p>CD4 T cells were enriched from the spleen of red fluorescent DsRed or CD69^−/− animals, both on the CD45.2⁺ B6 background. CD69-deficient CD4 T cells were labelled with green fluorescent CFSE. These cells were mixed in the approximate ratio 1∶1 and injected i.v. into CD45.1⁺ B6 mice. 18 or 72 h later cells were isolated from blood, mesenteric lymph nodes (MLN), small intestinal lamina propria (siLP) and colonic lamina propria (cLP) of the hosts and analyzed for the surface expression of CD4, CD45.2, DsRed and CFSE by multicolour flow cytometry (FCM). Numbers of DsRed and CFSE expressing CD45.2⁺ CD4⁺ cells recovered 18 h (<b>A</b>) or 72 h (<b>D</b>) after the transfer from the host tissues were determined with FCM and presented as mean (± SEM) total number of recovered cells per tissue for four mice analyzed. N.S. – not statistically significant; *p≤0.05. Homing index (HI) for every tissue 18 h (<b>B</b>) or 72 h (<b>E</b>) post-injection is calculated as: HI = number of CD4⁺CD45.2⁺CFSE⁺ cells/number of CD4⁺CD45.2⁺DsRed⁺ cells : IR (where IR is input ratio calculated before the injection as: IR = number of CD4⁺CD45.2⁺CFSE⁺ cells/number of CD4⁺CD45.2⁺DsRed⁺ cells). This value was normalized to the HI in the blood, so the potential retention of the injected cells in some of the periphery organs is eliminated. Mean (± SEM) of blood-normalized HI per tissue for four mice is presented. The deviation from the theoretical mean (TM = 1) was assessed (*p≤0.05). <b>C.</b> 18 h after the cell transfer sections of small intestine and colon of the host were stained with Alexa Fluor 350-conjugated wheat germ agglutinin (blue) analyzed for the number of green (CFSE⁺ CD69^−/−) and red (DsRed⁺ B6) CD4 T cells by confocal microscope (original magnification×40).</p

Increased susceptibility of CD69^−/− mice to DSS induced colitis is partially due to increased chemokine-dependent CD4 T cell migration to cLP.

<p>B6 or CD69^−/− mice are administrated 2% dextrane sodium sulphate (DSS) in the drinking water for 5 days and then provided with normal sterile water. One group of DSS-administrated CD69^−/− mice were treated with the mixture of CCL-1, CXCL-10 and CCL-19 blocking Abs by i.p. injection at days 0, 2, 4 and 6 (CD69^−/−+Ch-block group). The control group represents non-treated B6 animals. <b>A.</b> Mean (± SEM) of body weight loss (%) of at least 5 mice per group is shown. <b>B.</b> Colitis scores of the histological colon sections of DSS treated B6 and CD69^−/− mice, control and CD69^−/−+Ch-block groups. Mean (± SEM) for at least five mice per group are presented. <b>C.</b> Histopathological colon tissue samples taken from control B6 mice, DSS treated B6 or CD69^−/− animals and CD69^−/−+Ch-block group were embedded in paraffin, sectioned on a microtome, mounted on slides, and stained with H&E. Representative images of one individual mouse per group (from at least five mice per group analyzed) are shown (original magnification×20). RNA was isolated from the frozen colon tissue samples of control B6 animals and DSS treated B6 or CD69^−/− mice and reverse transcribed to complementary DNA. Relative expression of <i>CCL-1</i> (<b>D</b>), <i>CXCL-10</i> (<b>E</b>) and <i>CCL-19</i> (<b>F</b>) genes compared to <i>β-actin</i> gene is measured by qRT-PCR. Mean (± SEM) for at least five mice per group are presented. N.S. – not statistically significant; *p≤0.05; **p≤0.005.</p

Severe colitis in DSS treated CD69^−/− mice is associated with increased pro-inflammatory response and early influx of CD4 T cells devoid of Foxp3 Treg cell into the colon.

<p><b>A.</b> IL-17 concentration was determined by ELISA in the sera of control B6 mice and DSS treated B6 and CD69^−/− mice 7 days after the DSS administration. Mean (± SEM) of at least five mice per group is presented. <b>B.</b> RNA was isolated from frozen colon tissue samples of B6 and CD69^−/− mice treated with DSS, seven days after the DSS administration, or the control B6 mice and reverse transcribed to cDNA. Relative expression of <i>IFN-γ</i> gene was measured by qRT-PCR and presented as mean ± SEM of at least five mice per group. <b>C.</b> Mean (± SEM) total number of CD4⁺ T cells in blood, mesenteric lymph nodes (MLN) and colonic lamina propria (cLP) of 3 days DSS treated B6 and CD69^−/− mice are shown. At least five mice per each strain were analysed. <b>D.</b> The expression of Foxp3 by CD4 T cells from blood, MLN and cLP of B6 and CD69^−/− mice treated with DSS for 3 days was analysed by flow cytometry. Data from an individual, representative mouse (out of at least 5 mice per group analysed) are shown. Numbers indicate the percentage of CD4 T cells that express the transcriptional factor Foxp3. <b>E.</b> Mean (± SEM) total number of CD4⁺ Foxp3⁺ T cells in blood, MLN and cLP of 3 days DSS treated B6 and CD69^−/− mice are shown. At least five mice per each strain were analyzed. N.S. – not statistically significant; *p≤0.05; **p≤0.005.</p

Tissue-specific differentiation of a circulating CCR9- pDC-like common dendritic cell precursor

The ontogenic relationship between the common dendritic cell (DC) progenitor (CDP), the committed conventional DC precursor (pre-cDC), and cDC subpopulations in lymphoid and nonlymphoid tissues has been largely unraveled. In contrast, the sequential steps of plasmacytoid DC (pDC) development are less defined, and it is unknown at which developmental stage and location final commitment to the pDC lineage occurs. Here we show that CCR9(-) pDCs from murine BM which enter the circulation and peripheral tissues have a common DC precursor function in vivo in the steady state, in contrast to CCR9(+) pDCs which are terminally differentiated. On adoptive transfer, the fate of CCR9(-) pDC-like precursors is governed by the tissues they enter. In the BM and liver, most transferred CCR9(-) pDC-like precursors differentiate into CCR9(+) pDCs, whereas in peripheral lymphoid organs, lung, and intestine, they additionally give rise to cDCs. CCR9(-) pDC-like precursors which are distinct from pre-cDCs can be generated from the CDP. Thus, CCR9(-) pDC-like cells are novel CDP-derived circulating DC precursors with pDC and cDC potential. Their final differentiation into functionally distinct pDCs and cDCs depends on tissue-specific factors allowing adaptation to local requirements under homeostatic conditions

Comparison of NGS and MFC Methods: Key Metrics in Multiple Myeloma MRD Assessment

In order to meet the challenges in data evaluation and comparability between studies in multiple myeloma (MM) minimal residual disease (MRD) assessment, the goal of the current study was to provide a step-by-step evaluation of next-generation sequencing (NGS) and multicolor flow cytometry (MFC) data. Bone marrow (BM) sample pairs from 125 MM patients were analyzed by NGS and MFC MM MRD methods. Tumor load (TL) and limit of detection (LOD) and quantification (LOQ) were calculated. The best-fit MRD cut-off was chosen as 1 × 10−5, resulting in an overall 9.6% (n overall = 12 (NGS n = 2, MFC n = 10)) nonassessable cases. The overall concordance rate between NGS and MFC was 68.0% (n = 85); discordant results were found in 22.4% (11.2% (n = 14) of cases in each direction. Overall, 55.1% (n = 60/109) and 49.5% (n = 54/109) of patients with a serological response ≥ very good partial response (VGPR) showed BM MRD negativity by NGS and MFC, respectively. A good correlation in the TL assessed by both techniques was found (correlation coefficient = 0.8, n = 40, p &lt; 0.001). Overall, our study shows good concordance between MM BM MRD status and TL when comparing NGS and MFC at a threshold of 10–5. However, a sufficient number of analyzed events and calculation of MRD key metrics are essential for the comparison of methods and evaluability of data at a specific MRD cut-off