Cxcr5

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CXCR5 is a receptor protein that is expressed on the surface of certain immune cells, such as B cells and follicular helper T cells. It plays a key role in the migration and positioning of these cells within lymphoid tissues. CXCR5 responds to the chemokine CXCL13, which helps guide these cells to areas where they can interact and participate in immune responses.

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15 protocols using cxcr5

Multiparametric Immunofluorescence Profiling

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Immunofluorescence staining was performed using the following antibodies at the stated concentrations: BioLegend: CD3-BV421 (SK7; 1:25), CD4-AF594 (RPA-T4; 1:50), CD19–AF647 (SJ25C1; 1:25), CXCR4 (12G5; 1:100), CD21-FITC (Bu32; 1:100), PD-1-AF488 (NAT105; 1:100), BCL-6 (IG191E/A8; 1:50); Sigma-Aldrich: CXCR5 (Polyclonal; 1:100), CD83 (Polyclonal; 1:100); Thermo Fisher: AID (ZA001; 1:100), CD8-AF488 (AMC908; 1:100), CD20-eFluor 615 (L26; 1:100), CD20-eFluor 660 (L26; 1:100); BD Biosciences: Ki67-BV421 (B56; 1:5); Fisher Science: IgD-AF488 (IgD26; 1:10).

Wagar L.E., Salahudeen A., Constantz C.M., Wendel B.S., Lyons M.M., Mallajosyula V., Jatt L.P., Adamska J.Z., Blum L.K., Gupta N., Jackson K.J., Yang F., Röltgen K., Roskin K.M., Blaine K.M., Meister K.D., Ahmad I.N., Cortese M., Dora E.G., Tucker S.N., Sperling A.I., Jain A., Davies D.H., Felgner P.L., Hammer G.B., Kim P.S., Robinson W.H., Boyd S.D., Kuo C.J, & Davis M.M. (2021). Modeling human adaptive immune responses with tonsil organoids. Nature medicine, 27(1), 125-135.

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Comprehensive Immune Cell Profiling

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PBMCs were submitted to flow cytometry immunofluorescence assay using Attune Nxt Flow Cytometer (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommendation. After washing with PBS, Fc receptors were initially blocked using Human FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) for 30 min at 4 °C, followed by cell surface labeling by specific primary antibodies. The following antibodies were adapted into multiple panels: CD4, CD8, CD14, CD11c, CD16, CD19, CD25, CD62L, HLA-DR, CD56, CD45RA, CD45RO, CCR3, CCR5, CCR6, CCR10, CXCR3, and CXCR5 (Thermo Fisher Scientific, Waltham, MA, USA). Results were illustrated as the percentage of positive cells or as the ratio of the mean of fluorescence intensity (MFI) from the antibody of interest to the isotype control antibody.

Putri D.U., Wang C.H., Tseng P.C., Lee W.S., Chen F.L., Kuo H.P., Lee C.H, & Lin C.F. (2021). Profiles of Peripheral Immune Cells of Uncomplicated COVID-19 Cases with Distinct Viral RNA Shedding Periods. Viruses, 13(3), 514.

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Multiplex Fluorescent Immunohistochemistry for Immune Markers

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Multiplex fluorescent immunohistochemistry experiment was performed using the Opal™ 4-Color Manual IHC kit (PerkinElmer) according to the manufacturer’s instructions. Briefly, tonsil tissue samples fixed with 4% formalin for a minimum of 48 hours were paraffin-embedded. Exactly 4μm sections were cut, deparaffinized and stained with the following unlabelled primary antibodies: CD8 (clone: C8/144B, Dako), CD4 (clone: 4B12, Dako), p24 (clone: Kal-1, Dako), CXCR5 (clone: MU5UBEE, Thermofisher Scientific), GZMB (clone: 23 H8L20, Thermofisher Scientific), CD103 (clone: NBP1-88142, Novus Biologicals) and CD69 (clone: 15B5G2, Novus Biologicals). Opal fluorophores: FITC (Opal520) was used for p24 and CD103; Texas-Red (Opal570) was used for CD8 and CD4; then Cy5 (Opal690) was used for CD8, CXCR5, Granzyme-B and CD69 signal generation in the different IHC experiments performed. DAPI was used as the nuclear counterstain. Images were acquired on a Zeiss Axio Observer Z1 inverted microscope (Olympus) and analysed with TissueFAXS imaging and TissueQuest quantitation software (TissueGnostics).

Fardoos R., Nyquist S.K., Asowata O.E., Kazer S.W., Singh A., Ngoepe A., Giandhari J., Mthabela N., Ramjit D., Singh S., Karim F., Buus S., Anderson F., Porterfield J.Z., Sibiya A.L., Bipath R., Moodley K., Kuhn W., Berger B., Nguyen S., de Oliveira T., Ndung’u T., Goulder P., Shalek A.K., Leslie A, & Kløverpris H.N. (2022). HIV specific CD8+ TRM-like cells in tonsils express exhaustive signatures in the absence of natural HIV control. Frontiers in Immunology, 13, 912038.

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Quantifying Circulating Tfh Cells

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Thawed, cryopreserved PBMCs were assessed to determine the frequency of circulating T follicular helper (Tfh) using a panel which included the following antibodies: CD3 (BD Biosciences; clone SP34-2), CD4 (BD Biosciences; clone L200), CXCR5 (eBioscience; clone MU5UBEE), and PD-1 (BioLegend; clone EH12.2H7). Tfh cells were identified as CD3+/CD4+/CXCR5+/PD-1 +. Samples were acquired on a BD FACS Celesta flow cytometer and analysed using FlowJo v10.7 Software version (BD Life Sciences).

Walters J.N., Schouest B., Patel A., Reuschel E.L., Schultheis K., Parzych E., Maricic I., Gary E.N., Purwar M., Andrade V.M., Doan A., Elwood D., Eblimit Z., Nguyen B., Frase D., Zaidi F.I., Kulkarni A., Generotti A., Joseph Kim J., Humeau L.M., Ramos S.J., Smith T.R., Weiner D.B, & Broderick K.E. (2022). Prime-boost vaccination regimens with INO-4800 and INO-4802 augment and broaden immune responses against SARS-CoV-2 in nonhuman primates. Vaccine, 40(21), 2960-2969.

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Murine Immune Cell Profiling

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Single-cell suspensions of bone marrow, spleen, or peritoneal fluid were prepared and red blood cells were lysed. All analyzed mice were 6–12 weeks of age, unless indicated differently. Cells were incubated with various combinations of the following antibodies: CD3, CD4, CD5, CD8, CD19, CD21, CD23, CD24, CD62L, B220, AA4.1, CXCR5, FAS, GL7, Gr-1, IgD, IgM, PD1, and TCR-β (eBioscience and Tonbo). Samples were collected using FACS LSRII (BD) or sorted using FACSAria (BD), and data were analyzed with FlowJo software (Tree Star). Differences between groups were analyzed using unpaired t-test analysis (GraphPad Prism software).

Phillips N., Ke E., Nham A., Seidl M., Freeman B., Abadejos J.R., Xiao C., Nemazee D., Ku M, & Kirak O. (2020). Prediabetes Induced by a Single Autoimmune B Cell Clone. Frontiers in Immunology, 11, 1073.

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Multiparameter Flow Cytometry

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Fluorophore-conjugated ICOS, PD1, CXCR5, CD4, CD8, TCRb, CD44, CD62L, CD25, CD69, CD5, CD11b, CD11c, CD19, B220, Ter119, DX5, Gr1, IFNγ, IL-4, IL-17A, FoxP3, Gata3, and Bcl6 were purchased from eBioscience, BD Biosciences, BioLegend, and Tonbo Biosciences. Primary antibodies for phospho-flow cytometry were purchased from Cell Signaling Technologies: P-S6 S235/236 (2211), P-S6 S240/244 (2215), P-ERK (4377), and P-Akt (4058). Secondary antibody for phospho-flow cytometry was R-Phycoerythrin AffiniPure F(ab’)₂ Fragment Donkey Anti-Rabbit IgG (Jackson Immunoresearch).

Myers D.R., Norlin E., Vercoulen Y, & Roose J.P. (2019). Active Tonic mTORC1 Signals Shape Baseline Translation in Naive T Cells. Cell reports, 27(6), 1858-1874.e6.

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Comprehensive Flow Cytometry Staining Panel

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The following flow cytometry detection reagents were sourced from the indicated manufacturers, Biolegend (CA, USA): anti-mouse MHC-II (I-A/I-E, clone M5.114.15.2:PE-CF594, or PerCP-Cy5.5), CD11c (clone N418:PE-Cy7), CD49a (clone HMa1:APC) CD86 (clone GL1:PerCP-Cy5.5), podoplanin (PDPN, clone 8.1.1:APC), CD11b (clone M1/70:APC-Cy7 or BV421), CD24 (clone M1/69:PE or BV421, clone SN3:PerCP eFluor710), CXCR6 (CD186, clone SA051D1:PE), CCR4 (CD194, clone 2G12:PE), CD103 (clone 2E7:PE or BV510), B220 (clone RA3-6B2:BV711), CD64 (clone X54-5/7.1:APC), CXCR3 (CD183, clone CXCR3-173:PE), CD62L (clone Mel-14:PerCP-Cy5.5), CD44 (clone IM7:APC-Cy7), CXCR4 (CD194, clone L276F12:BV421), CD45.1 (clone A20:BV650, FITC, or Pacific Blue), CD45.2 (clone 104:APC-Cy7 or PE-Cy7). Becton Dickenson (BD, NJ, USA) and BD Pharmingen (SJ, USA): CD19 (clone 1D3:BUV737), CD4 (clone RM4-5:BV786), B220 (clone RA3-6B2:PECF594), CD69 (clone H1.2F3:PE-CF594), CD49a (clone HA31/8:BV510). eBioscience (SC, USA): CD45 Ab (clone 30-F11: PECy7), CXCR5 (clone SPRCL5:PeCy7), α4β7 (LPAM1, clone DATK32:APC).

Pejoski D., Ballester M., Auderset F., Vono M., Christensen D., Andersen P., Lambert P.H, & Siegrist C.A. (2019). Site-Specific DC Surface Signatures Influence CD4+ T Cell Co-stimulation and Lung-Homing. Frontiers in Immunology, 10, 1650.

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CD4+ CXCR5+ T Cell-Mediated B Cell Activation

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Mice CD4⁺T cells were isolated from the splenocytes by negative selection according to the manufacturer’s instructions (Miltenyi). For CD4⁺CXCR5⁺T cell sorting, negative selected CD4⁺T cells were stained with anti-CXCR5 antibody conjugated magnetic beads (Miltenyi), and then they were positive selected. B220⁺B cells were isolated from the splenocytes of DBA1/J mice by positive selection (Miltenyi).
B cells (3 × 10⁵ cells/well) were cocultured with CD4⁺CXCR5⁺T cells (1 × 10⁵ cells/ well) in the presence of 2 μg/ml anti-CD3e, 1 μgCD28 (eBioscience), 2.5 μg/ml CpG 2395 (Invitrogen), 50 ng/ml IL-4 (Peprotech), 5 μg/ml anti-IgM (Jackson ImmunoResearch Lab, West Grove, PA, USA) and 5 μg/ml anti-CD40 (eBioscience) in RPMI 1640 with 10% FBS for 5 days.

Liu R., Li X., Zhang Z., Zhou M., Sun Y., Su D., Feng X., Gao X., Shi S., Chen W, & Sun L. (2015). Allogeneic mesenchymal stem cells inhibited T follicular helper cell generation in rheumatoid arthritis. Scientific Reports, 5, 12777.

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Multiparameter Flow Cytometry

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Myers D.R., Norlin E., Vercoulen Y, & Roose J.P. (2019). Active Tonic mTORC1 Signals Shape Baseline Translation in Naive T Cells. Cell reports, 27(6), 1858-1874.e6.

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Multiparametric Flow Cytometry Analysis

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Single cell suspensions were immunostained with various combinations of fluorescent dye-conjugated antibodies against the following proteins: CD19, CD4, CXCR5, PD-1, CD138, and B220 (eBioscience, San Diego, CA, USA). For intracellular cytokine staining, single cell suspensions were simulated with 50 ng/ml of phorbol myristate acetate (Sigma-Aldrich), 1 µg/ml of ionomycin (Enzo, Farmingdale, NY, USA) and 2 µg/ml of monensin (Enzo) for 5 h. Then, the anti-IL-10 mAb (eBioscience) used to for intracellular staining of IL-10 according to the manufacturer’s instructions, and the stained cells were analyzed by flow cytometry using a FACS Calibur instrument (BD Biosciences, San Jose, CA, USA).

Wu X., Zhu D., Tian J., Tang X., Guo H., Ma J., Xu H, & Wang S. (2020). Granulocytic Myeloid-Derived Suppressor Cell Exosomal Prostaglandin E2 Ameliorates Collagen-Induced Arthritis by Enhancing IL-10+ B Cells. Frontiers in Immunology, 11, 588500.

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