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Aug 2021

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Protocol to Induce Follicular T Helper Cells, Germinal Centers, and Skin Lesions in Mouse Models for Skin Blistering Diseases
在皮肤起泡疾病小鼠模型中诱导滤泡 T 辅助细胞、生发中心和皮肤损伤的方法   

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Abstract

Autoreactive T cells in autoantibody-mediated autoimmune diseases can be divided into two major subsets: (i) follicular T helper cells (Tfh) that provide T cell help in germinal centers (GC) and (ii) effector T (Teff) cells that immigrate into peripheral tissue sites such as the skin and mediate local inflammation. To study the sequence of events leading to the loss of tolerance in autoantibody-mediated autoimmune diseases it is required to investigate both T cell subsets simultaneously. This approach is hampered mainly because the appearance of skin inflammation in mouse models is a random process, which makes it difficult to define the location of inflammation at the right time point. To overcome this problem, we developed a scratching technique for ear skins that leads to the establishment of chronic autoimmune wounds in the mouse model for the pemphigoid-like disease epidermolysis bullosa acquisita. By defining the exact place where the skin wounds should form, this protocol enables a detailed analysis of skin-immigrating Teff cells. Of note, this protocol induces GC in draining lymph nodes in parallel so that Tfh cells in GC can be investigated concurrently. This protocol is not restricted to T cells and can be used for any other skin-immigrating inflammatory cells.

Keywords: Germinal centers (生发中心), Follicular T helper cells (滤泡T辅助细胞), Skin immigrating T effector cells (皮肤迁移 T 效应细胞), Skin wounds (皮肤伤口), Mouse model (小鼠模型)

Background

Chronic tissue inflammation in autoantibody-mediated immune diseases is induced by the binding of IgG autoantibodies to their target structure (Ludwig et al., 2017). One main effect of bound autoantibodies is the formation of immune complexes that lead to uncontrolled recruitment of inflammatory cell infiltrates and subsequent tissue destruction. Typical examples are autoimmune skin diseases such as bullous pemphigoid or epidermolysis bullosa acquisita, in which autoantibodies specific for structural hemidesmosomal proteins of the skin such as type XVII or type VII collagen have been described, respectively (Tull and Benton, 2021). During the formation of these high-affinity autoreactive antibodies, autoantigen-specific Tfh cells, and B cells, closely interact with each other in GC within secondary lymphoid tissues (Vinuesa et al., 2009; Crotty, 2019). Furthermore, autoantigen-specific Teff cells get activated in T cell zones of the draining lymphoid organ, enter the circulation and infiltrate into the autoantibody-mediated inflamed peripheral tissues site (Chemin et al., 2019; Niebuhr et al., 2020). The presence of Teff cells in autoimmune wounds is well established, but their role is ill-defined. Especially, the assessment of autoantigen-specific T cells in tissues requires analysis even before wounds become clearly visible due to the dynamic migratory behavior of Teff cells (Ghani et al., 2009). It is therefore required to define the position of a developing autoimmune wound for detailed analysis. In this protocol, we describe a mouse model of type VII collagen-mediated skin inflammation (epidermolysis bullosa acquisita), in which (i) Tfh cells are induced in GC of the activated draining lymph nodes and (ii) the position of the autoimmune skin wounds is defined by slight scratching of the ear skin (Figure 1A–1C). This protocol has been applied successfully to study Tfh cells in GC and Teff cells in skin wounds in parallel (Niebuhr et al., 2020, 2021). Furthermore, we provide the details of subsequent immunohistochemical staining.



Figure 1. Induction of germinal centers and skin lesions in mice.

A. Experimental setup is shown. Autoantigen emulsified in TiterMax were injected into both footpads of one mouse. The inner side of the ears was slightly scratched with an electric nail fail. Germinal centers containing Tfh cells will develop in footpad-draining lymph nodes. B. Picture shows a skin wound early after scratching (white arrow). C. Germinal centers (black arrows) are visualized by immunohistological staining with anti-mouse Ki67 (red) and anti-mouse B220 (blue). Figure adapted from Niebuhr et al. (2021).

Materials and Reagents

  1. Female SJL/J mice (8 to 12 weeks old) were obtained from Charles River Laboratories (Sulzfeld, Germany)

  2. Reaction tubes, 1.5 mL, safe seal (Sarstedt, catalog number: 72.706.400)

  3. Coverslips thickness 1, 24 × 60 mm Gerhard Menzel GmbH (Carl Roth, catalog number: H878.2)

  4. 600 mL plastic beaker (BRAND® ETFE beaker with spout, low form (Merk, catalog number: BR87618)

  5. Aldrich® microscale syringe 1 mL (Sigma-Aldrich, catalog number: Z684309)

  6. Dako Pen (DAKO, catalog number: 5200230-2)

  7. Superfrost plus microscope slides (Thermo Scientific, catalog number: J1800AMNZ)

  8. TiterMax classic (Sigma-Aldrich, catalog number: H4397), store at 2–8°C

  9. Potassium chloride (KCl) (Roth, catalog number: 6781.1), store at room temperature

  10. Sodium dihydrogen phosphate monohydrate (NaH2PO4·H2O) (Merck, catalog number: 1.06346.1000), store at room temperature

  11. Sodium chloride (NaCl) (Roth, catalog number: 3957.1), store at room temperature

  12. Di-Sodiumhydrogenphosphate dodecahydrate (Na2HPO4·12H2O) (Merck, catalog number: 1.06579.1000), store at room temperature

  13. Sodium azide (NaN3) (Sigma-Aldrich, catalog number: S8032), store at room temperature in a dark and well-ventilated place

  14. Tween 20 (Merck, catalog number: 8.22184.0500), store at room temperature

  15. Tris base (Sigma-Aldrich, catalog number: T1503), store at room temperature

  16. Ketanest S (25 mg/mL) (Pfizer, catalog number: PZN 08707288), store at 2–8°C

  17. Rompun (Xylazine) (20 mg/mL) (Bayer Vital GmbH, catalog number: PZN 1320422), store at 4–30°C

  18. Bepanthen, eye and nose ointment (Bayer Ag, catalog number: PZN 01580241), store at room temperature

  19. Trichlormethan/Chloroform 99% (Roth, catalog number: Y015.1), store at room temperature in a ventilated place

  20. Acetone 99.8% (Roth, catalog number: 9372.5), store at room temperature in a ventilated place

  21. Paraformaldehyde (AppliChem, catalog number: A3813,1000), store at 2–8°C

  22. ExtrAvidin Alkaline Phosphatase (Sigma-Aldrich, catalog number: E2636), store in the dark at 2–8°C

  23. Fast Red TR Salt (Sigma-Aldrich, catalog number: 368881), store at room temperature

  24. Fast Blue BB Salt (Sigma-Aldrich, catalog number: F3378), store at -20°C

  25. Aquatex (Merck, catalog number: 1.08562.0050), store at 15–25°C

  26. Methanol 99.9% (Roth, catalog number: 4627.5), store at room temperature in a ventilated place

  27. ExtrAvidin Peroxidase (Sigma, catalog number: E2886), store in the dark at 2–8°C

  28. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153), store at 2–8°C

  29. Liquid DAB+ Substrate Chromogen System (Agilent Technologies, catalog number: K3468), store in the dark at 2–8°C

  30. Purified anti-mouse Ki-67 (clone 16A8) (BioLegend, catalog number: 652402), store at 2–8°C

  31. Naphthol AS-MX phosphate (Sigma-Aldrich, catalog number: N4875), store at -20°C

  32. Dimethylformamide (SERVA, catalog number: 20270), store at room temperature

  33. (-)-Tetramisole hydrochloride (Levamisole) (Sigma-Aldrich, catalog number: L9756), store at 2–8°C

  34. Purified Rat Anti-Mouse CD45R/B220 (clone RA3-6B2) (BD Biosciences, catalog number: 553084), store in the dark at 2–8°C

  35. Biotin hamster anti-mouse TCRβ chain (clone H57-597) (BD Biosciences, catalog number:553169), store undiluted at 4°C

  36. Rabbit Anti-Rat IgG(H+L), Human ads-BIOT (Southern Biotech, catalog number: 6185-08), store at 2–8°C

  37. Normal mouse serum (Invitrogen, catalog number: 10410), store at -20°C

  38. Recombinant mCOL7c-GST (see Recipes)

  39. 5 × PBS (phosphate-buffered saline) (see Recipes)

  40. Anesthetic solution (see Recipes)

  41. TBS-Tween (Tris base saline-tween) (see Recipes)

  42. Tris Buffer (0.1 M) (see Recipes)

  43. PFA 4% (paraformaldehyde) (see Recipes)

  44. Alkaline Phosphatase - Anti-Alkaline Phosphatase (APAAP) substrate (see Recipes)

  45. Antibody solution (see Recipes)

  46. Fast Red staining solution (see Recipes)

  47. Fast Blue staining solution (see Recipes)

Equipment

  1. Electrical nail file with a round grinding cone device and a micromotor providing a rotating speed of 2,000 rpm (Tchibo, TCM, Article number 66075, Germany)

  2. Vortex-Genie 2 (Scientific Industries INC, USA)

  3. MAULalpha 2000G, weight range 2 kg Readability (JAKOB MAUL GmbH, model·16420-90)

  4. LEICA® manual microtome (LEICA, model: CM3050S)

  5. pH211 Microprocessor pH Meter (HANNA Instruments)

  6. Refrigerator and freezer

  7. Nitrogen Tank

Procedure

  1. Induction of germinal centers and Tfh cells in mice

    Preparation of the injection solution and injections into mice
    1. 0.02 mM (20 µM) of the protein should be injected. It is important to quantify the protein by its molar concentration because only a small fraction of the entire protein is immunogenic. The molar concentration versus mass concentration can be calculated by equation 1.

      Equation 1:[M]=Ci/MW

      Where M is the molar concentration (μM), Ci is the mass concentration (μg/mL), and MW is molecular weight (kDa).

      Here, in this case, the molecular weight of mCOL7c-GST is 49.5 kDa. Thus, the mass concentration would be 0.02 mM × 49.5 kDa = 0.990 µg/µL; simplified = 1 µg/µL. Because this solution will be diluted at 1:2, a sterile stock solution of 2 µg/mL mCOL7c-GST in 1× PBS should be prepared shortly before usage and kept one ice.

    2. Pipette the adjuvant TiterMax into a sterile Eppendorf tube. Add an equal volume of the protein solution to get a final concentration of 0.01 mM (in case of mCOL7c-GST 1 µg/µL), in the proportion of 1:2 (for example, 25% 2 µg/µL mCOL7c-GST, 25% PBS 1× and 50% Titermax for the autoimmune group or 50% PBS 1× and 50% TiterMax for the control group).

    3. Vortex vigorously for 30–40 min to prepare a protein-TiterMax or PBS 1×-TiterMax emulsion. Keep the emulsions sterile at 4°C.

    4. Weigh the animals by putting them into a 600 mL plastic beaker that stands on a digital letter scale. Inject 100 µL anesthetic solution per 20 g body weight intraperitoneally (i.p.). Anesthesia should last 20–30 min. To prevent dryness, cover the eyes of anesthetized animals with Bepanthen.

    5. Hold the footpad for injection of the prepared emulsions. Inject 60 μL into the left and 60 µL right hind footpad subcutaneously slowly and carefully by using the entire footpad for injection (in case of mCOL7c-GST 120 µL (120 µg) per mouse).

    6. Sacrifice the mice 2–4 weeks after injection. Carefully open the skin to harvest activated popliteal and inguinal lymph nodes and shock freeze them immediately for immunohistological staining (by (i) quickly placing the sample into liquid nitrogen before (ii) storing it in a -80°C refrigerator) or place lymph nodes in cold PBS 1× on ice for isolation of cells for flow cytometric analysis.

      Note: GC reaction will induce the production of high affinity autoantibodies directed against mCOL7c-GST. These autoantibodies will bind to type VII collagen in the skin and induce skin wounds, which appear 4–5 weeks after injection. Mice in the control group, which receive only TiterMax, will develop GC reactions in activated lymph nodes but no skin pathology.


  2. Determine the place of a skin wound formation by scratching

    1. To induce skin wounds, scratch mouse ear skin slightly 1 week before wounds would usually appear by the binding of autoantibodies.

      Note: In our experiment, after injection of mCOL7c-GST, bound mCOL7c-specific autoantibodies and skin wounds can be observed at 4–5 weeks post-injection. Therefore, ear skin was scratched 3 weeks post-injection.

    2. Before scratching, mice must be anesthetized as described above (step A4). Attach the round grinding cone to the electrical nail drill machine (Figure 2A and B), switch it on, and adjust it to a rotational at speed of 2,000 rpm. Take the inner side of the ear on top of one of your fingers. Slightly and briefly touch the epidermis with the tip of the round grinding cone. This approach removes the epidermis, whereas the dermis remains intact (Figures 2C–2E).

      Note: A non-bleeding wound with a standardized size should appear (Figure 2C). This skin wound will not heal in mice injected with the autoantigen (here: mCOL7c-GST) in contrast to the control group that received vehicle only, in which the healing process becomes visible 14 days after scratching (Figure 3).



      Figure 2. Removal of the epidermal layer with an electric nail file.

      A. Image shows the electric nail drill machine and B. the grinding attachment at a higher magnification. C. The ear skin was briefly and gently touched with the grinding attachment, which removed the epidermal layer at one spot of approximately 1-mm diameter per ear. The dotted white line marks the ear. The black arrow points to the induced wound spot. D-E. Hematoxylin and eosin staining of skin sections show that only the epidermal layer was removed (white arrows in E). Bar 50 µm. Figure adapted from Niebuhr et al. (2020).



      Figure 3. Chronic wounds appear after removal of the epidermal layer.

      Representative pictures of ear wounds 7 days and 14 days after scratching are shown. Scratched-induced wounds become chronic in the autoimmune group. In contrast, the wound sizes decline in the control group. Figure adapted from Niebuhr et al. (2020).


    3. Sacrifice the mice one week after scratching (4 weeks post-injection of the autoantigen) and remove ears. Carefully isolate wounded skin areas from healthy unscratched skin, place wounded and unwounded skin in separate cryotubes and immediately snap freeze tissues using liquid nitrogen or store it short-term in 1× PBS at 4°C. For long-term storage, transfer cryotubes at -80°C. Extract skin pieces under clean and sterilized conditions.

      Note: This time point of 1 week was chosen to investigate migrated T eff. To characterize other infiltrating cells, adjust time points accordingly. For example, neutrophils will be found within hours.


  3. Visualization of Tfh cells and GC

    Tfh cells localize within the light zone of GC. To stain GC, T, and B cells for traditional light microscopy, prepare serial sections of the collected lymph nodes on two glass slides. Take the first slide and visualize GC by staining proliferating cells with mouse anti-Ki67 antibody and B cells with anti-mouse B220 antibody. Take the second slide and stain T cells and B cells with a primary biotinylated anti-mouse TCRβ antibody and a primary biotinylated anti-mouse B220 antibody, respectively (Figure 4A).

    1. Staining of proliferating cells

      1. Embed collected frozen lymph nodes in tissue freezing medium and cut until a plain cut-level is present. Cut 12 µm-sections of lymph nodes using a cryomicrotom. Distribute 6–10 adjacent sections onto two glass slides. Mount the 1st, 3rd and 5th, ... section on slide 1 and the 2nd, 4th and 6th, ... sections on slide 2 (Figure 4A). Dry slides at RT for at least 1 h.

      2. To fix tissues, incubate glass slide 1 in chloroform for 10 min and acetone for 10 min. Afterwards rinse tissues in TBS-Tween for 15 min and cover lymph node section with 4% PFA and incubate them at 4°C for 45 min.

      3. Rinse glass slide in TBS-Tween for 15 min.

      4. Draw a circle around tissue sections with the Dako Pen to confine the liquids during staining and incubate sections with the first primary antibody anti-mouse Ki-67 at room temperature overnight (1:100 dilution in antibody solution).

      5. Wash unbound antibodies away by incubation with TBS-Tween for 15 min and add the first secondary biotinylated anti-rat IgG antibody (1:500 dilution in PBS 1× with 5% normal mouse serum) for 30 min.

      6. Rinse with TBS-Tween for 30 min and cover tissue sections with ExtrAvidin Alkaline Phosphatase (1:100 in TBS-Tween) for 30 min.

      7. Rinse with TBS-Tween for 15 min and apply Fast Red staining solution for 25 min.

    2. Staining of B cells

      1. Wash glass slide with TBS-Tween for 10 min and add the second primary anti-mouse B220 antibody (1:200 dilution in antibody solution) for 1 h at RT.

      2. Rinse slide in TBS-Tween for 15 min to remove unbound antibody and add the second secondary antibody, the biotinylated anti-rat IgG antibody for 30 min.

      3. Wash slide in TBS-Tween for 30 min and add 200 µL ExtrAvidin Alkaline Phosphatase 1:100 for 30 min.

      4. Wash with TBS-Tween for 15 min and add Fast Blue staining solution for 10 min to visualize B cells.

      5. After rinsing in TBS-Tween for 15 min, mount sections using 150 µL of Aquatex and coverslips.

    3. Staining for T and B cells

      1. Take glass slide 2 with the mounted and dried lymph node sections (numbers 2, 4 and 6).

      2. To fix tissues, incubate glass slide in methanol:acetone (1:2) for 10 min at 20°C. Afterwards rinse tissues in TBS-Tween for 15 min.

      3. Incubate sections with the first primary biotinylated anti-mouse TCRβ antibody 1:50 for 1h and rinse glass slides in TBS-Tween for 30 min.

      4. Add 200 µL ExtrAvidin Peroxidase (1:100 in TBS-Tween) for 30 min and wash slide in TBS-Tween for 15 min.

      5. Incubate sections with Liquid DAB+ Substrate for 5 min to visualize the T cells.

      6. Wash slide with TBS-Tween for 15 min.

      7. Add the second primary anti-mouse B220 antibody (1:200 in antibody solution) for 1 h and proceed as described above (2b–e).



    Figure 4. Induction of germinal centers and Tfh cells after injection of antigen in TiterMax.

    A. Serial cryosections of draining popliteal lymph nodes were distributed on two slides, which were either stained for proliferating cells and B cells (left slide) or T and B cells (right slide). B–E. One example of two adjacent sections is shown. B. GC were visualized by staining the proliferating cells with anti-Ki67 (red) and B cells with anti-B220 (blue). C. Tfh cells were identified as T cells that locate within the light zone of the GC by staining with anti-TCRβ for T cells (brown) and anti-B220 (blue). Four GC were clearly visible (marked with numbers). D. The magnification shows the dark zone (DZ) and light zone (LZ) of the first GC depicted in B. E. Tfh cells can be identified as within the LZ of the GC (brown-colored cells within the area marked with a white line).

Recipes

  1. Recombinant mCOL7c-GST

    The recombinant protein mCOL7c-GST was generated as described by Sitaru et al. (2005). Prior utilization, the concentration, and purity of mCOL7c-GST were validated via denaturing polyacrylamide gel electrophoresis. The protein stock was stored in PBS 1× at -80°C. For immunization, an emulsion of mCOL7c-GST in the adjuvant TiterMax was prepared under sterile conditions. Each immunization batch was prepared immediately prior to application by vortexing for 30-40 min at 4°C until complete emulsification.

  2. 5× PBS (phosphate-buffered saline)

    The composition of 5× PBS is as follows: 90 g NaCl, 2.704 g NaH2PO4 monohydrate, 28.794 g Na2HPO4·12H2O, were diluted in laboratory-graded H2O with the final volume of 2 L. The pH was adjusted to 7.4. 1× PBS was used fresh after diluting 200 mL of 5× PBS in 800 mL of laboratory-graded H2O.

  3. Anesthetic solution

    10 mL of anesthetic solution was prepared by adding 3.5 mL Ketanest (25 mg/mL) and 2 mL Rompun (20 mg/mL) to 4.5 mL 0.9% NaCl solution to yield final concentrations of 8.75 mg/ml or 4 mg/mL, respectively. Inject 100 µL i.p. per 20 g body weight for a short-acting anesthesia (43.75 mg/kg Ketanest, 20 mg/kg Rompun).

  4. TBS-Tween (Tris base saline-tween)

    The TBS-Tween is prepared using a 10× TBS stock and a Tween-20 (5%) stock. The 10× TBS is prepared using 242.28 g Tris (hydroxymethylaminomethan) and 344.40 g NaCl, dissolved in laboratory grade H2O with a final volume of 4 L, the pH should be adjusted to 7.6 and the solution must be kept at 4°C. The Tween-20 (5%) is prepared by adding 190 mL laboratory-grade H2O to 10 mL of Tween-20. 1 L of 1× TBS-Tween is prepared by adding 890 mL laboratory-grade H2O to 100 mL of 10× TBS and 10 mL of Tween-20 (5%).

  5. Tris Buffer (0.1 M)

    The composition of the Tris Buffer (0.1 M) is as follows: 12.1 g Tris base is diluted in laboratory grade H2O with a final volume of 1 L, pH should be adjusted to 8.2 with HCl.

  6. PFA 4% (paraformaldehyde) solution

    The composition of the PFA solution is as follows: 8 g of paraformaldehyde in 200 mL of 1× PBS solution.

  7. Alkaline Phosphatase - Anti-Alkaline Phosphatase (APAAP) substrate

    The composition of the APAAP substrate is as follows: 20 mg Naphthol AS-MX phosphate, 2 mL N,N-Dimethylformamide, 100 µL Levamisole (0.24 g/mL), 98 mL Tris-buffer 0.1 M. The APAAP substrate is stable maximum for 40 days.

  8. Antibody solution

    The composition of antibody solution is as follows: 1× PBS +1% BSA + 0.1% NaN3.

  9. Fast Red staining solution

    The composition of the Fast Red staining solution is as follows: 0.01 g of Fast RedSalt in 3 mL of APAAP-substrate. After the dilution, the solution should be shaken gently for 5 min and then should be kept stand steal for an additional 5 min. When added to the samples, the Fast Red staining solution should be subjected to gentle shaking for 25 min. The waste should be considered as hazardous.

  10. Fast Blue staining solution

    The composition of the Fast Blue staining solution is as follows: 0.002 g of Fast Blue BB Salt in 4 mL of APAAP-substrate. The solution should be subjected to gentle shaking for 10 min. Prior to staining samples with the Fast Blue staining solution, the solution should be subjected to filtration. After treating the sample with the filtered solution, it should be subjected to gentle shaking for 10 min.

    The waste should be considered as hazardous.

Acknowledgments

This work was supported by grants from the German Research Foundation (DFG) within the framework of the Schleswig-Holstein Excellence Cluster I and I (EXC 306, Inflammation at Interfaces, project XTP4), the graduate school GRK 1727/2, GRK2633/1 and the TR-SFB654 project C4 at the University of Luebeck to KK and JW. Part of figures are adapted and modified from the studies of Niebuhr et al. (2020 and 2021).

Competing interests

The authors indicate no potential conflicts of interest.

Ethics

All experiments were approved by the Animal Care and Use Committee of the state Schleswig-Holstein (Ministerium fuer Energiewende, Landwirtschaft, Umwelt, Natur und Digitalisierung), proposals: V312-72241.122-1 (19-2/08), V312-72241.122-1 (92-7/09), V 312-72241.122-1 (104-10), V 242-45884/2016 (90-7/16) and 23/A11/05. All animal experiments were conducted by certified personnel.

References

  1. Chemin, K., Gerstner, C. and Malmstrom, V. (2019). Effector Functions of CD4+ T Cells at the Site of Local Autoimmune Inflammation-Lessons From Rheumatoid Arthritis. Front Immunol 10: 353.
  2. Crotty, S. (2019). T Follicular Helper Cell Biology: A Decade of Discovery and Diseases. Immunity 50(5): 1132-1148.
  3. Ghani, S., Feuerer, M., Doebis, C., Lauer, U., Loddenkemper, C., Huehn, J., Hamann, A. and Syrbe, U. (2009). T cells as pioneers: antigen-specific T cells condition inflamed sites for high-rate antigen-non-specific effector cell recruitment. Immunology 128(1 Suppl): e870-880.
  4. Ludwig, R. J., Vanhoorelbeke, K., Leypoldt, F., Kaya, Z., Bieber, K., McLachlan, S. M., Komorowski, L., Luo, J., Cabral-Marques, O., Hammers, C. M., et al. (2017). Mechanisms of Autoantibody-Induced Pathology. Front Immunol 8: 603.
  5. Niebuhr, M., Belde, J., Fahnrich, A., Serge, A., Irla, M., Ellebrecht, C. T., Hammers, C. M., Bieber, K., Westermann, J. and Kalies, K. (2021). Receptor repertoires of murine follicular T helper cells reveal a high clonal overlap in separate lymph nodes in autoimmunity. Elife 10: e70053.
  6. Niebuhr, M., Bieber, K., Banczyk, D., Maass, S., Klein, S., Becker, M., Ludwig, R., Zillikens, D., Westermann, J. and Kalies, K. (2020). Epidermal Damage Induces Th1 Polarization and Defines the Site of Inflammation in Murine Epidermolysis Bullosa Acquisita. J Invest Dermatol 140(9): 1713-1722 e1719.
  7. Sitaru, C., Mihai, S., Otto, C., Chiriac, M. T., Hausser, I., Dotterweich, B., Saito, H., Rose, C., Ishiko, A. and Zillikens, D. (2005). Induction of dermal-epidermal separation in mice by passive transfer of antibodies specific to type VII collagen. J Clin Invest 115(4): 870-878.
  8. Tull, T. J. and Benton, E. (2021). Immunobullous disease.Clin Med (Lond) 21(3): 162-165.
  9. Vinuesa, C. G., Sanz, I. and Cook, M. C. (2009). Dysregulation of germinal centres in autoimmune disease. Nat Rev Immunol 9(12): 845-857.

简介

摘要:自身抗体介导的自身免疫性疾病中的自身反应性 T 细胞可分为两个主要亚群:(i)在生发中心(GC)提供 T 细胞帮助的滤泡 T 辅助细胞(Tfh)和(ii)迁移的效应 T(Teff)细胞进入皮肤等外周组织部位并介导局部炎症。为了研究导致自身抗体介导的自身免疫性疾病耐受性丧失的事件序列,需要同时研究两个 T 细胞亚群。这种方法受到阻碍主要是因为小鼠模型中皮肤炎症的出现是一个随机过程,这使得很难在正确的时间点定义炎症的位置。为了克服这个问题,我们开发了一种用于耳部皮肤的刮擦技术,该技术导致在类天疱疮样疾病大疱性表皮松解症的小鼠模型中建立慢性自身免疫性伤口。通过定义皮肤伤口应该形成的确切位置,该协议能够对皮肤迁移的 Teff 细胞进行详细分析。值得注意的是,该协议在引流淋巴结中同时诱导 GC,以便可以同时研究 GC 中的 Tfh 细胞。该协议不限于 T 细胞,可用于任何其他皮肤迁移炎症细胞。


背景

自身抗体介导的免疫疾病中的慢性组织炎症是由 IgG 自身抗体与其靶结构的结合诱导的(Ludwig等,2017) 。结合自身抗体的一个主要作用是形成免疫复合物,导致炎症细胞浸润物不受控制地募集和随后的组织破坏。典型的例子是自身免疫性皮肤病,例如大疱性类天疱疮或获得性大疱性表皮松解症,其中已经分别描述了对皮肤结构半桥粒蛋白(例如 XVII 型或 VII 型胶原蛋白)特异的自身抗体(Tull 和 Benton,2021) 。在这些高亲和力自身反应性抗体的形成过程中,自身抗原特异性 Tfh 细胞和 B 细胞在次级淋巴组织内的 GC 中彼此密切相互作用(Vinuesa等,2009;Crotty,2019) 。此外,自身抗原特异性 Teff 细胞在引流淋巴器官的 T 细胞区被激活,进入循环并浸润到自身抗体介导的发炎外周组织部位(Chemin等人,2019 年;Niebuhr等人,2020 年) 。自身免疫性伤口中存在 Teff 细胞已得到充分证实,但它们的作用尚不明确。特别是,由于 Teff 细胞的动态迁移行为,甚至在伤口变得清晰可见之前,对组织中自身抗原特异性 T 细胞的评估就需要进行分析(Ghani等人,2009) 。因此,需要确定发展中的自身免疫伤口的位置以进行详细分析。在该协议中,我们描述了 VII 型胶原介导的皮肤炎症(大疱性表皮松解症)的小鼠模型,其中(i)在激活的引流淋巴结的 GC 中诱导 Tfh 细胞和(ii)自身免疫性皮肤的位置伤口是由耳朵皮肤的轻微刮伤定义的(图 1A -1C )。该方案已成功应用于并行研究 GC 中的 Tfh 细胞和皮肤伤口中的 Teff 细胞(Niebuhr等人,2020、2021) 。此外,我们提供了后续免疫组织化学染色的细节。




图 1. 小鼠生发中心和皮肤损伤的诱导。
A. 显示了实验装置。在 TiterMax 中乳化的自身抗原被注射到一只小鼠的两个脚垫中。耳朵内侧被电钉轻微划伤失败。含有 Tfh 细胞的生发中心将在足垫引流淋巴结中发育。 B. 图片显示刮伤后早期的皮肤伤口(白色箭头)。 C.用抗小鼠 Ki67(红色)和抗小鼠 B220(蓝色)进行免疫组织学染色,观察生发中心(黑色箭头)。图改编自Niebuhr等人。 (2021 年) 。

关键字:生发中心, 滤泡T辅助细胞, 皮肤迁移 T 效应细胞, 皮肤伤口, 小鼠模型

材料和试剂
雌性 SJL/J 小鼠 (8 至 12 周龄) 获自 Charles River Laboratories (Sulzfeld, Germany)
反应管,1.5 mL,安全密封(Sarstedt,目录号: 72.706.400)
盖玻片厚度1,24 × 60 mm Gerhard Menzel GmbH(Carl Roth,目录号: H878.2)
600 mL塑料烧杯(BRAND ® ETFE烧杯,带喷口,低型(Merk,目录号:BR87618)
Aldrich ®微型注射器1 mL(Sigma-Aldrich,目录号:Z684309)
Dako Pen(DAKO,目录号:5200230-2)
Superfrost plus 显微镜载玻片(Thermo Scientific,目录号:J1800AMNZ)
TiterMax 经典(Sigma-Aldrich, 目录号:H4397),储存在 2 – 8°C
氯化钾(KCl)(Roth,目录号:6781.1),室温储存
磷酸二氢钠一水合物(NaH 2 PO 4 · H 2 O)(Merck,目录号:1.06346.1000),室温储存
氯化钠(NaCl)(Roth,目录号:3957.1),室温储存
(Na 2 HPO 4 · 12H 2 O)(Merck,目录号:1.06579.1000),室温储存
叠氮化钠(NaN 3 )(Sigma-Aldrich,目录号:S8032),室温下储存在黑暗和通风良好的地方
Tween 20(Merck,目录号:8.22184.0500),室温储存
Tris碱(Sigma-Aldrich,目录号:T1503),室温储存
Ketanest S(25 mg/mL)(辉瑞,目录号:PZN 08707288),储存于 2 – 8°C
Rompun(甲苯噻嗪)(20 mg/mL)(Bayer Vital GmbH,目录号:PZN 1320422),储存于 4 – 30°C
Bepanthen,眼鼻软膏 (Bayer Ag,目录号:PZN 01580241),室温储存
三氯甲烷/氯仿99%(Roth,目录号:Y015.1),室温下储存在通风处
丙酮99.8%(Roth,目录号:9372.5),室温下储存在通风处
多聚甲醛(AppliChem,目录号:A3813,1000),储存于 2 – 8°C
ExtrAvidin 碱性磷酸酶(Sigma-Aldrich,目录号:E2636),在 2 – 8°C 下避光储存
Fast Red TR Salt(Sigma-Aldrich,目录号:368881),室温储存
坚牢蓝BB盐(Sigma-Aldrich,目录号:F3378),储存于-20°C
Aquatex(Merck,目录号:1.08562.0050),储存于 15 – 25°C
甲醇99.9%(Roth,目录号:4627.5),室温下储存在通风处
ExtrAvidin 过氧化物酶(Sigma,目录号:E2886),在 2 - 8°C的黑暗中储存
牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153),储存在 2 – 8°C
液体 DAB +底物显色剂系统(Agilent Technologies,目录号:K3468),在 2 – 8°C 下避光储存
纯化的抗小鼠 Ki-67(克隆 16A8)(BioLegend,目录号:652402),储存于 2 – 8°C
萘酚 AS-MX 磷酸盐(Sigma-Aldrich,目录号:N4875),-20°C 储存
二甲基甲酰胺(SERVA,目录号:20270),室温储存
(-)-盐酸四咪唑(左旋咪唑)(Sigma-Aldrich,目录号:L9756),储存于 2 – 8°C
纯化的大鼠抗小鼠 CD45R/B220(克隆 RA3-6B2)(BD Biosciences,目录号:553084),在 2 – 8°C 下避光储存
生物素仓鼠抗小鼠 TCR β链(克隆 H57-597)(BD Biosciences,目录号: 553169 ),未稀释于 4°C 储存
Rabbit Anti-Rat IgG(H+L),Human ads-BIOT(Southern Biotech,目录号:6185-08),储存于 2 – 8°C
正常小鼠血清(Invitrogen,目录号:10410),储存在-20°C
重组 mCOL7c-GST(见配方)
5 × PBS(磷酸盐缓冲盐水)(见食谱)
麻醉溶液(见食谱)
TBS-Tween(Tris base salt-Tween)(见食谱)
Tris 缓冲液 (0.1 M)(见配方)
PFA 4%(多聚甲醛)(见食谱)
碱性磷酸酶 - 抗碱性磷酸酶 (APAAP) 底物(参见食谱)
抗体溶液(见配方)
固红染色溶液(见配方)
固蓝染色溶液(见配方)




设备


带有圆形磨锥装置和转速为 2,000 rpm 的微型电机的电动指甲锉(Tchibo,TCM,商品编号 66075,德国)
Vortex-Genie 2(美国科学工业公司)
MAULalpha 2000G,重量范围 2 kg 可读性(JAKOB MAUL GmbH,型号·16420-90)
LEICA ®手动切片机(LEICA,型号:CM3050S)
pH211微处理器pH计(HANNA仪器)
冰箱和冰柜
氮气罐




程序


小鼠生发中心和 Tfh 细胞的诱导


注射液的制备和对小鼠的注射
应注入 0.02 mM (20 µM) 的蛋白质。重要的是通过其摩尔浓度来量化蛋白质,因为整个蛋白质中只有一小部分是免疫原性的。摩尔浓度与质量浓度的关系可以通过公式 1 计算。
等式1:[M]=Ci/MW
其中M是摩尔浓度 (μM), Ci是质量浓度 (μg/mL),MW 是分子量 (kDa)。
在这种情况下,mCOL7c-GST 的分子量为 49.5 kDa。因此,质量浓度为 0.02 mM × 49.5 kDa = 0.990 µg/µL;简化 = 1 µg/µL。由于该溶液将以 1:2 稀释,因此应在使用前不久制备2 µg/mL mCOL7c-GST 在 1 × PBS 中的无菌储备溶液,并保留一个冰块。
将佐剂 TiterMax 移液到无菌 Eppendorf 管中。添加等体积的蛋白质溶液以获得 0.01 mM 的最终浓度(如果 mCOL7c-GST 为 1 µg/µL),比例为 1:2(例如,25% 2 µg/µL mCOL7c-GST, 25% PBS 1 ×和 50% Titermax 用于自身免疫组或 50% PBS 1 ×和 50% TiterMax 用于对照组)。
剧烈涡旋 30 – 40 分钟以制备蛋白质-TiterMax 或 PBS 1 × - TiterMax 乳液。将乳液保持在 4°C 无菌。
将动物放入一个 600 mL 的塑料烧杯中,该烧杯位于数字字母秤上,为动物称重。每 20 g 体重腹膜内 (ip) 注入 100 μL 麻醉溶液。麻醉应持续 20 – 30 分钟。为防止干燥,用 Bepanthen 遮盖麻醉动物的眼睛。
握住脚垫注射准备好的乳液。通过使用整个脚垫进行注射(如果mCOL7c -GST 120 μL(120 μg )每只小鼠),将 60 μL 缓慢而小心地注入左后脚垫和 60 μL 右后脚垫。
注射后 2-4 周牺牲小鼠。小心地打开皮肤以收集激活的腘窝和腹股沟淋巴结并立即对其进行电击冷冻以进行免疫组织学染色(通过 (i) 将样品快速放入液氮中,然后 (ii) 将其储存在 -80°C 冰箱中)或放置淋巴结在冰上冷 PBS 1 ×中分离细胞进行流式细胞术分析。
注意:GC 反应会诱导产生针对 mCOL7c-GST 的高亲和力自身抗体。这些自身抗体会与皮肤中的 VII 型胶原蛋白结合并诱发皮肤伤口,这些伤口在注射后 4-5 周出现。对照组中仅接受 TiterMax 的小鼠将在活化的淋巴结中出现 GC 反应,但不会出现皮肤病变。


通过抓挠确定皮肤伤口形成的位置


为了诱发皮肤伤口,在伤口通常会因自身抗体的结合而出现前 1 周轻轻划伤小鼠耳部皮肤。
- GST 后,在注射后 4-5 周可以观察到结合的 mCOL7c 特异性自身抗体和皮肤伤口。因此,注射后 3 周刮伤了耳部皮肤。
在抓挠之前,必须如上所述对小鼠进行麻醉(步骤 A4)。将圆形磨锥连接到电动钉钻机(图 2A 和 B)上,将其打开,并将其调整为 2,000 rpm 的旋转速度。将耳朵内侧放在一根手指上。用圆形研磨锥的尖端轻轻地、短暂地接触表皮。这种方法去除了表皮,而真皮保持完整(图 2C - 2E)。
注意:应出现具有标准化尺寸的非出血伤口(图 2C)。与仅接受载体的对照组相比,这种皮肤伤口在注射了自身抗原(此处:mCOL7c-GST)的小鼠中不会愈合,其中愈合过程在刮伤后 14 天变得可见(图 3)。




 


图 2. 用电动指甲锉去除表皮层。 
A. 图像显示电动钉钻机和 B. 更高放大倍率的研磨附件。 C. 用磨削附件短暂而轻柔地接触耳部皮肤,磨削附件在每只耳朵约 1 毫米直径的一个点处去除表皮层。虚线白线标记耳朵。黑色箭头指向诱导伤口点。德。皮肤切片的苏木精和伊红染色表明仅去除了表皮层(E 中的白色箭头)。条形 50 µm。图改编自Niebuhr等人。 (2020 年) 。


 


图 3. 去除表皮层后出现慢性伤口。
显示了抓挠后 7 天和 14 天的耳朵伤口的代表性图片。刮伤引起的伤口在自身免疫组中变成慢性的。相比之下,对照组的伤口大小有所下降。图改编自Niebuhr等人。 (2020 年) 。


在抓挠后一周(注射自身抗原后 4 周)牺牲小鼠并去除耳朵。小心地将受伤的皮肤区域与未划伤的健康皮肤隔离开,将受伤和未受伤的皮肤放入单独的冷冻管中,并立即使用液氮快速冷冻组织或将其短期储存在 4°C的 1 × PBS 中。如需长期储存,请在 -80°C 下转移冷冻管。在清洁和消毒的条件下提取皮肤碎片。
注意:选择 1 周的这个时间点来调查迁移的 T eff。要表征其他浸润细胞,请相应调整时间点。例如,中性粒细胞将在数小时内被发现。


Tfh 细胞和 GC 的可视化


Tfh 细胞定位在 GC 的光区内。要为传统的光学显微镜染色 GC、T 和 B 细胞,请在两张载玻片上准备收集的淋巴结的连续切片。拍摄第一张幻灯片并通过用小鼠抗 Ki67 抗体染色增殖细胞和用抗小鼠 B220 抗体染色 B 细胞来可视化 GC。取第二张幻灯片,分别用初级生物素化抗小鼠 TCRβ 抗体和初级生物素化抗小鼠 B220 抗体染色 T 细胞和 B 细胞(图 4A)。
增殖细胞染色
将收集的冷冻淋巴结嵌入组织冷冻培养基中并切割直至出现普通切割水平。使用冷冻切片机切割 12 μm 的淋巴结部分。将6 – 10 个相邻部分分布到两个载玻片上。将第 1 、第3和第5 、... 部分安装在幻灯片 1 上,将第 2 、第4和第6 、... 部分安装在幻灯片 2 上(图 4A)。在 RT 干燥幻灯片至少 1 小时。
要固定组织,在氯仿中孵育载玻片 1 10 分钟,在丙酮中孵育 10 分钟。然后在 TBS-Tween 中冲洗组织 15 分钟,并用 4% PFA 覆盖淋巴结切片,并在 4°C 下孵育 45 分钟。
在 TBS-Tween 中冲洗载玻片 15 分钟。
用 Dako Pen 在组织切片周围画一个圆圈,以限制染色期间的液体,并在室温下用第一种一抗抗小鼠 Ki-67 孵育切片过夜(在抗体溶液中稀释1:100 )。
通过与 TBS-Tween 孵育 15 分钟来洗去未结合的抗体,然后加入第一个二级生物素化的抗大鼠 IgG 抗体(在 PBS 1 ×中 1:500 稀释,含 5% 正常小鼠血清)30 分钟。
用 TBS-Tween 冲洗 30 分钟,并用 ExtrAvidin 碱性磷酸酶(TBS-Tween 中 1:100)覆盖组织切片 30 分钟。
用 TBS-Tween 冲洗 15 分钟,然后涂上固红染色液 25 分钟。
B细胞染色
用 TBS-Tween 清洗载玻片 10 分钟,并在 RT 中加入第二个主要抗小鼠 B220 抗体(抗体溶液中 1:200 稀释)1 小时。
在 TBS-Tween 中冲洗载玻片 15 分钟以去除未结合的抗体并加入第二个二抗,即生物素化的抗大鼠 IgG 抗体 30 分钟。
在 TBS-Tween 中清洗载玻片 30 分钟,然后加入 200 µL ExtrAvidin 碱性磷酸酶 1:100 30 分钟。
用 TBS-Tween 洗涤 15 分钟,然后加入固蓝染色溶液 10 分钟以观察 B 细胞。
在 TBS-Tween 中冲洗 15 分钟后,使用 150 μL 的 Aquatex 和盖玻片安装部分。
T 和 B 细胞染色
将载玻片 2 与已安装和干燥的淋巴结部分(编号 2、4 和 6)一起取出。
要固定组织,请在甲醇:丙酮 (1:2) 中在 20°C 下孵育载玻片 10 分钟。然后在 TBS-Tween 中冲洗组织 15 分钟。
用第一个初级生物素化抗小鼠 TCR β抗体 1:50 孵育切片 1 小时,并在 TBS-Tween 中冲洗载玻片 30 分钟。
加入 200 μL ExtrAvidin 过氧化物酶(TBS-Tween 中 1:100)30 分钟,然后在 TBS-Tween 中清洗载玻片 15 分钟。
用液体 DAB+ 底物孵育切片 5 分钟以可视化 T 细胞。
用 TBS-Tween 清洗载玻片 15 分钟。
加入第二个一级抗小鼠 B220 抗体(抗体溶液中 1:200)1 小时,然后按上述(2b - e)进行。


 


图 4. 在 TiterMax 中注射抗原后生发中心和 Tfh 细胞的诱导。
A. 引流腘窝淋巴结的连续冷冻切片分布在两张载玻片上,对增殖细胞和 B 细胞(左载玻片)或 T 和 B 细胞(右载玻片)进行染色。 B – E. 显示了两个相邻部分的一个示例。 B. 通过用抗 Ki67(红色)染色增殖细胞和用抗 B220(蓝色)染色 B 细胞来观察 GC。 C. 通过对 T 细胞(棕色)和抗 B220(蓝色)进行抗 TCR β染色,将 Tfh 细胞鉴定为位于 GC 光区内的T 细胞。四个 GC 清晰可见(标有数字)。 D. 放大图显示 BE Tfh 细胞中描绘的第一个 GC 的暗区 (DZ) 和亮区 (LZ) 可以识别为 GC 的 LZ 内(用白线标记的区域内的棕色细胞) .




食谱


重组 mCOL7c-GST
如 Sitaru等人所述,产生重组蛋白 mCOL7c-GST。 (2005 年)。通过变性聚丙烯酰胺凝胶电泳验证了 mCOL7c-GST 的先前使用、浓度和纯度。蛋白质储备在-80°C下储存在 PBS 1 ×中。对于免疫,在无菌条件下制备佐剂 TiterMax 中的 mCOL7c-GST 乳液。每个免疫批次在应用前通过在 4°C 下涡旋 30-40 分钟直到完全乳化来制备。


5 × PBS(磷酸盐缓冲盐水)
5 × PBS的组成如下:90 g NaCl、2.704 g NaH 2 PO 4一水合物、28.794 g Na 2 HPO 4 · 12H 2 O ,用实验室分级H 2 O稀释,最终体积为2 L。将pH调节至7.4。在 800 mL 实验室分级 H 2 O中稀释 200 mL 5 × PBS后,新鲜使用1 × PBS。


麻醉液
将 3.5 mL Ketanest (25 mg/mL) 和 2 mL Rompun (20 mg/mL) 添加到 4.5 mL 0.9% NaCl 溶液中,制备 10 mL 麻醉溶液,最终浓度为 8.75 mg/ml 或 4 mg/mL,分别。每 20 g 体重注射 100 μL ip,用于短效麻醉(43.75 mg/kg Ketanest,20 mg/kg Rompun)。


TBS-Tween(Tris 基础盐水-Tween)
TBS-Tween 使用 10 × TBS 原料和 Tween-20 (5%) 原料制备。使用 242.28 g Tris(羟甲基氨基甲烷)和 344.40 g NaCl 制备10 × TBS,溶解在实验室级 H 2 O 中,最终体积为 4 L,pH 应调节至 7.6,溶液必须保持在 4 ° C . Tween-20 (5%) 是通过将 190 mL 实验室级 H 2 O 添加到 10 mL Tween-20 中制备的。通过将 890 mL 实验室级 H 2 O 添加到 100 mL 10 × TBS 和 10 mL Tween-20 (5%)中制备1 L 1 × TBS-Tween。


Tris 缓冲器 (0.1 M)
Tris Buffer (0.1 M) 的组成如下: 12.1 g Tris 碱在实验室级 H 2 O 中稀释,最终体积为 1 L,用 HCl 调节 pH 至 8.2。


PFA 4%(多聚甲醛)溶液
PFA 溶液的组成如下:在 200 mL 的 1 × PBS 溶液中加入 8 g 多聚甲醛。


碱性磷酸酶 - 抗碱性磷酸酶 (APAAP) 底物
APAAP 底物的组成如下:20 mg Naphthol AS-MX 磷酸盐、2 mL N,N-二甲基甲酰胺、100 µL Levamisole (0.24 g/mL)、98 mL Tris-buffer 0.1 M。APAAP 底物最稳定40天。


抗体溶液
抗体溶液的组成如下: 1 × PBS +1% BSA + 0.1% NaN 3 。


固红染色液
红染色溶液的组成如下:0.01 克固红盐在 3 毫升 APAAP 底物中。稀释后,溶液应轻轻摇动 5 分钟,然后再静置 5 分钟。加入样品时,快速红染色溶液应轻轻摇晃 25 分钟。废物应被认为是危险的。


固蓝染色液
固蓝染色溶液的组成如下:0.002 克固蓝 BB 盐在 4 毫升 APAAP 底物中。溶液应轻轻摇动 10 分钟。在用固蓝染色溶液染色样品之前,应对溶液进行过滤。用过滤后的溶液处理样品后,应轻轻摇动 10 分钟。
废物应被认为是危险的。




致谢


这项工作得到了德国研究基金会 (DFG) 在石勒苏益格-荷尔斯泰因卓越集群 I 和 I (EXC 306, Inflammation at Interfaces, project XTP4)、研究生院 GRK 1727/2、GRK2633/1 框架内的资助以及吕贝克大学的 TR-SFB654 项目 C4 到 KK 和 JW。部分数字是根据 Niebuhr等人的研究改编和修改的。 (2020 年和 2021 年)。



利益争夺


作者指出没有潜在的利益冲突。




伦理


所有实验均经石勒苏益格-荷尔斯泰因州动物护理和使用委员会(Ministerium fuer Energiewende、Landwirtschaft、Umwelt、Natur und Digitalisierung)批准,提案:V312-72241.122-1 (19-2/08)、V312-72241.122- 1 (92-7/09)、V 312-72241.122-1 (104-10)、V 242-45884/2016 (90-7/16) 和 23/A11/05。所有动物实验均由经过认证的人员进行。




参考


Chemin, K.、Gerstner, C. 和 Malmstrom, V. (2019)。 CD4 + T 细胞在局部自身免疫性炎症部位的效应功能——类风湿性关节炎的教训。 前免疫10:353。
Crotty, S. (2019)。 T 滤泡辅助细胞生物学:发现和疾病的十年。 豁免50(5):1132-1148。
Ghani, S.、Feuerer, M.、Doebis, C.、Lauer, U.、Loddenkemper, C.、Huehn, J.、Hamann, A. 和 Syrbe, U. (2009)。作为先驱的 T 细胞:抗原特异性 T 细胞调节发炎部位以进行高速率的抗原非特异性效应细胞募集。 免疫学128(1 增刊):e870-880。
Ludwig, RJ, Vanhoorelbeke, K., Leypoldt, F., Kaya, Z., Bieber, K., McLachlan, SM, Komorowski, L., Luo, J., Cabral-Marques, O., Hammers, CM等人_ (2017)。自身抗体诱导的病理学机制。 前免疫8:603。
Niebuhr, M., Belde, J., Fahnrich, A., Serge, A., Irla, M., Ellebrecht, CT, Hammers, CM, Bieber, K., Westermann, J. 和 Kalies, K. (2021) .小鼠滤泡 T 辅助细胞的受体库揭示了自身免疫中不同淋巴结中的高度克隆重叠。 生命10 : e70053 。
Niebuhr, M.、Bieber, K.、Banczyk, D.、Maass, S.、Klein, S.、Becker, M.、Ludwig, R.、Zillikens, D.、Westermann, J. 和 Kalies, K.( 2020)。表皮损伤诱导 Th1 极化并确定小鼠大疱性表皮松解症的炎症部位。 J Invest Dermatol 140(9): 1713-1722 e1719。
Sitaru, C.、Mihai, S.、Otto, C.、Chiriac, MT、Hausser, I.、Dotterweich, B.、Saito, H.、Rose, C.、Ishiko, A. 和 Zillikens, D. (2005 )。通过对 VII 型胶原蛋白特异性抗体的被动转移诱导小鼠真皮-表皮分离。 临床投资杂志 115(4):870-878。
Tull, TJ 和 Benton, E. (2021)。免疫大疱病。 临床医学(伦敦) 21(3):162-165。
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Copyright Bahreini et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Bahreini, F., Niebuhr, M., Belde, J., Bieber, K., Westermann, J. and Kalies, K. (2022). Protocol to Induce Follicular T Helper Cells, Germinal Centers, and Skin Lesions in Mouse Models for Skin Blistering Diseases. Bio-protocol 12(10): e4414. DOI: 10.21769/BioProtoc.4414.
  2. Niebuhr, M., Belde, J., Fahnrich, A., Serge, A., Irla, M., Ellebrecht, C. T., Hammers, C. M., Bieber, K., Westermann, J. and Kalies, K. (2021). Receptor repertoires of murine follicular T helper cells reveal a high clonal overlap in separate lymph nodes in autoimmunity. Elife 10: e70053.
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