SE Degn is a Lundbeckfonden Fellow and a Carlsberg Foundation Distinguished Fellow. This work was in part additionally supported by an NNF Biomedical Grant (SE Degn).

<ol>
	<li>Lerner, A., Jeremias, P., Matthias, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+World+Incidence+and+Prevalence+of+Autoimmune+Diseases+is+Increasing.">The World Incidence and Prevalence of Autoimmune Diseases is Increasing.</a> International Journal of Celiac Disease. 3 (4), 151-155 (2015).</li><li>Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+marker+based+on+the+UV-induced+green-to-red+photoconversion+of+a+fluorescent+protein.">An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (20), 12651-12656 (2002).</li><li>Mutoh, T., Miyata, T., Kashiwagi, S., Miyawaki, A., Ogawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+behavior+of+individual+cells+in+developing+organotypic+brain+slices+revealed+by+the+photoconvertable+protein+Kaede.">Dynamic behavior of individual cells in developing organotypic brain slices revealed by the photoconvertable protein Kaede.</a> Experimental neurology. 200 (2), 430-437 (2006).</li><li>Tomura, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+cellular+movement+in+vivo+with+photoconvertible+fluorescence+protein+&#8220;Kaede&#8221;+transgenic+mice.">Monitoring cellular movement in vivo with photoconvertible fluorescence protein &#8220;Kaede&#8221; transgenic mice.</a> Proceedings of the National Academy of Sciences. 105 (31), 10871-10876 (2008).</li><li>Tomura, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+and+quantification+of+dendritic+cell+migration+and+antigen+trafficking+between+the+skin+and+lymph+nodes.">Tracking and quantification of dendritic cell migration and antigen trafficking between the skin and lymph nodes.</a> Scientific Reports. 4, 6030 (2014).</li><li>Gurskaya, N. 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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+Evolution+of+Autoreactive+Germinal+Centers.">Clonal Evolution of Autoreactive Germinal Centers.</a> Cell. 170 (5), 913-926 (2017).</li><li>Berland, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+7-dependent+loss+of+B+cell+tolerance+in+pathogenic+autoantibody+knockin+mice.">Toll-like receptor 7-dependent loss of B cell tolerance in pathogenic autoantibody knockin mice.</a> Immunity. 25 (3), 429-440 (2006).</li><li>Chatterjee, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement+C4+maintains+peripheral+B-cell+tolerance+in+a+myeloid+cell+dependent+manner.">Complement C4 maintains peripheral B-cell tolerance in a myeloid cell dependent manner.</a> European journal of immunology. 43 (9), 2441-2450 (2013).</li><li>Toubai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+acute+GVHD+by+sex-mismatched+H-Y+antigens+in+the+absence+of+functional+radiosensitive+host+hematopoietic-derived+antigen-presenting+cells.">Induction of acute GVHD by sex-mismatched H-Y antigens in the absence of functional radiosensitive host hematopoietic-derived antigen-presenting cells.</a> Blood. 119 (16), 3844-3853 (2012).</li><li>Luzina, I. 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The authors have nothing to disclose.

A large number of murine models of autoimmunity are available, many of which present with spontaneous germinal centers16. However, many of the available models harbor complex genetic backgrounds or mutations in central regulators of lymphocyte proliferation or activation, rendering them poorly suited to intercrossing with reporter lines and studies of normal lymphocyte behavior in autoimmunity, respectively. The present model, on the contrary, allows a &#8216;plug-and-play&#8217; approach to in-depth analyses of autoreactive wild-type-derived germinal center B cells using any desired combination of transgenes, knock-outs and reporters, in the present case represented by photoactivatable GFP. Using in vivo labeling strategies, single germinal centers can be visualized in explanted lymphoid tissues and their cellular constituents photoactivated using a two-photon microscope. Photoactivated lymphocytes from single germinal centers can then be analyzed or sorted flow cytometrically, as single cells or in bulk. These cells may subsequently be subjected to additional downstream molecular and functional analyses to provide renewed insights in the field of autoimmunity.
There are some critical steps for successful performance of this procedure. As demonstrated by the representative results, the irradiation (1,100 Rad) and donor bone marrow reconstitution successfully replace the recipient bone marrow compartment yielding near-complete chimerism in the B cell compartment. This is an important point, as residual recipient-derived B cells would render a subset of the germinal center population &#8216;dark&#8217;. Regardless of the source used for irradiation, the dose/timing of irradiation has to be optimized to yield maximal myeloablative effect with minimal collateral tissue damage to the animals. For reconstitution, the bone-crush protocol and reconstitution with 20 million total donor cells has been found to robustly yield high reconstitution degrees. Working sterile and cold/on ice for the bone marrow extraction ensures high viability of the donor marrow. To reach the desired donor bone marrow ratios, it is essential to exercise great care when counting aliquots of the cells, both for the counting itself and when taking out the subsample of the bone marrow for counting. Mixing and co-pelleting the donor marrows, instead of centrifuging and resuspending separately and then mixing, serves to prevent any skew in donor ratios following the cell counting.
The mixed bone marrow chimera generation of the protocol can stand alone, and it enables generation of chimeras with autoreactive germinal centers with any desired reporter, transgene or knock-out. However, one limitation to this is the need to use histocompatible donors. The 564Igi strain is on a C57Bl/6J congenic background, and as a consequence, the other donor(s) and the recipients should have an H-2b congenic background (or alternatively, the 564Igi strain should be backcrossed to the desired strain and the autoimmune phenotype verified on the new background). The irradiation procedure tends to favor a tolerogenic environment17, and mismatches in some minor histocompatibility antigens may be tolerated. However, this aspect should be thoroughly considered, particularly if mixing male and female donors and/or recipients, due to the potential for female reactivity with male-restricted Y-antigens.
Similarly, the photoactivation aspect of the protocol can stand alone, and may be used in many different contexts. However, the PA-GFP reporter is currently only available with the UBC promotor, which is active in all hematopoietic lineage cells, but not in stromal cells. As mentioned in the Introduction, other photoactivatable, photoswitchable, or photoconvertible reporter strains are available, and may be substituted for PA-GFP, with appropriate adjustment of experimental conditions.
It is important to avoid inadvertent photoactivation of undesirable areas, by maintaining the laser well above 900 nm when imaging, as this wavelength will not photoactivate PA-GFP. For the photoactivation itself, specific settings, such as laser power and pixel dwell time, will depend on the depth in the tissue, the specific tissue used, and the imaging system, and each application has to be optimized for the specific imaging system used. Care should be taken not to photodamage the cells, but it is at the same time essential to get efficient photoactivation throughout the stack, in order to get a sufficient representation of activated cells for downstream analyses. Germinal center B cells generally constitute anywhere from 0.5% to ~2% of splenic or cutaneous lymph node B cells, and as can be seen from the representative results (Figure 5), photoactivated single germinal center B cells may make up ~1% of the total population present in a single spleen slice. Therefore, successful analysis or sorting of a significant number of cells requires processing a large number of events.

The incidence of autoimmune disease has risen rapidly in the past decades, particularly in Western societies. Today, autoimmune disease ranks third on the list of most prevalent causes of morbidity and mortality in the Western world1. Fundamental questions regarding the development and progression of autoimmune disease remain unanswered. One requirement for advancements in our understanding of the underlying disease mechanisms and cellular dynamics is the precise coupling of the microanatomical location of cell subsets with downstream molecular or functional analyses. In the past decade, the development of a number of stable photoconvertible, photoactivatable, or photoswitchable biological fluorophores and their integration into reporter strains has enabled the precise microanatomical labeling and tracking of cellular subsets in murine models.
Kaede, a photoconvertible fluorescent protein originating from a stony coral, undergoes irreversible photoconversion from green fluorescence to red fluorescence upon exposure to violet or ultraviolet light2. Initially employed to follow the dynamic behavior of individual cells in developing organotypic brain slices3, generation of a Kaede knock-in mouse subsequently allowed monitoring cellular movement in vivo, and the system was applied to analyses of immune cell migration to and from lymph nodes4. This approach was subsequently refined with a second-generation reporter5. A similar reporter is Dendra6, which was recently used to track lymph node metastases in vivo7.
The first photoactivatable protein developed was a green fluorescent protein (GFP) engineered with a single point mutation (T203H), leading to a very low absorbance in the wavelength region from 450 to 550 nm8. After photoactivation by violet light, this photoactivatable green fluorescent protein (PA-GFP) switches its absorption maximum from ~400 to ~500 nm, yielding an approximate 100-fold intensity increase when excited with a wavelength of 488 nm. The generation of transgenic mice in which all hematopoietic cells express PA-GFP allowed, for the first time, in-depth analyses of B cell selection in anatomically defined light and dark zones of the germinal center9.
Whereas photoactivation is an irreversible conversion from a non-fluorescent state to a fluorescent state, and photoconversion is a one-way transition from one wavelength to another,&#160; photoswitchable proteins are able to shuttle between both conditions10. This latter capacity was recently harnessed to engineer optical control of protein activity11.
Utilizing the PA-GFP reporter, we recently characterized the B cell repertoires of single germinal centers in a novel model of spontaneous lupus-like autoimmunity12. This model is based on mixed chimeras with 1 part bone marrow harboring an autoreactive B cell receptor knock-in with specificity for ribonuclear-protein complexes (564Igi13,14) combined with 2 parts bone marrow from any desired donor. At approximately 6 weeks post reconstitution, homeostatic conditions are achieved in which spontaneous autoreactive germinal centers are present in the spleen and the cutaneous lymph nodes. Notably, the germinal center B cell population is almost exclusively (~95%) composed of cells derived from the non-564Igi compartment, and these wild-type-derived B cells have become autoreactive. Therefore, the model allows a &#8216;plug-and-play&#8217; approach to analyses of autoreactive germinal center B cells using various transgenes, knock-outs and reporters. Here, we describe the procedure for generating mixed chimeras with spontaneous autoreactive germinal centers populated by lymphocytes carrying the PA-GFP reporter. Using in vivo labeling strategies, single germinal centers can be visualized in explanted lymphoid tissues and their cellular constituents photoactivated using a two-photon microscope. Photoactivated lymphocytes from single germinal centers can subsequently be analyzed by flow cytometry or sorted by fluorochrome-activated cell sorting (FACS) and subjected to additional downstream molecular and functional analyses. The ability to analyze autoreactive lymphocytes from single germinal centers may directly be applied to provide renewed insights in the field of autoimmunity, but the techniques and approaches described may additionally find relevant applications in studies of infectious diseases and tumor metastases.

<table><tbody><tr><td>Antibody, 9D11-A568</td><td>In-house generated</td><td></td><td>9D11 hybridoma, kindly provided by MC Carroll, labeled with kit: Biotium 92255</td></tr><tr><td>Antibody, 9D11-A647</td><td>In-house generated</td><td></td><td>9D11 hybridoma, kindly provided by MC Carroll, labeled with kit: Nordic Biosite ABD-1031</td></tr><tr><td>Antibody, FITC anti-mouse CD45.1</td><td>Biolegend</td><td>110705</td><td></td></tr><tr><td>Antibody, Pacific Blue anti-mouse/human GL7 Antigen (T and B cell Activation Marker)</td><td>Biolegend</td><td>144613</td><td></td></tr><tr><td>Antibody, PE anti-mouse CD169 (Siglec-1)</td><td>Biolegend</td><td>142403</td><td></td></tr><tr><td>Antibody, PE/Cy7 anti-mouse CD38</td><td>Biolegend</td><td>102717</td><td></td></tr><tr><td>Antibody, PerCP/Cy5.5 anti-mouse/human CD45R/B220</td><td>Biolegend</td><td>103235</td><td></td></tr><tr><td>Capillary tube, Mylar-wrapped, heparinized</td><td>Fisher Scientific</td><td>211766</td><td></td></tr><tr><td>Cell strainer, 70 &amp;micro;m</td><td>Falcon</td><td>352350</td><td></td></tr><tr><td>Conical tubes, 50 mL</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>Cover slip, square, 22x22 mm, 0.13-0.17 mm</td><td>Thermo Fisher Scientific</td><td>22X22-1</td><td></td></tr><tr><td>EDTA</td><td>Merck</td><td>1,084,180,250</td><td></td></tr><tr><td>Ethanol, 70%</td><td>VWR</td><td>8301.360</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Life Technologies</td><td>10270106</td><td></td></tr><tr><td>Flow cytometer, FACS Canto II</td><td>BD Biosciences</td><td>338962</td><td></td></tr><tr><td>Flow cytometer, LSRFortessa SORP</td><td>BD Biosciences</td><td>-</td><td>Special order product with 4 lasers (405 nm, 488 nm, 561 nm and 640 nm)</td></tr><tr><td>Grease, high vacuum, Dow Corning</td><td>VWR</td><td>DOWC1597418</td><td></td></tr><tr><td>Hemocytometer, Burker-T&amp;uuml;rk</td><td>VWR</td><td>630-1544</td><td></td></tr><tr><td>Isoflurane, IsoFlo vet.</td><td>Orion Pharma</td><td>9658</td><td></td></tr><tr><td>Lymphocyte separation medium (Lympholyte-M Cell Separation Media)</td><td>Cedarlane</td><td>CL5035</td><td></td></tr><tr><td>Microcentrifuge tube, 1.5 mL (Eppendorf)</td><td>Sarstedt</td><td>72.690.550</td><td></td></tr><tr><td>Microscope, Two-photon</td><td>Prairie Technologies (now Bruker)</td><td>-</td><td>Special order Ultima In Vivo Two Photon Microscope</td></tr><tr><td>Mortar w. lip, unglazed, 75 ml</td><td>VWR</td><td>410-0110</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Merck</td><td>1063290500</td><td></td></tr><tr><td>Needle, 18 gauge</td><td>BD Medical</td><td>304622</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>VWR</td><td>87,769,290</td><td></td></tr><tr><td>PBS</td><td>Sigma</td><td>d8537</td><td></td></tr><tr><td>Pestle homogenizer</td><td>VWR</td><td>47747-358</td><td></td></tr><tr><td>Pestle, unglazed, 175 mm</td><td>VWR</td><td>410-0122</td><td></td></tr><tr><td>Pipette, Serological, 10 ml</td><td>VWR</td><td>612-3700</td><td></td></tr><tr><td>Pipette, transfer, plastic</td><td>Sarstedt</td><td>861,172,001</td><td></td></tr><tr><td>Plastic paraffin film (Parafilm M)</td><td>Bemis</td><td>PM996</td><td></td></tr><tr><td>Plate, 96-well</td><td>Falcon</td><td>353910</td><td></td></tr><tr><td>Surgical forceps, Student Dumont #5 Forceps</td><td>FST - Fine Science Tools</td><td>91150-20</td><td></td></tr><tr><td>Surgical forceps, Student Dumont #7 Forceps</td><td>FST - Fine Science Tools</td><td>91197-00</td><td></td></tr><tr><td>Surgical scissors, Student Fine Scissors, Straight</td><td>FST - Fine Science Tools</td><td>91460-11</td><td></td></tr><tr><td>Syringe, 10 mL</td><td>Terumo</td><td>SS-10ES1</td><td></td></tr><tr><td>Syringe, 20 mL</td><td>Terumo</td><td>SS-20ES1</td><td></td></tr><tr><td>Syringe, 5 mL</td><td>Terumo</td><td>SS-05S1</td><td></td></tr><tr><td>Syringe, Insulin, 0.3 cc</td><td>BD Medical</td><td>324827</td><td></td></tr><tr><td>Tribrissen vet. 24% inj., containing 200 mg sulfadiazin and 40 mg trimethoprim/ml</td><td>MSD Animal health</td><td>431577</td><td></td></tr><tr><td>Trypan blue solution, 0.4%</td><td>VWR</td><td>K940-100ML</td><td></td></tr><tr><td>Viability dye, eBioscience Fixable Viability Dye eFluor 780</td><td>Thermo Fisher Scientific</td><td>65-0865-14</td><td></td></tr></tbody></table>

interrogating individual autoreactive germinal centers by photoactivation in a mixed chimeric model of autoimmunity

Autoimmune diseases present a significant health burden. Fundamental questions regarding the development and progression of autoimmune disease remain unanswered. One requirement for advancements in our understanding of the underlying disease mechanisms and cellular dynamics is the precise coupling of the microanatomical location of cell subsets with downstream molecular or functional analyses; a goal that has traditionally been difficult to achieve. The development of stable photoactivatable biological fluorophores and their integration into reporter strains has recently enabled precise microanatomical labeling and tracking of cellular subsets in murine models. Here, we describe how the ability to analyze autoreactive lymphocytes from single germinal centers may help to provide novel insights into autoimmunity, using the combination of a novel chimeric model of autoimmunity with a photoactivatable reporter as an example. We demonstrate a procedure for generating mixed chimeras with spontaneous autoreactive germinal centers populated by lymphocytes carrying a photoactivatable green fluorescent protein reporter. Using in vivo labeling strategies, single germinal centers can be visualized in explanted lymphoid tissues and their cellular constituents photoactivated by two-photon microscopy. Photoactivated lymphocytes from single germinal centers can then be analyzed or sorted flow cytometrically, as single cells or in bulk, and may be subjected to additional downstream molecular and functional analyses. This approach may directly be applied to provide renewed insights in the field of autoimmunity, but the procedure for generating bone marrow chimeras and the photoactivation procedure may additionally find broad application in studies of infectious diseases and tumor metastases.

Generation of mixed bone marrow chimeras
The present protocol robustly achieves mixed bone marrow chimeras with a near-complete chimerism in the B cell compartment as shown in the representative result in Figure 1 (for statistical significance please refer to 12). The serotyping reveals normalized B cell numbers at 6 weeks post reconstitution (Figure 1A), with a low frequency of 9D11 (idiotype) positive circulating B cells deriving from the 564Igi compartment (Figure 1B). Within the total lymphocyte gate, there is a low frequency of residual recipient-derived cells, ~6% CD45.1 (Q1), indicating an overall degree of chimerism of ~94% (Figure 1C). Within the donor compartment (CD45.1-, Q4+Q3) the ratio of 564Igi (Q4) to PA-GFP (Q3) is around 23% to 77%. This slightly lower than input 33% to 66% ratio is explained by the heavy negative selection of B cells derived from the 564Igi compartment 12. As seen in Figure 1D, there is virtually complete chimerism in the B cell compartment (99.9% CD45.1-) and dominance of PA-GFP bone marrow-derived B cells (Q3), which is a consequence of the heavy negative selection of 564Igi-derived B cells.
Harvest of tissues, processing and flow cytometric evaluation
Figure 2 and Figure 3 demonstrate procedures for and results of explanting freshly isolated lymph nodes and spleen slices. Figure 4 presents a representative result for in vivo labeling and photoactivation of a single germinal center area in an explanted spleen slice. As can be seen (Figure 4A), the in vivo labeling with CD169-PE has robustly labeled the marginal zone (red, indicated by &#8220;MZ&#8221;). The second harmonics signal is apparent in collagen-containing structural elements and major vessels (blue), including the central arteriole of the periarteriolar lymphoid sheath (PALS). Highly autofluorescent, activated tingible-body macrophages are associated with germinal center activity (arrowheads). Taken together, the identification of the marginal zone, the PALS, and tingible-body macrophages, allows identification of a region of interest which likely contains a single germinal center. The region of interest is photoactivated as illustrated in Figure 4B. As demonstrated, photoactivation is microanatomically precise9, yielding a defined area of activation. The presented results additionally serve as confirmation of a high density of PA-GFP+ lymphocytes in the reconstituted chimeras and presence of spontaneous germinal centers. Downstream flow cytometric evaluation further confirms normalized B cell compartment numbers (Figure 5D), a spontaneous germinal center population (Figure 5E), and the presence of a subset of germinal center B cells which have been photoactivated (Figure 5F).
Thus, the present protocol presents a robust method for generation of mixed bone marrow chimeras with spontaneous autoreactive germinal centers, which are predominantly composed of wild-type derived B cells carrying a photoactivatable reporter. This in turn allows for downstream analyses of individual germinal centers (graphical overview in Figure 6).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59397/59397fig1.jpg" /> 
Figure 1: Flow cytometric evaluation of degree of chimerism in blood of 564Igi (CD45.1-, PA-GFP-):PA-GFP (CD45.1-, PA-GFP+) mixed chimeras in lethally irradiated CD45.1 recipients (CD45.1+, PA-GFP-), 6 weeks post reconstitution. A) Plot showing gating of B220+ B cells, pre-gated on singlet lymphocytes. B) Subgate from plot A, showing 9D11+ (idiotype) frequency within the B cell population. C) Plot of PA-GFP versus CD45.1, pre-gated on singlet lymphocytes. D) Plot of PA-GFP versus CD45.1 in the B cell subgate from plot A. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59397/59397fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59397/59397fig2.jpg" /> 
Figure 2: Procedure for harvesting and mounting popliteal lymph nodes for imaging and photoactivation. A) An incision is made below the knee and extended up to the hip joint, and the edges are retracted to the sides, in order to expose the popliteal fossa (arrowhead). B) The overlying fatpad is opened and the popliteal lymph node is exposed (arrowhead). C) The popliteal lymph node is retrieved from the fossa. D) The procedure is repeated for the contralateral side and both nodes are mounted in a double-sided coverslip/vacuum-grease chamber filled with BM buffer. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59397/59397fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59397/59397fig3.jpg" /> 
Figure 3: Procedure for harvesting and mounting the spleen for imaging and photoactivation. A) An incision is made at the anterior medial line just below the ribcage and extended around the body to the posterior axillary line, and the edges are retracted to expose the tip of the spleen (arrowhead). B) The spleen is retracted and excised, and cut into thin (1-2 mm) slices, which are mounted in a double-sided coverslip/vacuum-grease chamber filled with BM buffer. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59397/59397fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59397/59397fig4.jpg" /> 
Figure 4: Photoactivation. A) Two-photon micrograph of a germinal center in the spleen before photoactivation. In vivo labeling with anti-CD169-PE was performed before harvest of the spleen to label the marginal zone (red, indicated by &#8220;MZ&#8221;). The second harmonics signal is apparent in collagen-containing structures associated with the integument and major vessels (blue). Arrowheads identify highly autofluorescent, activated tingible-body macrophages associated with germinal center activity. Imaging was performed at 940 nm excitation. The scale bar in the top left-hand corner indicates 200 &#181;m. B) As for A, but after photoactivation at 830 nm. Photoactivated cells are now visible (green) in a defined region of interest bounded by the marginal zone and encompassing the previously identified tingible-body macrophages. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59397/59397fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59397/59397fig5.jpg" /> 
Figure 5: Flow cytometric analysis of photoactivated germinal center B cells. A) Plot of forward versus side scatter and lymphocyte gate. B) Plot of forward scatter area as a function of forward scatter height within the lymphocyte gate, and resulting singlet gate. C) Viability dye exclusion plot within the singlet gate, and resulting live cell gate. D) Gating of B220+ B cells. E) Gating of germinal center B cells, identified as CD38lo GL7hi cells within the B220+ gate. F) Gating of photoactivated cells within the GC B cell population, identified as the subset of cells co-expressing non-activated and photoactivated PA-GFP. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59397/59397fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59397/59397fig6.jpg" /> 
Figure 6: Graphical overview of the protocol. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59397/59397fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Interrogating Individual Autoreactive Germinal Centers by Photoactivation in a Mixed Chimeric Model of Autoimmunity at JoVE.com

Interrogating Individual Autoreactive Germinal Centers by Photoactivation in a Mixed Chimeric Model of Autoimmunity

This protocol describes the generation of mixed murine bone marrow chimeras with spontaneous autoimmune germinal centers, in which autoreactive lymphocytes carry a photoactivatable green fluorescent protein (PA-GFP) reporter. This provides the ability to link cellular location in tissues with downstream molecular and functional analyses.

Autoimmune diseases present a significant health burden. Fundamental questions regarding the development and progression of autoimmune disease remain ...

interrogating-individual-autoreactive-germinal-centers

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

6.9K Views.  Aarhus University. This protocol describes the generation of mixed murine bone marrow chimeras with spontaneous autoimmune germinal centers, in which autoreactive lymphocytes carry a photoactivatable green fluorescent protein (PA-GFP) reporter. This provides the ability to link cellular location in tissues with downstream molecular and functional analyses.

Video: Interrogating Individual Autoreactive Germinal Centers by Photoactivation in a Mixed Chimeric Model of Autoimmunity

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-&#945; (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers+ cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IAs/PLP 139-151 dextramers (specific)/anti-CD4 and IAs/Theiler&#8217;s murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IAk/Myhc 334-352 dextramers/anti-CD4 and IAk/bovine ribonuclease (RNase) 43-56 dextramers (control)/anti-CD4. LSCM analysis of brain sections obtained from EAE mice showed the presence of cells positive for CD4 and PLP 139-151 dextramers, but not TMEV 70-86 dextramers suggesting that the staining obtained with PLP 139-151 dextramers was specific. Likewise, heart sections prepared from EAM mice also revealed the presence of Myhc 334-352, but not RNase 43-56-dextramer+ cells as expected. Further, a comprehensive method has also been devised to quantitatively analyze the frequencies of antigen-specific CD4 T cells in the &#8216;Z&#8217; serial images.

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

The protocol to detect self-reactive CD4 T cells in brain and heart by direct staining with major histocompatibility complex class II dextramers has been described in this report. For comprehensive analysis, a reliable method to enumerate the frequencies of antigen-specific CD4+ T cells in situ is also devised.

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels of autoantibodies to specific antigens are typically detected with the enzyme-linked immunosorbent assay (ELISA) technique. However, screening for multiple autoantibodies with ELISA can be time-consuming and requires a large quantity of patient sample. The antigen microarray technique is an alternative method that can be used to screen for autoantibodies in a multiplex fashion. In this technique, antigens are arrayed onto specially coated microscope slides with a robotic microarrayer. The slides are probed with patient serum samples and subsequently fluorescent-labeled secondary antibodies are added to detect binding of serum autoantibodies to the antigens. The autoantibody reactivities are revealed and quantified by scanning the slides with a scanner that can detect fluorescent signals. Here we describe methods to generate custom antigen microarrays. Our current arrays are printed with 9 solid pins and can include up to 162 antigens spotted in duplicate. The arrays can be easily customized by changing the antigens in the source plate that is used by the microarrayer. We have developed a two-color secondary antibody detection scheme that can distinguish IgG and IgM reactivities on the same slide surface. The detection system has been optimized to study binding of human and murine autoantibodies.

We describe here a method to generate customizable antigen microarrays that can be used for the simultaneous detection of serum IgG and IgM autoantibodies from humans and mice. These arrays allow for high-throughput and quantitative detection of antibodies against any antigens or epitopes of interest.

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

This protocol describes an imaging-based method to activate T lymphocytes using photoactivatable peptide-MHC, enabling precise spatiotemporal control of T cell activation.

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Analysis of Somatic Hypermutation in the JH4 intron of Germinal Center B cells from Mouse Peyer's Patches

Within the germinal centers of lymphoid organs, mature B cells alter their expressed immunoglobulin (Ig) by introducing untemplated mutations into the variable coding exons of the Ig heavy and light chain gene loci. This process of somatic hypermutation (SHM) requires the enzyme activation-induced cytidine deaminase (AID), which converts deoxycytidines (C), into deoxyuridines (U). Processing the AID-generated U:G mismatches into mutations by the base excision and mismatch repair pathways introduces new Ig coding sequences that may produce a higher affinity Ig. Mutations in AID or DNA repair genes can block or significantly alter the types of mutations observed in the Ig loci. We describe a protocol to quantify JH4 intron mutations that uses fluorescence activated cell sorting (FACS), PCR, and Sanger sequencing. Although this assay does not directly measure Ig affinity maturation, it is indicative of mutations in Ig variable coding sequences. Additionally, these methods utilize common molecular biology techniques which&#160;analyze&#160;mutations in Ig sequences of multiple B cell clones. Thus, this assay is an invaluable tool in the study of SHM and Ig diversification.

Presented here is an assay to quantify somatic hypermutation within the immunoglobulin heavy chain gene locus using germinal center B cells from mouse Peyer&#8217;s patches.

Within the germinal centers of lymphoid organs, mature B cells alter their expressed immunoglobulin (Ig) by introducing untemplated mutations into the ...

Analysis of Granulocyte-Macrophage Colony-Stimulating Factor-Producing T Helper Cells in a Mouse Model of Contact Hypersensitivity

Parallel to traditional Th1/Th2/Th17/Treg lineages, granulocyte-macrophage colony-stimulating factor-producing T helper (Th-GM) cells have been identified as a distinct subset of T helper cells (GM-CSF+ IFN-&#947;- IL-17A- IL-22- effector CD4+ T cells) in human and mice. Contact hypersensitivity (CHS) is considered an excellent animal model for allergic contact dermatitis (ACD) in human, manifesting an intact T cell-mediated immune response. To provide a standardized and comprehensive assay to analyze the Th-GM cell subset in the T cell-dependent immune response in vivo, a murine CHS model was induced by sensitization/challenge with a reactive, low-molecular-weight, organic hapten, 2,4-dinitrofluorobenzene (DNFB). The Th-GM subset in effector CD4+ T cells generated upon immunization with the hapten was analyzed by flow cytometry. We found that Th-GM was mainly expanded in lesions and draining lymph nodes in the DNFB-induced CHS mouse model. This method can be applied to further study the biology of Th-GM cells and pharmacological research of therapeutic strategies centered on GM-CSF in various conditions, such as ACD.

Here, we present a simple and standardized method of analyzing the granulocyte-macrophage colony-stimulating factor-producing T helper subset in vivo.

Parallel to traditional Th1/Th2/Th17/Treg lineages, granulocyte-macrophage colony-stimulating factor-producing T helper (Th-GM) cells have been identified ...

Investigating Humoral Response in Autoimmune Demyelination

Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological and molecular levels. EAE is induced by immunizing experimental animals via subcutaneous injection of short myelin peptides together with specific adjuvants to boost the immune response. Like the human counterpart, EAE mice develop demyelinating lesions, immune cell infiltration into the central nervous system (CNS), glia activation and neuronal injury. A consistent body of evidence also supports a mechanistic role for B cell dysfunction in the etiology of both MS and EAE. B cells can serve as antigen-presenting cells as well as a primary source of pro-inflammatory cytokines and autoantibodies. In EAE, antibodies are generated against the myelin peptides that were employed to induce the disease. Such autoantibodies have been shown to mediate either myelin loss or pathogenic T cell reactivation into the CNS. This article describes an efficient ELISA-based protocol to quantify autoantibodies in the serum of C57BL/6J mice immunized with the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide. The proposed method serves as a powerful tool to investigate the specificity and magnitude of the aberrant humoral response in the context of autoimmune demyelination.

Author Spotlight: Novel Assay for Studying B-Cell Responses in Multiple Sclerosis Research

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), which shares with the human disease a robust humoral autoimmune response. Here, we report a simple and flexible ELISA protocol to quantify autoantibodies in the serum of EAE immunized mice.

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological ...

Interrogating Individual Autoreactive Germinal Centers by Photoactivation in a Mixed Chimeric Model of Autoimmunity

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Chapters in this video

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Analysis of Somatic Hypermutation in the JH4 intron of Germinal Center B cells from Mouse Peyer's Patches

Analysis of Granulocyte-Macrophage Colony-Stimulating Factor-Producing T Helper Cells in a Mouse Model of Contact Hypersensitivity

Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis