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. G., 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=Engineering+of+a+monomeric+green-to-red+photoactivatable+fluorescent+protein+induced+by+blue+light.">Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light.</a> Nature biotechnology. 24 (4), 461-465 (2006).</li><li>Pereira, E. 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=Lymph+node+metastases+can+invade+local+blood+vessels,+exit+the+node,+and+colonize+distant+organs+in+mice.">Lymph node metastases can invade local blood vessels, exit the node, and colonize distant organs in mice.</a> Science (New York, NY). 359 (6382), 1403-1407 (2018).</li><li>Patterson, G. H., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photoactivatable+GFP+for+selective+photolabeling+of+proteins+and+cells.">A photoactivatable GFP for selective photolabeling of proteins and cells.</a> Science (New York, NY). 297 (5588), 1873-1877 (2002).</li><li>Victora, G. D., 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=Germinal+center+dynamics+revealed+by+multiphoton+microscopy+with+a+photoactivatable+fluorescent+reporter.">Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter.</a> Cell. 143 (4), 592-605 (2010).</li><li>Habuchi, S., 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=Reversible+single-molecule+photoswitching+in+the+GFP-like+fluorescent+protein+Dronpa.">Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (27), 9511-9516 (2005).</li><li>Zhou, X. X., Chung, H. K., Lam, A. J., Lin, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+control+of+protein+activity+by+fluorescent+protein+domains.">Optical control of protein activity by fluorescent protein domains.</a> Science (New York, NY). 338 (6108), 810-814 (2012).</li><li>Degn, S. 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. G., 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=Spontaneous+formation+of+germinal+centers+in+autoimmune+mice.">Spontaneous formation of germinal centers in autoimmune mice.</a> Journal of leukocyte biology. 70 (4), 578-584 (2001).</li><li>Sachs, D. H., Kawai, T., Sykes, M. <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+tolerance+through+mixed+chimerism.">Induction of tolerance through mixed chimerism.</a> Cold Spring Harbor perspectives in medicine. 4 (1), a015529 (2014).</li></ol>

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 &#181;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&#252;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>NaHCO3</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>NH4Cl</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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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

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

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

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 ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and ...

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 ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...