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 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.
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, 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 ‘plug-and-play’ 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.
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 ‘plug-and-play’ 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 ‘dark’. 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 authors have nothing to disclose.
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).
Antibody, 9D11-A568 | In-house generated | 9D11 hybridoma, kindly provided by MC Carroll, labeled with kit: Biotium 92255 | |
Antibody, 9D11-A647 | In-house generated | 9D11 hybridoma, kindly provided by MC Carroll, labeled with kit: Nordic Biosite ABD-1031 | |
Antibody, FITC anti-mouse CD45.1 | Biolegend | 110705 | |
Antibody, Pacific Blue anti-mouse/human GL7 Antigen (T and B cell Activation Marker) | Biolegend | 144613 | |
Antibody, PE anti-mouse CD169 (Siglec-1) | Biolegend | 142403 | |
Antibody, PE/Cy7 anti-mouse CD38 | Biolegend | 102717 | |
Antibody, PerCP/Cy5.5 anti-mouse/human CD45R/B220 | Biolegend | 103235 | |
Capillary tube, Mylar-wrapped, heparinized | Fisher Scientific | 211766 | |
Cell strainer, 70 µm | Falcon | 352350 | |
Conical tubes, 50 mL | Falcon | 352235 | |
Cover slip, square, 22×22 mm, 0.13-0.17 mm | Thermo Fisher Scientific | 22X22-1 | |
EDTA | Merck | 1,084,180,250 | |
Ethanol, 70% | VWR | 8301.360 | |
Fetal bovine serum | Life Technologies | 10270106 | |
Flow cytometer, FACS Canto II | BD Biosciences | 338962 | |
Flow cytometer, LSRFortessa SORP | BD Biosciences | – | Special order product with 4 lasers (405 nm, 488 nm, 561 nm and 640 nm) |
Grease, high vacuum, Dow Corning | VWR | DOWC1597418 | |
Hemocytometer, Burker-Türk | VWR | 630-1544 | |
Isoflurane, IsoFlo vet. | Orion Pharma | 9658 | |
Lymphocyte separation medium (Lympholyte-M Cell Separation Media) | Cedarlane | CL5035 | |
Microcentrifuge tube, 1.5 mL (Eppendorf) | Sarstedt | 72.690.550 | |
Microscope, Two-photon | Prairie Technologies (now Bruker) | – | Special order Ultima In Vivo Two Photon Microscope |
Mortar w. lip, unglazed, 75 ml | VWR | 410-0110 | |
NaHCO3 | Merck | 1063290500 | |
Needle, 18 gauge | BD Medical | 304622 | |
NH4Cl | VWR | 87,769,290 | |
PBS | Sigma | d8537 | |
Pestle homogenizer | VWR | 47747-358 | |
Pestle, unglazed, 175 mm | VWR | 410-0122 | |
Pipette, Serological, 10 ml | VWR | 612-3700 | |
Pipette, transfer, plastic | Sarstedt | 861,172,001 | |
Plastic paraffin film (Parafilm M) | Bemis | PM996 | |
Plate, 96-well | Falcon | 353910 | |
Surgical forceps, Student Dumont #5 Forceps | FST – Fine Science Tools | 91150-20 | |
Surgical forceps, Student Dumont #7 Forceps | FST – Fine Science Tools | 91197-00 | |
Surgical scissors, Student Fine Scissors, Straight | FST – Fine Science Tools | 91460-11 | |
Syringe, 10 mL | Terumo | SS-10ES1 | |
Syringe, 20 mL | Terumo | SS-20ES1 | |
Syringe, 5 mL | Terumo | SS-05S1 | |
Syringe, Insulin, 0.3 cc | BD Medical | 324827 | |
Tribrissen vet. 24% inj., containing 200 mg sulfadiazin and 40 mg trimethoprim/ml | MSD Animal health | 431577 | |
Trypan blue solution, 0.4% | VWR | K940-100ML | |
Viability dye, eBioscience Fixable Viability Dye eFluor 780 | Thermo Fisher Scientific | 65-0865-14 |