Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

Summary

Transgenic lck:eGFP zebrafish express GFP highly in T lymphocytes, and have been used to study T cell development and acute lymphoblastic leukemia. This line can be used to study B cells, which express lck at lower levels. This protocol describes purification of malignant and non-malignant B cells from lck:eGFP zebrafish.

Abstract

Zebrafish (Danio rerio) are a powerful model to study lymphocyte development. Like mammals, D. rerio possess an adaptive immune system that includes B and T lymphocytes. Studies of zebrafish lymphopoiesis are difficult because antibodies recognizing D. rerio cell surface markers are generally not available, complicating isolation and characterization of different lymphocyte populations, including B-lineage cells. Transgenic lines with lineage-specific fluorophore expression are often used to circumvent this challenge. The transgenic lck:eGFP line has been used to study D. rerio T cell development, and has also been utilized to model T cell development and acute lymphoblastic leukemia (T-ALL). Although lck:eGFP fish have been widely used to analyze the T-lineage, they have not been used to study B cells. Recently, we discovered that many zebrafish B cells also express lck, albeit at lower levels. Consequently, lck:eGFP B cells likewise express low levels of GFP. Based on this finding, we developed a protocol to purify B-lineage cells from lck:eGFP zebrafish, which we report here. Our method describes how to utilize a fluorescent-activated cell sorter (FACS) to purify B cells from lck:eGFP fish or related lines, such as double-transgenic rag2:hMYC; lck:eGFP fish. In these lines, B cells, particularly immature B cells, express GFP at low but detectable levels, allowing them to be distinguished from T cells, which express GFP highly. B cells can be isolated from marrow, thymus, spleen, blood, or other tissues. This protocol provides a new method to purify D. rerio B cells, enabling studies focused on topics like B cell development and B lymphocyte malignancies.

Introduction

Zebrafish offer powerful attributes, such as genetic manipulability, high fecundity, optical translucency, and rapid development that facilitate studying vertebrate development using genetic approaches. These advantages, together with the shared features of teleost and mammalian hematopoiesis, make D. rerio ideal for in vivo analyses of lymphopoiesis and lymphocyte function, from their earliest appearance in larvae throughout adulthood. Blood development in zebrafish relies upon well-conserved genetic processes that are shared with mammals, and these extend to the adaptive immune system. Additionally, molecular mechanisms governing lymphoid development are remarkably conserved between zebrafish and mammals¹.

Over the past 2 decades, transgenic D. rerio lines that label specific blood lineages and mutant lines deficient in these lineages have been created^2,3,4,5. One of these, the lck:eGFP transgenic line, uses the zebrafish lymphocyte protein tyrosine kinase (lck) promoter to drive GFP expression⁶. This gene, which is highly expressed by both T-lineage precursors and mature T lymphocytes, allows in vivo tracking of thymic T cell development and ex vivo purification of T-lineage cells by FACS⁷. Previously, we used this line in a forward-genetic ENU mutagenesis screen to identify germline mutants prone to T-ALL and to study somatically-acquired genetic events linked to T cell oncogenesis^8,9.

Recently, our laboratory further extended the utility of lck:eGFP zebrafish. In double-transgenic rag2:hMYC (human MYC), lck:eGFP D. rerio that are known to develop T-ALL ¹⁰, we discovered that B-lineage ALL also occur¹¹. Unlike T-ALL in this model, which fluoresce brightly due to high GFP expression, B-ALL are dimly-fluorescent due to low GFP levels, allowing fish with B-ALL to be distinguished grossly from those with T-ALL by fluorescent microscopy. This differential GFP expression also permits the separation of GFP^lo B-ALL cells from GFP^hi T-ALL cells using FACS¹¹. Moreover, low lck expression is not unique to zebrafish B-ALL, as human B-ALL also express low levels of LCK^11,12. Likewise, normal B-lineage cells of D. rerio, mice, and humans also express low levels of lck/Lck/LCK, with immature B cells having the highest expression^11,13. On a per cell basis, B-lineage cells in lckeGFP zebrafish or derivative lines express 1-10% as much GFP as T lymphocytes. These GFP^lo cells express characteristic B cell mRNAs such as pax5, cd79b, blnk, btk, ighm, ighz, and others, and can be purified from marrow, thymus, spleen, or peripheral blood¹¹. Therefore, both B- and T-lineage cells can be isolated from lckeGFP zebrafish, and in the case of rag2hMYC, lckeGFP animals, B- and T-ALL cells as well¹¹.

Here, we present our protocol to efficiently FACS-purify non-malignant B cells from lck:eGFP zebrafish, and non-malignant or malignant B cells of rag2hMYC;lck:eGFP fish, using various source tissues. Such cells can likewise be quantified by flow cytometry without FACS isolation, if desired. Discovery of low lck expression-and consequently, low GFP expression-by B cells opens new doors of experimental possibilities for lckeGFP zebrafish, such as in vivo B cell developmental studies. Thus, this transgenic line, first reported in 2004, has new life as we seek to utilize it to glean fresh insights concerning zebrafish adaptive immunity.

Protocol

All procedures involving zebrafish were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Oklahoma Health Sciences Center.

1. Isolating Non-malignant B and T Lymphocytes from Transgenic lck:eGFP Fish

1. Anesthetize the fish using 0.02% tricaine (MS-222) in fish system water.
2. Examine 2–6 month old fish for fluorescent thymi, which are located at the dorsomedial aspect of the branchial cavity of zebrafish and other teleosts¹⁴. Use an epifluorescence microscope (470/40 excitation wavelength and 525/50 emission filter) to detect GFP.
3. Prepare in advance 50 mL of filter-sterilized 1x Roswell Park Memorial Institute Medium (RPMI) containing 1% fetal bovine serum and 1% penicillin-streptomycin (sorting media). Unused sorting media can be stored at 4 °C for up to 2 months.
4. Euthanize the fish by placing it in a beaker containing 0.2% Tricaine for approximately 5 min, followed by ice bath immersion. Confirm death by the cessation of opercular (gill) movement.
5. Place the euthanized fish in a Petri dish and dissect lymphoid organs of interest, using the fluorescent microscope to dissect the GFP⁺ thymi¹⁴, and bright field microscopy settings to dissect the kidney marrow and spleen¹⁵. Results presented here were obtained using 3 month-old fish. Lymphocyte proportions and absolute numbers vary by age and genotype (see discussion for further detail).
6. Place each dissected organ in a 1.5 mL tube containing 500 µL of cold sorting media.
7. Homogenize the tissue on ice using a pestle micro-tube homogenizer.
8. Pass homogenized tissue through a 35 µm mesh filter to generate a single cell suspension. Keep the cells on ice until analysis.

2. Isolating Malignant Lymphocytes from Double-transgenic rag2:hMYC;lck:eGFP Zebrafish

1. Beginning at 2-4 months, microscopically screen rag2:hMYC; lck:eGFP fish for abnormal GFP patterns.
   NOTE: B cells are GFP^lo in the lck:eGFP background, so B-ALL requires practice to recognize; T-ALL is obvious, because T cells are GFP^hi. Consequently, the rag2:hMYC, lck:eGFP dual-transgenic line has two phenotypes: brightly-fluorescent T-ALL, which usually arise from the thymus and extend into the body, and dimly-fluorescent B-ALL.
   1. Using a fluorescent microscope, screen the fish using “low exposure” settings (200 ms, 2.4x gain) to identify bright T-ALL (Figure 1A) and “high exposure” (1.5 s, 3.4x gain) to identify dim B-ALL (Figure 1A). WT controls and pre-leukemic fish have GFP localized only in the thymus (Figure 1B).
2. Anesthetize and examine the fish as described in steps 1.1–1.2.
3. Categorize the fish based on the extent of GFP fluorescence, using a simple three category system:
   NOTE: Level 1: Fluorescence appears as a thymic tumor with only limited local spread.
   Level 2: Fluorescence appears beyond the thymus, involving <50% of the body.
   Level 3: Fluorescence extends beyond 50% of the body.
4. Separate the fish with ALL from those without cancer. Monitor pre-leukemic fish (i.e., fish without GFP⁺ tumors) once monthly for the development of new ALL. By ~9 months, all rag2:hMYC, lck:eGFP fish develop T-ALL, B-ALL, or both.
5. For T- or B-ALL cell isolation, select Level 3 fish (ALL involving >50% of the body), which yield more than 2 x 10⁶ ALL cells, and typically many more.
6. Euthanize the fish as in step 1.4.
   NOTE: There are two methods to obtain ALL cells: whole body homogenization and a peritoneal wash technique¹⁶. The methods differ in the absolute number of cells collected for sorting, and thus, the amounts of FACS time required and cost (see discussion for further detail).
7. For either method, first place the euthanized fish in a Petri dish and using a razor blade, remove the head including the thymic region. This can be processed separately, if desired, or used for histological staining.
8. Peritoneal Wash Method
   1. Using a P1000 pipette, wash the fish peritoneal cavity with 500 µL of cold sorting media, collecting the cells and media in a 5 mL tube.
   2. Using a fresh pipette tip, inject an additional 200–300 µL of cold sorting media into the body cavity. Then, using the tip of the pipette, apply gentle pressure to the fish body to extrude the cells out of the body cavity. Collect this media and add to the 5 mL tube.
   3. Repeat step 2.8.2 2–3 times. Keep the collected cells in sorting media on ice.
   4. Filter the cell suspension though a 35 µm mesh filter prior to flow cytometry/FACS and keep the cells on ice until analysis.
9. Whole Body Homogenization
   1. After removing the fish head, place the body in a 1.5 mL tube containing 200 µL of sorting media.
   2. Homogenize the body using a pestle micro-tube homogenizer.
   3. Add an additional 300 µL of cold sorting media. Filter the cell suspension though a 35 µm mesh filter. Add sorting media as needed to wash all cells through the filter, until only tissue debris remains on the filter. Keep the cells on ice until analysis.

3. Cytometric Analysis of Normal or Malignant B Lymphocytes

1. Set flow cytometric analysis and/or FACS parameters according to manufacturer’s guide.
2. Acquire desired number of events to initially characterize the sample. Analyze 1 x 10⁴ to 5 x 10⁴ events prior to sorting to determining specific gates for subsequent sorting steps.
   1. Determine gates: Define lymphocyte and progenitor cell gates using forward scatter (FSC) and side scatter (SSC) parameters, excluding cellular debris (Figure 2A-C)¹⁷. FSC and SSC correspond to cell size/diameter and granularity, respectively.
      NOTE: GFP^-, GFP^lo, and GFP^hi cells differ 10-to-100-fold in terms of their GFP fluorescence intensity, making separation of these populations straightforward (Figures 2-5). Live-dead discrimination can be assessed at this point using propidium iodine (PI) or 7-aminoactinomycin D (7-AAAD) viability staining. Previous experiments with PI typically demonstrate >95% viability of GFP⁺ cells after FACS.
   2. Exclude the cell doublets, according to the parameters of the FACS machine being used.
   3. Within the lymphoid and/or precursor gates, determine the number and percentage of GFP⁺ cells.
3. Use phycoerythrin (PE) and GFP intensities, define gate for GFP^- vs. GFP^lo vs. GFP^hi cells.
   NOTE: B cells exhibit dim GFP fluorescence and T cells show bright GFP in lck:eGFP lines. Many lymphoid organs or tumor samples contain both GFP⁺ populations. B-lineage cells/B-ALL are GFP^lo and T-lineage cells/T-ALL are GFP^hi. Define gates to distinguish between GFP^-, GFP^lo, and GFP^hi cells, and collect these populations separately (Figure 2A-C, Figure 3A-C, Figure 4A and Figure 5A).
4. Sort each cell population into different 15 mL polypropylene tubes containing 2 mL of sorting media, or directly into different 1.5 mL tubes containing an appropriate buffer for further analyses (e.g., RNA, DNA, or protein extraction, allo-transplantation, etc.).
5. Keep purified cells on ice prior to further analyses.

Results

We used flow cytometry to analyze and FACS to isolate GFP^lo and GFP^hi cells from thymus, kidney marrow, and spleen of lck:eGFP transgenic zebrafish. Analysis of 3-month-old fish revealed the thymus contained mostly GFP⁺ lymphocytes. GFP⁺ cells were largely confined to the lymphoid gate previously described by Traver et al.¹⁷. Two distinct GFP⁺ populations, GFP^loand GFP^hi,^can<...

Discussion

We developed and provide a protocol to isolate B cells from lck:eGFP transgenic zebrafish, adding this to other D. rerio models with B-lineage labels^3,4. Somewhat surprisingly, the identification of GFP^lo B cells in this line went unnoticed since its description in 2004.Generally, lck is considered to be T cell-specific⁶, but recent studies found unexpected lck expression by natural killer and...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35 µm mesh	Sefar Filter technology	7050-1220-000-13
5 mL Polystyrene round-Bottom tube with cell-strainer cap	Falcon Corning Brand	352235
50 mL conical tube	VWR international	525-0448
AZ APO 100 Fluorescent microscope	Nikon
Cytoflex	Beckman Coulter
DS-Qi1MC camara	Nikon
Ethyl 3-aminobenzoate methansesulfonate; MS-222	Sigma	E-10521
FACSJazz	BD Biosciences
Fetal bovine Serum	Thermo Fisher	10437028
FlowJo v10.2	FlowJo, LLC
lck:eGFP			See Langeneu et al., 2004
NIS Elements software	Nikon	Version 4.13
Penicilin-Streptomycin	Sigma	P4333
Pestle micro-tube homogenizers	Electron Microscopy Sciences	64788-20
Plastic Transfer pippetes
rag2:hMYC-ER			See Gutierrez et al., 2011
RPMI Media 1640 1x	Life Technologies	11835-030