Monitoring the Interaction Between Dendritic Cells and T Cells In Vivo with LIPSTIC

Abstract

Interactions between different cell types are key for immune function. Traditionally, interactions have been investigated in vivo by intravital two-photon microscopy, but the molecular characterization of the cells participating in a specific interaction is limited by the inability to retrieve the cells for downstream analysis. We recently developed an approach to label cells undergoing specific interactions in vivo, which we called LIPSTIC (Labeling Immune Partnership by Sortagging Intercellular Contacts). Here, we provide detailed instructions on how to track CD40-CD40L interactions between dendritic cells (DCs) and CD4⁺ T cells using genetically engineered LIPSTIC mice. This protocol requires expertise in animal experimentation and multicolor flow cytometry. Once mouse crossing has been achieved, it takes 3 days or more to complete, depending on the kinetics of the interactions that the researcher wishes to investigate.

1. Introduction

Cell-cell interactions are essential for several biological processes. In the immune system, multiple functions rely on direct cell-cell contacts, including positive and negative selection of T lymphocytes in the thymus, presentation of antigen to T cells by antigen-presenting cells (APCs), killing of infected cells by cytotoxic CD8⁺ T cells, and help to B cells provided by CD4⁺ T cells for antibody affinity maturation. Despite such an essential role of direct cell-cell signaling in many immunological processes, studying cell-cell interactions in vivo remains a challenge.

To facilitate the study of cell-cell interactions in vivo, we recently developed a novel technology called LIPSTIC (Labeling Immune Partnerships by SorTagging Intercellular Contacts) [1, 2] that allows one to enzymatically label cell-cell interactions in vivo and subsequently retrieve interacting cells for downstream analysis. Here, we describe the LIPSTIC approach and provide technical details for its use in monitoring CD40-CD40L interactions between dendritic cells (DCs) and CD4⁺ T cells in vivo using genetically engineered mice.

LIPSTIC is based on S. aureus enzyme Sortase A (SrtA), a transpeptidase capable of mediating the covalent ligation of a substrate containing the recognition sequence LPXTG (where X is any amino acids) to an N-terminal glycine residue. Upon recognition of the LPXTG motif, SrtA catalyzes the hydrolysis of the peptide bond between threonine and glycine and forms an acyl intermediate between the cysteine present on its catalytic pocket and the substrate threonine. The substrate can then be covalently ligated to an N-terminal glycine, thanks to the formation of a novel peptide bond [3]. For LIPSTIC, we genetically fused SrtA or a five-glycine (G5) tag to the extracellular portion of a ligand and receptor of interest. Upon ligand-receptor interaction, SrtA mediates the covalent ligation of a labeled substrate (e.g., a biotinylated or fluorescently labeled LPXTG peptide) to the G5-tagged molecule. After the interaction has occurred, the G5 expressing cell participating in interaction can be readily identified and retrieved by flow cytometry based on the presence on its surface of the covalently ligated label (Fig. 1) [1].

Fig. 1. LIPSTIC labeling to track cell-cell interactions. Schematic representation of the LIPSTIC approach.

We employed the LIPSTIC approach to monitor CD40-CD40L interaction between DCs and CD4⁺ T cells. In our published work [1], we generated two novel mouse lines: Cd40^G5, which constitutively expresses CD40 carrying a G5 tag at its extra-cellular portion, and Cd40lg^SrtA, which upon Cre recombination expresses CD40L fused to SrtA. Using these mice, it is possible to perform in vivo LIPSTIC labeling during the course of the immune response elicited by subcutaneous protein immunization or by transfer of antigen-pulsed DCs. Our published observations indicate that CD40-CD40L interactions between dendritic cells and CD4⁺ T cells take place in two different modalities during the initial phases of the immune response: an early phase (at ˜12 h post T cell transfer), when T cells label exclusively DCs that carry the cognate antigenic peptide, and a late phase (at ˜48 h after T cell transfer), when non-cognate contacts between T cells and bystander DCs can also be observed.

In the following protocol, we provide details on how to track the interactions between DCs and antigen-specific CD4⁺ T cells in the context of an immune response induced by immunization with ovalbumin (OVA) as model antigen. For this purpose, this protocol is divided into four experimental sections: (i) the adoptive transfer of OVA-specific Cd40lg^SrtA/SrtA CD45.1/1 CD4-Cre⁺ OT-II CD4⁺ T cells into Cd40^G5/G5 hosts, (ii) the induction of an immune response in Cd40^G5/G5 hosts by immunization with OVA, (iii) the injection of SrtA substrate to achieve in vivo LIPSTIC labeling, and (iv) the analysis of LIPSTIC-labeled cells by flow cytometry. A schematic representation of the experimental design is provided in Fig. 2a.

Fig. 2. LIPSTIC labeling of CD40-CD40L interactions between T cells and dendritic cells in the popliteal lymph node.

(a) Experimental design for (b), (c). CD45.1 is encoded by Ptprc^a; homozygotes are indicated as CD45.1/1. (b) Flow cytometric analysis of popliteal LN cells showing biotin labeling of endogenous dendritic cells at 12 h post immunization (left) or nonimmunized control (right). (c) Flow cytometric analysis of popliteal LN cells showing biotin labeling of adoptively transferred Cd40lg^SrtA/Y CD45.1/1 CD4-Cre OT-II CD4⁺ T cells at 12 h post immunization (left) or nonimmunized control (right)

2. Materials

2.1. Mouse Strains and Animal Procedures

1. Cd40^G5/G5 and Cd40lg^SrtA/SrtA CD45.1/1 CD4-Cre OT-II mice (see Note 1).

2. Rodent anesthesia system with isoflurane.

3. CO₂ euthanasia chamber (only if this euthanasia method is approved by your institution’s animal ethics committee).

4. Scissors and forceps for spleen and lymph node harvesting.

5. 1 mL insulin syringes with 28-G 1/2 in. needle.

2.2. Isolation and Transfer of Naïve CD4⁺ T Cells

1. PBE buffer: PBS, 0.5% bovine serum albumin (BSA), 2 mM EDTA.

2. Ammonium-chloride-potassium (ACK) lysis buffer for red blood cell lysis.

3. 70 μm strainers.

4. 3 mL syringes.

5. 50 mL Falcon tubes.

6. Naïve CD4⁺ T cell isolation kit (Miltenyi Biotec, see Note 2).

7. Hemocytometer.

2.3. Immunization

1. Imject Alum.

2. 1 mg/mL Ovalbumin solution (see Note 3).

3. PBS.

4. Rotator or shaker.

2.4. In Vivo LIPSTIC Labeling and Analysis of LIPSTIC-Labeled Cells by Flow Cytometry

1. 20 mM Biotin-LPETGS peptide in PBS (see Note 4). Dissolve lyophilized peptide in PBS to a final concentration of 20 mM. Sterilize solution by filtration trough a 0.22 μm filter. Aliquot solution and store at −80 °C for long-term storage. For in vivo labeling, prepare a 2.5 mM solution in PBS.

2. Collagenase digestion buffer: RPMI 2% fetal bovine serum (FBS), 20 mM HEPES, 400 U/mL type IV collagenase.

3. Plastic pestle for 1.5 mL microfuge tubes.

4. 1.5 mL microfuge tubes.

5. PBE buffer.

6. Antibodies and viability dyes for the analysis of interacting DCs and CD4⁺ T cells: CD16/32, CD11c, CD11b, MHC II, Biotin, XCR1, B220, NK1.1, CD3, Zombie NIR fixable viability dye (see Table 1 and Note 5).

7. Flow cytometers equipped with lasers and emission filters suitable for the analysis of cells stained with the dyes listed in the antibody panel (see Note 6).

8. Cytofix (BD Bioscience).

9. FlowJo software for flow cytometry data analysis.

Table 1. Antibodies for DCs and CD4⁺ T cell characterization by flow cytometry.

Marker	Conjugation	Clone	Dilution	Manufacturer	Cat. N.
CD16/32	/	2.4G2	1 Pg/mL	Bio X Cell	101242
CD11c	APC	N418	1:800	Biolegend	117310
CD11b	BV711	M1/70	1:800	Biolegend	101242
MHC-II	BV421	M5/114.15.2	1:400	Biolegend	107632
Biotin	PE		1:11	Miltenyi Biotec	130-090-756
XCR1	PerCP/Cy5.5	ZET	1:200	Biolegend	148208
CD45.1	PE/Cy7	A20	1:200	Biolegend	110730
B220	BV785	RA3-6B2	1:400	Biolegend	103246
NK1.1	BV785	PK136	1:400	Biolegend	108749
CD3	BV785	17A2	1:400	Biolegend	100355
Zombie	NIR	/	1:1000	Biolegend	423106

3. Methods

3.1. Adoptive Transfer of Ovalbumin-Specific Naïve Cd40lg^SrtA/SrtA CD45.1/1 CD4-Cre OT-II CD4⁺ T Cells into Cd40^G5/G5 Hosts (See Note 7)

1. Euthanize Cd40lg^SrtA/SrtA CD45.1/1 CD4-Cre OT-II mice using the method approved by your institution’s animal ethics committee.

2. Harvest the spleen using scissor and forceps.

3. Mesh the spleen through 70 μm strainer into 50 mL falcon tube using the plunger of a 3 mL syringe.

4. Rinse the strainer with PBE buffer and spin cells at 300 × g for 5 min.

5. Remove the supernatant and lyse the red blood cells by adding 1 mL of ACK lysis buffer per spleen. Incubate for 3–5 min at room temperature (RT). Stop the lysis by adding 4 mL of PBE buffer. Filter the cell suspension through 70 μm strainer.

6. Spin the cells at 300 × g for 5 min.

7. Perform the isolation of CD4⁺ T cells using Miltenyi Biotec CD4⁺ T cell isolation kit. Follow exactly the Miltenyi Biotec CD4⁺ T cell isolation kit protocol.

8. Using the hemocytometer, count the number of CD4⁺ T cells retrieved. The typical yield is 8–10 × 10⁶ cells/spleen.

9. Carefully resuspend cells in PBS at a final concentration of 3 × 10⁶ cells/mL.

10. Using an insulin syringe, inject 3 × 10⁵ naïve (100 μL) CD4⁺ T cells into Cd40^G5/G5 mouse intravenously using the method approved by your institution’s animal ethics committee.

3.2. Immunization of Cd40^G5/G5 Hosts (See Note 8)

In general, alum should be one third of the final immunization mix. Here, we provide details for a 600 μL of immunization mix.

1. Vortex alum extensively.

2. Mix 200 μL of alum with 240 μL of ovalbumin (OVA) (1 mg/mL) and 160 μL of PBS.

3. Incubate the immunization mix at 4 °C on the rotator or shaker for at least 30 min.

4. For immunization of popliteal lymph nodes, using an insulin syringe inject 25 μL (10 μg of OVA) of immunization mix subcutaneously into the hind footpad of a Cd40^G5/G5 mouse.

3.3. Injection of SrtA Substrate to Achieve In Vivo LIPSTIC Labeling (See Note 9)

In order to capture solely antigen-specific T cell-DC interactions labeling needs to be performed within 12 h of immunization [1].

1. Briefly anesthetize the mice with isoflurane before each injection.

2. Inject Biotin-LPETG substrate subcutaneously into the hind footpad (20 μL of 2.5 mM substrate solution in PBS, equivalent of 50 nmol of substrate).

3. Repeat steps 1 and 2 for a total of six times with 20 min interval between injections. Wait 40 min after the last injection before euthanasia.

3.4. Analysis of LIPSTIC-Labeled Cells by Flow Cytometry

1. Euthanize mice using the method approved by your institution’s animal ethics committee.

2. Using forceps, carefully harvest popliteal lymph nodes and place them in 1.5 mL microfuge tube containing 200 μL of collagenase digestion buffer.

3. Incubate for 30 min at 37 °C.

4. Using a plastic pestle, gently macerate the LN against the wall of a 1.5 mL microfuge tube (see Note 10).

5. Add 600 μL of PBE buffer.

6. Pass the digested lymph node through a 70 μm strainer into a 1.1 mL microtube.

7. Spin cells down at 300 × g for 5 min. Remove supernatant.

8. Resuspend cells in 100 μL of PBE buffer with 1 μg/mL of anti-CD16/32 antibody for 5 min at room temperature.

9. Without washing, add 100 μL of 2× antibody solution in PBE buffer, for a final volume of 200 μL. Incubate at 4 °C for 15 min.

10. Add 800 μL of PBS. Spin cells down at 300 × g for 5 min. Remove supernatant.

11. Stain cells in PBS with Zombie fixable viability dye according to the manufacturer’s instructions.

12. Fix cells with Cytofix (BD Biosciences) according to the manufacturer’s instructions. If cells will be sorted for subsequent RNA extraction or functional assays, omit the fixation step.

13. Acquire samples using a flow cytometer equipped with appropriate lasers. Collect 1–2 million events per sample.

14. Analyze data using FlowJo software. To identify classical endogenous DCs, apply the following gating strategy (Fig. 3a, b):

    • Exclude doublets based on FSC and SSC parameters (area A, height H, width W).
    • Exclude dead cells based on Zombie Dye (ZD) fluorescence intensity. Live cells are ZD⁻.
    • Gate on Lin⁻ (B220⁻, CD3⁻ and NK1.1⁻)MHC-II⁺ cells.
    • Based on this gating strategy in the mouse lymph node two populations of conventional DCs with different expression of CD11c and MHC-II can be identified: CD11c^high MHC-II^int and CD11c^int MHC-II^high.
    • In the identified DC populations, analyze biotin fluoresce signal as well as CD11b and XCR1 to characterize labeled DCs.
    To identify adoptively transferred CD45.1/1 Cd40lg^SrtA/SrtA CD4-Cre OT-II T cells apply the following gating strategy (Fig. 3a, c):

    • Exclude doublets based on FSC and SSC parameters (area A, height H, width W).
    • Exclude dead cells based on Zombie Dye (ZD) fluorescence intensity. Live cells are ZD⁻.
    • Gate on MHC-II⁻ Lin⁺ cells.
    • Gate on CD45.1⁺ cells. This population corresponds to adoptively transferred T cells.

Fig. 3. Gating strategy for the analysis of endogenous dendritic cells and adoptively transferred T cells.

(a) Gating strategy to exclude doublets and dead cells. ZD: Zombie viability dye. (b) In the “Live” gate, conventional DCs are identified as Lin⁻ (B220⁻, NK1.1⁻, CD3⁻) MHC-II⁺. In the mouse lymph node, two populations of conventional DCs with differential expression of CD11c and MHC-II can be identified: CD11c^highMHC-II^int and CD11c^highMHC-II^int. (c) In the “Live” gate, adoptively transferred T cells are identified as Lin⁺ MHC-II⁻ CD45.1⁺