Mice were housed and bred in specific pathogen-free (Spf) conditions in the animal facilities at Columbia University Irving Medical Center (CUIMC). OT-II (B6.Cg.Tg(TcraTcrb)425Cbn/J) 26 and CD45.1(B6.SJL-PtprcaPepcb/BoyJ) mice were purchased from Jackson Laboratories while C57BL/6 mice were purchased from Charles River. TCF-1 (p45) transgenic mice expressing the long isoform of TCF-133 were generously provided by Dr. Hai-Hui Xue and Dr. Werner Held. CD45.1 mice were bred to generate CD45.1 congenic hosts and CD45.2 mice were bred to use for phosphoflow studies. OT-II mice were also crossed to CD45.1 mice and TCF-1 Tg OT-II mice were generated by breeding TCF-1 Tg male to OT-II females. All infant mice were used at 10–14 days of age, and adult mice were used at 6 weeks and older. Infections were performed in BSL-2 level biocontainment animal facilities. All animal studies were approved by Columbia University IACUC.
T cell adoptive transfer and Influenza infection
To isolate CD4 T cells from infant and adult mice, spleen and lymph nodes were harvested and processed to generate a single cell suspension. Single litters of at least 5 pups were combined to generate sufficient numbers of CD4+ T cells for each experiment. CD4 T cells were purified by negative magnetic selection (Stemcell Technologies, Cambridge, MA). For co-transfers, 250,000 cells in 100µl of PBS containing 1:1 ratio of adult and infant OT-II T cells were transferred into adult congenic B6 or CD45.1 host mice retro-orbitally one-day prior (day − 1) to infection. For 4-day in vivo proliferation experiments, OT-II T cells were labeled with cell proliferation dye (CPD) (ThermoFisher) and 500,000 each of infant and adult T cells were transferred into host mice. At day 0, host mice were infected intranasally (i.n.) with 2000 TCID50 of a recombinant PR8-OVA strain expressing the OVA323–339 peptide (sequence ISQAVHAAHAEINEAGR; provided by Dr. Paul Thomas, St. Jude Children’s Research Hospital, Memphis, TN)54.
Mediastinal lymph nodes and lungs were harvested at indicated days of infections and processed to generate single cell suspension as previously described12, 55. A list of all antibodies used for flow cytometery in this study is in Supplemental Table 2. In general, cells were first stained with surface markers at 30 min room temperature (RT) and an additional 30 min on ice. Cells were then fixed and permeabilized with Foxp3 fix/perm (Tonbo) before being stained for intracellular markers for 30 min on ice. For OTII TCR specific tetramer staining, cells were incubated with I-A(b)AAHAEINEA tetramers in 50µl for 1hr in 37ºC incubator before addition of other surface markers with additional 30 min at RT before fixation as described above. For Annexin V staining, cells were stained for surface markers and washed before staining for Annexin V for 20 min at RT. Cells were washed and quickly analyzed in a cytometer. All flow cytometry samples were acquired using LSRII cytometry (BD) with FACSDiva software. All cell sorting were performed in BD Influx cell sorter. Data were analyzed using FSC Express (DeNovo software).
Human donor samples
Human adult lymph nodes were obtained from deceased (brain dead) organ donors through an approved protocol and material transfer agreement with LiveOnNY as described 56. Human infant lymph nodes were obtained from deceased (brain dead) organ donors under the Human Atlas for Neonatal Development and Early Life – Immunity (HANDEL-I) tissue procurement program. A list of all donors used and their ages is in Supplemental Table 1. Tissues were obtained and processed for single-cell suspensions, as previously described 57. The study does not qualify as human subjects research as determined by Columbia University institutional review board (IRB), because tissue samples were obtained from deceased individuals.
Mouse T cell stimulations
For activation of OT-II CD4+T cells, OT-II cells were isolated from spleens and LNs of infant and adult mice as described above, labeled with CPD and cultured 1:1 with antigen presenting cells (APC; T-depleted splenocytes from adult CD45.1 or C57BL/6 mice in the presence of chicken ovalbumin peptide epitope recognized by the OT-II TCR (ISQAVHAAHAEINEAGR) (InVivoGen) in complete RPMI media (10% fetal bovine serum (FBS), 1% penicillin – streptomycin-glutamine (PSQ), 25mM HEPES). Controls were T cell and APC cultured without peptide. For short-term stimulations for assessing TCR signaling, infant or adult OT-II cells were stimulated with 10µg of peptide-pulsed T cell-depleted splenocytes. For measuring cytokine production by lung T cells, total lung cells were isolated as described 12, 55 from mice at day 11 post-infection and stimulated in the presence of 10µg of peptide for 6–16 hr in complete RPMI. Protein transport inhibitor (ThermoFisher) was added 2 hours into stimulation. After stimulation cells were first stained with surface markers, then fixed and permeabilized with Foxp3 fix/perm (Tonbo) before being stained for intracellular cytokine for 30 min on ice.
Human T cell stimulation.
Naïve (CD45RA+CCR7+) CD4+ T cells were sorted from lymph nodes (LN), labeled with cell proliferation dye (CPD), and allowed to rest overnight before stimulation. Cells (100,000 cells/well) were cultured with carboxylate modified latex (CML) microbeads (ThermoFisher) functionalized with 10µg/mL of anti-CD28 (BioXcell) and anti-CD2 (ThermoFisher) monoclonal antibodies and 0.1–10µg/mL of anti-CD3 monoclonal antibody (BioXcell), at 1:1 in complete AIM V media (10% human serum, 1% PSQ, Glutamax, and IL-2 (50ng/ml)). Preparation of CML microbeads used for stimulation was done as described 58, 59, 60.
For short-term (0-8hr) stimulation, total lymph node T cells were pre-stained with surface makers CD4, CD3, CD45RA, and CCR7 before being plated in complete RPMI media. Cells were stimulated using beads coated with 10µg/mL of anti-CD3/ anti-CD28/ anti-CD2 (Miltenyi) for indicated time. At each time point, cells were harvested and further stained for surface marker (CD3) before being fixed and permeabilized for intracellular marker Nur77.
TCR signaling analysis using Phosphoflow
C57BL/6 wild-type (WT) mice were used for examination of phosphoERK1/2 expression upon stimulation. Cells were isolated with magnetic negative enrichment using CD3 isolation kit (StemCell). CD3 enriched cells were kept ice cold until time of stimulation in 37ºC water bath. Cells were first labeled with anti-CD3ε (10µg/ml) and anti-CD28 (5µg/ml) (BioXcell) antibodies in ice cold PBS for 30min on ice. Cells were washed and then stained with cross-linker goat anti-Armenian hamster IgG (Jackson Immuno Research Laboratories) (20µg/ml) and anti-CD44/ anti-CD62L antibodies for additional 30min on ice. Tubes were then placed in water bath to start stimulation and lyse/fix buffer (BD) was added to stop stimulation at indicated times. Cells were permeabilized with Perm III buffer (BD) before being stained for CD3, CD4, CD8, and pERK1/2.
Analysis of T cell and APC conjugates
To image immunological synapse (IS) formation between OT-II T cells and APC, CPD labeled OT-II cells were pre-stained with anti-CD4 antibodies at 4oC and washed with ice cold PBS before stimulation. OT-II cells were then placed with 10µg of peptide pulsed T cell-depleted CFSE-labeled splenocytes and spun down at 100 x g for 1min to enable conjugation. Stimulation was started by placing tubes into 37ºC water bath. After each stimulation time point, cells were fixed with cytofix (BD) and placed on ice overnight. Next day, cells were washed and then permeabilized with 1x Triton-X solution (Sigma-Aldrich) for 15min RT. After wash, cells were stained with the rest of the markers in RT before being analyzed by ImageStream X (Amnis, Seattle, WA).
Images were acquired at 60x magnification on a four-laser ImageStream X (Amnis, Seattle, WA) imaging flow cytometer with INSPIRE software. Data was analyzed using IDEAS v6. Using Area measurement, doublet cells were gated and acquired for all samples. From doublet cells, events with both CPD and CFSE dye were gated to identify conjugates of T cell: APC. Previously published analysis from Au-Wabnitz et al and Abrahamsen et al was adapted 49, 50. Using the ‘threshold’ mask function on CPD channel, T cell were highlighted. With ‘interface’ mask function on CPD, the immune synapse of T cells was highlighted. Formula used to quantify accumulation of molecules in the IS:
To adjust for background differences existing before stimulation, quantification was normalized to unstimulated controls.
All data were compiled and statistical analysis performed using Prism software (GraphPad Software). Results are shown with SEM unless otherwise indicated. Significance was determined using Student’s t-test or two-way repeated measure ANOVA with multiple comparison testing using the Sidak method. Result was designated significant when p value was p ≤ 0.05.