Mapping Infant Immunity with Minimal Input: Integrative Single-Cell and Multiomic Profiling

Our research defines how early-life immunity adapts after birth using longitudinal high-dimensional multiomic profiling from small blood volumes. This protocol can be applied to neonatal and pediatric studies for longitudinal immune profiling from limited biospecimens across health, infection and inflammation. To begin, quickly thaw a vial containing lysed infant red blood cells in a 37-degrees-Celsius water bath.

Transfer the thawed cells to a 15-milliliter conical tube containing 10 milliliters of warm complete RPMI medium at 37 degrees Celsius. Centrifuge the tube at 400 g for five minutes at room temperature, and discard the supernatant. Re-suspend the cell pellet in three milliliters of complete RPMI medium and count the total number of cells.

Dilute the sample if the concentration is too high to count. After counting the cells, repeat the centrifugation step and re-suspend the cells in complete RPMI medium to obtain a concentration of one million cells per milliliter. Transfer 100 microliters containing 0.1 million cells into each well of a 96-well V-bottom plate.

Centrifuge the plate at 764 g for two minutes at four degrees Celsius. Discard the supernatant. Next, add 100 microliters of live dead dye stock diluted one to 2, 000 in PBS with FC block and monocyte block diluted one to 100 to all samples except the unstained control.

Incubate the samples for 15 minutes at four degrees Celsius. To wash the cells, add 100 microliters of PBS. Centrifuge the plate at 764 g for two minutes at four degrees Celsius, and carefully discard the supernatant.

Add 100 microliters of surface antibody cocktail, diluted one to 200 in fluorescence-activated cell sorting, or FACS buffer, to all samples except the unstained control. Incubate for 30 minutes at four degrees Celsius. Then wash the cells with 100 microliters of FACS buffer, centrifuge and discard the supernatant.

Fix the cells by adding 100 microliters of 4%paraformaldehyde to each sample. Incubate for 15 minutes at room temperature in the dark, then wash the cells again, and re-suspend in 200 microliters of FACS buffer. Prepare UltraComp compensation beads as single-color controls.

Stain the single-color control beads using the same procedure as the samples, including fixation if the samples were fixed. Also, prepare fluorescence minus one or FMO controls in the same manner. Run the samples, single-color controls, unstained controls and fluorescent-minus-one controls on a conventional or spectral flow cytometer.

Open FlowJo and select the sample of interest to analyze. Set gates using appropriate isotype and FMO controls. Gate leukocytes using FSC versus SSC to exclude debris, and select cells by size and granularity.

Exclude doublets using FSCA versus FSCH. Gate live cells using a viability dye to exclude dead cells. From live single cells, divide into CD33-negative lymphocytes and CD-33-positive myeloid cells.

Within CD33-positive cells, identify CD14-positive monocytes. Then within CD33-positive cells, identify CD14-negative, CD11C-positive dendritic cells. Now, among CD33-negative lymphocytes gait CD3-positive T cells and CD3-negative, CD56-positive natural killer cells.

Within CD3-positive T cells, identify CD8-positive and CD4-positive subsets and identify CD3-negative, CD56-negative, CD19-positive B cells. To begin, thaw one vial of cryo-preserved red blood cell lysed cells in a 37-degrees-Celsius water bath. Transfer the suspension to a 15-milliliter conical tube containing 10 milliliters of T-cell media and centrifuge at 430 g for five minutes at four degrees Celsius.

Discard the supernatant. After re-suspending the cell pellet with one milliliter of PBS, add another nine milliliters of PBS to the sample. Centrifuge the tube at 430 g for five minutes at four degrees Celsius and discard the supernatant.

Count the cells and assess viability using trypan blue exclusion. If viability is less than 70%proceed to use a dead cell removal kit as follows. First, prepare 1x-binding buffer by diluting 20x-binding buffer stock solution with sterile double-distilled water.

Re-suspend the cell pellet in 100 microliters of dead cell removal microbeads per 10 to the power of seven total cells. Mix well and incubate for 15 minutes at room temperature. Place the magnetic separation column into the appropriate magnetic stand, along with a collection tube.

Then equilibrate the column by rinsing with 500 microliters of 1x-binding buffer. Bring the cell suspension volume to 500 microliters with 1x binding buffer. After replacing the collection tube, apply the cell suspension to the column and collect the flow-through in the new 1.7-milliliter microcentrifuge tube.

Next, wash the column two times with 500 microliters of binding buffer and collect the flow-through in the same microcentrifuge tube. Centrifuge the eluate at 300 g for 10 minutes and discard the supernatant. Count the cells and assess viability again using trypan blue exclusion.

Aliquot 20, 000 viable cells into a new 1.7-milliliter microcentrifuge tube. Then centrifuge the cells at 300 g for 10 minutes and remove the supernatant. Re-suspend the cells with 40 microliters of PBS containing 0.04%BSA.

Finally, place the tube containing the cells on ice and send it immediately to a genomic core facility for library generation and sequencing. Collect the sequencing results in the format of cell ranger outputs. Dried blood spot samples from preterm infants and controls were processed.

And the normalized protein expression of 92 immune-related proteins was quantified. The expression of lymphocyte-activation gene three and signaling threshold-regulating transmembrane adapter 1 were significantly higher in peripheral blood from preterm infants at two months of age compared with term cord blood. A comprehensive gating strategy was used to identify leukocyte populations from red blood cell lysed white blood cell samples.

Within the T-cell population, CD4-positive and CD8-positive subsets were distinguished. After filtering, over 120, 000 cells were captured and profiled in the single-cell ribonucleic acid sequencing dataset. Dimensionality reduction and clustering analysis identified 14 distinct cell clusters.

Cell clusters were annotated based on differentially expressed genes and known cell markers. 12 of the 14 clusters corresponded to immune cell lineages, including T cells, natural killer cells, B cells, myeloid cells, and neutrophils. A stacked bar graph illustrated the relative proportion of cells per cluster across one week, one month, and two months of age.

This study enables measurement of cellular, transcriptomic, proteomic, and functional immune signatures longitudinally from limited-volume blood samples. Future studies can utilize these methods to define healthy immune trajectories and uncover dysregulated early-life immune responses linked to diseases.