We conducted this study on CD127 expression by mouse monocytes, and compared our results with a recent study conducted by Zhang et al. Furthermore, we functionally evaluated CD127 expression in response to the addition of IL-7 on CD127+ monocytes/macrophages. We also measured CD127 expression in naïve and mice with EAE to determine whether expression of this receptor changes in inflammatory conditions like EAE.

All mice used in this study were on C57BL/6J genetic background. Mice were obtained from The Jackson Laboratories (10–12 weeks old). C57BL/6J mice were housed in isolated, ventilated cages (maximum of 5 mice per cage) at the specific pathogen–free facility at Thomas Jefferson University. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University.

Blood samples from healthy subjects were obtained at the Department of Neurology, Thomas Jefferson University. All subjects provided informed consent before their participation in the current study, and all human studies were approved by the Institutional Review Board (IRB) of Thomas Jefferson University. Consent forms were signed and reported yearly to Institutional Review Boards (IRB).

Whole blood was collected into EDTA tubes (BD vacutainer system) and peripheral blood mononuclear cells (PBMCs) were obtained by density centrifugation (Ficoll Paque). The blood was first diluted 2-4 times with phosphate-buffered saline (PBS); the suspension was added to Ficoll-Paque solution at room temperature. After centrifugation at room temperature (1200 rpm, 30 min), the buffy coat layer containing the PBMC was collected, and a lysis step with ammonium chloride solution (Biolegend) was implemented to remove red blood cells (RBCs). For isolation of mononuclear cells from the spleen and lymph nodes of mice, tissues were homogenized by maceration through a 70 μm sterile filter with syringe plunger. Homogenized cells from the spleen were centrifuged at 1500 RPM (300 x g) for 10 min, followed by RBC lysis. To isolate CNS mononuclear cells, mice were perfused with ice-cold PBS, and brains and spinal cords were collected and digested in Liberase (Sigma-Aldrich) for 30 min at 37°C. Brains and spinal cords were then mechanically dissociated, and mononuclear cells were isolated using Percoll gradient (GE Healthcare).

Monocytes were enriched by magnetic separation using an Isolation Kit (130-049-601, Miltenyi Biotec). Briefly, the PBMCs were magnetically labeled with CD11b Microbeads for mouse monocytes, and CD14 Microbeads for human monocytes. The cell suspension was then loaded onto a MACS^® Column which was placed in the magnetic field of a MACS Separator. The magnetically labeled cells were retained on the column. After removal of the column from the magnetic field, the magnetically retained labeled cells were eluted as the positively selected cell fraction. Isolated CD11b+ cells had a purity of almost 85% ( Supplementary Figure 1 ).

Cells were rested overnight (16 hours) at 37°C, 5% CO₂ in 5 ml non-adherent polypropylene cell-culture tubes (BD Biosciences) prior to stimulation assays. Given that we did not observe enough CD127+ mouse monocytes in plastic plates, probably due to monocyte adhesion to the plates, we used Falcon 352063 polypropylene tubes for this experiment. Cells were cultured in IMDM (Gibco) supplemented with 20% FBS (Gibco), and 1% penicillin/streptomycin with and without LPS (Escherichia coli O127:B8; Sigma-Aldrich) in a dosage of 100 ng/ml for mice and 10 ng/ml for human at 37°C under an atmosphere containing 5% CO₂. After culture, the supernatants were removed, and monocytes were incubated in PBS containing 10 mM EDTA and 0.5% BSA on ice for 15 minutes to detach adherent cells. For the CD127 expression assay, we measured the receptor at 0, 6, and 18 hours. To assess CD127 expression following IL-7 addition, recombinant IL-7 (20 ng/ml; R&D Systems) was added for the last 2 hours of culture.

EAE was induced by immunization with 1:1 emulsion of PBS solution and complete Freund’s adjuvant (CFA) containing 5 mg/mL heat-killed M. tuberculosis (BD Biosciences) and 1 mg/mL MOG_35-55 peptide (Genscript). Mice were immunized on both flanks by subcutaneous injection of the emulsion for a total of 200 µL. Pertussis toxin was intraperitoneally (i.p.) injected on days 0 and 2 post immunization (p.i.) at 200 ng per dose. Mice were scored according to the following scale: 0 - No clinical symptoms; 0.5 - Partial paralysis of the tail or waddling gait; 1.0 - Full paralysis of the tail; 1.5 - Full paralysis of the tail and waddling gait; 2.0 - Partial paralysis in one leg; 2.5 - Partial paralysis in both legs or one leg paralyzed; 3.0 - Both legs paralyzed; 3.5 - Ascending paralysis; 4.0 - Paralysis above the hips; 4.5 – Moribund; mouse being unable to right itself for 30 seconds; 5.0 - Death. EAE Mice were sacrificed at the peak of disease (14–15 days post-induction). Th score procedure was blindly done.

The fluorochrome-conjugated antibodies for targets of interest and fluorochrome-conjugated isotype control antibodies were as follows: anti-mouse CD3ϵ antibody [PE (clone 145-2C11, 100307; Biolegend), FITC (clone 145-2C11, 100306; Biolegend)], anti-mouse CD45 [Alexa Flour 700, clone I3/2.3, Biolegend], anti-mouse CD11b [Brilliant Violet 785 (clone M1/70, 101243; Biolegend), Brilliant Violet 421 (clone M1/70, 101251; Biolegend)], anti-mouse CD127 (IL-7Rα) [PE/Cyanine5 (clone A7R34, 135016; Biolegend), APC (clone A7R34, 135012,Biolegend), PE (clone A7R34, 135010; Biolegend)], anti-mouse Ly6C [Brilliant Violet 510 (clone HK1.4, 128033; Biolegend), Brilliant Violet 650 (clone HK1.4, 128049; Biolegend), Brilliant Violet 421 (clone HK1.4, 128032; Biolegend)], anti-mouse Ly6G [PE/Cyanine7, clone 1A8, 127618, Biolegend], anti-mouse I-A/I-E (MHCII) [PerCP/Cyanine 5.5 (clone M5/114.15.2, 107624; Biolegend), APC/Cyanine 7 (clone AF6-120.1, 116426; Biolegend)], anti-mouse F4/80 (MHCII) [PE/Cyanine 7, clone BM8, 123114; Biolegend], anti-mouse CD86 [FITC, clone GL-1, 105006, Biolegend], anti-mouse CD80 [PE, clone 16-10A1, 104708, Biolegend]. For human antibodies, the fluorochrome-conjugated antibodies were as follows: anti-human CD14 [APC/Cyanine 7, clone M5E2, 301820, Biolegend], anti-human CD14 [FITC, clone 3G8, 302006, Biolegend], anti-human CD3 [Brilliant Violet 650, clone OKT3, 317324, Biolegend], anti-mouse CD127 (IL-7Rα) [PE/Cyanine5 (clone A019D5, 351324; Biolegend). To optimize the concentration of antibodies used for flow cytometry experiments, titration of antibodies was performed.

Cells of interest were collected and washed with staining buffer (PBS containing 3% FBS). Cell surface antigens were stained with Abs in 100 μL of PBS/3%FBS for 20-30 min at 4°C. For blocking non-specific binding of immunoglobulin to the Fc receptors, we used FC blocker (TruStain FCx, Biolegend) before staining. After staining, cells were washed two times with staining buffer and resuspended in PBS/3%FBS. Cells were washed and fixed with 100 μL Fix and Perm Medium A (Thermo Fisher) for 20 min at room temperature and washed again. Cells were then washed twice and resuspended in 500 μL PBS, and data was obtained by FACSAria Fusion (BD Biosciences). Data analysis was implemented using FlowJo software (TreeStar).

Statistical comparison between two groups was done by student’s t-test and Mann-Whitney based on normality. One-way ANOVA was done for multiple group comparisons; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; NS, not statistically different. Prism 9.0 (GraphPad Software) was used for analyses.

We measured CD127 expression on mouse monocytes from blood at different time points during LPS stimulation (0, 6, 18 hours). Monocytes within LPS-stimulated PBMCs upregulated CD127 expression at 6 hours (20.5±10.9%) and 18 hours (16.8±9.7%) as compared to before stimulation [0 hours (4.15±2.1%)] ( Figures 1A B ). Supplementary Figure 2 shows the detailed gating strategy for CD127 expression analysis on mouse monocytes. We measured CD127 expression on T cells as positive control, and assessed MHC II expression on monocytes, as an activation marker induced by LPS ( Figure 1C ). The upregulated CD127 on monocytes within LPS-stimulated PBMCs was significantly higher in terms of percentage and mean fluorescence intensity ( Figure 1D ). Zhang et al. reported that LPS induces CD127 expression on human monocytes, but not on murine monocytes (9). To better characterize murine monocytes, we also stained for Ly6C and found an inducible CD127 expression on blood CD11b+Ly6C+ monocytes after LPS activation ( Figures 1A, B ). Given that CD11b could be expressed on CD8+ T cells (12) and Ly6G+ neutrophils (13), we also determined CD127 expression on CD3-Ly6G-CD11b+ monocytes. Likewise, we found induced CD127 expression after LPS activation ( Figure 1E ).

CD127 expression on blood monocytes after LPS stimulation prompted us to evaluate its expression on splenic monocytes as well. Splenic monocytes also upregulated CD127 expression after LPS stimulation, especially after 18 hours of treatment ( Figures 1F, G ). Next, we analyzed CD127 expression on splenic macrophages (CD45+CD11b+Ly6C+MHCII+CD86+CD80+) at different time points (0, 6, 18 hours) after LPS treatment. Similarly, macrophages upregulated CD127 expression following LPS activation ( Figures 1H, I ).

Overall, we found that mouse blood and splenic monocytes can upregulate CD127 expression in culture containing LPS; however, if LPS is responsible for the CD127 upregulation needs to be addressed.

Upregulation of CD127 expression on isolated human monocytes has been reported (8, 9). We also consistently observed upregulation of CD127 expression on isolated classic, intermediate and non-classic human monocytes after LPS activation ( Figures 2A–C ). Given that LPS induces CD127 expression on human monocytes, and we also found CD127 upregulation on mouse monocytes following LPS activation ( Figure 1 ), we then tested if LPS is responsible for CD127 upregulation on mouse monocytes. Hence, we cultured splenic cells with or without LPS. Surprisingly, mouse monocytes upregulated CD127 expression in the splenic leukocyte cell mixture culture with and without LPS treatment compared with before culture ( Figures 2D, E ). CD127 expression was also upregulated in blood mouse monocytes that were not treated with LPS ( Supplementary Figure 3 ). This finding is in contrast with the observations that human monocytes upregulated CD127 expression only after LPS treatment. We hypothesized that upregulation of CD127 on mouse monocytes may be induced by other splenic immune cells that present in the culture. To test this, we isolated CD11b+ monocytes and cultured them with or without adding LPS for 6 and 18 hours. Interestingly, we did not find any upregulation of CD127 expression irrespective of LPS stimulation ( Figures 2F, G ), indicating that upregulation of CD127 on mouse monocytes is induced by other immune cells. Supplementary Figure 4 shows gating strategy for CD127 expression analysis on mouse CD11b+ and isolated CD11b+ cells from spleen.

To examine whether upregulated CD127 expression on mouse monocytes are functional, we treated them with recombinant IL-7 (20 ng/ml). IL-7 treatment caused a significant loss of CD127 from the surface of monocytes ( Figures 3A, B ). As a control, we treated T cells with IL-7 and observed similar reduction in CD127 levels ( Figure 3C ).

We next explored whether CD127 expression on mouse monocytes changes during inflammation in vivo. Given that CD127 is expressed at the minimal level on blood and splenic monocytes in homeostatic conditions, we assessed CD127 expression on CNS and lymph node monocytes as well. We have not found CD127 expression on CD11b+ cells from the CNS, including microglia; surprisingly, we have detected CD127 on lymph node monocytes of naïve mice ( Figure 3D ). CD127 was expressed significantly higher on lymph node monocytes (mean=13.4%) than on those from the blood (mean=3.2%), spleen (mean=3.0%) and CNS (mean=0.8%) in steady state ( Figure 3E ). Hence, we compared CD127 expression of lymph node monocytes between naïve and mice with EAE. We immunized naïve mice with MOG_35-55 peptide and M. tuberculosis to induce EAE, and collected cells at the peak of disease. Compared with naïve mice, the CD127 expression was significantly lower in lymph node monocytes of mice with EAE ( Figures 3F, G ). There was no significant change of CD127 expression on spleen, blood and CNS monocytes of mice with EAE (data not shown).