Fig 1.
CD8+ T cells are resistant to mAb-mediated depletion and more difficult to identify post-depletion.
(A-C) Naïve mice were treated i.p. with a total of 10μg of: anti-CD8α only, anti-CD8β only, or 5μg of both. Splenocytes were isolated and stained the next day. CD8+ T cells were considered CD3+ CD4- B220-. (A) CD8+ T cells as a percentage of lymphocytes and total numbers were calculated. (B) Remaining CD8+ T cell surface bound depleting mAb was determined by staining with Goat anti-Rat IgG. (C) Levels of unbound CD8α and CD8β on surviving CD8+ T cells was determined by staining with the same clones used to deplete. (D) Unmanipulated splenocytes were kept on ice and stained with either anti-CD8α or –β and then stained with the other. Data representative of at least 3 independent experiments. Each point represents the average value of a mouse with 3–5 mice per group.
Fig 2.
CD8+ T cells that survive depletion participate in immune responses.
(A-B) 106 CD45.1 OT1 T cells were transferred i.v. into CD45.2+ mice, treated i.p. with 500μg of anti-CD8α or –β the next day, and immunized or infected. Spleens and lymph nodes were collected on day 5 post-infection or on day 7 post-immunization. CD8+ T cells were considered as CD45.1+ B220-. (A) Surface bound depleting mAb on OT1s was determined by staining with Goat anti-Rat IgG. (B) Remaining OT1 T cells as a percentage of lymphocytes and total numbers were calculated per spleen or pooled lymph nodes. Data representative of at least 3 independent experiments. Each point represents the average value of a mouse with 3–4 mice per group.
Fig 3.
CD8+ T cells that survive depleting mAb treatment acquire distinct differentiation phenotypes.
(A) Flow cytometry gating strategy: after gating on singlet and live lymphocytes, B cells were gated away and the CD45.1+ adoptively transferred OT1 T cells were gated on. Subsequently OT1 T cells were assessed for IL-7Rα and KLRG1 expression. (B) Percent of OT1 T cells that are of MPEC phenotype (IL-7Rα+ KLRG1-) and gMFI of IL-7Rα expression of the MPEC cell only. (C) % of OT1 T cells that are SLEC phenotype (IL-7Rα- KLRG1+) and gMFI of KLRG1 of the SLEC cells only. Representative data shown from 3 independent experiments for immunization and 2 experiments for infection. Each point represents the average value of a mouse with 3–4 mice per group.
Fig 4.
CD8+ T cells surviving depletion differentially localize dependent on mAb used for depletion and type of challenge.
(A-B) 106 CD45.1+ OT1 T cells were transferred i.v. into CD45.2+ mice, treated with anti-CD8α or –β the next day, and immunized or VV-ova infected the day after. Spleens were harvested on day 7 post-infection or immunization and either used for flow cytometry or fixed for immunofluorescence imaging. (A) Post-depletion OT1 T cells were stained for levels of CXCR3 and CD62L. (B) CD45.1+ OT1 T cells were quantified as either white pulp (WP) or red pulp (RP) localized and plotted as a ratio. Points represent the average value per mouse determined from at least 3 sections, pooled from 2 experiments. (C) Representative images of spleen sections 7 days after immunization. Top: blue IgM staining is used to highlight the architecture and delineate RP and WP and false color spots were superimposed to show OT1 T cells scored as WP localized (green) and RP localized (red). White bar represents 100μm. Bottom: images magnified to show individual WP regions; blue MOMA-1 (CD169) staining shows metallophilic marginal macrophages and outlines WP regions and red CD45.1 staining depicts CD45.1+ OT1 T cells.
Fig 5.
Anti-CD8 mAbs have direct effects on target recognition and indirect effects on cytotoxic function.
(A-B) IncuCyte cytotoxicity assays presented as the ratio of target cell death/total target cells over time. Activated OT1 T cells were added to previously plated B16.OVA.RFP tumor target cells. (A) in vitro SIINFEKL activated OT1 T cells were plated at an effector to target ratio of 10:1. Anti-CD8 mAbs were then added at multiple concentrations with at least 4 technical replicates per condition. Data is representative of 3 independent experiments. (B) OT1 T cells were adoptively transferred into naïve mice that were anti-CD8 treated the next day. The following day the mice were immunized and the OT1 T cells FACS isolated from pooled lymph nodes and spleens 7 days later. OT1 T cells were then plated at an effector to target ratio of 1:1 with at least 3 technical replicates per treatment group. Data is representative of 2 independent experiments.
Fig 6.
CD8+ T cells that survive anti-CD8α or -β mAb treatment form memory T cells with differing phenotypes.
(A-B) 106 CD45.1 OT1 T cells were transferred i.v. into CD45.2+ mice, treated with anti-CD8α or –β the next day, and immunized the day after. Mice were allowed to rest, unmanipulated for 62–67 days before staining of splenocytes by flow cytometry. (A) Representative flow plots of memory CD45.1+ OT1 T cells surviving treatment with depleting anti-CD8α or -β mAb with quantification in total numbers and as a percent of CD8+ B220- cells. (B) Comparison of select markers associated with memory and differentiation of cells depicted in A. Black control line in histograms depicts CD8+ B220- T cell population as a whole for reference, except for CD44 where black line depicts B220- cells. Data are representative of 2 independent experiments pooled with 6 mice total.
Fig 7.
Anti-CD8α boosts the metabolism of CD8+ T cells during activation.
(A-D) CD8+ T cells were isolated from unmanipulated mice and anti-CD3ε /anti-CD28 stimulated for 2–3 days in the presence of either anti-CD8α or –β prior to metabolic flux profiling. Dashed line represents control value. (A) Resting ECAR and OCR of blasting CD8+ T cells. (B) Maximum glycolysis was obtained by injection of oligomycin and glycolytic reserve was then calculated by subtracting basal ECAR from the maximum. (C) Maximum respiration was obtained by injection of FCCP and spare capacity was then calculated by subtracting basal OCR from the maximum. (D) Plot of ECAR and (E) OCR measurements overtime with drug injections indicated. Data are representative of 3 experiments performed with at least 3 technical replicates per treatment group.