Research ArticleT CELL DIFFERENTIATION

TCR signal strength controls the differentiation of CD4+ effector and memory T cells

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Science Immunology  20 Jul 2018:
Vol. 3, Issue 25, eaas9103
DOI: 10.1126/sciimmunol.aas9103
  • Fig. 1 Memory-biased TCRs induce weaker TCR signals than effector-biased TCRs in vitro.

    (A) Eleven T cell hybridoma lines, each expressing a unique GP61–80-specific TCR, an NFAT GFP reporter, and an NFκB CFP reporter were stimulated with GP61–80-pulsed DCs (pepDCs) or PMA/ionomycin (ION) for 24 hours. Representative flow plots show GFP expression for three of the cell lines. Bar graphs show GFP expression after stimulation with PMA/ionomycin or pepDCs for 24 hours. (B) Bar graphs depict GFP and CFP mean fluorescence intensity (MFI) after 24 hours of stimulation, comparing TCRs that are present at reduced frequency at memory time points in vivo (Memlo) to TCRs that were present at equal or increased frequencies at memory time points (Memhi), as compared with the peak of the effector response. (C) Plot indicates the correlation of GFP expression to either CD25 surface expression after 24 hours of stimulation with pepDCs, as determined by Pearson’s correlation. Throughout the study, error bars indicate the SEM, and statistical significance was determined by Student’s t test. *P < 0.05 (n = 3 biological replicates per group, representative of three independent experiments).

  • Fig. 2 TCR signal strength and CD25 expression correspond to CD4+ Tfh effector differentiation and memory T cell formation in vivo.

    (A) At day 5 post-infection with LCMV, CD4+ splenocytes from SMα mice were stained with I-Ab/GP66–77 tetramer (Tet), CD25, and either Vβ7 or Vβ14. Representative plots show tetramer staining (gated on CD4+) and CD25 staining (gated on CD4+tetramer+). The bar graph indicates the ratio frequency of Vβ7+ or Vβ14+ cells within the CD25hi versus CD25lo tetramer+ cells. (B) Bar graph shows the T-bet MFI for Vβ7+ and Vβ14+ tetramer-binding cells. (C) Four retrogenic (GFP+) CD4+ T cell lines were adoptively transferred (1 × 105 for analysis at day 3 and 1 to 3 × 104 for analysis at days 8 and 42) into B6 hosts that were subsequently infected with LCMV. At day 3 post-infection, GFP+ T cells were analyzed for the presence of pZAP-70 and expression of CD25 by flow cytometry. The induction of ZAP-70 phosphorylation was calculated by subtracting the pZAP-70 MFI of the total CD4+ T cell population from the pZAP-70 MFI of the GFP+ Rg T cells (ΔpZAP-70). (D) The numbers of GFP+ retrogenic T cells in the spleen were calculated at days 8 and 42 post-infection, and percent survival between these two time points was calculated (% survival). The plots indicate the correlation of ΔpZAP-70 MFI or CD25 expression (%CD25hi) at day 3 post-infection to % survival for each Rg TCR, as determined by Pearson’s correlation. Error bars indicate the SEM, and statistical significance was determined by Student’s t test (n = 3 to 5 mice per group, representative of at least two independent experiments). *P < 0.05, **P < 0.01.

  • Fig. 3 CD25 surface expression and TCR signal strength predict T helper differentiation and memory potential of early effector T cells in vivo.

    (A) SMARTA T cells (Thy1.1+) were adoptively transferred (3 × 104) into B6 hosts (Thy1.2+), followed by LCMV infection. Representative flow histograms indicate the CD25 surface expression by SMARTA T cells at days 0 to 5 (d0 to d5) after LCMV infection. (B) The representative flow histogram shows expression of CD25 by SMARTA cells at day 3 post-infection and expression of Tim-3 and Ly6C by CD25hi and CD25lo subsets. (C) The bar graphs indicate the MFI of CXCR5, T-bet, TCF-1, and Bcl-6 in CD25hi and CD25lo SMARTA CD4+ T cells in the spleen at day 3 post-infection. (D) The bar graph indicates ΔpZAP-70 MFI of CD25hi (“High”) and CD25lo (“Low”) SMARTA CD4+ T cells in the spleen at days 3 and 5 post-infection. Error bars indicate the SEM, and statistical significance was determined by Student’s t test (n = 4 mice per group, representative of four independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 4 CD25 expression predicts effector and memory differentiation.

    (A) Schematic depicts isolation and transfer of CD25hi and CD25lo early effector SMARTA CD4+ T cells infection-matched secondary hosts. (B) Graph indicates the frequency of SMARTA cells (Thy1.1+) in the blood of secondary hosts at the indicated time points after transfer of CD25hi (“High”) and CD25lo (“Low”) SMARTA cells at day 5 post-infection (p.i.). (C) Bar graph indicates the total number of SMARTA memory cells (day 42 post-infection) in the spleen and liver after transfer of equal numbers of CD25hi or CD25lo early effector cells into infection-matched hosts at either days 3 or 5 post-infection. (D) Plots indicate the frequency of CXCR5hiPD-1hi Tfh effector cells derived from CD25lo and CD25hi effector SMARTA that were transferred at day 5 and analyzed at day 8. (E) Bar graphs depict the frequency of Tfh (CXCR5hiPD-1hi) and TH1 (Ly6Chi) day 8 effector cells derived from CD25hi and CD25lo early effector cells isolated and transferred at day 5 post-infection. Remaining bar graphs depict the MFI of Bcl-6 and T-bet at day 8. (F) Bar graphs show the frequency of CXCR5+ SMARTA cells in the spleen and the MFI after intracellular antibody staining for the presence of Bcl-6 and T-bet. Error bars indicate SEM, and statistical significance was determined by Student’s t test (n = 3 to 5 mice per group, representative of at least three independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 5 CD4+ memory T cells derived from either CD25hi or CD25lo effector cells respond robustly to secondary challenge.

    (A) Schematic depicts the isolation and transfer of SMARTA memory cells (day 42) derived from either day 5 CD25hi or CD25lo effector populations into naïve B6 hosts, followed by secondary challenge with LCMV. (B) Bar graph indicates the fold expansion of each SMARTA population, as measured by splenic cell numbers at day 5 postsecondary challenge. (C) Representative flow plots show the expression of PD-1 and CXCR5 on SMARTA T cells 5 days after LCMV infection. (D) Bar graphs indicate the expression levels of CXCR5, PD-1, Ly6C, Bcl-6, and T-bet in either frequency or MFI via flow cytometry. (E) Bar graphs show the frequency of single and multicytokine-producing SMARTA T cells (day 5 post-infection) after ex vivo peptide restimulation. Error bars indicate SEM, and statistical significance was determined by Student’s t test (n = 3 mice per group).

  • Fig. 6 CD25 expression identifies two transcriptionally distinct subsets of very early effector cells.

    (A) CD25hi and CD25lo SMARTA cells were isolated at day 5 post-infection, followed by RNA-seq (n = 3). Hierarchical clustering indicated unique gene expression patterns and significantly up-regulated (red) or down-regulated (blue) genes. (B) Bar graph indicates a list of selected genes with significantly increased expression in CD25hi (red) and CD25lo (blue) populations. The x axis indicates the difference in the number of transcripts on a log2 scale. (C) Bar graphs show RT-PCR–based confirmation of differences in gene expression for the selected genes between day 5 CD25hi (“High”) and CD25lo (“Low”) SMARTA CD4+ T cells. SEM (n = 3 samples per group). *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 7 SHP-1 KD induces a bias toward effector and memory TH1 cells.

    We generated SMARTA bone marrow chimeras expressing either a SHP-1–specific shRNA or an EV control, along with a GFP reporter. At 8 to 10 weeks, SMARTA T cells (Thy1.1+) were adoptively transferred into B6 recipient and infected with LCMV. GFP+ (SHP-1 KD or EV) and GFP (nontransduced, WT) effector SMARTA cells from the spleen were analyzed. (A) Representative flow plots show CD25 and Tim-3 expression on SHP-1 KD (black) and WT (gray) SMARTA T cells 3 days after LCMV infection. Line graphs show the difference in the frequency of GFP+ and GFP SMARTA expressing each marker within the same mouse. (B) Representative flow plots show the surface expression of CD44 and CD62L on SMARTA T cells at day 8 post-infection. (C) Representative flow histograms show CXCR5 expression by SHP-1 KD (GFP+) and WT (GFP) SMARTA cells within the same mouse. Line graphs depict the differences in the frequency of CXCR5-expressing SMARTAs at days 8 and 42 post-infection between GFP+ and GFP SMARTA cells in the spleen in both SHP-1 KD and EV recipients. (D) Representative flow plots show the expression of CXCR5 and Ly6C by SHP-1 KD (GFP+) and WT (GFP) SMARTA cells in the spleen at day 8 post-infection. Numbers indicate the frequency of CXCR5+Ly6C (Tfh) and the CXCR5Ly6C+ (TH1) SMARTA effector cells. The line graph shows the change of frequency of CXCR5+Ly6C SMARTA cells in the spleen when comparing SHP-1 KD (GFP+) and WT (GFP) SMARTA in the same host. (E) The line graph shows the change of frequency of IFN-γ–secreting SMARTA cells in the spleen. The bar graph shows the IFN-γ MFI of SHP-1 KD (GFP+) and WT (GFP) SMARTA IFN-γ–producing cells from the spleen. Error bars indicate the SEM. Pairwise comparisons and statistics were performed on GFP+ and GFP SMARTA cells that were analyzed in the same recipient mouse. Results are representative of at least two independent experiments (n = 3 to 4 mice per group). *P < 0.05, **P < 0.01, ****P < 0.0001.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/25/eaas9103/DC1

    Materials and Methods

    Fig. S1. Creation and stimulation of hybridoma T cell lines with NFAT and NFκB reporters.

    Fig. S2. Stimulation and characterization of C7 and C26 transgenic CD4+ T cells in vitro.

    Fig. S3. Analysis of CD25 expression in an endogenous CD4+ T cell repertoire early after viral infection.

    Fig. S4. Characterization of C7 and C26 transgenic CD4+ T cells via adoptive transfer and LCMV infection.

    Fig. S5. Early expression of CD25 by clone 18 retrogenic CD4+ T cells predicts effector and memory differentiation.

    Fig. S6. SHP-1 KD validation and effect on TCR signaling in vitro.

    Fig. S7. Flow cytometry gating strategies.

    Table S1. Primary source data.

    Table S2. Complete list of genes with significantly changed expression when comparing CD25lo to CD25hi day 5 SMARTA effector cells, as determined by RNA-seq.

  • Supplementary Materials

    The PDF file includes:

    • Materials and Methods
    • Fig. S1. Creation and stimulation of hybridoma T cell lines with NFAT and NFκB reporters.
    • Fig. S2. Stimulation and characterization of C7 and C26 transgenic CD4+ T cells in vitro.
    • Fig. S3. Analysis of CD25 expression in an endogenous CD4+ T cell repertoire early after viral infection.
    • Fig. S4. Characterization of C7 and C26 transgenic CD4+ T cells via adoptive transfer and LCMV infection.
    • Fig. S5. Early expression of CD25 by clone 18 retrogenic CD4+ T cells predicts effector and memory differentiation.
    • Fig. S6. SHP-1 KD validation and effect on TCR signaling in vitro.
    • Fig. S7. Flow cytometry gating strategies.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Primary source data.
    • Table S2 (Microsoft Excel format). Complete list of genes with significantly changed expression when comparing CD25lo to CD25hi day 5 SMARTA effector cells, as determined by RNA-seq.

    Files in this Data Supplement:

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