Research ArticleT CELLS

Modulation of asymmetric cell division as a mechanism to boost CD8+ T cell memory

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Science Immunology  12 Apr 2019:
Vol. 4, Issue 34, eaav1730
DOI: 10.1126/sciimmunol.aav1730
  • Fig. 1 ACD is a feature of CD8+T cell subtypes that retain stemness.

    (A) Confocal images from fixed samples of CD8+ T cells 32 to 36 hours after in vitro (re-)activation on (+/-) Fc-ICAM-1–, α-CD3–, and α-CD28–coated plates. Naïve cells (n = 63) were obtained from wild-type C57BL/6 or P14 mice. Effector (n = 60), memory (n = 61), and exhausted (n = 24) cells were obtained upon sorting of adoptively transferred P14 cells after LCMV (WE or C13 strain) infection. Mitotic cells were identified on the basis of nuclear and β-tubulin structures and imaged during late anaphase or cytokinesis, where daughter cells could be distinguished. dpi, days post infection. (B) ACD rates are higher in subsets that retain stemness, namely, naïve and memory cells (30 days after LCMV WE infection). CD8 staining was quantified in both daughter cells, and P1 was arbitrarily set up as the pole (daughter cell) with higher amounts of CD8. Mitosis was classified as asymmetric when the amount of CD8 was 50% higher in one daughter cell in comparison with the other one, defining the threshold of 0.2 (dotted line in the graph). Data are represented as mean ± SEM. (C) Percentage of asymmetrically dividing cells from (B). (D) Stimulation in the absence of Fc-ICAM-1 leads to symmetric mitosis. Naïve CD8+ T cells were activated in α-CD3– and α-CD28–coated plates in the presence or absence of human Fc-ICAM-1. Data are represented as mean ± SEM. (E) Further subdivision of differentiation subsets revealed that SLECs and PD-1hi/low cells lack the ability to divide asymmetrically. Different cell subsets were obtained upon sorting of adoptively transferred P14 cells 8 days after LCMV WE infection (SLECs, n = 38; MPECs, n = 30), 30 days after LCMV WE infection (EM, n = 23; CM, n = 26) and >30 days after LCMV high-dose C13 infection (PD-1int, n = 47; PD-1hi, n = 32). (F) ACD rates were higher in subsets that retain stemness, MPECs, and MPs. Data are represented as mean ± SEM. (G) Percentage of asymmetrically dividing cells from (F). Statistical analysis was performed using the unpaired two-tailed Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (see also fig. S1).

  • Fig. 2 Transient mTOR inhibition enhances and restores ACD capability.

    (A) Confocal microscopy images from naïve C57BL/6 or P14 mice CD8+ T cells 32 to 36 hours after stimulation on α-CD3– and α-CD28–coated plates in the presence or absence of Fc-ICAM-1 [−ICAM, n = 48; −ICAM R, n = 28; +ICAM, n = 90; +ICAM R, n = 80; +ICAM Akt i (Akt-kinase inhibitor), n = 69]. (B) Percentage of asymmetrically dividing cells from (C). (C) ACD rates are higher in cells that were submitted to transient mTOR inhibition with both rapamycin or Akt-kinase inhibitor. Data are represented as mean ± SEM. (D) Confocal microscopy images of mitosis from SLECs (n = 12), MPECs (n = 25), EM (n = 13), CM (n = 20), PD-1int (n = 32), and PD-1hi (n = 24) CD8+ T cells after restimulation. (E) Percentage of asymmetrically dividing cells from (F). Frequencies of symmetrically and asymmetrically dividing cells from conditions with no pharmacological intervention (+ICAM) are depicted, respectively, in white and gray. Frequencies of asymmetrically dividing cells upon transient rapamycin treatment (+ICAM R) are shown in black. (F) ACD is increased upon transient mTOR inhibition in MPECs, EM, and CM CD8+ T cells, and reestablished in PD-1int CD8+ T cells. Asymmetry rates shown in Fig. 1F (white) are depicted on the left side of data from the same subsets obtained upon transient mTOR inhibition (black). Data are represented as mean ± SEM. (G) Confocal microscopy images of mitoses of PD-1int TCF-1lo or TCF-1hi cells submitted or not to transient mTOR inhibition during stimulation (TCF-1lo, n = 16; TCF-1lo R, n = 10; TCF-1hi, n = 15; TCF-1hi R, n = 11). Statistical analysis was performed using the unpaired two-tailed Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (see also figs. S3 and S4).

  • Fig. 3 Higher ACD rates lead to better memory potential and viral clearance.

    (A) Experimental setup. Naïve P14 cells were stimulated under different conditions, harvested after 36 hours, and adoptively transferred into wild-type recipients. Each recipient mouse received 10,000 cells and was infected intravenously with LCMV WE 30 days later. ffu, focus forming units. (B) Frequency of P14 cells within the CD8+ T cell population in the blood of recipients that were infected with LCMV WE 30 days after adoptive transfer. (C) Frequency and numbers of adoptively transferred P14 cells within CD8+ T cells in the spleens of recipient mice. (D) Representative plots of KLRG1 and IL-7Rα expressing splenic P14 cells stimulated on human Fc-ICAM-1–, α-CD3–, and α-CD28–coated plates in the presence or absence of rapamycin for 30 to 36 hours before adoptive transfer (left); percentages of KLRG1+ IL-7Rα and KLRG1 IL-7Rα+ P14 cells in the spleens of recipient mice (right). (E) Frequencies and numbers of IFN-γ– and TNF-producing cells. (F) Top: Experimental setup. P14 naïve CD8+ T cells were stimulated under different experimental conditions for 36 hours and further adoptively transferred to wild-type recipients. Each recipient mouse received 10,000 cells and was infected intravenously with LCMV WE or LCMV C13 30 days after transfer. Bottom: Increased memory potential derived from higher ACD rates led to better viral clearance upon LCMV infection. LCMV titers obtained 4 days after infection in spleens of recipient mice that were infected with LCMV WE and 12 days after infection in blood and spleens of recipient mice that were infected with LCMV C13. (G) Frequency and numbers of adoptively transferred P14 cells within CD8+ T cells in the spleens of recipient mice. (A to C) Data pooled from five independent experiments. (D) Data pooled from three independent experiments. (E) Representative data from one of two experiments. (F and G) Representative data from one of four experiments. Data are depicted as mean + SEM. Statistical analysis was performed using the unpaired two-tailed Student’s t test or, when data did not pass the normality test, the unpaired Mann-Whitney U test. *P < 0.05; **P < 0.01; ***P < 0.001 (see also figs. S4 to S6).

  • Fig. 4 The polarisome is required for ACD modulation by transient mTOR inhibition.

    (A) Inhibition of phosphorylated extracellular signal–regulated kinase (P-ERK) was used as a readout for AuTM efficiency on PKCζ inhibition. (B) Confocal microscopy images from stimulated naïve CD8+ T cells obtained from P14 mice (+ICAM R, n = 52; +ICAM AuTM, n = 33; +ICAM AuTM R, n = 32). (C) CD8 and T-bet asymmetry upon transient PKCζ inhibition. Data are represented as mean ± SEM. (D) Experimental setup. P14 naïve CD8+ T cells were stimulated under different experimental conditions for 36 hours and adoptively transferred to wild-type recipients. Each mouse received 10,000 cells and was infected intravenously with LCMV WE 30 days after transfer. (E) Frequencies and numbers of adoptively transferred P14 cells within CD8+ T cells in the spleens and lymph nodes (LNs) of recipient mice 10 days after LCMV infection. Data are depicted as mean + SEM. (A to C) Data pooled from two independent experiments. (D and E) Representative data from one of two experiments. Statistical analysis was performed using the unpaired two-tailed Student’s t test or, when data did not pass the normality test, the unpaired Mann-Whitney U test. *P < 0.05; **P < 0.01; ****P < 0.0001.

  • Fig. 5 Exhausted CD8+T cells expressing intermediate levels of the co-inhibitory receptor PD-1 show recovered memory potential when ACD capacity is reestablished.

    (A) Experimental setup. P14 exhausted CD8+ T cells (PD-1int CD44hi cells isolated from spleens of recipient mice >30 days after LCMV C13 infection) were stimulated under different experimental conditions for 36 hours and further adoptively transferred to wild-type recipients. Each mouse received 10,000 cells and was infected with LCMV WE 30 days after transfer. (B) Frequency of P14 cells within the CD8+ T cell population in the blood. (C) Frequency and numbers of adoptively transferred P14 cells within CD8+ T cells in the spleens and lungs. (D) Representative plots of KLRG1 and IL-7Rα expressing splenic P14 cells stimulated on human Fc-ICAM-1–, α-CD3–, and α-CD28–coated plates in the presence or absence of rapamycin for 30 to 36 hours before adoptive transfer (left); percentages of KLRG1+ IL-7Rα and KLRG1 IL-7Rα+ P14 cells in the spleens of recipient mice (right). (E) Frequencies and numbers of IFN-γ– and TNF-producing cells. Data from two pooled experiments. Data are depicted as mean + SEM. Statistical analysis was performed using the unpaired two-tailed Student’s t test or, when data did not pass the normality test, the unpaired Mann-Whitney U test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

  • Fig. 6 Human naïve and CM CD8+T cells show higher ACD rates upon transient mTOR inhibition.

    (A) Confocal images from fixed samples of human naïve CD8+ T cells 32 to 36 hours after in vitro stimulation. Transient mTOR inhibition (12 hours after activation until fixation) was achieved by treatment with 20 nM rapamycin. Stimulation was done on α-CD3– and α-CD28–coated plates in the presence or absence of Fc-ICAM-1 (−ICAM, n = 11; −ICAM R, n = 10; +ICAM, n = 37; +ICAM R, n = 42). (B) ACD rates in the presence or absence of transient mTOR inhibition with rapamycin. Data are represented as mean ± SEM. (C) Percentage of asymmetrically dividing cells from (B). (D) Confocal images from fixed samples of human CM CD8+ T cells 32 to 36 hours after in vitro stimulation. Transient mTOR inhibition (12 hours after activation until fixation) was obtained upon treatment with 20 nM rapamycin. Stimulation was done on α-CD3– and α-CD28–coated plates in the presence or absence of Fc-ICAM-1 (−ICAM, n = 7; −ICAM R, n = 7; +ICAM, n = 15; +ICAM R, n = 19). (E) ACD rates in cells that were submitted to transient mTOR with rapamycin. Data are represented as mean ± SEM. (F) Percentage of asymmetrically dividing cells from (E). Pooled data from peripheral blood mononuclear cells of four different healthy donors. Statistical analysis was performed using the unpaired two-tailed Student’s t test. *P < 0.05; **P < 0.01 (see also fig. S5).

  • Fig. 7 ACD generates progenies with strengthened memory signatures.

    (A) Naïve P14 cells were stimulated on human Fc-ICAM-1–, α-CD3–, and α-CD28–coated plates in the presence or absence of rapamycin (initiated 12 hours after stimulation) for 30 to 36 hours. Cell trace yellow (CTY) dilution was used to identify first daughter cells, from which CD8lo and CD8hi cells were sorted and transferred to recipient mice. Thirty days after adoptive transfer, recipients were infected with LCMV WE. Frequencies of P14 cells in the blood of mice (bottom); CD8 expression in first daughter cells that were submitted or not to rapamycin treatment before adoptive transfer (top). Representative data from one of two experiments. Data are depicted as mean ± SEM. Statistical analysis was performed using unpaired two-tailed Student’s t test or, when data did not pass the normality test, the unpaired Mann-Whitney U test. ns, not significant. (B) Differential expression of genes among CD8lo and CD8hi first daughter cells (top 100 differentially expressed genes based on the lowest P value) presented as a heatmap. (C) Expression of selected transcription factors previously associated with CD8+ T cell differentiation into effector or memory cells. (D) Expression of selected genes associated with survival and homing to secondary lymphoid organs, features of long-lived memory cells. (E) Principal components analysis of 50 selected genes previously associated with CD8+ T cell differentiation. Green, effector gene signatures; blue, memory gene signatures. (F) Differential expression of the 50 selected genes among CD8lo and CD8hi first daughter cells derived from SCD, standard ACD, and enforced ACD.

  • Fig. 8 Enforced ACD generates daughter cells with improved homing features to T cell zones of secondary lymphoid organs.

    (A) Experimental setup. Naïve P14 cells were stimulated for 30 to 36 hours on α-CD3– and α-CD28–coated plates, in the presence or absence of plate-bound Fc-ICAM-1 and/or transient mTOR inhibition by rapamycin (starting 12 hours after activation). CTY dilution was used to identify first daughter cells, from which CD8lo and CD8hi cells were sorted and transferred to recipient mice. Each recipient mouse received 1 × 105 to 3 × 105 P14 cells. Organs were harvested 6 to 7 hours after transfer. (B) Frequencies of P14 cells within the CD8+ T cell population in spleen, inguinal lymph nodes, lungs, and liver of recipient mice. (C) Right: Confocal images from 10-μm splenic sections. Tissues were stained for the localization of metallophilic macrophages (CD169), B cells (B220), and CD8+ T cells (CD8). P14 cells could be identified by their preserved CTY staining. Left: Quantification of P14 cells present in the imaged sections of T cell zones from different recipients (−ICAM CD8lo, n = 35; +ICAM R CD8lo, n = 50; −ICAM CD8hi, n = 30; +ICAM R CD8hi, n = 25). (D) Naïve P14 cells were stimulated under different conditions and harvested after 36 hours, and progenies were adoptively transferred into wild-type naïve recipients (1 × 105 cells per mouse). (E) Frequencies and numbers of transferred cells were assessed 4 weeks later in spleen and lymph nodes of recipients. (A and B) Pooled data from five independent experiments. (C) Representative data from one of two experiments. (D and E) Representative data from one of three experiments. Data are depicted as mean + SEM. Statistical analysis was performed using the unpaired two-tailed Student’s t test or, when data did not pass the normality test, the unpaired Mann-Whitney U test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/34/eaav1730/DC1

    Material and Methods

    Fig. S1. Stimulation of P14 CD8+ T cells on α-CD3–, α-CD28–, and Fc-ICAM-1–coated plates or in a coculture with dendritic cells leads to comparable ACD rates.

    Fig. S2. Transient mTOR inhibition was obtained by treatment with rapamycin or Akt-kinase inhibitor.

    Fig. S3. Transient mTOR inhibition modulates T-bet asymmetry.

    Fig. S4. Transient but not continuous mTOR inhibition is a requirement for ACD modulation in CD8+ T cells.

    Fig. S5. Decreased ACD rates in P14 CD8+ T cells submitted to FTY720 treatment led to reduced proliferation upon LCMV challenge.

    Fig. S6. CM CD8+ T cells produce progenies with enhanced memory potential after enforcing ACD.

    Fig. S7. Murine and human rapamycin-treated naïve CD8+ T cells produce progenies that can survive better in the presence of limited amounts of IL-15.

    Fig. S8. CD8lo and CD8hi daughter cells inherit distinct cyclin expression profiles.

    Fig. S9. ACD generates progenies with strengthened memory signatures.

    Table S1. Raw data files.

    References (4755)

  • Supplementary Materials

    The PDF file includes:

    • Material and Methods
    • Fig. S1. Stimulation of P14 CD8+ T cells on α-CD3–, α-CD28–, and Fc-ICAM-1–coated plates or in a coculture with dendritic cells leads to comparable ACD rates.
    • Fig. S2. Transient mTOR inhibition was obtained by treatment with rapamycin or Akt-kinase inhibitor.
    • Fig. S3. Transient mTOR inhibition modulates T-bet asymmetry.
    • Fig. S4. Transient but not continuous mTOR inhibition is a requirement for ACD modulation in CD8+ T cells.
    • Fig. S5. Decreased ACD rates in P14 CD8+ T cells submitted to FTY720 treatment led to reduced proliferation upon LCMV challenge.
    • Fig. S6. CM CD8+ T cells produce progenies with enhanced memory potential after enforcing ACD.
    • Fig. S7. Murine and human rapamycin-treated naïve CD8+ T cells produce progenies that can survive better in the presence of limited amounts of IL-15.
    • Fig. S8. CD8lo and CD8hi daughter cells inherit distinct cyclin expression profiles.
    • Fig. S9. ACD generates progenies with strengthened memory signatures.
    • References (4755)

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Raw data files.

    Files in this Data Supplement:

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