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Transcriptional downregulation of S1pr1 is required for the establishment of resident memory CD8+ T cells

Abstract

Cell-mediated immunity critically depends on the localization of lymphocytes at sites of infection. While some memory T cells recirculate, a distinct lineage (resident memory T cells (TRM cells)) are embedded in nonlymphoid tissues (NLTs) and mediate potent protective immunity. However, the defining transcriptional basis for the establishment of TRM cells is unknown. We found that CD8+ TRM cells lacked expression of the transcription factor KLF2 and its target gene S1pr1 (which encodes S1P1, a receptor for sphingosine 1-phosphate). Forced expression of S1P1 prevented the establishment of TRM cells. Cytokines that induced a TRM cell phenotype (including transforming growth factor-β (TGF-β), interleukin 33 (IL-33) and tumor-necrosis factor) elicited downregulation of KLF2 expression in a pathway dependent on phosphatidylinositol-3-OH kinase (PI(3)K) and the kinase Akt, which suggested environmental regulation. Hence, regulation of KLF2 and S1P1 provides a switch that dictates whether CD8+ T cells commit to recirculating or tissue-resident memory populations.

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Figure 1: Differences in the expression of KLF2 and S1P1 by memory CD8+ T cells in lymphoid tissues and NLTs.
Figure 2: KLF2 is downregulated during the seeding of NLTs by CD8+ T cells.
Figure 3: Distinct KLF2-GFP expression by recirculating memory CD8+ T cells in NLTs.
Figure 4: Forced expression of S1P1 prevents the establishment of TRM cells.
Figure 5: Accumulation of activated CD8+ T cells in inflamed skin is dependent on downregulation of S1P1 expression.
Figure 6: The KLF2lo phenotype of TRM cells does not correlate with sustained engagement of the TCR.
Figure 7: Diverse cytokines induce downregulation of KLF2 expression in activated CD8+ T cells, through a PI3K/Akt sensitive pathway.

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Acknowledgements

We thank J. Cyster (University of California, San Francisco) for the vector MSCV-S1PR1-hCD4; J. Chen (Massachusetts Institute of Technology) for the retroviral vector MSCV-IRES-Thy1.1 (MiT); K. Walkowiak and J. Schenkel for input on parabiosis; L. Mackay and D. Kaplan for advice on DNFB studies; and the Jamequist laboratory for intellectual support. Supported by the US National Institutes of Health (R37 AI38903 to S.C.J.; R37 AI39560 to K.A.H.; T32 AI07313 to C.N.S.; and T90 DE022732 to K.G.A.).

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Authors

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C.N.S., J.-Y.L., D.M., K.A.H. and S.C.J. designed the experiments; C.N.S., J.-Y.L. and K.G.A. did experiments; and C.N.S., J.-Y.L. and S.C.J. wrote the manuscript.

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Correspondence to Stephen C Jameson.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 CD69 expression and KLF2 downregulation correlate with parenchymal P14 T cells in NLTs.

Analysis of KLF2-GFP expression in adoptively transferred P14 cells 8 days (a) or 30 days post LCMV infection (b). (A) IV anti-CD8 antibody was used to distinguish P14 cells in tissue parenchyma (black) versus vascular-associated (red). Data are overlaid with wildtype P14 cells (grey filled) as control. Representative of n=9 from 3 independent experiments. (b) Gated on live congenically marked P14 cells isolated from different tissues. Showing CD8 IV versus CD69 (left) or KLF2-GFP (right). Horizontal dotted line represents the separation between red pulp and white pulp of the spleen, based on previous studies (25). Representative of 4 independent experiments with 3 mice each. Abbreviations as in Fig. 1.

Supplementary Figure 2 Kinetics of KLF2 downregulation in lymphoid and nonlymphoid tissues.

Wildtype (grey filled) and KLF2-GFP P14 cells (black or red) were adoptively co-transferred and host mice infected with LCMV. (a) KLF2-GFP expression on live, non-vascular-associated P14 cells at the indicated days following LCMV infection, in lymphoid tissues (black) and NLT (red) (representative of n=9 from 3 independent experiments). Cells in blood exhibit slightly lower KLF2-GFP levels relative to spleen/LN, especially at memory timepoints, for unclear reasons. (b) GFP MFI from KLF2-GFP P14 cells isolated from various tissues 30 days post LCMV infection. GFP MFI from Wildtype P14 cells was subtracted from KLF2-GFP MFI to eliminate background variability from tissue to tissue (this graph was compiled from 2 independent, representative experiments; n=6).

Supplementary Figure 3 Schematic for parabiosis experiments.

Congenically distinct KLF2-GFP P14 cells were transferred into normal C57BL/6 mice, which were infected with LCMV the following day. A parallel set of mice were infected but did not receive P14 adoptive transfer. At 30-65 days post-infection, paris of transferred and non-transferred mice underwent parabiotic surgery, and 13-17 days after surgery, paried animals were sacrificed and tissues harvested. The mice originally receiving the KLF2-GFP P14 cell population is termed the “Donor” while the other animal in a parabiotic pair is termed the “Parabiont”.

Supplementary Figure 4 Retroviral transduction system used for forced expression of S1PR1 and KLF2.

(a) Retroviral constructs used for transduction contained S1PR1 cDNA in the first expression casette, or lacked a gene at this site (Empty vector, “EV”). A similar construct was used for forced KLF2 expression. In all cases, Thy-1.1 was used as a transduction marker. (b-d) Characterization of transduction system using S1PR1 vector. (b) Mock, EV or S1PR1-transduced P14 cells incubated in vitro for 2 days with 10 ng/ml hIL-2 and 250 nM gp33 peptide. Cells were stained for Thy-1.1 (the transduction marker) and for CD69 or the Flag-epitope (which was cloned into the N-terminus of the retroviral S1PR1). Expression of the retroviral S1PR1 is indicated by cell surface Flag-epitope staining, and loss of staining for CD69 (which competes with S1PR1 for surface expression). Gated on live CD8+ cells. (c) Mock (grey), S1PR1 (red), and EV (black) transduced P14 cells were cultured in vitro for additional days with 20ng/ml hIL-2. Data show the fold-change in live cell numbers over 2 day increments. Data were compiled from at least 3 independent experiments (n=5-8). (d) Transduction efficiency of live S1PR1 (top) and EV (bottom) transduced P14 cells cultured in vitro as in (b). Each line is from an independent experiment (n=7). Note that proliferation is not impaired in the S1PR1 transduced population (relative to mock or EV transduced) (c), and that there is no selective disadvantage of S1PR1 transduced cells for expansion (d).

Supplementary Figure 5 Gating strategy and number of bulk and transduced P14 cells for transduction model in vivo.

(a) Gating strategy for calculating percent transduction per tissue and equation for calculating normalized transduction relative to the spleen. Adoptively co-transferred P14 cells were identified using congenic markers, non-vascular-associated cells were detected using CD8b IV administration, and percent transduction was calculated using the Thy1.1 marker. (b) Number of total non-vascular-associated P14 T cells from spleen and LN that underwent S1PR1 (red) or EV (black) transduction prior to adoptive transfer. The date ranges are the times of sacrifice following in vivo LCMV infection. (c) Number of transduced, non-vascular-associated P14 T cells for EV (black) or S1PR1 (red) vectors in indicated tissues within the time ranges following LCMV infection in vivo. (b-c) Data are compiled from 4 independent experiments (n=9-15).

Supplementary Figure 6 Compared to empty vector–transduced P14 cells, there is no skewing toward CD103 expression on the few P14 cells -tranduced to express S1P1 that remain in nonlymphoid tissues.

(a) Shows percent transduction (relative to spleen) of EV (black) and S1PR1 (red) vector-transduced P14 cells in the parenchyma of IEL 28-60 days post LCMV. The frequency of S1PR1 transduced cells was more variable in the IEL versus other non-lymphoid tissue (see figure 4b,4d). N=15-18 from at least 5 independent experiments. (b) The percentage of CD103+ P14 cells transduced by EV (black) or S1PR1 (red) vectors in the indicated tissue parenchyma isolated at 5, 8 and 30 days post LCMV infection (n=9 from 3 independent experiments). All analyses used gating on live, non-vascular-associated CD8+ P14 T cells.

Supplementary Figure 7 TGF-β and IL-33 induce loss of KLF2 expression in a PI(3)K-Akt-dependent pathway.

(a-c, e) Wildtype and KLF2-GFP P14 cells were co-transferred and recipients infected with LCMV for 4.5 days before sacrifice and splenocyte preparation. (a) Splenocytes were cultured with TGF-β and IL-33 (grey) or no additional cytokines (black) (set as one) for 10-40 hours (n=11-13 from at least 4 independent experiments). (b) Splenocytes were cultured with the indicated cytokines for 40 hrs ex vivo (as in Fig. 7)(n=8 from 3 independent experiments). The graph shows mean (+/- SEM) of KLF2-GFP expression in P14 cells, normalized to P14 cells cultured with no added cytokines. (c) Splenocytes were exposed to TGF-β and IL-33 (left) or no additional cytokines (right) in the presence of indicated LY294002 concentrations for 40 hrs ex vivo). Graph shows KLF2-GFP mean fluorescent intensity of P14 cells (representative of 4 independent experiments (n=8)). (d) Wildtype and KLF2-GFP P14 CD8+ T cells were activated in vitro for 48 hours and then cultured with cytokines and inhibitors as in Fig. 7d. Data compiled from 4 independent experiments (e) Percentage of CD103+ P14 cells from splenocytes cultured in indicated cytokines and inhibitors (similar to Fig. 7c). Data are compiled from 4 experiments (n=12).

Supplementary Figure 8 In vivo administration of the PI(3)K inhibitor LY294002 leads to upregulation of KLF2 in the salivary gland and LPL; Foxo1 expression is reduced in memory P14 CD8+ T cells from NLTs versus lymphoid sites.

(a) Wildtype and KLF2-GFP P14 CD8+ T-cells were co-transferred into C57BL/6 animals which were then infected with LCMV. Four days post infection, animals were treated with LY294002 or vehicle only, as in Fig. 7f. N=12 from 5 independent experiments. (a) KLF2-GFP gMFI (minus wildtype gMFI) was calculated and analyzed (with vehicle only group set as 1). (b) P14 cells were isolated from spleen and salivary gland 30-60 days post LCMV and stained for intracellular Foxo1 protein (n=9 from 3 independent experiments). Data are normalized on isotype control and gated on live P14 CD8+ T cells. Statistical analysis for (a) used ANOVA, while (b) used Student's t-test.

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Skon, C., Lee, JY., Anderson, K. et al. Transcriptional downregulation of S1pr1 is required for the establishment of resident memory CD8+ T cells. Nat Immunol 14, 1285–1293 (2013). https://doi.org/10.1038/ni.2745

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