Research Article

The protein LEM promotes CD8+ T cell immunity through effects on mitochondrial respiration

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Science  29 May 2015:
Vol. 348, Issue 6238, pp. 995-1001
DOI: 10.1126/science.aaa7516
  • Fig. 1 Retro mutant mice exhibit enhanced CD8 T cell responses to chronic LCMV infection.

    (A) Flow cytometry on spleen cells from day 8 post infection (p.i.) with LCMV C13 of WT and Retro homozygous mutant (R) mice. Staining with tetramers, refolded with either np396 or gp33 peptides, and PD-1 is shown (percentage next to gate) on CD8+ gated cells. (B) Number of tetramer+ CD8+ splenocytes on day 8 p.i. (C) Percentage of PD-1+ of tetramer+ CD8+ splenocytes on day 8 p.i. (D) Number of PD-1+ tetramer+ CD8+ splenocytes on day 8 p.i. (E) Percentage of specific CTL lysis from Cr51-release assays for splenocytes on day 8 p.i. (F) Number of CD107α+ CD8+ splenocytes on day 8 p.i. (G) Titer of LCMV C13 in the spleen on day 8 p.i. (H) Kaplan-Meier survival graph after LCMV C13 infection. (I) Percentage of BrdU+ of np396+ CD8+ splenocytes on day 8 p.i. Mean ± SEM; N = 6 to 12 mice. ***P < 0.0001; **P < 0.0005; *P < 0.001. Two-tailed Student’s t tests were used for all, except that Gehan-Breslow-Wilcoxon tests were used for survival curves. Each data set is representative of at least three individual experiments.

  • Fig. 2 Retro improves immunity against tumors and acute viral infections.

    Mice were injected intravenously with B16 F10 melanoma cells. (A) Percentage of CD3+ CD8+ CD44hi cells from lungs, day 35 p.i. (B) Foci of melanomas in the lung on day 35 p.i. (C) Number of melanomas per lung on day 35 p.i. Mice were infected with LCMV Armstrong. (D) Mean percentage of (left) np396+ and (right) gp33+ CD8+ peripheral blood leukocytes (PBL). Mean ± SEM; N = 6 to 10 mice; ***P < 0.0001. Two-tailed Student’s t tests were used for all. Each data set is representative of at least three individual experiments.

  • Fig. 3 Retro mutation identifies LEM.

    BC055111WT/WT and BC055111WT/KO mice were infected with LCMV C13. (A) Western blot on CTLs for BC055111 (49 kD) on total CD8 T cells from the spleen on day 4 p.i. (B) Bar graph of BC055111 signal strength from (A). BC055111WT/WT and BC055111WT/KO mice were infected with LCMV C13. (C) Number of np396+ CD8+ splenocytes on day 8 p.i. (D) LCMV titer in the spleen on day 8 p.i. (E) Percentage of specific CTL lysis from Cr51-release assays for splenocytes on day 8 p.i. (F) Bar graph for mean Lem mRNA in total CD8 T cells from the spleens of WT or R mice after infection with LCMV C13. Level of mRNA normalized to the t = 0 WT level. (G) Bar graphs for mean Lem mRNA in CD8 T cells cultured for 3 days in cytokines, then incubated with actinomycin D or emitine. Level of mRNA normalized to the t = 0 WT level. [(A) to (G)] Mean ± SEM; N = 6 mice. Each data set is representative of at least three individual experiments. (H) Bar graphs for mean relative Lem or Gapdh mRNA from two independent immunoprecipitation experiments performed on N = 2 mice each time. mRNA levels are relative to WT and are from CD8 T cells cultured for 3 days in cytokines. (I) Western blot for SF2 (27 kD) after immunoprecipitation in (H). (J) Mean number of green fluorescent protein–positive (GFP+) CTLL-2 cells cultured in IL-2 (N = 4 wells) from three independent transductions with MIGR1 alone (V-black), or MIGR1 encoded Lem ORF (WT, blue sequence; R, red sequence). ***P < 0.005; **P < 0.01; *P < 0.05. Two-tailed Student’s t tests were used.

  • Fig. 4 The Retro mutation increases LEM expression.

    Mice were infected with LCMV C13. (A) Western blots for LEM (49 kD) on total CD8 T cells from the spleen. (B) Bar graph of signal strength from (A). (C to G) Mean fluorescence intensity (MFI) after staining with antibody to LEM in gated (C) CD44hi CD62Llo CD8+ or (D) CD44lo CD62Lhi CD8+ splenocytes after infection with LCMV C13, or on total CD8 T cells that were activated in vitro with either (E) antibody to CD3/CD28, (F) IL-2, or (G) IL-15. [(A) to (G)] Mean ± SEM; N = 6 mice. (H) Western blots for human LEM, 49 kD on antibody to CD3/CD28-activated human CD8 T cells. (I) Bar graph of signal strength from (H). (J) Mean number of GFP+ human CD8 T cell blasts (N = 4 wells) after three independent transductions with MIGR1 alone (V-black), or MIGR1 encoded hLEM ORF (gray). ***P < 0.0001; **P < 0.0005; *P < 0.001. Two-tailed Student’s t tests were used. Each data set is representative of at least three individual experiments.

  • Fig. 5 LEM interacts with CRIF1 to control the activity of OXPHOS proteins.

    WT and R mice were infected with LCMV C13, and on day 4 p.i. total CD8 T cells were purified. (A) Western blots directly on cell extracts (input) or after immunoprecipitation for LEM (LEM, 49 kD; CRIF1, 25 kD; β actin, 42 kD). (B) Bar graph of signal strengths from (A). (C) CIM from staining with antibodies against LEM (red) or CRIF1 (green). Nuclei stained with 4′,6-diamidino-2-phenylindole (blue). Examples of colocalization (yellow) are indicated by arrows. Scale bar, 2.5 μm. (D) Colocalization of LEM and CRIF1 as measured by Pearson’s correlation coefficient in multiple optical cell slices (N = 62). Mean = 0.68Embedded Image 0.11. WT mice were infected with LCMV C13, and on day 4 p.i. total CD8 T cells were purified. (E) Western blots directly on cell extracts (input) or after immunoprecipitation with LEM antibody or with control rabbit immunoglobulin G (MRPL23, 18 kD; ND1, 36 kD; COX1, 57 kD; ATP5A1, 60 kD). LemWT/WT, LemR/R, or LemWT/KO mice were infected with LCMV C13, and on day 4 p.i. total CD8 T cells were purified. (F) Western blots for OXPHOS proteins (UQCRC2, 49 kD.) (G) Bar graph of signal strength from (F). WT and R mice were infected with LCMV C13, and on day 4 p.i. total CD8 T cells were purified. (H) Immunocapture-enzyme assays for OXPHOS complexes I and IV. (I) Bar graphs for mean specific activity (signal strength per mg protein) from (H). Donor CD8 T cells harboring vector alone of Crif1-AS were purified from WT recipient mice on day 4 p.i. with LCMV C13. (J) Immunocapture-enzyme assays for OXPHOS complexes I and IV. (K) Bar graphs for mean specific activity from (J). Mean ± SEM; N = 6 mice. ***P < 0.005; **P < 0.01; *P < 0.05. Two-tailed Student’s t tests were used. Each data set is representative of at least three individual experiments.

  • Fig. 6 LEM controls production of mitogenic mROS.

    LemWT/WT, LemR/R, or LemWT/KO mice were infected with LCMV C13, and on day 4 p.i. total CD8 T cells were purified from the spleen. (A and B) Oxygen consumption rate (OCR) after sequential injection of oligomycin, FCCP, and antimycin A/rotenone. (C) LemWT/WT, LemR/R, or LemWT/KO mice were infected with LCMV C13, then on day 8 p.i. np 396+ CD8+ splenocytes were gated, then the MFI of MitoSOX red staining was determined. R mice were infected with LCMV C13, then over the course of 8 days were injected with MnTBAP or not. Gated np 396+ CD8+ splenocytes were examined on day 8 p.i. for (D) MitoSOX red staining and (E) % BrdU-positive cells. In the same spleens: (F) Number of tetramer-positive cells and (G) Titer of LCMV. Mean ± SEM. For (A) to (F), N = 6 mice, and for (G), N = 9 mice. ***P < 0.001; **P < 0.005; *P < 0.01. Two-tailed Student’s t tests were used. Each data set is representative of at least three individual experiments.

Supplementary Materials

  • The protein LEM promotes CD8+ T cell immunity through effects on mitochondrial respiration

    Isobel Okoye, Lihui Wang, Katharina Pallmer, Kirsten Richter, Takahuru Ichimura, Robert Haas, Josh Crouse, Onjee Choi, Dean Heathcote, Elena Lovo, Claudio Mauro, Reza Abdi, Annette Oxenius, Sophie Rutschmann, Philip G. Ashton-Rickardt

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to 19
    • Tables S1 to S3
    • References
    Correction (9 October 2015): Fig. S12 has been replaced. For the preparation of figures, we cropped the original Western blots to generate the appropriate figure panels with the relevant lanes. Panels resulting from cropped Westerns were boxed in as per the instructions to authors and assembled in the final composite figures. In the process of generating the revised version of the paper, we mistakenly used the wrong Western blots to generate the cropped figure panels for Fig. S12B. For each experiment in the paper, Western blots were replicated, and on each Western we ran positive and negative controls. The oversight was due to large number of similar-looking Westerns that were used to produce the figure panels.
    The original version is accessible here.

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