. 2015 Oct 9;11(10):e1005193.

doi: 10.1371/journal.ppat.1005193. eCollection 2015 Oct.

IL-4 Induced Innate CD8+ T Cells Control Persistent Viral Infection

Ara Lee¹, Seung Pyo Park², Chan Hee Park¹, Byung Hyun Kang³, Seong Hoe Park⁴, Sang-Jun Ha¹, Kyeong Cheon Jung⁵

Affiliations

"VSports手机版" Affiliations

¹ Department of Biochemistry, College of Life Science & Biotechnology, Yonsei University, Seoul, Korea.
² Transplantation Research Institute, Medical Research Center, Seoul National University College of Medicine, Seoul, Korea.
³ Graduate School of Translational Medicine, Seoul National University College of Medicine, Seoul, Korea.
⁴ Transplantation Research Institute, Medical Research Center, Seoul National University College of Medicine, Seoul, Korea; Graduate School of Translational Medicine, Seoul National University College of Medicine, Seoul, Korea.
⁵ Transplantation Research Institute, Medical Research Center, Seoul National University College of Medicine, Seoul, Korea; Graduate School of Translational Medicine, Seoul National University College of Medicine, Seoul, Korea; Department of Pathology, Seoul National University College of Medicine, Seoul, Korea.

PMID: 26452143
PMCID: "VSports" PMC4599894
DOI: 10.1371/journal.ppat.1005193

IL-4 Induced Innate CD8+ T Cells Control Persistent Viral Infection

"V体育官网入口" Ara Lee et al. PLoS Pathog. 2015.

. 2015 Oct 9;11(10):e1005193.

doi: 10.1371/journal.ppat.1005193. eCollection 2015 Oct.

Authors

Ara Lee¹, Seung Pyo Park², Chan Hee Park¹, Byung Hyun Kang³, Seong Hoe Park⁴, Sang-Jun Ha¹, Kyeong Cheon Jung⁵

Affiliations

¹ Department of Biochemistry, College of Life Science & Biotechnology, Yonsei University, Seoul, Korea.
² Transplantation Research Institute, Medical Research Center, Seoul National University College of Medicine, Seoul, Korea.
³ Graduate School of Translational Medicine, Seoul National University College of Medicine, Seoul, Korea.
⁴ Transplantation Research Institute, Medical Research Center, Seoul National University College of Medicine, Seoul, Korea; Graduate School of Translational Medicine, Seoul National University College of Medicine, Seoul, Korea.
⁵ Transplantation Research Institute, Medical Research Center, Seoul National University College of Medicine, Seoul, Korea; Graduate School of Translational Medicine, Seoul National University College of Medicine, Seoul, Korea; Department of Pathology, Seoul National University College of Medicine, Seoul, Korea.

Abstract

Memory-like CD8+ T cells expressing eomesodermin are a subset of innate T cells initially identified in a number of genetically modified mice, and also exist in wild mice and human. The acquisition of memory phenotype and function by these T cells is dependent on IL-4 produced by PLZF+ innate T cells; however, their physiologic function is still not known. Here we found that these IL-4-induced innate CD8+ T cells are critical for accelerating the control of chronic virus infection. In CIITA-transgenic mice, which have a substantial population of IL-4-induced innate CD8+ T cells, this population facilitated rapid control of viremia and induction of functional anti-viral T-cell responses during infection with chronic form of lymphocytic choriomeningitis virus. Characteristically, anti-viral innate CD8+ T cells accumulated sufficiently during early phase of infection. They produced a robust amount of IFN-γ and TNF-α with enhanced expression of a degranulation marker. Furthermore, this finding was confirmed in wild-type mice. Taken together, the results from our study show that innate CD8+ T cells works as an early defense mechanism against chronic viral infection VSports手机版. .

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The authors have declared that no competing interests exist.

VSports在线直播 - Figures

**Fig 1. Clone 13 infection caused the immunopathology in CIITA^Tg mice with abundant IL-4-induced innate CD8⁺ T cells.**
(A) Cells were isolated from the thymus and spleen of wild-type (WT) and CIITA^Tg mice and expression of Eomes and T-bet on CD8 single positive (CD8SP) thymocytes and splenic CD8⁺ T cells were analyzed by flow cytometry. Numbers in the plots indicate the percentages of cells in each quadrant. (B) CXCR3, CD124, CD122, CD44, and CD24 expression levels were compared on CD8SP thymocytes of wild-type and CIITA^Tg mice. (C) Wild-type and CIITA^Tg mice were infected with 2 x 10⁶ PFU of LCMV CL–13. Survival of wild-type and CIITA^Tg mice were monitored at indicated time points after chronic infection with LCMV CL–13. P value = 0.0013, wild-type versus CIITA^Tg mice upon conventional dose of LCMV CL–13 infection (log-rank (Mantel-Cox) test). (D) Lung pathology in wild-type and CIITA^Tg mice at 8 days post-infection (DPI). Data are pooled from two experiments or are representative of at least two experiments (n≥3 per group in each experiment). Original magnification x100, bar = 200 μm.

**Fig 2. Accelerated viral control in CIITA^Tg mice during LCMV clone 13 infection.**
(**A-E**) Wild-type (WT) and CIITA^Tg mice were infected with 5 x 10⁵ PFU of LCMV CL–13 per mouse. Peripheral blood mononuclear cells (PBMCs) were collected at indicated days post infection (DPI). Frequency of GP_33-41 (GP33) tetramer-positive cells among CD8⁺ T cells were analyzed by flow cytometry (A) and the numbers of GP33 tetramer-positive CD8⁺ T cells per 10⁶ PBMCs during the course of LCMV CL–13 infection are represented (B). Numbers in plots indicate percentage of the tetramer-positive cells among CD8⁺ T cells. PD–1 (**C and D**) and CD127 (E) expression by GP33 tetramer-positive cells after gating on CD8⁺ T cells were also analyzed, and summary showing the PD–1 expression level on GP33 tetramer-positive CD8⁺ T cells is represented by mean fluorescence intensity (MFI) (D). The percentage of CD127⁺ cells among GP33 tetramer-positive CD8⁺ T cells is also summarized (E). Line graph shows mean ± SD. Data are representatives of four independent experiments (n≥3 per group in each experiment). (F) Serum were collected at indicated DPI from the infected wild-type and CIITA^Tg mice, and viral titer were checked. Dashed line indicates the virus detection limit. Undetectable samples were given a half of detection limit. Line graph shows mean ± SD. Data of (**A-E**) are representative of three independent experiments, and data of (F) is pooled from two independent experiments (n≥3 per group in each experiment).*P<0.05; **P<0.01; ***P<0.001.

**Fig 3. Phenotype and function of virus-specific CD8⁺ T cells in tissues of LCMV infected CIITA^Tg mice.**
(**A-C**) Lymphocytes were isolated from the spleen and lungs of LCMV CL–13 (5 x 10⁵ PFU per mouse) infected wild-type and CIITA^Tg mice at 31 DPI and analyzed by flow cytometry. Total numbers of GP33 and GP_276-286 (GP276) tetramer-positive CD8⁺ T cells (A) and PD–1 expression level on tetramer-specific CD8⁺ T cells (B) in the spleen and lungs of wild-type and CIITA^Tg mice are represented. Percentages of CD127⁺ cells among virus-specific CD8⁺ T cells in the indicated tissues are also summarized in the bar graph (C). (**D and E**) Splenocytes were restimulated *in vitro* with GP33, GP276, or LCMV peptide pool (GP33, GP276, GP_118-125, GP_92-101, and GP_70-77) and stained for CD8⁺ T cells. Frequency of IFN-γ- and TNF-α-producing CD8⁺ T cells were analyzed by flow cytometry (D), and absolute numbers of CD8⁺ T cells producing IFN-γ and producing both IFN-γ and TNF-α in the spleen are summarized, respectively (E). Numbers in the plots indicate the percentage of TNF-α⁺ and TNF-α^- CD8⁺ T cells producing IFN-γ, respectively. (F) Viral titers were checked in the spleen and kidney extracted from LCMV CL-13-infected mice at 31 DPI. Dashed line indicates the virus detection limit. Undetectable samples were given a half of detection limit. Bar graphs show mean + SD. Data are representative of three independent experiments (n≥3 per group in each experiment). NS, not significant; *P<0.05; **P<0.01; ***P<0.001.

**Fig 4. CD8⁺ T cells responses in IL-4-deficient CIITA^Tg and control mice during LCMV clone 13 infection.**
**(A)** Cells were isolated from the thymus of CIITA^Tg and IL-4^KOCIITA^Tg mice and expression of CXCR3 and CD44 on CD8 single positive (CD8SP) thymocytes were analyzed by flow cytometry. Numbers in the plots indicate the percentages of cells in each quadrant. (**B-F**) IL-4^KO and IL-4^KOCIITA^Tg mice were infected with 5 x 10⁵ PFU of LCMV CL–13 per mouse. PBMCs were collected at the indicated DPI for analysis. The numbers of GP33 tetramer-positive CD8⁺ T cells per 10⁶ PBMCs (B) and frequency of GP33 tetramer-specific CD8⁺ T cells and their PD–1 expression among CD8⁺ T cells (C) were assessed during the course of LCMV CL–13 infection by flow cytometry. Numbers in the plots indicate percentage of PD–1⁺ or PD–1^- GP33 tetramer-positive cells among CD8⁺ T cells. PD–1 expression level on GP33 tetramer-positive CD8⁺ T cells in PBMCs represented by MFI value (D). (E) Lymphocytes isolated from the spleen of mice at 31 DPI were restimulated *in vitro* with GP33, GP276, or LCMV peptide pool for CD8⁺ T cells. Frequency of IFN-γ- and TNF-α-producing CD8⁺ T cells were analyzed by flow cytometry. (F) Serum viral titer in IL-4^KO and IL-4^KOCIITA^Tg mice at the indicated DPI. Dashed line indicates the virus detection limit. Line graph shows mean ± SD. Data of (**B-D**) are data pooled from two independent experiments. Data of (**E and F**) are representative of four independent experiments (n≥3 per group in each experiment). NS, not significant.

**Fig 5. Generation and characterization of virus-specific IL–4 induced innate CD8⁺ T cells.**
(A) Wild-type (WT) and CIITA^TgPIV^KO host mice (Thy1.2⁺) were sublethally irradiated. Irradiated wild-type and CIITA^TgPIV^KO mice were reconstituted for 10 weeks with P14 (Thy1.1⁺) BM (T-E P14) and a 1:1 mixture of P14 (Thy1.1⁺) and CIITA^Tg (Thy1.2⁺) BM (T-T P14), respectively. (**B and C**) Lymphocytes were isolated from the spleen of T-E and T-T P14 mice. Eomes expression on P14 cells were analyzed by flow cytometry (B). Numbers in the plots indicate MFI values of Eomes staining on T-E and T-T P14 cells. MFI values are summarized in the bar graphs. CD127 and CD44 expression level on P14 cells of the spleen in T-E and T-T P14 mice were also analyzed (C). Numbers in the plots indicate CD127 and CD44 MFI value on T-E and T-T P14 cells. MFI values of CD127 and CD44 are also summarized in the bar graph. (D) Splenocytes from the BM chimeric mice were stimulated *in vitro* with GP33 for P14 cells for 5 hours, and stained with anti-Thy1.1, CD107a, and IFN-γ. Numbers in quadrants indicate the percentage of CD107a⁺ IFN-γ-producing or nonproducing P14 cells. Data were representative from more than two independent experiments and summarized data are shown; bars indicate the mean + SD. **P<0.01; ***P<0.001.

**Fig 6. Control of chronic LCMV infection by innate CD8⁺ T cells.**
(A) T-E and T-T P14 cells were isolated from the spleens of each BM chimera as shown in Fig 5 and transferred into Thy1.2⁺ congenic C57BL/6 mice. One day after transfer, the mice were infected with 5 x 10⁵ PFU of LCMV CL–13 per mouse. **(B-D)** Thy1.1⁺ CD8⁺ donor P14 cells were gated for following analysis. **(B)** CellTrace Violet (CTV)-labelled donor T-E and T-T P14 cells were analyzed at 2.5 DPI by flow cytometry. Frequency of Thy1.1⁺ CD8⁺ P14 cells among splenocytes are depicted. The gated donor Thy1.1⁺ CD8⁺ P14 cells are examined for CTV dilution along with CD44 expression. Division time and frequency of CD44^hi cell population are indicated in histogram plot. (C) Lymphocytes were isolated from the spleens of T-E and T-T P14 chimeric mice at 5 and 18 DPI and analyzed by flow cytometry. The number of Thy1.1⁺ P14 cells in the spleen and PD–1 expression among CD8⁺ T cells were analyzed at indicated DPI. Numbers in the plots indicate PD–1⁺ or PD–1^- Thy1.1⁺ transferred cells. PD–1 expression level (MFI) on Thy1.1⁺ transferred cells is summarized in the graph. (D) Splenocytes of T-E and T-T P14 chimeric mice were restimulated *in vitro* with GP33 for P14 cells, and frequency of IFN-γ- and TNF-α-producing Thy1.1⁺ transferred cells were analyzed. Numbers in the plots indicate the percentage of TNF-α⁺ and TNF-α^- CD8⁺ T cells producing IFN-γ, respectively. Frequency of Thy1.1⁺ transferred cells producing both IFN-γ and TNF-α was summarized in the graph. (E) Serum viral titers were checked in T-E and T-T P14 chimeras at the indicated DPI. Dashed line indicates the virus detection limit. Undetectable samples were given a half of detection limit. **(F-G)** CD8⁺ T cells were gated for following analysis. **(F)** PD–1 expression on GP276 tetramer-positive CD8⁺ T cells was analyzed at indicated DPI. Numbers in the plots indicate PD–1⁺ or PD–1^- GP276 tetramer-positive cells among CD8⁺ T cells. PD–1 expression level (MFI) on GP276 tetramer-positive CD8⁺ T cells is summarized in the graph. **(G)** Splenocytes of T-E and T-T P14 chimeric mice were restimulated *in vitro* with GP276 peptide for endogenous CD8⁺ T cells, and frequency of IFN-γ- and TNF-α-producing CD8⁺ T cells was analyzed. Numbers in the plots indicate the percentage of TNF-α⁺ and TNF-α^- CD8⁺ T cells producing IFN-γ, respectively. Frequency of CD8⁺ T cells producing both IFN-γ and TNF-α was summarized in the graph. Bar graphs show mean + SD. Data are representative of more than two independent experiments (n≥3 per group in each experiment). NS, not significant; *P<0.05; **P<0.01; ***P<0.001.

**Fig 7. Co-adoptive transfers of T-E and T-T P14 cells.**
**(A)** T-E P14 (Thy1.1⁺Ly5.1⁺) and T-T P14 (Thy1.1⁺Ly5.2⁺) cells were isolated from the spleens of each BM chimera as shown in Fig 5 and transferred into Thy1.2⁺Ly5.2⁺ congenic C57BL/6 mice. One day after transfer, the mice were infected with LCMV CL–13. **(B)** Lymphocytes were isolated from the spleens of recipient mice at 5 DPI and analyzed by flow cytometry. Numbers in the plot indicates the frequency of transferred T-E or T-T P14 cells among CD8⁺ T cells in the spleen and absolute numbers of Thy1.1⁺Ly5.1⁺ and Thy1.1⁺Ly5.2⁺ cells in the spleen are summarized in the graph. **(C)** Splenocytes of T-E and T-T P14 chimeric mice were restimulated *in vitro* with GP33 for P14 cells, and frequency of IFN-γ- and TNF-α-producing P14 cells were analyzed. Numbers in the plots indicate the percentage of TNF-α⁺ and TNF-α^- P14 cells producing IFN-γ, respectively. Frequency of P14 cells producing both IFN-γ and TNF-α was summarized in the graph. Bar graphs show mean + SD. n≥4 per group in each experiment. **P<0.01; ***P<0.001.

**Fig 8. Accelerated viral control in BALB/c wild-type mice compared to BALB/c-CD1d^KO mice.**
(**A-C**) CD8⁺ T cells were enriched from the spleens of BALB/c-CD1d^KO and BALB/c wild-type by using magnetic sorting. NP118 tetramer⁺ or tetramer^- CD8⁺ T cells were analyzed by flow cytometry. (A) NP118 tetramer⁺ cells are gated in blue and NP118 tetramer^- cells are gated in green. The numbers in the plots indicated the frequency of NP118 tetramer⁺ cells among CD8⁺ T cells. Absolute number of NP118 tetramer⁺ cells in the spleen is represented in the bar graph. (**B and C**) Eomes expression on NP118 tetramer⁺ or tetramer^- cells in BALB/c-CD1d^KO and BALB/c wild-type. Numbers in the histogram plots indicate MFI values of Eomes stained on NP118 tetramer⁺ or tetramer^- cells. MFI value is summarized in the bar graph. Numbers in the contour plots indicate the frequency of each population in the quadrants depending on CD44 and CXCR3 expression. The frequency of CD44^hiCXCR3⁺ cells are summarized in the bar graph. (**D-I**) BALB/c-CD1d^KO and BALB/c wild-type mice were infected with 2 x 10⁵ PFU per mouse of LCMV CL–13. (**D and E**) Virus titers were checked in serum at the indicated DPI and in spleen and kidney extracted at 29 DPI. Dashed line indicates the virus detection limit. Undetectable samples were given a half of detection limit. (F) Absolute numbers of NP118 tetramer-positive CD8⁺ T cells in the spleen and lung of CL–13 infected mice were analyzed at 29 DPI. **(G)** Numbers in plots indicate percentage of PD–1⁺ or PD–1^- CD8⁺ T cells. PD–1 expression by NP118 tetramer-positive cells after gating on CD8⁺ T cells were also analyzed, and summary showing the PD–1 expression level on NP118 tetramer-positive CD8⁺ T cells is represented by mean fluorescence intensity (MFI). (**H and I**) Splenocytes were restimulated in vitro with NP118, GP_283-291 (GP283), or NP_313-322 (NP313), and frequency of IFN-γ- and TNF-α-producing CD8⁺ T cells were analyzed by flow cytometry (H). Numbers in the plots indicate the percentage of TNF-α⁺ and TNF-α^- CD8⁺ T cells producing IFN-γ, respectively. Absolute numbers of CD8⁺ T cells producing IFN-γ or both IFN-γ and TNF-α in the spleen are summarized (I). Bar graphs show mean + SD. Data are representative of two independent experiments (n≥3 per group in each experiment). *P<0.05; **P<0.01; ***P<0.001.

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IL-4 Induced Innate CD8+ T Cells Control Persistent Viral Infection

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IL-4 Induced Innate CD8+ T Cells Control Persistent Viral Infection

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Abstract

"V体育官网" Conflict of interest statement

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V体育2025版 - References

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