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Access of protective antiviral antibody to neuronal tissues requires CD4 T-cell help

Abstract

Circulating antibodies can access most tissues to mediate surveillance and elimination of invading pathogens. Immunoprivileged tissues such as the brain and the peripheral nervous system are shielded from plasma proteins by the blood–brain barrier1 and blood–nerve barrier2, respectively. Yet, circulating antibodies must somehow gain access to these tissues to mediate their antimicrobial functions. Here we examine the mechanism by which antibodies gain access to neuronal tissues to control infection. Using a mouse model of genital herpes infection, we demonstrate that both antibodies and CD4 T cells are required to protect the host after immunization at a distal site. We show that memory CD4 T cells migrate to the dorsal root ganglia and spinal cord in response to infection with herpes simplex virus type 2. Once inside these neuronal tissues, CD4 T cells secrete interferon-γ and mediate local increase in vascular permeability, enabling antibody access for viral control. A similar requirement for CD4 T cells for antibody access to the brain is observed after intranasal challenge with vesicular stomatitis virus. Our results reveal a previously unappreciated role of CD4 T cells in mobilizing antibodies to the peripheral sites of infection where they help to limit viral spread.

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Figure 1: Intranasal immunization confers B-cell-dependent neuron protection following genital HSV-2 challenge.
Figure 2: Antibody-mediated neuroprotection depends on CD4 T cells but not on FcRn-mediated transport.
Figure 3: Memory CD4+ T cells are required for antibody access to neuronal tissues.
Figure 4: α4-Integrin-dependent recruitment of memory CD4+ T cells is required for antibody access to neuronal tissues.

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Acknowledgements

We thank H. Dong, S. L. Fink and K. Hashimoto-Torii for animal care support and technical help. We thank R. Medzhitov for discussions. This study was supported by awards from National Institutes of Health grants AI054359, AI062428, AI064705 (to A.I.). A.I. is an investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

Authors

Contributions

N.I. and A.I. planned the project, designed experiments, analysed and interpreted data and wrote the manuscript. N.I. performed experiments.

Corresponding author

Correspondence to Akiko Iwasaki.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 In the absence of TRM, B cells are required for the protection of the host against genital HSV-2 challenge.

a, C57BL/6 mice and μMT mice were immunized intravaginally or intranasally with TK HSV-2. Five weeks later, vaginal tissue sections were stained for CD4+ cells (red) and MHC class II+ cells (green). Blue labelling depicts nuclear staining with DAPI (blue). Images were captured using a ×10 or ×40 objective lens. Scale bars, 100 μm. Data are representative of three similar experiments. bd, BALB/c mice and JHD mice were immunized with TK HSV-2 (105 p.f.u.) intranasally or intravaginally. Six weeks later, the number of total CD4+ T cells and HSV-2-specific IFN-γ+ CD4+ T cells in the vagina (b), spleen (c) and peripheral blood (d) were analysed by flow cytometry. Percentages and total number of IFN-γ+ cells among CD4+CD90.2+ cells are shown. Data are mean ± s.e.m. *P < 0.05; **P < 0.001; ***P < 0.001 (two-tailed unpaired Student’s t-test). e, C57BL/6 mice were immunized intravaginally (naive → D7) or intranasally (WT/i.n. → D0) with TK HSV-2 virus. At the indicated time points (D7: 7 days after immunization; WT/i.n. → D0: 6 weeks after immunization), total viral genomic DNA in the vaginal tissues, DRG and spinal cord were measured by quantitative PCR. fh, Intravaginally immunized C57BL/6 (WT), μMT and HEL-BCR Tg mice (left partner) were surgically joined with naive WT mice (right partner). Three weeks after parabiosis, the naive partner was challenged with a lethal dose of WT HSV-2 intravaginally. Mortality (e), clinical score (f) and virus titre in vaginal wash (g) following viral challenge are depicted.

Extended Data Figure 2 Mucosal TK HSV-2 immunization generates higher levels of virus-specific IgG2b and IgG2c compared with intraperitoneal immunization.

WT mice were immunized with TK HSV-2 (105 p.f.u. per mouse) via intravaginal, intraperitoneal or intranasal routes. Six weeks later, these mice were challenged with a lethal dose of WT HSV-2 intravaginally. At the indicated days after challenge, HSV-2-specific Ig (a) and total Ig (b) in serum were analysed by ELISA. Data are mean ± s.e.m. *P < 0.05 (Mann–Whitney U-test).

Extended Data Figure 3 IFN-γ enhances antibody access to the DRG.

WT mice immunized with TK HSV-2 (105 p.f.u. per mouse) intranasally 6 weeks earlier were challenged with a lethal dose of WT HSV-2 intravaginally. Six days after challenge, after extensive perfusion, HSV-2-specific (a) and total Ig (b) in DRG homogenates were analysed by ELISA. Depletion of CD4 T cells or neutralization of IFN-γ was performed on days −4, and −1, 2 and 4 days after challenge by intravenous injection of anti-CD4 (GK1.5) or anti-IFN-γ (XMG1.2), respectively. Data are mean ± s.e.m. *P < 0.05; **P < 0.001 (two-tailed unpaired Student’s t-test).

Extended Data Figure 4 Neutralization of IFN-γ, α4-integrin or depletion of CD4 T cells has no impact on circulating immunoglobulin levels.

a, b, WT mice immunized intranasally with TK HSV-2 6–8 weeks earlier were challenged with a lethal dose of WT HSV-2. Depletion of CD4 T cells or neutralization of IFN-γ was performed on days −4, and −1, 2 and 4 days after challenge by intravenous injection of anti-CD4 (GK1.5) or anti-IFN-γ (XMG1.2), respectively. At time points indicated, HSV-2-specific Ig in the blood (n = 4) (a) and total Ig in the blood (n = 4) (b) were measured. c, d, WT mice immunized intranasally with TK HSV-2 6 weeks earlier were challenged with a lethal dose of WT HSV-2. Neutralization of α4-integrin was performed on days 2 and 4 after challenge by intravenous injection of anti-α4-integrin/CD49b antibody. Six days later, HSV-2-specific antibody (c) and total antibody (d) in the blood were measured. Data are representative of three similar experiments.

Extended Data Figure 5 An irrelevant immunization fails to increase the levels of total antibodies in neuronal tissues.

a, C57BL/6 mice were immunized with a sublethal dose of influenza A/PR8 virus (10 p.f.u. per mouse) intranasally. Three weeks later, Flu-specific IFN-γ+ CD4+ T cells in spleen and neuronal tissues (DRG and spinal cord) (CD45.2+) following co-culture with HI-Flu/PR8 loaded splenocytes (CD45.1+) were analysed by flow cytometry. As a control, lymphocytes isolated from spleen of TK HSV-2 intranasally immunized mice 6 weeks after vaccination were used for co-culture. (***P < 0.001; two-tailed unpaired Student’s t-test). bd, C57BL/6 mice were immunized with a sublethal dose of influenza A/PR8 virus (10 p.f.u. per mouse). Four weeks later, these mice were challenged with a lethal dose of WT HSV-2 (104 p.f.u. per mouse) intravaginally. Six days after challenge, total antibodies in lysate in DRG (b), spinal cord (c) and blood (d) were measured by ELISA.

Extended Data Figure 6 Most CD4 T cells recruited to the DRG and spinal cord of immunized mice are localized in the parenchyma of neuronal tissues.

a, C57BL/6 mice were immunized intranasally with TK HSV-2. Six days after challenge of immunized mice 6 weeks prior, neuronal tissue sections (DRG and spinal cord) were stained for CD4+ cells and VCAM-1+ cells or CD31+ cells (red or green). Blue labelling depicts nuclear staining with DAPI (blue). Images were captured using a ×10 or ×40 objective lens. Scale bars, 100 μm. b, C57BL/6 mice were immunized intranasally with TK HSV-2. Six weeks later, mice were challenged with WT HSV-2 intravaginal and neuronal tissues were collected 6 days later. DRG and spinal cord were stained for CD4+ cells (red) and MHC class II+ cells, CD11b+ cells or Ly6G+ cells (green). Blue labelling depicts nuclear staining with DAPI (blue). Images were captured using a ×10 or ×40 objective lens. Scale bars, 100 μm. Data are representative of at least three similar experiments.

Extended Data Figure 7 Intravascular staining reveals localization of CD4 T cells in the parenchyma of neuronal tissues.

a, b, C57BL/6 mice immunized intranasally with TK HSV-2 6 weeks previously were challenged with lethal WT HSV-2. Six days after challenge, Alexa Fluor 700-conjugated anti-CD90.2 antibody (3 μg per mouse) was injected intravenously (tail vain) into immunized mice. Five minutes later, these mice were killed for fluorescence-activated cell sorting analysis of intravascular versus extravascular lymphocytes. Data are representative of at least two similar experiments.

Extended Data Figure 8 Recombinant IFN-γ is sufficient to increase epithelial and vascular permeability in vaginal tissues.

a, WT mice immunized with TK HSV-2 (105 p.f.u.) intranasally 6 weeks earlier were injected intravaginally with recombinant mouse IFN-γ (10 μg per mouse) (n = 3) or PBS (n = 3). At the indicated time points, HSV-2-specific Ig (a) and total Ig (b) in vaginal wash were measured by ELISA. c, Two days after rIFN-γ treatment, vaginal tissue sections were stained for VCAM-1+ cells (red) or CD4+ cells (green) and CD31+ cells (green). Blue labelling depicts nuclear staining with DAPI (blue). Images were captured using a ×10 or ×40 objective lens. Scale bars, 100 μm. Data are representative of at least three similar experiments.

Extended Data Figure 9 Vascular permeability in DRG and spinal cord is augmented following WT HSV-2 challenge.

a, C57BL/6 mice were immunized intranasally with TK HSV-2. Six days after challenge of mice immunized 6 weeks previously, neuronal tissue sections (DRG and spinal cord) were stained for CD4+ cells (red) and mouse albumin (green). Blue labelling depicts nuclear staining with DAPI (blue). b, C57BL/6 mice were immunized intranasally with TK HSV-2. Six weeks later, these mice were challenged with lethal WT HSV-2. Six days after challenge, Oregon green 488-conjugated dextran (70 kDa) (5 mg ml−1, 200 μl per mouse) was injected intravenously into intranasally immunized mice. Forty-five minutes later, these mice were killed for immunohistochemical analysis. GM, grey matter; WM, white matter. Data are representative of three similar experiments.

Extended Data Figure 10 Memory CD4+ T cells are required for the increase in antibody levels and vascular permeability in the brain following VSV immunization and challenge.

a, C57BL/6 mice were immunized intravenously with WT VSV (2 × 106 p.f.u. per mouse). Five weeks later, these mice were challenged intranasally with WT VSV (1 × 107 p.f.u. per mouse). Six days after challenge, VSV-specific IFN-γ+ CD4+ T cells in spleen (CD45.2+) following co-culture with HI-VSV loaded splenocytes (CD45.1+) or HI HSV-2 loaded splenocytes were analysed by flow cytometry. Data are mean ± s.e.m. *P < 0.05; **P < 0.001 (two-tailed unpaired Student’s t-test). b, c, Five weeks after VSV immunization, these mice were challenged intranasally with WT VSV (1 × 107 p.f.u. per mouse). Six days after challenge, VSV-specific antibodies in lysate of brain (b) and serum (c) were measured by ELISA. Depletion of CD4 T cells was performed on −4, −1, 2 and 4 days after challenge by intravenous injection of anti-CD4 (GK1.5). d, Albumin levels in tissue homogenates were analysed by ELISA. Data are mean ± s.e.m. *P < 0.05; *P < 0.01; ***P < 0.001 (Mann–Whitney U-test).

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Iijima, N., Iwasaki, A. Access of protective antiviral antibody to neuronal tissues requires CD4 T-cell help. Nature 533, 552–556 (2016). https://doi.org/10.1038/nature17979

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