T cell memory. Resident memory CD8 T cells trigger protective innate and adaptive immune responses

Abstract

The pathogen recognition theory dictates that, upon viral infection, the innate immune system first detects microbial products and then responds by providing instructions to adaptive CD8 T cells. Here, we show in mice that tissue resident memory CD8 T cells (T(RM) cells), non-recirculating cells located at common sites of infection, can achieve near-sterilizing immunity against viral infections by reversing this flow of information. Upon antigen resensitization within the mouse female reproductive mucosae, CD8(+) T(RM) cells secrete cytokines that trigger rapid adaptive and innate immune responses, including local humoral responses, maturation of local dendritic cells, and activation of natural killer cells. This provided near-sterilizing immunity against an antigenically unrelated viral infection. Thus, CD8(+) T(RM) cells rapidly trigger an antiviral state by amplifying receptor-derived signals from previously encountered pathogens.

Fig 1. T_RM reactivation induces memory CD8 T cell and B cell recruitment through IFNγ-dependent VCAM-1 upregulation

(A) VCAM-1 mean fluorescence intensity (MFI) on CD31+ vascular endothelium in the FRT 12h after OT-I Ifng^-/- or OT-I Ifng^+/+ T_RM were reactivated by SIINFEKL peptide deposited t.c. (B) Representative images of VCAM-1 (red) and CD31 (blue) staining. 12 hours after t.c. challenge, VCAM-1 expression was quantified on FRT vascular endothelium after (C) P14 immune chimeras were t.c. challenged with either VV-gp33, VV-OVA or left untreated or (D) when P14 immune chimeras were injected with anti-Thy1.1 antibody or left untreated 5 days prior to t.c. deposition of gp33 peptide. (E) OT-I CD8 T cells were transferred i.v. into P14 immune chimeras that were treated with anti-VCAM-1, anti-CD49d or isotype control antibody and were then challenged with gp33 t.c. 48h later, OT-I T cells per coronal section were enumerated. (F) B cells in the FRT were enumerated 48h after t.c. gp33 peptide challenge of P14 immune chimeras, and (G) shows a representative image. B cells in the FRT were also enumerated in (H) P14 immune chimeras that were t.c. challenged with either VV-gp33, VV-OVA or left untreated, in (I) LCMV immune mice that never received P14 cells, and (J) in P14 immune chimeras that were injected with anti-Thy1.1 antibody 5d prior to t.c. gp33 peptide challenge. (K) B cells within the FRT of OT-I Ifng^-/- or OT-I Ifng^+/+ immune chimeras were enumerated 48h after t.c. SIINFEKL challenge. (L) B cells in the FRT were quantified 48h after P14 immune chimeras were challenged t.c. with gp33 peptide in the presence of VCAM-1 or CD49d blocking antibodies. Each experiment shown includes 3-6 mice and data is representative of 2-3 independent experiments. Scale bars=20μm. *=p<0.05, **=p<0.01, ***=p<.001, unpaired two-tailed t-test, error bars indicate mean±SEM.

Fig 2. T_RM reactivation induces DC maturation

CD86 and CCR7 expression was evaluated on CD11c⁺/MHC-II⁺ DC in the FRT 12h after t.c challenge of (A) P14 immune chimeras challenged with gp33 peptide, (B) LCMV immune mice (that never received P14 cells) challenged with gp33 peptide, (C) P14 immune chimeras that were t.c. challenged with either VV-gp33 or VV-OVA, or (D) P14 immune chimeras that were injected i.p. with 1 μg of anti-Thy1.1 antibody five days prior to gp33 challenge. (E) Intracellular TNFα expression was evaluated in P14 CD8 T cells from the FRT by flow cytometry 12h after t.c. gp33 challenge. (F&G) DC phenotype was evaluated as in (A), but the indicated mice were pretreated with TNFα blocking antibody. Representative of 2-3 experiments totaling 6-14 mice/group. *=p<0.05, **=p<0.01, ***=p<.001, unpaired two-tailed t-test, error bars indicated mean±SEM.

Fig 3. T_RM reactivation induces NK cell activation

(A) P14 immune chimeras were challenged t.c. with gp33 to reactivate T_RM. 12h later, intracellular granzyme B expression was evaluated in bystander CD8 T cells (P14 CD8 T cells were excluded from analysis) and NK cells isolated from the FRT (grey line=without gp33 challenge, black=gp33 challenge). Intracellular granzyme B expression within NK cells isolated from the FRT was evaluated 12h after (B) P14 immune chimeras were t.c. challenged with either VV-gp33 or VV-OVA or left untreated or (C) P14 immune chimeras were previously treated with anti-Thy1.1 antibody before t.c. gp33 peptide challenge. (D) 12h after t.c. gp33 peptide challenge, intracellular IL-2 expression by P14 CD8 T cells isolated from the FRT of P14 immune chimeras was evaluated. Intracellular granzyme B expression by (E) NK cells and (F) CD8 T cells isolated from the FRT of P14 chimeras 12h after gp33 peptide challenge when mice were pre-treated with IL-2Rβ blocking antibody. n=3, representative of 3 experiments. *=p<0.05, **=p<0.01, ***=p<.001,unpaired two-tailed t-test,mean±SEM.

Fig 4. T_RM reactivation induces antiviral state

(A) P14 immune chimeras or control naïve mice were challenged t.c. with 4×10⁶ PFU of antigenically unrelated VV-OVA in the presence or absence of gp33 reactivating peptide and/or IFNγ, TNFα, and IL-2Rβ blocking antibodies. Two days later, viral titers were evaluated by plaque assay from homogenized FRT. Data pooled from two independent experiments totaling 6-11 mice per group. (B) As in A, however gp33 peptide was delivered 12h prior to viral challenge. Data pooled from two independent experiments totaling 8 or 9 mice per group. (C) LCMV immune mice (without P14 transfer) were transcervically challenged with 1×10⁶ PFU of VV-OVA in the presence or absence of gp33 peptide. Two days after infection, viral titers were evaluated. Data pooled from two independent experiments totaling 6 mice per group. *=p<.05, **=p<.01,***=p<.001, unpaired two-tailed t-test, mean±SEM.