Role of novel type I interferon epsilon in viral infection and mucosal immunity

Abstract

Intranasal infection with vaccinia virus co-expressing interferon epsilon (VV-HIV-IFN-ε) was used to evaluate the role of IFN-ε in mucosal immunity. VV-HIV- IFN-ε infection induced a rapid VV clearance in lung that correlated with (i) an elevated lung VV-specific CD8(+)CD107a(+)IFN-γ(+) population expressing activation markers CD69/CD103, (ii) enhanced lymphocyte recruitment to lung alveoli with reduced inflammation, and (iii) an heightened functional/cytotoxic CD8(+)CD4(+) T-cell subset (CD3(hi)CCR7(hi)CD62L(lo)) in lung lymph nodes. These responses were different to that observed with intranasal VV-HA-IFN-α(4) or VV-HA-IFN-β infections. When IFN-ε was used in an intranasal/intramuscular heterologous HIV prime-boost immunization, elevated HIV-specific effector, but not memory CD8(+)T cells responses, were observed in spleen, genito-rectal nodes, and Peyer's patch. Homing marker α4β7 and CCR9 analysis indicated that unlike other type I IFNs, IFN-ε could promote migration of antigen-specific CD8(+)T cells to the gut. Our results indicate that IFN-ε has a unique role in the mucosae and most likely can be used to control local lung and/or gut infections (i.e., microbicide) such as tuberculosis, HIV-1, or sexually transmitted diseases.

Figure 1

Anti-vaccinia CD8⁺ T-cell response in spleen and lung at 7 days post IN infection of type I IFNs. Mice (n=4–5) per group were infected with 5 × 10⁶ PFU of VV-HIV, VV-HIV-IFN-ɛ, VV-HA, VV-HA-IFN-β, and VV-HA-IFN-α₄. (a) Spleen and (d) lung cells were stimulated with vaccinia-specific K^dA52R₇₅₋₈₃ (KYGRLFNEI) peptide described as in Methods, and IFN-γ-producing T-cell responses were measured by IFN-γ ELISpot and/or ICS. (b) Picture represents four wells of an ELISpot plate, dots represent IFN-γ spot-forming unit (SFU) in spleen, and (c) bar chart represents IFN-γ protein levels measured by enzyme-linked immunosorbent assay (ELISA) following VV-HIV-IFN-ɛ or VV-HIV infection, upon re-stimulation with K^dA52R_75−83. (e) Histogram analysis of CD69 and CD103 surface markers on lung CD8⁺ T cells (gated on total CD8⁺ T cells). (f) Lung VV titers at days 3 and 6 following IN VV-HIV or VV-HIV-IFN-ɛ infection. The dotted line indicates the limit of detection of the assay, which is <10² PFU. (g) Lung H&E staining of uninfected naive control and VV-HIV and VV-HIV-IFN-ɛ IN-infected mice, (h) Lung H&E staining of VV-HA, VV-HA-IFN-β, and VV-HA-IFN-α₄ IN-infected mice 7 days PI. Images were analyzed by Olympus Microscope at × 4 (top) and × 60 (bottom) magnification, respectively. The × 60 magnification represents the boxed area at the top. Error bars represent ±s.d. APC, allophycocyanin; HA, hemagglutinin; H&E, hematoxylin and eosin; ICS, intracellular cytokine staining; IFN, interferon; IN, intranasal; PI, post infection; intranasal; VV, vaccinia virus.

Figure 2

Intranasal VV-HIV-IFN-ɛ infection can induce functional and cytotoxic CD8⁺CD4⁺ double-positive CTL in LLN. BALB/c mice (n=5–6) per group were IN infected with 5 × 10⁶ PFU of VV-HIV, VV-HIV-IFN-ɛ, VV-HA, VV-HA-IFN-β, and VV-HA-IFN-α₄, and unimmunized mice were used as naive controls. (a) FACS plots represent one pooled experiment (left to right: naive, VV-HIV, and VV-HIV-IFN-ɛ), and bar chart on the right indicates average values from three individual pooled experiments. Upper right quadrants (R3) indicate the percentage of CD8⁺CD4⁺ DP T cells out of total CD8⁺ T-cell population (R2) in LLN. (b) FACS plot represent percentage of IFN-γ⁺CD8⁺CD4⁺ T cells (out of total DP population–R3) at 7 days PI (28 days is not shown), following VV-HIV (left) and VV-HIV-IFN-ɛ (right) infections. The cells were stimulated for 16 hrs with K^dA52R₇₅₋₈₃-specific peptide (bottom FACS plots) and unstimulated cells were used as background controls (top FACS plots). The bar chart below represents the percentage of IFN-γ⁺CD8⁺CD4⁺ T cells at 7 and also 28 days PI, and the plotted data indicate A52R₇₅₋₈₃ stimulated minus the unstimulated values. (c) CD107a, CD3, CCR7 and CD62L expression on CD8⁺CD4⁺ DP T cells (R3) following VV-HIV-IFN-ɛ infection. (d) The left graph indicates the percentage of CD8⁺CD4⁺ T cells in LLN following IN, IM, and IP VV infection. The right graph represents the percentage of CD8⁺CD4⁺ T cells in the LLN at 3, 7, and 28 days post IN infection. (e) Percentage of CD8⁺CD4⁺ T cells (left), and IFN-γ⁺CD8⁺CD4⁺ T cells (right) in lung at 7 and 28 days post IN infection. (f) FACS plots from left to right represent percentage of LLN CD8⁺CD4⁺ population obtained with VV-HA, VV-HA-IFN-β, and VV-HA-IFN-α₄-infected groups, respectively. (g and h) Surface markers CD107a, CD3, CCR7, and CD62L expression on CD8⁺CD4⁺ DP T cells (R3) following VV-HA-IFN-β and VV-HA-IFN-α₄ IN infection. Except where stated, data represent pooled values, error bars represent ±s.d., and experiments were repeated for 2–3 times. DP, double positive; FACS, fluorescence-activated cell sorting; HA, hemagglutinin; IFN, interferon; IN, intranasal; IM, intramuscular; IP, intraperitoneal; LLN, lung lymph node; PFU, plaque-forming unit; PI, post infection; VV, vaccinia virus.

Figure 3

Co-expression of IFN-ɛ induced a higher level of gut-specific homing markers on K^dA52R₇₅₋₈₃-specific CD8⁺ T cell in PP following IN infection. BALB/c mice (n=5 per group) were infected IN with 5 × 10⁶ PFU of (a) VV-HIV, (b) VV-HIV-IFN-ɛ, (d) VV-HA, (e) VV-HA-IFN-β, and (f) VV-HA-IFN-α₄. Seven days post infection, the percentage of K^dA52R₇₅₋₈₃-specific CD8⁺ T cells that expressed gut-specific homing markers (α4β7 and CCR9) were analyzed. (c and g) The expression frequencies of homing markers on K^dA52R₇₅₋₈₃-specific CD8⁺ T cells following IN IFN-ɛ, IFN-β, and IFN-α₄ infections were calculated as indicated in Methods. All FACS plots, the upper right quadrants (R3) represent gates R1+R2. Data represent pooled values, and experiments were repeated two times. FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; HA, hemagglutinin; IFN, interferon; IN, intranasal; PFU, plaque-forming unit; VV, vaccinia virus.

Figure 4

HIV-specific CD8⁺ T effector cell function in systemic and mucosal compartments following rFPV/rVV prime-boost immunization. BALB/c mice (n=4–5 per group) were immunized with 1 × 10⁷ PFU of FPV-HIV/VV-HIV or FPV-HIV-IFN-ɛ/VV-HIV-IFN-ɛ using (a) combined mucosal systemic (IN/IM) or (b) pure systemic (IM/IM) immunization. Two weeks post boost, the percentage of K^dGag_197−205-specific CD8⁺ T cells were evaluated in the spleen, lung, genito-rectal nodes, and Peyer's patch using tetramer staining. Spleen data represent average and ±s.d. of five mice (IN/IM, *P=0.033) and due to small sample size mucosal data represent pooled values, spleen error bars represent ±s.d. All experiments were repeated at least three times. HA, hemagglutinin; IFN, interferon; IM, intramuscular; IN, intranasal; PFU, plaque-forming unit; rFPV, recombinant fowlpox; rVV, recombinant vaccinia virus; VV, vaccinia virus.

Figure 5

Gut-specific homing marker expression on K^dGag_197−205-specific CD8⁺ T cell in Peyer's patch. BALB/c mice (n=6 per group) were immunized with 1 × 10⁷ PFU of FPV-HIV/VV-HIV or FPV-HIV-IFN-ɛ/VV-HIV-IFN-ɛ using (a) combined mucosal-systemic (IN/IM) and/or (b) pure systemic (IM/IM) immunization. Two weeks post boost, the percentage of K^dGag_197−205-specific CD8⁺ T cells that expressed gut-specific homing markers (α4β7 and CCR9) were analyzed. (c) Comparison of the frequency of homing marker expression on K^dGag_197−205-specific CD8⁺ T cells following IN/IM and IM/IM strategies, and percentage was calculated as indicated in Methods. All fluorescence-activated cell sorting plots, the upper right quadrants (R3) represent gates R1+R2 (similar to Figure 3). Data represent pooled values, and experiments were repeated two times. HA, hemagglutinin; IFN, interferon; IM, intramuscular; IN, intranasal; PFU, plaque-forming unit; FPV, fowlpox; VV, vaccinia virus.

Figure 6

Magnitude and avidity of HIV-specific effector T cells in systemic and mucosal compartments following rFPV/rVV (IN/IM and IM/IM) prime-boost immunization. BALB/c mice (n=4–5) were (a) primed IN with 1 × 10⁷ PFU FPV-HIV or FPV-HIV-IFN-ɛ and boosted IM with 1 × 10⁷ PFU VV-HIV or VV-HIV- IFN-ɛ or (b) primed IM with 1 × 10⁷ PFU FPV-HIV or FPV-HIV-IFN-ɛ and boosted IM with 1 × 10⁷ PFU VV-HIV or VV-HIV-IFN-ɛ. IFN-γ production was assessed by intracellular cytokine staining following H-2K^d AMQMLKETI Gag peptide stimulation. (c) Following IN/IM immunization, the number of IL-2-producing CD8⁺ T cells were evaluated by ELISpot. (d) Avidity of K^dGag_197−205-specific CD8⁺ T splenocytes measured by tetramer dissociation assay. In all graphs (a, b, and c), unstimulated cells were used as background controls and were subtracted from each sample before plotting the data. Spleen data represent average and ±s.d. of five mice, and due to small sample size mucosal data represent pooled values. All experiments were repeated at least three times. HA, hemagglutinin; IFN, interferon; IL, interleukin; IM, intramuscular; IN, intranasal; PFU, plaque-forming unit; rFPV, recombinant fowlpox; rVV, recombinant vaccinia virus; SFU, spot-forming unit; VV, vaccinia virus.

Figure 7

HIV-specific memory T-cell responses in systemic and mucosal compartments following IN/IM prime-boost immunization. BALB/c mice (n=5) were primed IN with 1 × 10⁷ PFU FPV-HIV or FPV-HIV-IFN-ɛ and boosted IM with 1 × 10⁷ PFU VV-HIV or VV-HIV-IFN-ɛ. Mice were killed 8 weeks post booster immunization, and (a) the percentage of K^dGag_197−205-specific memory CD8⁺ T cell was measured by tetramer staining in the spleen, lung, Peyer's patch, and genito-rectal nodes following 16 h stimulation with H-2K^d-binding AMQMLKETI Gag peptide, (b) the HIV-specific IFN-γ-producing CD8⁺ T-cell responses were measured by intracellular cytokine staining. All above mucosal data represent pooled values, with the exception of spleen data. Experiments were repeated at least three times. HA, hemagglutinin; IFN, interferon; IM, intramuscular; IN, intranasal; PFU, plaque-forming unit; FPV, fowlpox; VV, vaccinia virus.