Near-Infrared 1064 nm Laser Modulates Migratory Dendritic Cells To Augment the Immune Response to Intradermal Influenza Vaccine

Abstract

Brief exposure of skin to near-infrared (NIR) laser light has been shown to augment the immune response to intradermal vaccination and thus act as an immunologic adjuvant. Although evidence indicates that the NIR laser adjuvant has the capacity to activate innate subsets including dendritic cells (DCs) in skin as conventional adjuvants do, the precise immunological mechanism by which the NIR laser adjuvant acts is largely unknown. In this study we sought to identify the cellular target of the NIR laser adjuvant by using an established mouse model of intradermal influenza vaccination and examining the alteration of responses resulting from genetic ablation of specific DC populations. We found that a continuous wave (CW) NIR laser adjuvant broadly modulates migratory DC (migDC) populations, specifically increasing and activating the Lang⁺ and CD11b^-Lang^- subsets in skin, and that the Ab responses augmented by the CW NIR laser are dependent on DC subsets expressing CCR2 and Langerin. In comparison, a pulsed wave NIR laser adjuvant showed limited effects on the migDC subsets. Our vaccination study demonstrated that the efficacy of the CW NIR laser is significantly better than that of the pulsed wave laser, indicating that the CW NIR laser offers a desirable immunostimulatory microenvironment for migDCs. These results demonstrate the unique ability of the NIR laser adjuvant to selectively target specific migDC populations in skin depending on its parameters, and highlight the importance of optimization of laser parameters for desirable immune protection induced by an NIR laser-adjuvanted vaccine.

Figure 1. Effect of the laser adjuvant on anti-influenza immune responses

(a–f) Influenza-specific humoral responses 4 days after challenge. Mice were vaccinated with 1 µg of inactivated influenza virus (A/PR/8/34) with or without laser illumination or representative chemical adjuvant (alum or AddaVax) and challenged intranasally with live homologous virus 4 weeks after vaccination. Titer of influenza-specific serum IgG subclass was determined by ELISA, plates were coated with inactivated influenza virus. (a) IgG, (b) IgG1 and (c) IgG2c titers. (d) HAI titers. (e) IgG2c/IgG1 ratio. (f) IgE titers. (g–h), Systemic T-cell responses were measured 4 days after challenge by re-stimulating 1×10⁶ splenocytes with inactivated influenza vaccine antigen. Levels of (g) IFN-γ and (h) IL-4 in splenocyte culture supernatants are shown. Experimental and control groups: (a–c, e) n = 30, 29, 26, 27, 10, 8, 18, 5, (d) n = 14, 22, 22, 22, 10, 7, 10, 5, (f) n = 5, 5, 5, 5, 5, 3, 5, 5, (g–h) n = 20, 19, 18, 17, 5, 8, 9, 5, for no vaccine, vaccine i.d., vaccine i.d. + CW 1064 nm, vaccine i.d. + PW 1064 nm, vaccine i.d. + PW 532 nm, vaccine i.m., vaccine + Alum i.d., and vaccine + AddaVax i.d. vaccine groups, respectively. Results were pooled from three independent experiments and analyzed using two-way ANOVA with Tukey’s correction.

Figure 2. The effect of NIR laser adjuvant on DCs within the skin draining lymph nodes

DCs in skin-draining lymph nodes (skin-dLN) were processed and stained for multi-parameter flow cytometry 24 hours after intradermal vaccination with 40 µg Alexa Fluor-488-labeled OVA with or without one minute CW or PW 1064 nm NIR laser treatment. (a) Representative gates of plasmacytoid DCs (pDC), classical lymphoid tissue-resident DCs (cDCs), and migratory DCs (migDC); numbers indicate percent of total lymphocytes. (b) Cell counts. (c) Representative histograms of CD86 expression. (d) Median fluorescent intensity of CD86 expression for pDC, cDC, and migDC population. (e) Representative gates of migDC subsets. (f) Cell counts of migDC subpopulation within skin-dLN. (g) Representative gates of Lang⁺migDC subsets, numbers indicate percent parent. (h) Cell counts of Lang⁺migDC subpopulation within skin-dLN. (i) Representative gates of cDC subsets, number representing percent parent. (j) Cell counts of cDC subpopulation within skin-dLN. (k) Representative gates of CD11b⁺Ly6C⁺ monocytes. (l) Cell counts of CD11b⁺Ly6C⁺ monocytes within skin-dLN. Data were analyzed with (b, f, h, j and l) two-way ANOVA followed by the Tukey's honestly significant difference (HSD) tests or (d) Kruskal-Wallis with Dunn’s correction for multiple comparisons. Experimental and control groups: (a–j) n = 16, 16, 17, (k–l) n = 6, 6, 7, for OVA i.d., OVA i.d. + CW 1064 nm, OVA i.d. + PW 1064 nm, respectively. Data are derived from three independent experiments.

Figure 3. The effect of NIR laser adjuvant on antigen-bearing DCs within the skin draining lymph nodes

(a) Representative gates of pDC, cDC, and migDC bearing OVA antigen, number indicates cell count of positive gate. (b) Cell counts of OVA⁺ pDC, cDC, and migDC population within skin-dLN. (c) Representative histograms of OVA⁺migDCs. (d) Cell counts of OVA⁺migDC subpopulation within skin-dLN. (e) Cell counts of OVA⁺Lang⁺migDC subpopulation within skin-dLN. (b, d, e) Data were analyzed with two-way ANOVA followed by the Tukey's honestly significant difference (HSD) tests. Experimental and control groups: (a–j) n = 15, 16, 16, 17 for no vaccine, OVA i.d., OVA i.d. + CW 1064 nm, OVA i.d. + PW 1064 nm, respectively. Data are derived from three independent experiments.

Figure 4. The effect of NIR laser on emigration of migDC subsets

Mice were shaved, depilated, and painted with 1% FITC solution on the flank skin 4 hours before vaccination with 40 µg of OVA with or without NIR laser treatment. At the times indicated, single-cell suspensions from skin-dLN were labeled and analyzed based on surface markers and FITC fluorescence by flow cytometry. (a) Representative gates of FITC⁺ migDC emigrating into the skin-dLN after FITC painting. (b) Cell counts of in the skin-dLN after FITC painting. (c) Representative gates of migDC subsets. (d) Cell counts of migDC subpopulation within skin-dLN after FITC painting. (e) Representative gates of Lang⁺migDC subsets. (f) Cell counts of Lang⁺migDC subpopulation within skin-dLN after FITC painting. (b, e, f) Two-way ANOVA followed by the Tukey's HSD tests. Experimental and control groups: n = 6–7, 6–7, 4 for vaccine i.d., vaccine i.d. + CW 1064 nm, vaccine i.d. + PW 1064 nm, respectively. Data are derived from three independent experiments.

Figure 5. Depletion of Lang+ DCs removes CW laser induced population changes

Lang⁻DTR/GFP mice were treated with diphtheria toxin 1 day prior to four intradermal injections of 10 µg of A488-labeled ovalbumin (OVA) with or without laser adjuvant illumination. DCs in skin-dLN were processed and stained for multi-parameter flow cytometry 24 hours after intradermal vaccination with 40 µg Alexa Fluor-488-labeled OVA with or without one minute CW or PW 1064 nm NIR laser treatment. (a) Representative gates of plasmacytoid DCs (pDC), classical lymphoid tissue-resident DCs (cDCs), and migratory DCs (migDC); numbers indicate percent of total lymphocytes. (b) Cell counts. (c) Representative gates of migDC subsets, numbers indicate percent parent. (f) Cell counts of migDC subpopulation within skin-dLN. (i) Representative gates of cDC subsets, number indicating percent parent. (j) Cell counts of cDC subpopulation within skin-dLN. (f, j) Data were analyzed with two-way ANOVA followed by the Tukey's honestly significant difference (HSD) tests. Experimental and control groups: (a–j) n = 4, 4, 3 for OVA i.d., OVA i.d. + CW 1064 nm, OVA i.d. + PW 1064 nm, respectively.

Figure 6. Effect of the laser adjuvant on anti-influenza immune responses in CCR2^−/− or CCR7^−/− mice

(a–e) Influenza-specific humoral responses in post-challenge (4 days after challenge). C57BL/6 WT, CCR2^−/−, or CCR7^−/− mice were vaccinated with 1 µg of inactivated influenza virus (A/PR/8/34) with or without laser illumination and challenged intranasally with live homologous virus 4 weeks after vaccination. Titer of influenza-specific serum IgG subclass was determined by ELISA. (a) IgG, (b) IgG1 and (c) IgG2c titers. (d) HAI titers. (e) IgG2c/IgG1 ratio. All experiments were repeated 3 times and pooled to show results. (f–g) Systemic T-cell responses were measured 4 days after challenge by re-stimulating 1×10⁶ splenocytes with inactivated influenza vaccine antigen for 60 hours. Levels of (f) IFN-γ and (g) IL-4 in splenocyte culture supernatants are shown. Data were analyzed with two-way ANOVA followed by the Tukey's HSD tests. WT data from Fig. 1 are shown for comparison. See the Material and Methods section for strategy used for statistical analysis. Experimental and control groups: (a–c, e) n = 30, 29, 26, 27, 9, 9, 8, 10, 9, 7, (d) 14, 22, 22, 22, 9, 9, 8, 7, 6, 4 (f–g) n = 20, 19, 18, 17, 9, 9, 8, 10, 9, 7 for no vaccine in WT, vaccine i.d. in WT, vaccine i.d. + CW 1064 nm in WT, vaccine i.d. + PW 1064 nm in WT, vaccine i.d. in CCR2^−/−, vaccine i.d. + CW 1064 nm in CCR2^−/−, vaccine i.d. + PW 1064 nm in CCR2^−/−, vaccine i.d. in CCR7^−/−, vaccine i.d. + CW 1064 nm in CCR7^−/−, vaccine i.d. + PW 1064 nm in CCR7^−/−groups, respectively.

Figure 7. Effect of the laser adjuvant on anti-influenza immune responses in Lang⁺ cell-depleted mice

(a–e) Influenza-specific humoral responses in post-challenge (4 days after challenge). Lang-DTR/GFP mice were treated with diphtheria toxin 1 day prior to vaccination with 1 µg of inactivated influenza virus (A/PR/8/34) with or without laser illumination and challenged intranasally with live homologous virus 4 weeks after vaccination. Titer of influenza-specific serum IgG subclass was determined by ELISA. (a) IgG, (b) IgG1 and (c) IgG2c titers. (d) HAI titers. (e) IgG2c/IgG1 ratio. All experiments were repeated 3 times and pooled to show results. (f–g) Systemic T-cell responses were measured 4 days after challenge by re-stimulating 1×10⁶ splenocytes with inactivated influenza vaccine antigen for 60 hours. Levels of (f) IFN-γ and (g) IL-4 in splenocyte culture supernatants are shown. Data were analyzed with two-way ANOVA followed by the Tukey's honestly significant difference (HSD) tests. WT data from Fig. 1 are shown for comparison. See the Material and Methods section for strategy used for statistical analysis. Experimental and control groups: (a–c, e) n = 30, 29, 26, 27, 11, 11, 10, (d) n = 14, 22, 22, 22, 8, 11, 10, (f–g) n = 20, 19, 18, 17, 8, 8, 7 for no vaccine in WT, vaccine i.d. in WT, vaccine i.d. + CW 1064 nm in WT, vaccine i.d. + PW 1064 nm in WT, vaccine i.d. in Lang/DTR + DT, vaccine i.d. + CW 1064 nm in Lang/DTR + DT, vaccine i.d. + PW 1064 nm in Lang/DTR + DT groups, respectively. (h) Kaplan-Meier survival plots of influenza-vaccinated mice for 15 days following lethal challenge. Data were analyzed with Gehan-Breslow-Wilcoxon test. EID₅₀, the 50% egg infectious dose. Experimental and control groups: n = 10, 15, 8, 5, 4 for no vaccine in WT, vaccine i.d. in WT, vaccine i.d. + CW 1064 nm in WT, vaccine i.d. in Lang/DTR + DT, vaccine i.d. + CW 1064 nm in Lang/DTR + DT, respectively.