In vivo antigen expression regulates CD4 T cell differentiation and vaccine efficacy against Mycobacterium tuberculosis infection

Abstract

New vaccines are urgently needed against Mycobacterium tuberculosis (Mtb), which kills more than 1.4 million people each year. CD4 T cell differentiation is a key determinant of protective immunity against Mtb, but it is not fully understood how host-pathogen interactions shape individual antigen-specific T cell populations and their protective capacity. Here, we investigated the immunodominant Mtb antigen, MPT70, which is upregulated in response to IFN-γ or nutrient/oxygen deprivation of in vitro infected macrophages. Using a murine aerosol infection model, we compared the in vivo expression kinetics of MPT70 to a constitutively expressed antigen, ESAT-6, and analysed their corresponding CD4 T cell phenotype and vaccine-protection. For wild-type Mtb, we found that in vivo expression of MPT70 was delayed compared to ESAT-6. This delayed expression was associated with induction of less differentiated MPT70-specific CD4 T cells but, compared to ESAT-6, also reduced protection after vaccination. In contrast, infection with an MPT70-overexpressing Mtb strain promoted highly differentiated KLRG1⁺CX3CR1⁺ CD4 T cells with limited lung-homing capacity. Importantly, this differentiated phenotype could be prevented by vaccination and, against the overexpressing strain, vaccination with MPT70 conferred similar protection as ESAT-6. Together our data indicate that high in vivo antigen expression drives T cells towards terminal differentiation and that targeted vaccination with adjuvanted protein can counteract this phenomenon by maintaining T cells in a protective less-differentiated state. These observations shed new light on host-pathogen interactions and provide guidance on how future Mtb vaccines can be designed to tip the immune-balance in favor of the host.

Keywords: ESAT-6; M. tuberculosis; MPT70; T cell differentiation; Vaccination; in vivo expression.

Figure 1.. In vivo antigen expression and immune recognition of MPT70 is delayed during Mtb Erdman infection.

CB6F1 mice were infected by the aerosol route with Mtb Erdman. (a) MPT70 and ESAT-6 in vivo gene expression were assessed pre-infection (week 0) and 4 and 13 weeks post-infection (p.i.) (n=4). The expression pre-infection was below detection levels (b.d.). Shown as average mean ± SEM. Paired t-test, two-tailed. (b, left) At week 3, 12, and 20 post Mtb infection, lungs were harvested for immunological analyses. Frequency of cytokine-producing CD3⁺CD4⁺ T cells specific for MPT70, ESAT-6 for the same time points as in c, exp1 (medium cytokine production subtracted) analysed by flow cytometry using antibody panel 2 (n=4). Shown as average mean ± SEM. One-way ANOVA with Tukey’s Multiple Comparison test (b, right) Fold change in cytokine-producing CD4 T cells from baseline. (c) Lung cells from infected mice were restimulated in vitro with media, MPT70 or ESAT-6 for five days. Culture supernatant was harvest and measured for IFN-γ levels in two individual experiments (n=4). Values were log-transformed and shown as average mean ± SEM. Paired t-test, two-tailed.

Figure 2.. MPT70-specific CD4 T cells maintain a low differentiation state compared to ESAT-6.

(a) The functional differentiation score (FDS) of MPT70 and ESAT-6-specific CD4 T cells over the course of Mtb Erdman infection (n=4). The FDS is defined as the ratio of all IFN-γ producing CD4 T cell subsets divided by subsets producing other cytokines (IL-2, TNF-α), but not IFN-γ (high FDS = high IFN-γ production). Multiple t-tests with correction for multiple testing using the Holm-Sidak method. Shown as average mean ± SEM. Flow Cytometry gating as depicted in Figure S1, using antibody panel 2. (b) Frequencies of KLRG1 expressing MPT70 and ESAT-6-specific CD4 T cells throughout infection (n=4). Shown as average mean ± SEM. Multiple t-tests with correction for multiple testing using the Holm-Sidak method. (c) Frequency of CD45-labelled MPT70 and ESAT-6 specific CD4 T cells in the lung-associated vasculature (CD45⁺) 20 weeks post-infection (p.i.) with Mtb (n=4). Paired t-test, two-tailed. (d, upper) Schematic representation of custom-made I-A^b:MPT70_38–52 MHC-II tetramer. (d, lower) Representative concatenated FACS plots showing frequencies of I-A^b:MPT70_38–52 and I-Ab:ESAT-6_4–17 tetramer⁺ CD4 T cells or corresponding hClip tetramer⁺ CD4 T cells in lungs of mice 12 weeks post Mtb infection (n=4). (e) Frequency of I-A^b:MPT70_38–52 and I-Ab:ESAT-6⁴⁻¹⁷ CD4 T cells 12–16 weeks post Mtb infection expressing CXCR3, KLRG1, and T-bet. Parametric, paired t-test, two-tailed (n=12). Flow Cytometry gating as depicted in Figure S3 using antibody panel 1. (f) Concatenated FACS plot of CX3CR1⁺KLRG1⁺ co-expressing ESAT-6⁴⁻¹⁷ CD4 T cells (n=4).

Figure 3.. Vaccination with MPT70 has a lower impact on CD4 T cell differentiation than ESAT-6.

Female CB6F1 mice were immunised with either MPT70 or ESAT-6 recombinant protein three times s.c. and challenged with Mtb Erdman six weeks post the third immunisation. (a) Frequency of MPT70 and ESAT-6 specific CD4 T cells in the spleen two weeks post the third vaccination (n=4). (b) Frequency of MPT70 and ESAT-6 specific CD4 T cells in the lung week 3, 12, and 20 post Mtb infection (n=4). Shown as average mean ± SEM. (c) Functional differentiation score (FDS) of MPT70 and ESAT-6-specific CD4 T cells pre-infection in the spleen (n=4). (d) FDS of MPT70 and ESAT-6-specific CD4 T cells 3 and 20 weeks post Mtb infection in lungs of vaccinated and saline mice (n=4). Shown as average mean ± SEM. Flow Cytometry gating as depicted in Figure S1, using antibody panel 2. (e) The bacterial burden was determined in the lungs of saline, MPT70 and ESAT-6 vaccinated mice at 3–4 weeks post Mtb infection (n=26–28). The graph represents four individual experiments of which experiment 4 is already published in (29). One-Way ANOVA with Tukey’s multiple comparison test.

Figure 4.. Overexpression of MPT70 accelerates T cell differentiation and improves vaccine efficacy.

(a) In vitro fold gene expression of sigK, rskA, ESAT-6, MPT70, and MPT83 in H37Rv::mpt70^high compared to WT H37Rv. All genes were tested in technical duplicates and normalised to esxA expression using primers in Table 1. (b) In vivo fold gene expression of MPT70 and ESAT-6 in lungs ofH37Rv::mpt70^high infected mice compared to WT H37Rv infected mice 3 weeks post Mtb challenge (n=5). Genes were analysed in technical duplicates using primers and probes in Table 2, normalised to 16s rRNA expression, and shown as fold increase from WT H37Rv. Unpaired t-test, two-tailed. (c) Bacterial burden in lungs of PBS-vaccinated mice at day1, week 3, week 12, and week 22 after infection with either H37Rv:: mpt70^high or WT H37Rv infection (n=5). Shown as average mean ± SEM. Multiple t-tests with correction for multiple testing using the Holm-Sidak method. (d) Frequency of lung MPT70 and ESAT-6-specific CD4 T cells 4 weeks post Mtb infection (n=10). Shown as box plots with whiskers indicating the minimum and maximum values. Mean indicated with ‘+’. Unpaired, two-tailed t-test. (e) Frequency of lung MPT70-specific CD4 T cells 3 weeks post Mtb infection in PBS vaccinated (white boxes) and MPT70 vaccinated (blue boxes, n=5). Unpaired, two-tailed t-test. (f) Representative concatenated FACS plots (n=10) showing the expression of CX3CR1, CXCR3, KLRG1 or CD45 on MPT70-specific CD4 T cells 4 weeks post H37Rv:: mpt70^high infection (blue) or H37Rv infection (grey). Flow Cytometry gating as depicted in Figure S1, using antibody panel 3. (g) Bacterial numbers were determined in the lungs of PBS, MPT70 and ESAT-6 vaccinated mice at day 1, week 3, week 12, and week 22 post WT H37Rv infection (left) or H37Rv::mpt70^high infection (right) (n=4–5). One mouse was excluded from the week 12 timepoint (H37Rv::mpt70^high, MPT70 vaccinated), as the mouse was very sick, had high weight loss, and met the study’s predefined humane endpoints (p-value=0.67, if included). Shown as average mean ± SEM. Statistical differences were assessed using One-Way ANOVA with Tukey’s Multiple Comparison Test.