key: cord-0836135-t0d6h68i
authors: Clemmensen, Helena Strand; Dube, Jean-Yves; McIntosh, Fiona; Rosenkrands, Ida; Jungersen, Gregers; Aagaard, Claus; Andersen, Peter; Behr, Marcel A.; Mortensen, Rasmus
title: In vivo antigen expression regulates CD4 T cell differentiation and vaccine efficacy against Mycobacterium tuberculosis infection
date: 2021-02-03
journal: bioRxiv
DOI: 10.1101/2021.02.02.429488
sha: 9603d456fc7d6470cdb9994f7d84b2b41f09546a
doc_id: 836135
cord_uid: t0d6h68i

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. Importance Tuberculosis, caused by Mtb, constitutes a global health crisis of massive proportions and the impact of the current COVID-19 pandemic is expected to cause a rise in tuberculosis-related deaths. Improved vaccines are therefore needed more than ever, but a lack of knowledge on protective immunity hampers their development. The present study shows that constitutively expressed antigens with high availability drive highly differentiated CD4 T cells with diminished protective capacity, which could be a survival strategy by Mtb to evade T cell immunity against key antigens. We demonstrate that immunisation with such antigens can counteract this phenomenon by maintaining antigen-specific T cells in a state of low differentiation. Future vaccine strategies should therefore explore combinations of multiple highly expressed antigens and we suggest that T cell differentiation could be used as a readily measurable parameter to identify these in both preclinical and clinical studies.

1.4 million people each year. CD4 T cell differentiation is a key determinant of protective immunity 23 against Mtb, but it is not fully understood how host-pathogen interactions shape individual antigen-24 specific T cell populations and their protective capacity. Here, we investigated the immunodominant 25 Mtb antigen, MPT70, which is upregulated in response to IFN-γ or nutrient/oxygen deprivation of in 26 vitro infected macrophages. Using a murine aerosol infection model, we compared the in vivo expres-27 sion kinetics of MPT70 to a constitutively expressed antigen, ESAT-6, and analysed their correspond- 28 ing CD4 T cell phenotype and vaccine-protection. For wild-type Mtb, we found that in vivo expression 29 of MPT70 was delayed compared to ESAT-6. This delayed expression was associated with induction of 30 less differentiated MPT70-specific CD4 T cells but, compared to ESAT-6, also reduced protection after 31 vaccination. In contrast, infection with an MPT70-overexpressing Mtb strain promoted highly differ-32 entiated KLRG1 + CX3CR1 + CD4 T cells with limited lung-homing capacity. Importantly, this differentiat-33 ed phenotype could be prevented by vaccination and, against the overexpressing strain, vaccination 34 with MPT70 conferred similar protection as ESAT-6. Together our data indicate that high in vivo anti- of the current COVID-19 pandemic is expected to cause a rise in tuberculosis-related deaths. Im- 43 proved vaccines are therefore needed more than ever, but a lack of knowledge on protective immuni- 44 ty hampers their development. The present study shows that constitutively expressed antigens with 45 high availability drive highly differentiated CD4 T cells with diminished protective capacity, which 46 could be a survival strategy by Mtb to evade T cell immunity against key antigens. We demonstrate 47 that immunisation with such antigens can counteract this phenomenon by maintaining antigen-48 specific T cells in a state of low differentiation. Future vaccine strategies should therefore explore 49 combinations of multiple highly expressed antigens and we suggest that T cell differentiation could be 50 used as a readily measurable parameter to identify these in both preclinical and clinical studies. T cells are able to enter the lung parenchyma   74  and inhibit Mtb growth (25, 26) while terminally differentiated CD4 T cells co-expressing   75   CX3CR1 + KLRG1 + accumulate in the lung vasculature and provide no pulmonary control of Mtb infec-76 tion (27, 28) . Individual differences in antigen expression are suggested to shape T cell phenotype (22) 77 and may therefore be a key determinant of vaccine protection. 78 The goal of this study was to investigate the impact of in vivo antigen expression on antigen recogni-79 tion kinetics and adaptive immunity during Mtb infection, with MPT70 as a unique tool. Using the 80 well-described 6kDa early secretory antigenic target (ESAT-6) as prototypic immunodominant model 81 antigen (22, (29) (30) (31) , we show that MPT70 displays delayed in vivo antigen expression as well as de-82 layed immune recognition. This is associated with the induction of less differentiated CD4 T cells, but 83 also lower protection in mice vaccinated with MPT70. Based on these observations, we hypothesise 84 that high constitutive antigen expression is associated with increased T cell differentiation, but also 85 improved vaccine capacity. In support of this, we demonstrate that artificial overexpression of MPT70 86 leads to accelerated CD4 T cell differentiation and diminished lung-homing capacity. However, vac-87 cination with MPT70 counteracts this by stabilising a low degree of T cell differentiation and increases 88 protection substantially in the MPT70 overexpressing strain compared to wild-type (WT) Mtb. Our 89 study therefore reveals that antigen expression kinetics regulates CD4 T cell differentiation during 90 infection and establishes a link between in vivo antigen expression, T cell differentiation, and vaccine 91 protective capacity. This has implications for rational vaccine design, and future efforts in TB antigen 92 discovery might use antigen-specific T cell differentiation as a readily measurable proxy for high in 93 vivo antigen expression and increased vaccine potential.

Delayed in vivo antigen transcription results in late immune recognition of MPT70 97 Previous studies indicate that MPT70 expression by Mtb is very low during in vitro cultivation (7, 11) 98 but that expression is induced upon IFN-γ activation (5, 6) or nutrient-deprivation (8). Based on these 99 studies we hypothesised that in vivo transcription and immune recognition of MPT70 would be de-100 layed compared to ESAT-6, a constitutively expressed virulence factor (32, 33). 101 To map the kinetics of MPT70 expression in vivo, we infected a group of CB6F1 mice with Mtb Erd-102 man, which we expected to produce low amounts of MPT70 (7). RNA was extracted from the post-103 caval lobe and cDNA was quantified by real-time qPCR using dual-labelled probes and normalised to 104 16srRNA. Expression levels of MPT70 and ESAT-6 mRNA were analysed prior to infection (week 0), at 105 an early time point (week 4), and at a late time point (week 13). As expected, expression levels were 106 below detection level prior to infection (Figure 1a) . At week 4, MPT70 expression was low and signifi-107 cantly lower than ESAT-6 but as the infection progressed to week 13, MPT70 expression increased 108 and approached levels of ESAT-6, indicating a delayed expression profile (Figure 1a ). 109 We next investigated the kinetics of the immune recognition to the two antigens during the course of . This was in contrast to ESAT-6 responses that were greater at week 3 and 12, after which they 117 plateaued. 118 Together, these data indicate that in vivo expression of MPT70 is delayed compared to ESAT-6 and 119 that this difference in kinetics is associated with delayed onset of specific CD4 T cell responses. to ESAT-6, and this difference was sustained throughout the entire infection (Figure 2b) . Investigating 140 the ability of these CD4 T cell subsets to enter the infected lung tissue by CD45 iv staining further sup-141 ported that a smaller fraction of MPT70-specific CD4 T cells were retained in the lung-associated vas-142 culature (CD45 iv + ) compared to ESAT-6 CD4 T cells (Figure 2c ). 143 We next wanted to confirm these observations using an MHC-II tetramer. In contrast to ICS, tetramers 144 identify antigen-specific T cells without the risk of affecting the expression of certain markers due to 145 ex vivo stimulation. We therefore epitope mapped the MPT70 protein (29) and developed a murine The impact of vaccinating with MPT70 is lower than for ESAT-6 160 The previous data showed that T cells specific for MPT70 were less differentiated than those specific Danchuk for technical assistance and fruitful discussion on RNA extractions and qPCR assays. 348 We thank Vivi Andersen, Ming Liu Olsen, and Camilla Haumann Rasmussen at SSI for their excellent 349 technical assistance. We also gratefully acknowledge the mouse work done by the competent veteri- 

To determine in vitro mRNA levels of MPT70, SigK, RskA, and ESAT-6, we performed an RT-qPCR using 436 Maxima SYBR green kit (Thermo Scientific, Cat.no.K0223) with the use of the following primers (Table   437 1). mRNA levels were normalised to EsxA and fold gene expression from Mtb H37Rv was plotted as 2 - Splenocytes or lung cells were adjusted to a cell concentration of 2x10 5 cells/well and restimulated in 526 the presence of recombinant protein or peptides in round-bottom plates for 3 days as previously de-527 scribed (29). A sandwich ELISA was performed on the culture supernatants to determine the concen-528 tration of total IFN-γ. In brief, microtiter plates were coated with primary IFN-γ antibody, blocked with 529 2% skimmed milk, and incubated overnight with pre-diluted supernatants. IFN-γ was detected with a 530 secondary IFN-γ antibody, followed by an HRP-conjugated antibody and the reaction was developed 531 using TMB substrate (TMB Plus; Kementec). Plates were read at 450 nm with 620 nm background cor-532 rection using an ELISA reader (Tecan Sunrise). 

Global tuberculosis report 2020. World Health Organization

Dormancy phenotype displayed by extracellular Mycobacterium tuberculosis within artificial 549 granulomas in mice

Expression of mycobacterial cell 551 division protein, FtsZ, and dormancy proteins, DevR and Acr, within lung granulomas throughout 552 guinea pig infection

Transcriptional Adaptation of Mycobacterium tuberculosis within

Macrophages: Insights into the Phagosomal Environment

Rv0444c, the gene encoding anti-SigK, explain high level expression of MPB70 and MPB83 in

Mycobacterium bovis

MPB70, a unique antigen of Mycobacterium bovis BCG

Mycobacterium tuberculosis persistence by gene and protein expression profiling

Cellular and humoral immune responses of cattle to 565 purified Mycobacterium bovis antigens

Antigen Availability Shapes T Cell Differentiation and Function during

Role of antigen persistence and dose for CD4+

T-cell exhaustion and recovery

Cutting edge: control of Mycobacterium tuberculosis infection by a subset of lung parenchyma-homing

The chemokine receptor CXCR3 attenuates 610 the control of chronic Mycobacterium tuberculosis infection in BALB/c mice

Early granuloma formation after aerosol Mycobacterium tuberculosis infection is 616 regulated by neutrophils via CXCR3-signaling chemokines

Rescuing ESAT-6 Specific CD4 T Cells From Terminal Differentiation Is Critical for

ESAT-6 (EsxA) and TB10.4 (EsxH) based vaccines for pre-and post-exposure tuberculosis 622 vaccination

ESAT-6-specific CD4 T cell responses to aerosol

Mycobacterium tuberculosis infection are initiated in the mediastinal lymph nodes

Expression levels of

Mycobacterium tuberculosis antigen-encoding genes versus production levels of antigen-specific T cells 629 during stationary level lung infection in mice

Effect of growth state on transcription levels of genes encoding 631 major secreted antigens of Mycobacterium tuberculosis in the mouse lung

Cationic liposomes formulated with 638 synthetic mycobacterial cordfactor (CAF01): a versatile adjuvant for vaccines with different 639 immunological requirements

RskA Is a Dual Function 641

The Rate of CD4 T Cell Entry 644 into the Lungs during Mycobacterium tuberculosis Infection Is Determined by Partial and Opposing 645 Effects of Multiple Chemokine Receptors

IFNgamma/TNFalpha specific-cells and effector memory phenotype associate with active tuberculosis

The temporal expression profile of 652 <em>Mycobacterium tuberculosis</em> infection in mice

Subunit 654 vaccine H56/CAF01 induces a population of circulating CD4 T cells that traffic into the Mycobacterium 655 tuberculosis-infected lung

Expression of Th1-mediated immunity in mouse 660 lungs induces a Mycobacterium tuberculosis transcription pattern characteristic of nonreplicating 661 persistence

A booster vaccine expressing a 663 latency-associated antigen augments BCG induced immunity and confers enhanced protection against 664 tuberculosis

Multi-stage 666 subunit vaccines against Mycobacterium tuberculosis: an alternative to the BCG vaccine or a BCG-667 prime boost?

A multistage tuberculosis vaccine that confers efficient protection before and after 670 exposure

Mouse models of human TB pathology: roles in the analysis of necrosis and 674 the development of host-directed therapies

Mycobacterium tuberculosis Clinical Isolates and Resulting Outcomes of Tuberculosis Infection and 677

Overexpression of heat-shock proteins reduces survival of Mycobacterium tuberculosis in the 683 chronic phase of infection

Orme 685 IM, Barnes PF. 2004. The principal sigma factor sigA mediates enhanced growth of Mycobacterium 686 tuberculosis in vivo

T-and B-cell epitopes in the secreted

Mycobacterium bovis antigen MPB70 in mice

MPT70 and 693 ESAT-6 in vivo gene expression were assessed pre-infection (week 0) and 4 and 13 weeks post-694 infection (p.i.) (n=4). The expression pre-infection was below detection levels (b.d.). Shown as average 695 mean ± SEM. Paired t-test, two-tailed. (b, left) At week 3, 12, and 20 post Mtb infection, lungs were 696 harvested for immunological analyses

Shown as average mean ± SEM. One-way ANO-699 VA with Tukey's Multiple Comparison test (b, right) Fold change in cytokine-producing CD4 T cells 700 from baseline. (c) Lung cells from infected mice were restimulated in vitro with media, MPT70 or 701 ESAT-6 for five days. Culture supernatant was harvest and measured for IFN-γ levels in two individual 702 experiments (n=4)

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

The functional differentiation score (FDS) of MPT70 and ESAT-6-specific CD4 T cells over the course of

The FDS is defined as the ratio of all IFN-γ producing CD4 T cell subsets 708 divided by subsets producing other cytokines (IL-2, TNF-α), but not IFN-γ (high FDS = high IFN-γ pro-709 duction). Multiple t-tests with correction for multiple testing using the Holm-Sidak method. Shown as ± SEM. Flow Cytometry gating as depicted in Figure S1

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-714 associated vasculature (CD45 + ) 20 weeks post-infection (p.i.) with Mtb (n=4). Paired t-test, two-tailed. 715 (d, upper) Schematic representation of custom-made I-A b :MPT70 38-52 MHC

Representative concatenated FACS plots showing frequencies of I-A b :MPT70 38-52 and I-Ab

tetramer + CD4 T cells or corresponding hClip tetramer + CD4 T cells in lungs of mice 12 weeks post Mtb 718 infection (n=4). (e) Frequency of I-A b :MPT70 38-52 and I-Ab:ESAT-6 4-17 CD4 T cells 12-16 weeks post Mtb 719 infection expressing CXCR3, KLRG1, and T-bet. Parametric, paired t-test, two-tailed (n=12). Flow Cy-720 tometry gating as depicted in Figure S3 using antibody panel 1. (f) Concatenated FACS plot of

Fe-724 male CB6F1 mice were immunised with either MPT70 or ESAT-6 recombinant protein three times s.c. 725 and challenged with Mtb Erdman six weeks post the third immunisation. (a) Frequency of MPT70 and 726 ESAT-6 specific CD4 T cells in the spleen two weeks post the third vaccination (n=4). (b) Frequency of 727 MPT70 and ESAT-6 specific CD4 T cells in the lung week 3, 12, and 20 post Mtb infection (n=4)

Shown as box plots with whiskers 747 indicating the minimum and maximum values. Mean indicated with '+'. Unpaired, two-tailed t-test. 748 (e) Frequency of lung MPT70-specific CD4 T cells 3 weeks post Mtb infection in PBS vaccinated (white 749 boxes) and MPT70 vaccinated (blue boxes, n=5). Unpaired, two-tailed t-test. (f) Representative con-750 catenated FACS plots (n=10) showing the expression of CX3CR1

Bacterial numbers were deter-753 mined in the lungs of PBS, MPT70 and ESAT-6 vaccinated mice at day 1, week 3, week 12, and week 754 22 post WT H37Rv infection (left) or H37Rv::mpt70 high infection (right) (n=4-5)

Tukey's Multiple Comparison Test

Spleens 762 and lungs were harvested from mice and prepared as single-cell suspensions. Shown as representa-763 tive gating from sample WT H37Rv, ESAT-6-specific T cells 3 weeks post Mtb infection using antibody 764 panel 3. Cells were gated as singlets, lymphocytes

CD4 T cells were analysed for their intracellular production of IFN-γ, TNF-α, IL-2, and IL-17A. The fre-766 quency of antigen-specific CD4 T cells was determined by a make or gate for IFN-γ

T cells can produce one or more of the cytokines). T cell differentiation degrees were ana-768 lysed with a combination gate for IFN-γ, TNF-α, and IL-2, characterising the cytokine subsets of T cells

The frequency of CD45 + , KLRG1, PD-1, CXCR3, and CX3CR1 was assessed on antigen-specific T cells

Fluorescence minus one (FMO) controls were used to set boundaries gates for CD44, KLRG1, PD-1, 771 CXCR3, and CX3CR1

Epitope mapping and design of an MPT70 tetramer. (a) Splenocytes of MPT70-vaccinated 774 mice were in vitro restimulated with overlapping peptides of 15 amino acids in length for 3 days (n=4)

The dominant epitope required for 776 binding is highlighted in bold blue text and the predicted core epitope in bold black text. (b, left) The 777 minimal epitope of the 38-53 sequence of MPT70 was investigated with varying lengths of peptides in 778 MPT70 vaccinated mice, 20 weeks post-infection (n=4). (b, right) Comparison of the response to me-779 dium, the chosen 38-53 epitope, and recombinant MPT70

Gating strate-782 gy for tetramer-positive CD4 T cells. Lung cells of vaccinated and infected mice were prepared as sin-783 gle-cell suspensions and analysed by flow cytometry. Shown as representative gating for tetramer-784 positive CD4 T cells exemplified with saline mouse A6

Tetramer positive CD4 T cells were further characterised for their expression of KLRG1, T-bet, 788 and CXCR3. Fluorescence minus one (FMO) controls were used to set boundaries gates for KLRG1

Shown as box plots with whiskers indicat-795 ing the minimum and maximum values. (b) The bacterial burdens were determined in the lungs of 796 saline and vaccinated mice 19 or 20 weeks post Mtb infection (n=28). The graph represents four indi-797 vidual experiments

mRNA levels were normalised to 16s rRNA. Shown as box plots with whiskers 802 indicating the minimum and maximum values. Paired t-test, two-tailed. (b) In vitro growth of WT 803 H37RV, H37Rv::mpt70 high (rskA and sigK insert of M.orygis origin), and H37Rv::Rv (rskA and sigK insert 804 of Mtb origin). Strains were grown in 7H9 medium for 4 days and the OD 600 was measured every 24 805 hours (n=3)

KLRG1 + CX3CR1 + expressing MPT70 specific CD4 T cells in PBS vaccinated and 807 MPT70-vaccinated mice 3 weeks post WT H37RV and H37Rv::mpt70 high infection (n=5)

Shown as box plots with whiskers indicating the minimum and maximum values. Two independent 811 experiments