TCR affinity controls the dynamics but not the functional specification of the Th1 response to mycobacteria

TL;DR: A distinct yet cooperative role for IL-12 and TCR signalling in Th1 differentiation is revealed and it is suggested that the temporal activation of clones with different TCR affinity is a major strategy to coordinate immune surveillance against persistent pathogens.

Abstract: The quality of T cell responses depends on the lymphocytes’ ability to undergo clonal expansion, acquire effector functions and traffic to the site of infection. Although TCR signal strength is thought to dominantly shape the T cell response, by using TCR transgenic CD4+ T cells with different pMHC binding affinity, we reveal that TCR affinity does not control Th1 effector function acquisition nor the functional output of individual effectors following mycobacterial infection. Rather, TCR affinity calibrates the rate of cell division to synchronize the distinct processes of T cell proliferation, differentiation and trafficking. By timing cell division-dependent IL-12R expression, TCR affinity controls when T cells become receptive to Th1-imprinting IL-12 signals, determining the emergence and magnitude of the Th1 effector pool. These findings reveal a distinct yet cooperative role for IL-12 and TCR signalling in Th1 differentiation and suggests that the temporal activation of clones with different TCR affinity is a major strategy to coordinate immune surveillance against persistent pathogens.

Summary

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Figures

Figure. 6. High affinity TCRs accelerate Th1 cell egress from the spleen to expedite the antimycobacterial Th1 tissue response at the primary site of infection.

Figure. 3. High affinity TCRs accelerate the IL-12-dependent generation of Th1 effector populations. (A-D) Purified C7 and C24 CD4+ Tg cells were cultured with CD4-depleted splenocytes from naïve B6 mice in the presence or absence of E6 peptide. In some E6-stimulated cultures, mAbs to IL-12p70, IL-4 and IFN-g were added (TCR-driven). In others, mAbs to IL-4 and IFN-g, together with recombinant IL-12p70, were included (IL-12-driven). (A) Flow cytometry plots showing the levels of T-bet expression in Tg cells in the absence (TCRdriven) or presence of IL-12 (IL-12-driven). (B) Changes in the percentage of T-bet high (T-bethi) cells under different polarization conditions analysed by flow cytometry. Data shown are the mean percentage of T-bethi Tg cells ± sd of triplicate cultures. (C) Representative flow cytometry plot showing intracellular IFN-γ expresseion in T-bethi cell populations. E6 peptide-activated C7 and C24 T cells were cultured under IL-12 driven culture conditions for 66 h and intracellular T-bet and IFN-γ expression was analyzed without further re-stimulation. BFA was added to the culture 6h before analysis. (D) Expansion of T-bethi Tg cell populations in the presence of IL-12, anti-IL-4 and anti-IFN-g. Data shown are the mean numbers of T-bethi Tg cells ± sd of triplicate cultures. (E-F) Purified CD4+ T cells from C7xGFPxRag1—/— or C24xGFPxRag1—/— mice were transferred i.v. into separate naive B6 or Il12p40—/— recipients 1 day prior to i.v. inoculation with BCG-E6. Data are representative of two independent experiments with similar results (4 mice / group). Symbols denote individual mice and open bars represent group means. (E) Proportion of C7 and C24 cells that express T-bet in the spleen at day 2 p.i.. (F) Representative flow cytometry plots and summary data showing the percentage of IFN-γ expressing Tg cells in the liver 6 d.p.i.. Hepatic leukocytes were re-stimulated in the presence or absence of E6 peptide (1 μg/mL) for 5 h ex vivo before flow cytometry analysis. For all data, the lines, symbols or FACS plots in black and red represent C7 and C24 cells, respectively. Data are representative of three independent experiments with similar results. In (E) and (F), closed circles represent individual mice and bars denote the group mean. Statistical differences between C7 and C24 cells were determined by Student t-test analysis, (*p<0.05, **p < 0.01, ***p < 0.001).

Figure. 1. CD4+ T cells with high affinity TCRs undergo enhanced accumulation in lymphoid and non-lymphoid organs following mycobacterial infection. (A) Schematic diagram illustrating the adoptive transfer and infection model. Magnetically-purified CD4+ T cells from C7xGFPxRag1—/— or C24xGFPxRag1—/— TCR transgenic (Tg) mice were transferred i.v. into separate intact, naïve C57BL/6 (B6) recipients (105 / mouse) 1 d prior to i.v. inoculation with BCG-E6 (106 CFU / mouse). The liver-draining lymph node (dLN), spleen, liver and spleen was collected at various time-points p.i.. (B) Flow cytometric detection of GFP-expressing TCR Tg T cells in the spleens of recipient mice that did not receive or received transgenic T cells 3 days after BCG-E6 infection. (C) Flow cytometric analysis of CTV dilution in GFP-expressing CD4+ C7 and C24 cells in the dLN, liver and spleen at day 1, 2, 3 and 6 p.i.. Black and red FACS plots represent C7 and C24 CD4+ T cells, respectively. (D) Frequency of GFP+ C7 or C24 cells in the dLN, liver and spleen at the indicated time-points. Data are pooled from three independent experiments with similar trends; Data shown are the mean percentage ± sem (n > 8 mice / group / time point). Symbols or lines in black and red represent C7 and C24 cells, respectively. Statistical differences between C7 and C24 cells were determined by Student’s t-test (*p<0.05, **p < 0.01).

Figure. 5. TCR signal-driven IL-12Rb2 upregulation is cell division-dependent. Purified C7 and C24 CD4+ Tg cells were cultured with CD4-depleted splenocytes from naïve B6 mice in the presence or absence of E6 peptide. (A) Flow cytometry plots showing IL-12Rβ2 and T-bet expression in Tg cells 48 h after stimulation. In some E6-stimulated cultures, mAbs to IL-12p70, IL-4 and IFN-g were added (TCRdriven) while in others, mAbs to IL-4 and IFN-g, together with recombinant IL-12p70, were included (IL-12driven). (B) Kinetic analysis of the percentage and absolute number of IL-12Rβ2-expressing Tg cells by flow cytometry. Data shown are the mean ± sd of triplicate cultures. Statistical differences between C7 and C24 cultures were determined by Students t-test (*p<0.05, **p < 0.01, ***p<0.001, ns., not statistically significant). The difference in the proportion of IL-12Rb2+ C7 and C24 cells was statistically significant (at least p<0.05) for all time points, unless indicated otherwise. (C-E) CTV-labelled C7 and C24 T cells were stimulated with E6 peptide and IL-12 in the presence of anti-IL-4 and anti-IFN-γ mAbs. Where indicated, paclitaxel (200 nM) was added at the beginning of the cultures. (C) Flow cytometric analysis of T-bet and IL-12Rβ2 expression on CTVlabelled C7 and C24 cells 66 h after peptide stimulation. The percentage of (D) Tbethi and (E) T-betint populations in C7 and C24 cultures with or without paclitaxel examined by flow cytometry at 52 h after peptide stimulation. Bars that are black or red represent C7 and C24 populations, respectively. Data shown are mean ± sd of triplicate cultures. Statistical differences between untreated and paclitaxel-treated cultures were determined by Students t-test (*p<0.05, **p < 0.01). (F) Flow cytometric analysis of the effect of paclitaxel treatment on IL-12 versus IFN-g augmented T-bet expression. As specified, cells were exposed to TCR-driven (anti-IL-12p70, anti-IL-4 and anti-IFN-g mAbs), IL-12-driven (5 ng/mL IL-12p70 and anti-IFN-g and anti-IL-4 mAbs) or IFN-g-driven (anti-IL-12p70, anti-IL-4 mAbs) culture conditions and analyzed at 66 h. All data are representative of at least two independent experiments with similar results.

Figure. 2. Strong TCR signaling accelerates the activation, proliferation and differentiation of Th1 cells. (A-F) CTV-labelled C7 and C24 cells were transferred into naive B6 recipients 1 day prior to inoculation with BCG-E6. The activation and proliferation of TCR Tg cells in the spleen was determined using flow cytometry. (A) Flow cytometric analysis of CD44 and CD25 expression on C7 and C24 cells at the indicated time-points. Data are pooled from at least three independent experiments with similar trends. Data shown are the mean percentage ± sem (n > 10 mice / group / time point). (B) Frequency of Tg cells in each division 3 d.p.i. determined by flow cytometry. Data are representative of four independent experiments with similar results. Symbols and bars denote the group mean ± sem (n = 3 mice / group), respectively. The difference in the proportion of C7 and C24 cells in each division was statistically significant (at least p<0.05) for all division numbers, unless indicated otherwise. (C) Percentage of Ki-67-expressing Tg cells in the spleen 3 days after BCG-E6 inoculation determined by flow cytometry. Closed circles represent individual mice and bars denote the group mean. Data are representative of two independent experiments with similar results (n = 4 mice / group). (D) Time to first division determined by CTV dilution in vivo. Best-fit log normal probability distribution describing the mean time to first division (C7: µ = 71.1 h, sd. = 0.2 h; C24: µ = 65.2 h, sd. = 0.2 h). Data are representative of two independent experiments (n = 5 mice / group). (E) Flow cytometric analysis of T-bet and CXCR3 expression in donor TCR Tg cells (GFP+CD4+) at 2 d.p.i.. Endogenous naïve CD4+ cells (GFP—CD4+CD44—) within the same recipient spleen were used as controls. (F) Percentage of T-bet expression at indicated time points p.i.. Data are pooled from three independent experiments Symbols and bars denote the group mean ± sem ( 3-10 mice / group). (G-L) Purified C7 and C24 Tg cells were CTV-labelled and cultured with CD4-depleted splenocytes isolated

Figure. 4. TCR affinity does not determine the activation status or effector function of divided CD4+ T cells. (A-C) CTV-labelled C7 and C24 Tg cells were stimulated with E6 peptide in the presence of recombinant IL-12p70 and anti-IFN-γ mAb for 72 h. (A) Flow cytometric analysis of the MFI of CD44, CD25, IRF4 and CXCR3 as well as FSC-A (cell size) in each division of C7 and C24 populations at 42 h after peptide stimulation. (B-C) Tg cells were stimulated with E6 peptide in the presence of the graded concentrations of recombinant IL-12p70 and T-bet expression determined by flow cytometry. (B) T-bet expression in C7 and C24 Tg cell populations 60 h after stimulation. Numbers outside and within parentheses represent the percentage of T-bet+ cells and the MFI of T-bet expression in T-bet+ populations, respectively. (C) MFI of T-bet in each division of CTV-labelled C7 and C24 cells 66 h after stimulation. Data shown are representative of two independent experiments with similar results. (D-E) Purified C7 and C24 CD4+ T cells were stimulated with E6 peptide under non-polarizing (anti-IL-12, anti-IFN-g and anti-IL-4 mAbs), Th1 (IL-12p70 with anti-IFN-g and anti-IL-4 mAbs) or Th2 (IL-4 with anti-IFN-g and anti-IL-12 mAbs) culture conditions for 96 h. Following expansion in IL-2-containing medium for 48 h, an equal number of T cells were re-stimulated with immobilized anti-CD3 in the presence (D) or absence (E) of BFA for 6 h. (D) Intracellular IFN-g and IL-4 expression under the indicated culture conditions was analyzed by flow cytometry. (E) Quantification of IFN-g and IL-4 produced by Th1 and Th2 Tg cells by Cytokine Bead Array. Data shown are the mean ± the sd of triplicate cultures. For all data the bars, lines, symbols or FACS plots in black and red represent C7 and C24 cells, respectively.

Full text

1
TCR affinity controls the dynamics but not the functional specification
1
of the Th1 response to mycobacteria
2
Nayan D Bhattacharyya
1,2
, Claudio Counoupas
3,2
, Lina Daniel
1,2
, Guoliang Zhang
4,1,2
, Stuart J
3
Cook
5
, Taylor A Cootes
1,2
, Sebastian A Stifter
1,2
, David G Bowen
7,8
, James A Triccas
3,2,6
, Patrick
4
Bertolino
7,8
, Warwick J Britton
2
& Carl G Feng
1,2,6*
5
6
Affiliations:
7
1
Immunology and Host Defense Group, Department of Infectious Diseases and Immunology,
8
School of Medical Sciences, Faculty of Medicine & Health, The University of Sydney, NSW,
9
2006, Australia.
10
2
Tuberculosis Research Program, Centenary Institute, Royal Prince Alfred Hospital,
11
Camperdown, NSW, 2050, Australia.
12
3
Microbial Pathogenesis and Immunity Group, Department of Infectious Diseases and
13
Immunology, School of Medical Sciences, Faculty of Medicine & Health, University of Sydney,
14
NSW, 2006, Australia.
15
4
National Clinical Research Center for Infectious Diseases, Guangdong Key Laboratory of
16
Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Southern
17
University of Science and Technology, Shenzhen, China.
18
5
Immune Imaging Program, Centenary Institute, Royal Prince Alfred Hospital, Camperdown,
19
NSW, 2050, Australia.
20
6
Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney,
21
Sydney, NSW, 2006, Australia.
22
7
Liver Immunology Program, Centenary Institute, Royal Prince Alfred Hospital, Camperdown,
23
NSW, 2050, Australia.
24
8
AW Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Camperdown,
25
NSW, 2050, Australia.
26
27
* Corresponding author: Carl G Feng: carl.feng@sydney.edu.au
28
29
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted October 26, 2020. ; https://doi.org/10.1101/2020.10.25.353763doi: bioRxiv preprint

2
Abstract:
30
The quality of T cell responses depends on the lymphocytes’ ability to undergo clonal expansion,
31
acquire effector functions and traffic to the site of infection. Although TCR signal strength is
32
thought to dominantly shape the T cell response, by using TCR transgenic CD4
+
T cells with
33
different pMHC binding affinity, we reveal that TCR affinity does not control Th1 effector
34
function acquisition nor the functional output of individual effectors following mycobacterial
35
infection. Rather, TCR affinity calibrates the rate of cell division to synchronize the distinct
36
processes of T cell proliferation, differentiation and trafficking. By timing cell division-dependent
37
IL-12R expression, TCR affinity controls when T cells become receptive to Th1-imprinting IL-12
38
signals, determining the emergence and magnitude of the Th1 effector pool. These findings reveal
39
a distinct yet cooperative role for IL-12 and TCR signalling in Th1 differentiation and suggests
40
that the temporal activation of clones with different TCR affinity is a major strategy to coordinate
41
immune surveillance against persistent pathogens.
42
43
Keywords:
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TCR affinity, TCR signal, Th1 response, cell division, IL-12, mycobacteria.
45
46
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted October 26, 2020. ; https://doi.org/10.1101/2020.10.25.353763doi: bioRxiv preprint

3
Introduction:
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The successful containment of invading pathogens requires the rapid generation of large numbers
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of antigen-specific T cells with the correct effector function. This involves the activation of distinct
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programs, including T cell proliferation, differentiation, and migration. Failure in activating or
50
regulating these programs results in impaired host defense. The majority of studies have focused
51
on one or a limited number of CD4
+
T cell programs, such as, the magnitude of population
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expansion or expression of master regulators of transcription. There is little information available
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as to whether these processes, which operate at different biological scales spanning from the
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molecule to the tissue, are individually or cooperatively regulated in vivo. Indeed, in vitro studies
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have already suggested a link between cell division and differentiation, demonstrating that cell
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division progression is associated with increased expression of signature Th cytokines (1-3).
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The pool of naive T cells in vivo is diverse and contains clones that express distinct TCRs
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recognizing different peptide:MHC (pMHC) complexes. It is estimated that there are anywhere
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between twenty and one thousand naive T cells that possess the same pMHC specificity (4, 5),
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each with different binding affinities. The strength of TCR signals, regulated by the TCRs affinity,
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the density of pMHC and co-stimulatory molecules on antigen presenting cells (APCs), regulates
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downstream T cell activation and function (6, 7). While high affinity TCR signals in cytotoxic
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CD8
+
T lymphocytes accelerate cell division and prolong population expansion (8, 9), it delays
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their migration from secondary lymphoid organs (SLOs) (9, 10) resulting in impaired pathogen
65
control (10). Similarly, strong TCR signals enhance the expansion of CD4
+
T cell populations (11-
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13).
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Defining the role of TCR signaling strength in the CD4
+
lymphocyte response is
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challenging because of the functional heterogeneity in helper T cell populations. The effector
69
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted October 26, 2020. ; https://doi.org/10.1101/2020.10.25.353763doi: bioRxiv preprint

4
function of Th populations is instructed by signals from the TCR as well as from pathogen-
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conditioned accessory cells and APCs. Historically, investigations into Th cell differentiation have
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focused on “qualitative” T cell-extrinsic cytokine signals (7, 14). In the case of Th1 differentiation,
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the innate cytokine IL-12 promotes the generation of interferon-γ (IFN-γ)-producing effectors (15,
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16) and host survival following infection with intracellular pathogens (17). Recent studies have
74
suggested a role for “quantitative” differences in TCR signal strength in regulating CD4
+
T cell
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differentiation (12, 18-20). Potent TCR signaling is associated with the generation of Th1 (18, 19)
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or Tfh cells (11, 20, 21). Mechanisms proposed to mediate strong TCR signal-driven Th lineage
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commitment vary depending on experimental settings. For example, IL-2 (12, 13, 19) and IL-12
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receptor signaling (18) have each been suggested to contribute to the generation of Th1 populations
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following potent TCR stimulation. The model-dependent function of strong TCR signaling
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suggests a potential interplay between quantitative TCR and qualitative environmental signals in
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instructing Th differentiation. Currently, the relative role of TCR and innate cytokine signals in
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lineage commitment and the mechanisms integrating these signals is unknown.
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In this study, we developed a T cell adoptive transfer model using CD4
+
T cells from two
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TCR transgenic (Tg) mouse lines that recognize the same epitope of the Mycobacterium
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tuberculosis protein Early Secretory Antigenic Target 6 (ESAT-6, E6), with different binding
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affinities. Following T cell transfer, WT or IL-12-deficient recipient mice were infected with a
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recombinant Mycobacterium bovis Bacillus Calmette-Guérin-expressing E6 (BCG-E6). By
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tracking transgenic CD4
+
T cells across multiple time-points and in different tissues following
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intravenous (i.v.) BCG infection, we reveal that by adjusting the rate of cell division, a major
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function of TCR affinity is to determine the speed and magnitude of the CD4
+
T cell response.
91
Moreover, TCR affinity plays a minimal role in specifying T helper cell effector function.
92
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted October 26, 2020. ; https://doi.org/10.1101/2020.10.25.353763doi: bioRxiv preprint

5
However, by regulating cell division-dependent IL-12Rb2 expression, TCR affinity controls when
93
T cells become receptive to IL-12 and acquire Th1 effector function. Since high affinity CD4
+
T
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cells also migrate to infected non-lymphoid tissues faster than their low affinity counterparts, our
95
findings show that TCR affinity coordinates multiple programs to determine the overall potency
96
of the Th cell response to infection. They also suggest that the temporal activation of distinct T
97
cell clones is a mechanism controlling the initiation and maintenance of Th1 immunity against
98
persistent infection.
99
100
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted October 26, 2020. ; https://doi.org/10.1101/2020.10.25.353763doi: bioRxiv preprint