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. 2010 Nov 9;107(45):19408-13.
doi: 10.1073/pnas.1006298107. Epub 2010 Oct 20.

Distinct functions of antigen-specific CD4 T cells during murine Mycobacterium tuberculosis infection

William W Reiley  1 , Shahin Shafiani, Susan T Wittmer, Glady's Tucker-Heard, James J Moon, Marc K Jenkins, Kevin B Urdahl, Gary M Winslow, David L Woodland
Affiliations

Distinct functions of antigen-specific CD4 T cells during murine Mycobacterium tuberculosis infection

William W Reiley et al. Proc Natl Acad Sci U S A. .

Abstract

The immune response elicited after Mycobacterium tuberculosis (Mtb) infection is critically dependent on CD4 T cells during both acute and chronic infection. How CD4 T-cell responses are maintained throughout infection is not well understood, and evidence from other infection models has suggested that, under conditions of chronic antigen stimulation, T cells can undergo replicative exhaustion. These findings led us to determine whether subpopulations of CD4 T cells existed that displayed markers of terminal differentiation or exhaustion during murine Mtb infection. Analysis of antigen-specific effector CD4 T cells revealed that programmed death-1 (PD-1) and the killer cell lectin-like receptor G1 (KLRG1) delineated subpopulations of T cells. PD-1-expressing CD4 T cells were highly proliferative, whereas KLRG1 cells exhibited a short lifespan and secreted the cytokines IFNγ and TNFα. Adoptive transfer studies demonstrated that proliferating PD-1-positive CD4 T cells differentiated into cytokine-secreting KLRG1-positive T cells, but not vice versa. Thus, proliferating PD-1-positive cells are not exhausted, but appear to be central to maintaining antigen-specific effector T cells during chronic Mtb infection. Our findings suggest that antigen-specific T-cell responses are maintained during chronic mycobacterial infection through the continual production of terminal effector cells from a proliferating precursor population.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The proliferative capacity of antigen-specific cells diminishes during Mtb infection. (A) BrdU was administered to C57BL/6 mice on day 30 after low-dose aerosol infection with Mtb (strain H37Rv) and BrdU incorporation in ESAT64–17/I-Ab antigen-specific CD44hi cells gated on CD4hi T cells in the lung was measured 1 d later. (B) The percentages of ESAT64–17/I-Ab antigen-specific CD4 T cells that incorporated BrdU on the indicated days postinfection are shown. Data are presented from the lungs of five mice analyzed on each day. Error bars indicate SD from the mean, and are shown from two experiments.
Fig. 2.
Fig. 2.
Antigen-specific cells display markers of terminal differentiation or senescence during Mtb infection. (A) Flow cytometric gating strategy used to analyze KLRG1 and PD-1 expression on ESAT64–17/I-Ab antigen-specific CD4 T cells. (B) The number of KLRG1- and PD-1–expressing ESAT64–17/I-Ab antigen-specific CD4 T cells during Mtb infection. Data are presented from the lungs of five mice analyzed on each day. The error bars indicate the SD. The data are representative of two independent experiments. (C) The dot plots are representative of the CD69 and PD-1 or CD69 and KLRG1 expression on ESAT64–17/I-Ab antigen-specific CD4 T cells isolated from lungs of mice on day 30 postinfection.
Fig. 3.
Fig. 3.
PD-1–PDL1 blockade does not affect the number or function of antigen-specific T cells. Mtb-infected mice were treated with anti-PDL1, from day 30 to day 45 postinfection. (A) The numbers of ESAT64–17/I-Ab antigen-specific CD4 T cells among each of the indicated populations of KLRG1- and PD-1–expressing cells in the lung are shown. Each datum represents an individual mouse. (B) Bacterial burdens in the lung, mediastinal lymph node, spleen, and liver, were determined on day 46 postinfection. The data are representative of two experiments of similar design.
Fig. 4.
Fig. 4.
Differentiation and proliferation of PD-1– and KLRG1-expressing CD4 T cells. (A and E) B6.SJL-Ptprca Pep3b/BoyJ mice were infected, lymphocytes from the spleen, mediastinal lymph node, and lung were isolated on day 25 postinfection, and PD-1– or KLRG1-expressing cells were purified by flow cytometric cell sorting. CFSE-labeled PD-1–expressing cells (2.3 × 104) or KLRG1-expressing cells (2.5 × 104) were transferred on day 25 to infected congenic C57BL/6 recipient mice. (B and F) Representative dot plots of KLRG1 and PD-1 expression on CD4+/CD45.1+ donor cells in the lung on days 6 and 14 following transfer. Gates were set on the host population CD4 T cells. (C and G) A representative histogram of CFSE staining on CD4+/CD45.1+ donor T cells in the lung on days 6 and 14 after transfer. (D and H) The number of CD4+/CD45.1+ donor cells in the lung and spleen are shown. Differences with a P value <0.05 were considered significant and are denoted by an asterisk.
Fig. 5.
Fig. 5.
Model of antigen-specific CD4 T-cell differentiation and self-renewal during Mtb infection. We hypothesize that PD-1–expressing effector CD4 T cells differentiate into KLRG1-expressing cells, as delineated by the bold arrow. In this model, a self-renewing population of either PD-1lo/int– or PD-1hi–expressing effector cells maintains antigen-specific T-cell numbers. PD-1lo/int cells can differentiate into cells that express higher levels of PD-1. Presently, it is unclear if the surface expression of PD-1 on antigen-specific CD4 T cells, once up-regulated, can be down-regulated to a low or intermediate phenotype. The KLRG1-expressing T cells secreted both IFNγ and TNFα, underwent only moderate proliferation, and exhibited a shorter life span, all characteristics of terminally differentiated cytokine-secreting cells.
Fig. 6.
Fig. 6.
KLRG1 expression identifies terminally differentiated cytokine secreting cells. Lymphocytes isolated from the lung of Mtb-infected mice were stimulated with ESAT61–20 peptide for 5 h in the presence of brefeldin A. (A) Representative dot plots of IFNγ-and TNFα-producing KLRG1lo PD-1hi, KLRG1hiPD-1lo, and KLRG1lo PD-1lo/int CD4 T cells on day 60 postinfection. (B) The number of ESAT6-specific IFNγ- and TNFα-producing KLRG1- or PD-1–expressing CD4 T cells is shown for the indicated days postinfection. Data are presented from the lungs of five mice analyzed on each day. The error bars indicate the SD. The analysis was conducted from two experiments of similar design. (C) The frequencies of ESAT64–17/I-Ab tetramer-positive cells within the KLRG1- or PD-1–expressing populations were plotted against the frequency of ESAT61–20/I-Ab IFNγ- and TNFα-producing cells within these same populations in each mouse. The best-fit line was generated using least-squares regression analysis. (D) A representative histogram of ESAT64–17/I-Ab tetramer expression and CD44 expression on KLRG1 (filled)- or PD-1 (open)–expressing CD4 T cells, on day 60 postinfection. The MFI of ESAT64–17/I-Ab tetramer-positive cells was 2,147 ± 371 for PD-1–expressing cells and 1,280 ± 439 for KLRG1-expressing cells. The MFI of CD44 was 6,555 ± 935 for PD-1–expressing cells and 2,617 ± 352 for KLRG1-expressing cells. The data are representative of mice analyzed at several different time points in two experiments. Differences with a P value <0.05 were considered significant and are denoted by an asterisk.
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