The kinetics of CD4+Foxp3+ T cell accumulation during a human cutaneous antigen-specific memory response in vivo
Milica Vukmanovic-Stejic, Elaine Agius, Nicola Booth, Padraic J. Dunne, Katie E. Lacy, John R. Reed, Toni O. Sobande, Steven Kissane, Mike Salmon, Malcolm H. Rustin, Arne N. Akbar
Milica Vukmanovic-Stejic, Elaine Agius, Nicola Booth, Padraic J. Dunne, Katie E. Lacy, John R. Reed, Toni O. Sobande, Steven Kissane, Mike Salmon, Malcolm H. Rustin, Arne N. Akbar
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Research Article

The kinetics of CD4+Foxp3+ T cell accumulation during a human cutaneous antigen-specific memory response in vivo

Abstract

Naturally occurring CD4+CD25hiFoxp3+ Tregs (nTregs) are highly proliferative in blood. However, the kinetics of their accumulation and proliferation during a localized antigen-specific T cell response is currently unknown. To explore this, we used a human experimental system whereby tuberculin purified protein derivative (PPD) was injected into the skin and the local T cell response analyzed over time. The numbers of both CD4+Foxp3– (memory) and CD4+Foxp3+ (putative nTreg) T cells increased in parallel, with the 2 populations proliferating at the same relative rate. In contrast to CD4+Foxp3– T cell populations, skin CD4+Foxp3+ T cells expressed typical Treg markers (i.e., they were CD25hi, CD127lo, CD27+, and CD39+) and did not synthesize IL-2 or IFN-γ after restimulation in vitro, indicating that they were not recently activated effector cells. To determine whether CD4+Foxp3+ T cells in skin could be induced from memory CD4+ T cells, we expanded skin-derived memory CD4+ T cells in vitro and anergized them. These cells expressed high levels of CD25 and Foxp3 and suppressed the proliferation of skin-derived responder T cells to PPD challenge. Our data therefore demonstrate that memory and CD4+ Treg populations are regulated in tandem during a secondary antigenic response. Furthermore, it is possible to isolate effector CD4+ T cell populations from inflamed tissues and manipulate them to generate Tregs with the potential to suppress inflammatory responses.

Authors

Milica Vukmanovic-Stejic, Elaine Agius, Nicola Booth, Padraic J. Dunne, Katie E. Lacy, John R. Reed, Toni O. Sobande, Steven Kissane, Mike Salmon, Malcolm H. Rustin, Arne N. Akbar

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Figure 7

Anergy induction leads to the development of a suppressive phenotype.

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Anergy induction leads to the development of a suppressive phenotype. (A...
(A) Cells recovered from a suction blister induced over a MT site 21 days following the induction were stimulated in vitro with PPD every 14 days (indicated by arrows). Peripheral blood CD4+ T cells from the same donor were cultured in parallel. (B) Dot plot shows IFN-γ production by the cell line in response to PPD stimulation. The percentage of cells expressing IFN-γ is indicated. (C) PPD-specific CD4+ T cell line was stimulated with immobilized anti-CD3 mAb. The cells were then washed and stimulated with PPD and antigen-presenting cells for 3 days, and proliferation was measured by [3H]-thymidine incorporation. The mean of triplicate wells ± SEM is shown (representative of 3 separate experiments). PPD-specific CD4+ T cells that were not anergized were used as control. (D) Anergized PPD-specific cells described in C were mixed with an equal number of autologous non-anergized PPD-specific cells and stimulated with PPD and autologous antigen-presenting cells. Proliferation was measured on day 3 and is expressed as mean ± SEM. Responders, non-anergized PPD cell lines cultured alone; control, responders with non-anergized PPD-specific cells. Results are representative of 2 separate experiments. (E) Foxp3 expression was measured by real-time quantitative RT-PCR using 18s rRNA as an internal control. Relative quantity values are plotted in a log-scale bar chart using the triplicate values to estimate SD. Cells recovered from the blister are indicated as “blister cells ex vivo.” Blister cells expanded in vitro prior to anergy induction were used as a control.