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. 2012;6(8):e1789.
doi: 10.1371/journal.pntd.0001789. Epub 2012 Aug 14.

Phenotypic characterization of peripheral T cells and their dynamics in scrub typhus patients

Affiliations

Phenotypic characterization of peripheral T cells and their dynamics in scrub typhus patients

Bon-A Cho et al. PLoS Negl Trop Dis. 2012.

Abstract

Background: Scrub typhus, caused by Orientia tsutsugamushi infection, is one of the main causes of febrile illness in the Asia-Pacific region. Although cell-mediated immunity plays an important role in protection, little is known about the phenotypic changes and dynamics of leukocytes in scrub typhus patients.

Methodology/principal findings: To reveal the underlying mechanisms of immunological pathogenesis, we extensively analyzed peripheral blood leukocytes, especially T cells, during acute and convalescent phases of infection in human patients and compared with healthy volunteers. We observed neutrophilia and CD4(+) T lymphopenia in the acute phase of infection, followed by proliferation of CD8(+) T cells during the convalescent phase. Massive T cell apoptosis was detected in the acute phase and preferential increase of CD8(+) T cells with activated phenotypes was observed in both acute and convalescent phases, which might be associated or correlated with elevated serum IL-7 and IL-15. Interestingly, peripheral Treg cells were significantly down-regulated throughout the disease course.

Conclusions/significance: The remarkable decrease of CD4(+) T cells, including Treg cells, during the acute phase of infection may contribute to the loss of immunological memory that are often observed in vaccine studies and recurrent human infection.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Profiles of peripheral blood leukocytes and T lymphocytes in scrub typhus patients.
A. The frequencies (%) of neutrophils, lymphocytes, and monocytes in blood leukocytes from healthy controls (HC, n = 9, open circle) were determined using a hematology analyzer and compared to frequencies in scrub typhus patients during acute phase (AP, n = 17, gray circle) or convalescent phase (CP, n = 17, black circle) of infection. B. Peripheral blood mononuclear cells (PBMCs) from scrub typhus patients and healthy controls were stained with anti-CD3 and CD8 antibodies and analyzed by flow cytometry (see Figure S1). The frequencies (% in lymphocytes) and absolute numbers (cells/mm2) of CD4+ or CD8+ T cells in PBMCs of the patients (AP, n = 15 and CP, n = 15) and healthy controls (HC, n = 9) were compared. The absolute number of each leukocyte subset was calculated based on the leukocyte differential counts and the frequency information obtained from flow cytometry. For example, CD4+ T cells count = (lymphocyte count) x (% of CD4+ T cells)/100. The data of each individual person are presented in Table S3. Red bars indicate the mean value and p values were obtained using the Mann-Whitney U test or Wilcoxon signed-rank test. Statistically significant p values (<0.05) are shown in bold.
Figure 2
Figure 2. Apoptosis and proliferation of T cells during Orientia infection.
A and B. PBMCs were stained with antibodies against CD4, CD8, Annexin V, or Ki-67 and then analyzed by flow cytometry. The frequencies of Annexin V- (A) or Ki-67- (B) positive cells in CD4+ and CD8+ T cells were compared among healthy controls (HC, n = 6, open circle) and scrub typhus patients at acute phase (AP, n = 13–15, gray circle) or convalescent phase (CP, n = 6, black circle). Red bars indicate the mean value. C. The CD8/CD4 ratio of apoptotic or proliferating cells were compared among the patients and healthy volunteers. D. The levels of IL-7 and IL-15 (pg/ml) in the sera were measured and compared. Error bars indicate standard error from the mean value (C and D). p values were obtained using the Mann-Whitney U test. Statistically significant p values (<0.05) are shown in bold.
Figure 3
Figure 3. Profiles of type 1 and type 2 T cells in peripheral blood of scrub typhus patients.
PBMCs were stained with antibodies against CXCR3, CCR4, CD4, and CD8 and then analyzed on a flow cytometer. A. The frequencies of Th1 (CD4+CXCR3+) or Th2 (CD4+CCR4+) within CD4+ T cells were compared among healthy controls (HC, n = 6, open circle) and the patients in acute phase (AP, n = 12, gray circle) or convalescent phase (CP, n = 5, black circle). B. The frequencies of Tc1 (CD8+CXCR3+) and Tc2 (CD8+CCR4+) within CD8+ T cells were compared among healthy controls and the patients as in A. Red bars indicate the mean value and p values were obtained using the Mann-Whitney U test. Statistically significant p values (<0.05) are shown in bold.
Figure 4
Figure 4. Profiles of CD4+Foxp3+ or CD4+CD25++ regulatory T cells in scrub typhus patients.
A. PBMCs were stained with antibodies against CD4 and CD25 or Foxp3 and then analyzed on a flow cytometer. Representative dot plots show the identification of CD25++ T cells (upper panels) or Foxp3+ T cells (lower panels) within CD4+ T cells. Numbers in the plots indicate the frequencies (%) of the gated cells in total PBMCs. B. The frequency of CD4+CD25++ or CD4+Foxp3+ T cells were compared between healthy controls (HC, n = 7, open circle) and scrub typhus patients at acute phase (AP, n = 10, gray circle) or convalescent phase (CP, n = 12, black circle). C. PBMCs were stained with antibodies against CD4 and Fas or CCR4, followed by intracellular staining of Foxp3 and/or CTLA-4 after fixation and permeablization. Mean fluorescent Intensities (MFI) representing CTLA-4, Fas, or CCR4 expression in CD4+Foxp3+ regulatory cells from healthy controls (HC, n = 7–8, open circle) or the patients (AP, n = 7–10, gray circle and CP, n = 9–12, black circle) were compared. Red bars indicate the mean value and p values were obtained using the Mann-Whitney U test or Wilcoxon signed-rank test. Statistically significant p values (<0.05) are shown in bold.
Figure 5
Figure 5. Changes of effector and memory T cell subsets in scrub typhus patients.
A. Representative contour plots show the frequencies of each subsets within CD4+ (upper panels) or CD8+ (lower panels) T cell populations. Numbers within the plots indicate the percentage of each subset. B. The frequencies of naïve (CCR7+CD45RA+), central memory (CM, CCR7+CD45RA−), and effector memory (EM, CCR7−CD45RA−), CD45RA+ EM (EMCD45RA+, for CD8+ T cells), or CCR7−CD45RA+ (for CD4+ T cells) T-cell subsets in healthy controls (HC, n = 9) and scrub typhus patients at acute phase (AP, n = 15) or convalescent phase (CP, n = 17) were compared. Error bars indicate standard error of mean values. p values were obtained using the Mann-Whitney U test or Wilcoxon signed-rank test. *, p<0.05; **, p<0.01; and ***, p<0.001 (compared with that of healthy control); †, p<0.05 and ††, p<0.01 (compared with that of the patients at acute phase).
Figure 6
Figure 6. Analysis of activated CD8+ T cells in peripheral bloods of scrub typhus patients.
PBMCs were stained with antibodies against CD8 and PD-1 or IL-7Rα and then analyzed on a flow cytometer. A and B. Representative histograms show the frequencies of IL-7Rαlow cells (A) and PD-1high cells (B) within CD8+ T cells. Gray histogram, isotype control. C. The frequency of IL-7RαlowCD8+ T cells from healthy controls (HC, n = 9, open circle) is compared with that of scrub typhus patients at acute phase (AP, n = 7, gray circle) or convalescent phase (CP, n = 8, black circle). D. The frequencies of PD-1highCD8+ T cells were compared among healthy controls (n = 9) and the patients (AP, n = 13; CP, n = 15). Red bars indicate the mean value and p values were obtained using the Mann-Whitney U test or Wilcoxon signed-rank test. Statistically significant p values (<0.05) are shown in bold.

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