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. 2012 Mar;2(1):25-39.
doi: 10.4236/oji.2012.21004. Epub 2011 Dec 30.

Innate-like CD4 T cells selected by thymocytes suppress adaptive immune responses against bacterial infections

Yu Qiao  1 , Brian M Gray, Mohammed H Sofi, Laura D Bauler, Kathryn A Eaton, Mary X D O'Riordan, Cheong-Hee Chang
Affiliations

Innate-like CD4 T cells selected by thymocytes suppress adaptive immune responses against bacterial infections

Yu Qiao et al. Open J Immunol. 2012 Mar.

Abstract

We have reported a new innate-like CD4 T cell population that expresses cell surface makers of effector/memory cells and produce Th1 and Th2 cytokines immediately upon activation. Unlike conventional CD4 T cells that are selected by thymic epithelial cells, these CD4 T cells, named T-CD4 T cells, are selected by MHC class II expressing thymocytes. Previously, we showed that the presence of T-CD4 T cells protected mice from airway inflammation suggesting an immune regulatory role of T-CD4 T cells. To further understand the function of T-CD4 T cells, we investigated immune responses mediated by T-CD4 T cells during bacterial infection because the generation of antigen specific CD4 T cells contributes to clearance of infection and for the development of immune memory. The current study shows a suppressive effect of T-CD4 T cells on both CD8 and CD4 T cell-mediated immune responses during Listeria and Helicobacter infections. In the mouse model of Listeria monocytogenes infection, T-CD4 T cells resulted in decreasedfrequency of Listeria-specific CD8 T cells and the killing activity of them. Furthermore, mice with T-CD4 T cells developed poor immune memory, demonstrated by reduced expansion of antigen-specific T cells and high bacterial burden upon re-infection. Similarly, the presence of T-CD4 T cells suppressed the generation of antigen-specific CD4 T cells in Helicobacter pylori infected mice. Thus, our studies reveal a novel function of T-CD4 T cells in suppressing anti-bacterial immunity.

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Figures

Figure 1
Figure 1
Reduced anti-listerial responses in Tg mice during primary infection. Tg and WT littermates were infected intravenously with rLM-OVA (5 × 104) or PBS. Mice were euthanized and analyzed 7 days after infection. (a) Num- bers of the total splenocytes and of the indicated cell popula- tions are shown; (b) and (c) Frequencies of IFN-γ-producing rLM-OVA-specific CD8 (b) and CD4 (c) T cells. The values in representative FACS profiles are percentages of the total splenocyte population; the graphs on right show the percentage of antigen specific CD8 (IFN-γ+ CD8/total CD8) (b) and CD4 (IFN-γ+ CD4/total CD4) (c) T cells. The bars indicate the median value; (d) In vivo killing assay. A mixture of OVA peptide-loaded target cells (CFSEhi) and control cells (CFSElo) were injected into recipient mice. Mice were euthanized 3 hours later and the composition of the injected cells in the spleenswere analyzed by flow cytometry. The numbers above histograms indicate the percentages of CFSEhi and CFSElo.
Figure 2
Figure 2
Poor memory response against listeria in Tg mice. Tg and WT littermates were inoculated intravenously with 4 × 103 rLM-OVA and rested for one month before challenge with 5 × 105 rLM-OVA or PBS as a control. All the mice were euthanized and analyzed 3 days after the secondary infection. (a) Numbers represent the total splenocytes and the indicated cell populations; (b) and (c) Frequencies of IFN-γ producing rLMOVA-specific CD8 (b) and CD4 (c) T cells. Experiments and data analyses were done as described in Figure 1; (d) Numbers of viable bacteria from liver homogenates are depicted. The bars indicate median values. The colony forming units from mice treated with PBS were below the detection limit of 100 CFU/mouse liver.
Figure 3
Figure 3
Effector CD8 T cell generation is compromised in the presence of T-CD4 cells. (a) A scheme of the experimental protocol. The infection dose was 2 × 104 rLM-OVA; (b) The composition of the CD45.1+ input cell population prior to the transfer; (c) Numbers of the indicated cells originating from the donor on day 7; (d) T- and E-CD4 ratio after co-transfer into naïve mice. Cell populations were tracked using congenic markers CD45.1 and CD45.2 on the indicated days and the T/E-CD4 ratios were normalized to the input ratio on day 0. N = 3. (e) and (f) Frequencies of IFN-γ-producing rLM-OVA-specific CD8 (e) and CD4 (f) T cells. The FACS data shown were gated on donor populations. The values in representative FACS profiles are percentages of total donor splenocytes; the graphs on right represent percentages of total donor CD8 (e) or CD4 (f) T cells that produced IFN-γ. The bars indicate the median value; (g) In vivo killing assay. Experiments were performed as described in Figure 1(d), except that mice were sacrificed 24 hours after cell transfer.
Figure 3
Figure 3
Effector CD8 T cell generation is compromised in the presence of T-CD4 cells. (a) A scheme of the experimental protocol. The infection dose was 2 × 104 rLM-OVA; (b) The composition of the CD45.1+ input cell population prior to the transfer; (c) Numbers of the indicated cells originating from the donor on day 7; (d) T- and E-CD4 ratio after co-transfer into naïve mice. Cell populations were tracked using congenic markers CD45.1 and CD45.2 on the indicated days and the T/E-CD4 ratios were normalized to the input ratio on day 0. N = 3. (e) and (f) Frequencies of IFN-γ-producing rLM-OVA-specific CD8 (e) and CD4 (f) T cells. The FACS data shown were gated on donor populations. The values in representative FACS profiles are percentages of total donor splenocytes; the graphs on right represent percentages of total donor CD8 (e) or CD4 (f) T cells that produced IFN-γ. The bars indicate the median value; (g) In vivo killing assay. Experiments were performed as described in Figure 1(d), except that mice were sacrificed 24 hours after cell transfer.
Figure 4
Figure 4
T-CD4 cells inhibit development of memory CD8 effector cells. (a) A scheme of the experimental protocol. Mice were infected with 2 × 103 and 5 × 105 rLM-OVA for primary and secondary infection, respectively, and then sacrificed three days after the second infection; (b) The composition of the CD45.1+ input cell populations prior to the transfer; (c) The numbers represent total splenocytes and the indicated cells originating from the donor; (d) and (e) Frequencies of IFN-γ-producing rLM-OVA-specific CD8 (d) and CD4 (e) T cells (N = 5). Experimental design and data analysis were performed as described in Figures 3(e) and (f); (f) Numbers represent viable bacteria isolated from liver homogenates. The bars indicate median values. The CFU counts of PBS-treated mice were below the detection limit.
Figure 5
Figure 5
T-CD4 T cells does not change Treg populations ((a) and (b)) Mice were infected intravenously with 100,000 rLM-OVA and sacrificed on day 3 to assess cell populations (a) and to measure cytokine expression (b) from the spleen of Tg and WT mice. The RNA expression of the indicated cytokine genes were measured by real time PCR after reverse transcription. Relative expression of each cytokine was normalized to GAPDH. The data are representative of 4 mice in each group. (c) Representative profiles of Foxp3 expression in E- and T-CD4 T cells from [Tg→Aβ-/-] and [WT→WT] chimeric mice. (d) and (e) WT and Tg mice were infected as in Figure 1. Seven days after infection, freshly isolated CD4 T cells were stained for Foxp3 expression (d) or stimulated in the presence of rLM-OVApeptides for 5 hours followed by staining of Foxp3 and IFN-γ (e).
Figure 6
Figure 6
The presence of T-CD4 T cells inhibits the generation of Helicobacter-specific effector CD4 T cells. (A) A scheme of the experimental protocol; (B) Recovery of T cells from Helicobacter infected mice. Flow cytometric analysis of cells recovered from the spleens of SCID recipients of adoptive transfers of CD4 T cells; (C) CD4 T cells were isolated from mice and then stimulated with anti-CD3/CD28 or with H. pylori lysate. Supernatants were used forELISA. Bars are the average of 3 replicates of each condition, and each replicate used CD4 T cells pooled from 5 mice; (D) Hematoxylin and eosin-stained sections of gastric mucosa from H. pylori-infected SCID recipient mice. (a) WT CD4 recipient. Inflammatory infiltrate (arrowheads) consists of neutrophils, lymphocytes, and macrophages. Bracket indicates metaplastic glands. (b) Tg CD4 recipient. Arrow indicates a gland abscess. Bars = 50 μm; (E) Relative mRNA levels of cytokine genes from the gastric mucosa of uninfected and infected SCID mice that were adoptively transferred with either WT or Tg CD4 cells. Expression of cytokine genes normalized to GAPDH expression, and multiplied by 10,000 for ease of visualization.
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