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. 2009 Sep 1;183(5):2921-31.
doi: 10.4049/jimmunol.0801191. Epub 2009 Jul 31.

C3 Promotes Expansion of CD8+ and CD4+ T Cells in a Listeria Monocytogenes Infection

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Free PMC article

C3 Promotes Expansion of CD8+ and CD4+ T Cells in a Listeria Monocytogenes Infection

Yumi Nakayama et al. J Immunol. .
Free PMC article

Abstract

It is known that C3 is required for optimal expansion of T cells during acute viral infections. However, it is not yet determined whether T cell responses to intracellular bacterial infections require C3. Therefore, we have investigated the requirement for C3 to elicit potent T cell responses to Listeria monocytogenes (LM). We show that expansion of Ag-specific CD8 and CD4 T cells during a primary response to LM was markedly reduced in the absence of C3 activity. Further studies indicated that, unlike in an influenza virus infection, the regulation of LM-specific T cell responses by C3 might not involve the downstream effector C5a. Moreover, reduced T cell responses to LM was not linked to defective maturation of dendritic cells or developmental anomalies in the peripheral T cell compartment of C3-deficient mice. Experiments involving adoptive transfer of C3-deficient CD8 T cells into the C3-sufficient environment of wild-type mice showed that these T cells do not have intrinsic proliferative defects, and a paracrine source of C3 will suffice for clonal expansion of CD8 T cells in vivo. However, stimulation of purified C3-deficient CD8 T cells by plastic-immobilized anti-CD3 showed that C3 promotes T cell proliferation directly, independent of its effects on APC. On the basis of these findings, we propose that diminished T cell responses to LM in C3-deficient mice might be at least in part due to lack of direct effects of C3 on T cells. These studies have furthered our understanding of C3-mediated regulation of T cell immunity to intracellular pathogens.

Figures

Figure 1
Figure 1. Activation of CD8 T cells is compromised in C3-deficient mice
+/+ and C3−/− were infected with rLM/GP33. On the seventh day after infection, splenocytes were stained with anti-CD8 and anti-CD44 antibodies. A. Dot plots are gated on total viable splenocytes and the numbers are the percentages of naive or activated CD8 T cells amongst splenocytes. B. Total numbers of activated (CD44hi) and naïve (CD44lo) CD8 T cells in spleen on day 7 PI. Data are the averages of three to five mice per group ± SD.
Figure 2
Figure 2. Reduced expansion of antigen-specific CD8 T cells in C3-deficient mice
+/+ and C3−/− mice were infected with rLM/GP33, and on day 7 PI, splenocytes were stained with anti-CD8, anti-CD44, and the indicated MHC I tetramers. Dot plots in A are gated on total CD8 T cells, and the numbers represent the percentages of tetramer-binding CD8 T cells of splenocytes; numbers in parentheses are the percentages amongst total CD8 T cells. B. Total number of epitope-specific CD8 T cells in spleen. Data are from 3–5 mice/group ± SD. C. Epitope-specific IFNγ-producing CD8 T cells in spleen of +/+ and C3−/− mice. Splenocytes were stimulated with the indicated LCMV epitope peptides, and IFNγ-producing CD8 T cells were detected by intracellular staining and flow cytometry. The dot plots are gated on total splenocytes and numbers in the dot plots are the percentages of antigen-specific IFNγ producing cells amongst total splenocytes. The FACS histograms are gated on IFNγ-producing CD8 T cells, and numbers are the mean fluorescence intensities (MFI) of staining for IFNγ. D. Total number of epitope-specific IFNγ−producing CD8 T cells in spleen of +/+ and C3−/− mice. Data are the averages of three to five mice/group ± SD.
Figure 3
Figure 3. Effect of C3 deficiency on generation of CD8 T cell responses to an unprocessed epitope expressed by Listeria monocytogenes
+/+ and C3−/− mice were infected with rLM/NP. On day 7 after infection, the number of CD8 T cells that are specific to the NP396 epitope was determined by staining with MHC I tetramers (A, B) or intracellular staining for IFNγ (C). A. Splenocytes were stained with anti-CD8, anti-CD44, and Db/NP396 tetramers. Dot plots in A are gated on total CD8 T cells, and numbers are the percentages of tetramer-binding cells of splenocytes; numbers in parentheses are percentages amongst total CD8 T cells. B. Absolute numbers of NP396 tetramer positive cells. C. Splenocytes were stimulated with NP396 peptide and number of IFNγ-producing CD8 T cells was determined by intracellular staining. The total number of NP396-specific CD8 T cells in the spleens is shown. Data is from 3–5 mice/group ± SD.
Figure 4
Figure 4. Activation of CD4 T cells is compromised in C3-deficient mice
+/+ and C3 −/− mice were infected rLM/GP33 or rLM/NP, and on day 7 PI, activation of CD4 T cells was assessed in the spleen. A and B. Activation of CD4 T cells. Splenocytes were stained with anti-CD4 and anti-CD44 and the number of activated (CD44hi) and naive (CD44lo) CD4 T cells was determined by flow cytometry. Dot plots in A are gated on total viable splenocytes and the numbers are percentages of cells in the respective quadrant of total splenocytes. Data in B are from 3–5 mice/group ± SD. C. Activation of antigen-specific CD4 T cells. Splenocytes from rLM/GP33- or- rLM/NP-infected mice were stimulated ex vivo with the MHC II-restricted peptide LLO190-201, and the number of IFNγ-producing CD4 T cells was determined by intracellular staining. Each symbol in C and D represents data from individual mice.
Figure 5
Figure 5. Effect of treatment with C5aR antagonist on CD8 T cell responses to LM in mice
C57BL/6 mice were infected with rLM/GP33 and treated with the C5aR antagonist (C5aRa) or vehicle PBS on days 1, 3, and 5 post-infection. A. On day 7 PI, splenocytes were stained with anti-CD8 and anti-CD44 antibodies and the number of activated (CD44hi) and naive (CD44lo) CD8 T cells were determined by flow cytometry. Data in A are from 5 mice/group ± SD. B. Antigen-specific CD8 T cells in spleen. On day 7 PI, splenocytes were stained with anti-CD8 and Db/GP33 tetramers, and the number of tetramer-binding CD8 T cells was enumerated by flow cytometry. Data in B are from 5 mice/group ± SD.
Figure 6
Figure 6. Effect of treatment with C5aR antagonist on CD4 T cell responses to LM in mice
Cohorts of rLM/GP33-infected C57BL/6 mice were treated with vehicle control PBS or C5aRa on days 1, 3, and 5 PI. A. On day 7 PI, the total number of activated (CD44hi) and naïve (CD44lo) CD4 T cells in spleen was determined by flow cytometry. B. Total number of LLO190-specific CD4 T cells in spleen was quantitated by intracellular cytokine staining. Data are derived from 5 mice/group ± SD.
Figure 7
Figure 7. CD8 and CD4 T cell responses to LM in C5aR-deficient mice
Groups of wild type C57BL/6 (+/+) and C5aR−/− mice were infected with rLM/GP33. On day 7 PI, activation of CD8 T cells (A) and CD4 T cells (B) was assessed in the spleen. A. Splenocytes were stimulated ex vivo with the indicated epitope peptides, and the number of epitope-specific IFNγ-producing CD8 T cells was determined by intracellular staining. B. The number of LLO190-specific CD4 T cells was quantitated by intracellular staining for IFNγ. Data are the mean of 4–5 mice/group ± SD.
Figure 8
Figure 8
LM-induced maturation of DCs in C3-deficient mice. Groups of wild type (+/+) and C3−/− mice were left uninfected or infected with rLM/GP33. At day 4 after infection, DCs were isolated from spleens and stained with anti-CD11c, anti-B220, anti-Db (MHC I), anti-IAb (MHC II), anti-CD80, anti-CD86, and anti-CD40. Following staining, the expression of MHC I, MHC II, CD80, CD86, and CD40 on plasmacytoid (pDCs; CD11c+veB220+ve) and conventional (cDCs; CD11c+veB220−ve) DCs was assessed by flow cytometry. The data are the mean fluorescent intensities (MFIs) of staining for the indicated molecule from analysis of 3 mice/group.
Figure 9
Figure 9
Activation and expansion of adoptively transferred TCR transgenic CD8 T cells in LM-infected C3-deficient mice. Purified naïve GP33-specific P14/Ly5.1 CD8 T cells were adoptively transferred into congenic +/+/Ly5.2 or C3−/−/Ly5.2 mice, which were subsequently infected with rLM/GP33. On the seventh day after infection, splenocytes were stained with anti-CD8, anti-Ly5.1, and Db/GP33 tetramers. The number of Ly5.1+ve tetramer-binding P14 CD8 T cells was quantitated by flow cytometry. Dot plots in A are gated on total splenocytes, and the numbers are the percentages of P14 CD8 T cells among splenocytes. B. Total number of P14 CD8 T cells in +/+ and C3−/− mice. Data in B are from 4 mice/group and representative of two independent experiments.
Figure 10
Figure 10
Characterization of T cells, B cells, and DCs in spleen of uninfected C3-deficient mice. A. Single cell suspensions of splenocytes from uninfected +/+ or C3−/− mice were stained with anti-CD8, anti-CD4, anti-CD44, anti-CD19, or anti-B220 antibodies. The numbers of naïve (CD44lo) or activated/memory (CD44hi) phenotype CD4 or CD8 T cells, and CD19+veB220+ve B cells were enumerated by flow cytometry. To stain for regulatory T cells, splenocytes were stained for cell surface CD4 and intracellular Foxp3 using a commercially available kit (e-bioscience, San Diego, Ca). B. Splenocytes were stained with anti-CD11c, anti-CD8, and anti-B220 antibodies. Following staining, the percentages of DC subsets were quantitated by flow cytometry. Data are from 3 mice/group.
Figure 11
Figure 11
Activation and expansion of adoptively transferred C3-deficient CD8 T cells. CD8 T cells were purified from spleen of +/+ or C3−/− mice by magnetic bead separation (Milltenyi). 4.8 × 106 Ly5.2+ve CD8 T cells from +/+ or C3−/− mice were adoptively transferred into congenic Ly5.1/C57BL/6 mice, which were subsequently infected with rLM/GP33; Ly5.1 mice receiving CD8 T cells from +/+ and C3−/− mice are labeled as +/+ and C3−/− respectively. At day 7 after infection, the numbers of Ly5.1+ve (CD8 T cells of endogenous or recipient origin) and Ly5.2+ve (donor-derived CD8 T cells) GP33-specific CD8 T cells were quantitated in spleen by intracellular cytokine staining for IFN-γ. Data are from 4 mice/group and representative of two independent experiments.
Figure 12
Figure 12
Proliferative responses of C3-deficient CD8 T cells to anti-CD3 stimulation in vitro. CD8 T cells were purified from spleen of +/+ or C3−/− mice by negative selection using magnetic bead separation kit (Milltenyi). Purified CD8 T cells were unstimulated or stimulated with plastic-immobilized anti-CD3 antibodies in a 96-well round-bottomed plate. Cultures were pulsed with 3[H] Thymidine between 24–48 hours after stimulation. 3[H] incorporation by proliferating CD8 T cells was assessed at 48 hours after stimulation. Each symbol represents data from individual mice, and the data is representative of two independent experiments.

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