Vasoactive intestinal peptide receptor 1 is downregulated during expansion of antigen-specific CD8 T cells following primary and secondary Listeria monocytogenes infections

Abstract

As regulation of CD8 T cell homeostasis is incompletely understood, we investigated the expression profile of the vasoactive intestinal peptide (VIP) receptors, VPAC1 and VPAC2, on CD8 T cells throughout an in vivo immune response. Herein, we show that adoptively transferred CD8 T cells responding to a Listeria monocytogenes infection significantly downregulated, functionally active VPAC1 protein expression during primary and secondary expansion. VPAC1 mRNA expression was restored during contraction and regained naïve levels in primary, but remained low during secondary, memory generation. VIP co-administration with primary infection suppressed CD8 T cell expansion (≈ 50%). VPAC2 was not detected at any time points throughout primary and secondary infections. Collectively, our data demonstrate that functionally active VPAC1 is dynamically downregulated to render expanding CD8 T cells unresponsive to VIP.

Figure 1. VIP receptor mRNA levels are downregulated by in vivo activation conditions

Mice were retro-orbitally injected with 10 µg of Armenian hamster isotype control antibody, or anti-CD3 for 24 hours followed by magnetic bead isolation of splenic T cells and qRT-PCR analysis (Materials and Methods). Data is representative of 2–4 experiments utilizing at least four mice per experiment. A. Flow cytometric analysis of indicated activation markers for isotype control (grey line) or anti-CD3 treated mice (black line) in CD4 and CD8 T cells. Gate frequencies are percent positive of total population. B–C. qPCR measurements for VIP receptor mRNA expression normalized to HPRT from isotype control, or anti-CD3 treatment for hours. Relative levels of VIP receptor expression in CD4 cells (B) and CD8 T cells (C) are shown. Left panels show VPAC1 expression and right panels show VPAC2, however VPAC2 was not detected (N.D.) in CD8 T cells as indicated.

Figure 1. VIP receptor mRNA levels are downregulated by in vivo activation conditions

Mice were retro-orbitally injected with 10 µg of Armenian hamster isotype control antibody, or anti-CD3 for 24 hours followed by magnetic bead isolation of splenic T cells and qRT-PCR analysis (Materials and Methods). Data is representative of 2–4 experiments utilizing at least four mice per experiment. A. Flow cytometric analysis of indicated activation markers for isotype control (grey line) or anti-CD3 treated mice (black line) in CD4 and CD8 T cells. Gate frequencies are percent positive of total population. B–C. qPCR measurements for VIP receptor mRNA expression normalized to HPRT from isotype control, or anti-CD3 treatment for hours. Relative levels of VIP receptor expression in CD4 cells (B) and CD8 T cells (C) are shown. Left panels show VPAC1 expression and right panels show VPAC2, however VPAC2 was not detected (N.D.) in CD8 T cells as indicated.

Figure 2. Schematic representation of LM-OVA infection time line and example of Thy1.1⁺/OT-I purification

A. One day prior to infection with LM-OVA, approximately 500 Thy1.1⁺/OT-I CD8 T cells were adoptively transferred by retro-orbital injection into recipient Thy1.2⁺ C57Bl/6J mice. Primary and secondary infections were monitored by purifying CD8 OT-I (Thy1.1⁺) cells at the indicated days post infection (p.i.). CD8 T cell expansion, contraction and memory phases are indicated for primary and secondary responses. B. Representative magnetic bead purification of CD8⁺CD90.1⁺ (Thy 1.1⁺) adoptively transferred T cells isolated from recipient mice on day 5 p.i. (Materials and Methods). Data is representative of 4 independent experiments of at least 3 mice each. A two-tailed, paired student T test was conducted to determine statistical significance (* indicates a p≤0.05).

Figure 3. Appropriate kinetics and activation profile of adoptively transferred OT-I mice

A. Splenocytes were collected at the indicated times p,i, that corresponded to expansion, contraction, and memory time points following primary and secondary infections. Representative dot plots are shown of anti-Thy 1.1 and anti-CD8 staining. Gate frequencies are percent positive of the total splenocyte populations. B. Total cell number of Thy.1.1⁺ CD8 T cells per spleen throughout primary and secondary responses represented by a line graph versus days after challenge. Gray circles indicate when mice were infected with LM-OVA. C. Histogram plots showing staining of splenocytes collected from recipient mice isolated at the indicated days p.i.. Cells were stained for two activation markers as indicated and analyzed by flow cytometry. Gray lines represent the unactivated endogenous CD8⁺ population, and black lines represent the Thy1.1⁺ CD8 T cells at the indicated day p.i.. Gate frequencies are percent positive of previously gated Thy1.1⁻/CD8^hi cells (gray lines) or previously gated Thy1.1⁺/CD8⁺ cells (black lines). All data is representative from 2–4 independent experiments using three mice per experiment.

Figure 4. Mapping of VPAC receptor expression throughout a primary and secondary CD8 T cell response

Thy.1.1⁺ CD8 T cells were purified by magnetic beads (Materials and Methods) at various times p.i., and used to measure VPAC1 (square; solid black line) and VPAC2 (diamond; dotted black line) mRNA levels normalized to HPRT as assessed by qPCR. Data is representative of 2–4 independent experiments using three mice per time point per experiment. Total number of Thy.1.1⁺ CD8 T cells per spleen (right Y-axis) in Figure 3B is also shown (circle; dashed black line) for comparison. Arrows indicate primary and secondary infection with LM-OVA.

Figure 5. Functional VPAC1 protein levels are undetectable during peak infection

A. Thy1.1⁺CD8 T cells adoptively transferred into Thy1.2⁺ recipient mice (day 0) and splenocytes from day 5 p.i. were stained with rabbit serum (grey line) and anti-mVPAC1 pAb (black line). Data shown was previously gated on CD8⁺/Thy1.1⁺ cells. Representative histogram plots are shown from two independent experiments with similar data using at least three mice per experiment. B. Day 0 and day 5 p.i. Thy1.1⁺ CD8 T cells were treated for 15 minutes with 10⁻⁶ M VIP ligand or vehicle control (water). Forskolin (50 uM) and vehicle control (DMSO) were used as positive and negative controls with day 5 p.i. cells. Cells were assayed directly using a competitive cAMP ELISA (Materials and Methods). A two-tailed, paired student T test was conducted to determine statistical significance (* indicates a p≤0.05 for day 0, vehicle control versus +VIP). Data is presented as averages +/−SEM from two independent experiments. C. Cells were gated on CD8⁺/Thy1.1 or Thy1.2, and incubated anti-CD44 and rabbit serum (grey line) or anti-mVPAC1 pAb (black line). Protein expression was compared between resting (CD44^low/CD8^high) and activated (CD44^high/CD8^mid) Thy1.1⁺ versus Thy1.2⁺ CD8⁺ T cells.

Figure 6. Exogenously administered VIP suppresses CD8 T cell expansion

Thy1.1⁺ CD8 T cells were adoptively transferred into recipient mice 1 day prior to LM-OVA infection +/− administration of 5 nmols of VIP by i.v. injection. Mice were sacrificed on day 5 p.i. A. The percentage of gated Thy1.1⁺ CD8 T cells from mice receiving VIP (+VIP) versus PBS vehicle control (−VIP) were measured by flow cytometry. Dot plots are representative of four mice analyzed in two independent experiments (top row = 1^st experiment, bottom row = 2^nd experiment). Gate frequencies are of total splenocyte population. B. Total numbers of OT-I/CD8 T cells −VIP and +VIP treatment at day 5 p.i.. A two-tailed, paired student T test was conducted to determine statistical significance (** indicates a p≤0.1). C. CD127 expression on OT-I/CD8 T cells treated with LM-OVA −/+VIP at 5 days p.i. is shown. Grey lines represent isotype control, and black lines represent anti-CD127 staining from two independent experiments with four mice each (n=1 top, n=2 bottom). D. VPAC1 mRNA levels normalized to HPRT (day 5 p.i.). qPCR data is presented as averages +/− SEM.