LAG-3 regulates plasmacytoid dendritic cell homeostasis

Abstract

Lymphocyte activation gene 3 (LAG-3) is a CD4-related, activation-induced cell surface molecule expressed by various lymphoid cell types and binds to MHC class II with high affinity. We have previously shown that LAG-3 negatively regulates the expansion of activated T cells and T cell homeostasis, and is required for maximal regulatory T cell function. In this study, we demonstrate for the first time that LAG-3 is also expressed on CD11c(low)/B220(+)/PDCA-1(+) plasmacytoid dendritic cells (pDCs). Lag3 expression, as determined by real time PCR, was approximately 10-fold greater in pDCs than in either regulatory T cells or activated T effector cells. Activated pDCs also generate approximately 5 times more sLAG-3 than activated T cells. LAG-3-deficient pDCs proliferate and expand more than wild-type pDCs in vivo in response to the TLR9 ligand, CpG. However, the effect of LAG-3 appears to be selective as there was no effect of LAG-3 on the expression of MHC class II, TLR9, and chemokine receptors, or on cytokine production. Lastly, adoptive transfer of either Lag3(+/+) or Lag3(-/-) T cells plus or minus Lag3(+/+) or Lag3(-/-) pDCs defined a role for LAG-3 in controlling pDC homeostasis as well as highlighting the consequences of deregulated Lag3(-/-) pDCs on T cell homeostasis. This raised the possibility of homeostatic reciprocity between T cells and pDCs. Collectively, our data suggests that LAG-3 plays an important but selective cell intrinsic and cell extrinsic role in pDC biology, and may serve as a key functional marker for their study.

FIGURE 1

LAG-3 is expressed on pDCs. A, The gating scheme used for the sorting of the spleen of a Rag1^−/− mice for Figure 1B. The splenocytes were stained with anti-CD11c, anti-B220 (CD45R), anti-Gr1 (Ly6G) and anti-Mac1 (CD11b). The cells were sorted based on the presence (+) or absence (-) of the various markers. The sorted cells were lysed, RNA isolated and cDNA generated. B, Lag3 was detected by qPCR using a Lag3 specific primer/probe set. The fold increase in expression was determined by comparing the levels of Lag3 in the different cell types to a resting CD4⁺ T cell population devoid of T_regs (Resting T cells - CD4⁺/CD25⁻). C, Lag3 expression determined by qPCR in B cells (CD11c⁻/B220⁺), conventional DCs (CD11c⁺/B220⁻), IKDCs (CD11c⁺/NK1.1 [C57BL/6 and Rag1^−/−] or panNK [BALB/c]), and pDCs (CD11c^lo/PDCA-1⁺/B220⁺) from C57BL/6, BALB/c and Rag1^−/− mice. D, Relative level of Lag3 mRNA as determined by qPCR in purified pDCs versus resting and activated CD4⁺/CD25⁻ and CD4⁺/CD25⁺ T cells (T_regs). T cells were activated with anti-CD3ε (1μg/ml). pDCs were activated with CpG 1688 (10 μM conc). For all qPCR analysis a primer/probe set specific for 18s RNA was used as an endogenous control. E, Splenocytes from Lag3^+/+ and Lag3^−/− mice were stained with anti-PDCA-1, anti-CD11c and anti-LAG-3 and the percentage of CD11c^lo/PDCA-1⁺ cells shown for the Lag3^+/+ cells (left) and LAG-3 surface expression on the CD11c^lo/PDCA-1⁺ cells from Lag3^+/+ and Lag3^−/− mice determined (right).

FIGURE 2

LAG-3 functions as a negative regulator of antigen induced pDC activation/expansion. A, Rag1^−/− and Lag3^−/−;Rag1^−/− mice were injected i.v. with 100 μl of CpG 1688 (100 μM). At the various time points indicated, the mice were sacrificed. Single cell suspensions were made from the spleens, stained for CD11c, B220 and PDCA-1 expression and the number of pDCs determined. Data represent the mean ± SE of seven independent experiments with 2-14 mice per group. B, The mean surface level of MHC class II on B220⁺/PDCA-1⁺ (pDCs) cells 24 hr post CpG stimulation as determined by FACS. The data represents the mean ± SE of 2 independent experiments with 3-4 mice per group. C&D, Rag1^−/− and Lag3^−/−;Rag1^−/− mice were pretreated with BrdU. They were then injected with CpG and 24 hr later spleens and lymph nodes removed and processed. C, The number of BrdU⁺ B220⁺/PDCA-1⁺ cells was determined in the spleen. D, The total percentage of B220⁺/PDCA-1⁺ cells (top panel) and the percentage of BrdU⁺ PDCA-1⁺/B220⁺ cells (lower panel) in the lymph nodes following CpG activation. Data represent the mean ± SE of two independent experiments with 6-7 mice per group. *,p < 0.05, **,p< 0.005

FIGURE 3

pDC LAG-3 surface expression and sLAG-3 production is increased on pDCs following stimulation with CpG. A & B, Rag1^−/− and Lag3^−/−;Rag1^−/− mice were injected with CpG. At 24 hr post activation the mice were sacrificed and single cell suspensions were made from the spleens, stained for B220, PDCA-1 and LAG-3. The expression of LAG-3 was determined on B220⁺/PDCA-1⁺ cells. A, A representative histogram of the level of LAG-3 expression on resting and activated pDCs directly ex vivo. B, Figure depicting LAG-3 expression on multiple mice 24 hr post CpG injection. Data represent the mean ± SE of two experiment with 3-6 mice per group. C, T cells and pDCs were sorted from spleens and lymph nodes of Lag3^+/+ and Lag3^−/− mice and incubated at various cell concentration with either anti-CD3ε/CD28-coupled beads (T cells) or CpG (pDCs). Following a 72 h incubation, supernatants were collected and sLAG-3 ELISAs performed using a purified sLAG-3 standard to determine the concentration of sLAG-3. ND = not detectable. Data represent the mean ± SD of 2-4 experiments. *,p < 0.05, **,p< 0.005

FIGURE 4

The control of homeostatic maintenance of pDCs by LAG-3 has a secondary effect on T cell homeostatic expansion. A, Lag3^+/+;Rag1^+/+, Lag3^−/−;Rag1^+/+, Lag3^+/+;Rag1^−/− and Lag3^−/−;Rag1^−/− mice were sacrificed at 16 weeks of age and the spleens were removed. Single cells suspensions were made and the cells counted. Splenocytes were stained with anti-PDCA-1 and anti-CD11c to determine the total number of pDCs. B, CD4⁺ T cells were purified from the spleens of Lag3^+/+ and Lag3^−/− mice by negative MACS depletion and 5×10⁶ cells transferred into Rag1^−/− or Lag3^−/−;Rag1^−/− mice via the tail vein. Seven days later the mice were sacrificed and the number of CD4⁺ T cells determined. The data represent the mean ± SE 6-7 mice. C. pDCs were purified from CD45.1:Lag3^+/+ and CD45.1:Lag3^−/− mice by FACS and transferred into Rag1^−/−:MHCII^Δ/Δ mice (CD45.2). The next day purified CD4⁺ T cells from Lag3^+/+ mice were transferred into the pDC recipients. Day 7 post transfer, spleens were removed and the total CD4⁺ T cells determined. The data are the mean ± SE of a representative of 3 independent experiments with 5-6 mice per group. D, pDCs or DCs (CD11c⁺ cells devoid of pDCs) were purified from CD45.1:Lag3^+/+ and CD45.1:Lag3^−/− mice by FACS and transferred into Lag3^−/−;Rag1^−/−;MHCII^Δ/Δ mice. Day 7 post transfer the spleens where removed and the total number of CD45.1 positive pDCs (top panel) or CD45.1 DCs (bottom panel) was determined. Data represent the mean ± SE of 2 independent experiments with 2-4 mice per group. *,p < 0.05, **,p< 0.005