Lymphocyte Circadian Clocks Control Lymph Node Trafficking and Adaptive Immune Responses

Abstract

Lymphocytes circulate through lymph nodes (LN) in search for antigen in what is believed to be a continuous process. Here, we show that lymphocyte migration through lymph nodes and lymph occurred in a non-continuous, circadian manner. Lymphocyte homing to lymph nodes peaked at night onset, with cells leaving the tissue during the day. This resulted in strong oscillations in lymphocyte cellularity in lymph nodes and efferent lymphatic fluid. Using lineage-specific genetic ablation of circadian clock function, we demonstrated this to be dependent on rhythmic expression of promigratory factors on lymphocytes. Dendritic cell numbers peaked in phase with lymphocytes, with diurnal oscillations being present in disease severity after immunization to induce experimental autoimmune encephalomyelitis (EAE). These rhythms were abolished by genetic disruption of T cell clocks, demonstrating a circadian regulation of lymphocyte migration through lymph nodes with time-of-day of immunization being critical for adaptive immune responses weeks later.

Keywords: adaptive immune response; circadian rhythm; immunization; leukocyte trafficking; lymph node egress; lymph node homing.

Graphical abstract

Figure 1

Lymphocyte Numbers Exhibit Circadian Oscillations in Lymph Nodes (A) Lymphocyte oscillations in blood (left panel) and inguinal lymph node (middle and right panels) over 24 hr. Zeitgeber time (ZT, time after light onset) 1 is double-plotted to facilitate viewing; n = 4–49 mice, one-way ANOVA, WBC: white blood cells. (B) Lymph node oscillations under light-dark (LD), dark-dark (DD) and inverted, dark-light (DL) conditions, normalized to peak times; CT, circadian time in constant darkness conditions; n = 3–15 mice, one-way ANOVA. (C) Oscillations across multiple lymph nodes, axi: axillary, sup: superficial cervical, ing: inguinal, mes: mesenteric, com: combined counts; n = 3–19 mice, one-way ANOVA, counts are plotted per single lymph node. (D) Lymph node counts after treatment with FTY720 (egress block) or integrin-blocking antibodies (homing block); n = 3–5 mice, one-way ANOVA with Tukey’s multiple comparisons test. (E) Lymphocyte subpopulations after homing block (left) and egress block (right); n = 3 mice. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001. All data are represented as mean ± SEM. See also Figure S1.

Figure 2

Rhythmic Lymphocyte Homing Is Dependent on Oscillations in Both Lymphocytes and Microenvironment (A) Lymph node homing of lymphocyte populations over the course of the day, normalized to peak times; n = 3–17 mice, one-way ANOVA. (B) Adoptive transfer of lymphocyte populations using donor and recipient mice kept at ZT5 or ZT13; n = 6–17 mice, one-way ANOVA with Tukey’s multiple comparisons test. (C) Oscillations of CCR7 surface expression on lymphocyte subpopulations in LN; n = 3–5 mice, one-way ANOVA. (D) Q-PCR analysis of LN Ccl21 amounts over 24 hr; n = 3–5 mice, one-way ANOVA. (E) Quantification and images of expression of CCL21 on HEV over 24 hr in constant darkness (CT, circadian time: the corresponding light and dark phase are indicated); n = 3–18 mice, one-way ANOVA. Scale bar represents 50 μm. (F) LN homing of lymphocytes harvested at ZT5 or ZT13 and treated with or without pertussis toxin (PTX); n = 5–11 mice, one-way ANOVA with Tukey’s multiple comparisons test. (G) Lack of oscillations in LN cellularity of Ccr7^−/− mice; n = 4 mice, unpaired Student’s t test. (H) Lack of rhythmic LN homing of Ccr7^−/− cells into WT hosts; n = 5–6 mice, unpaired Student’s t test. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001. All data are represented as mean ± SEM. See also Figure S2.

Figure 3

Oscillations of Circadian Clock Genes in Lymph Nodes Control Cellularity (A) Q-PCR analysis of circadian clock genes in LN over 24 hr; n = 3–5 mice, one-way ANOVA. (B) Circadian clock gene mRNA profiles in sorted CD4⁺ T cells from Bmal1^flox/floxxCd4-cre and control animals; n = 3–10 mice, two-way ANOVA. (C) Lymph node CD4 and CD8 T cell counts in control and T cell specific Bmal1^−/− mice, n = 3–9 mice, one-way and two-way ANOVA. (D) LN homing of lymphocytes harvested from control or T cell-specific Bmal1^−/− mice at ZT5 or ZT13 into WT hosts; n = 10–34 mice, one-way ANOVA with Tukey’s multiple comparisons test. (E) CCR7 surface expression on T lymphocyte subpopulations in LN of control and T cell-specific Bmal1^−/− mice; n = 3–5 mice, one-way ANOVA. (F) Q-PCR analysis of CD4⁺ T cell Ccr7 over 24 hr in control and T cell-specific Bmal1^−/− mice; n = 4–8 mice, one-way and two-way ANOVA. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001. All data are represented as mean ± SEM. See also Figure S2.

Figure 4

Circadian Oscillations in Lymphocyte Egress from LNs (A) Oscillations of total leukocyte (left panel) and lymphocyte (right panel) counts in efferent lymph over 24 hr; n = 6–33 mice, one-way ANOVA. (B) Lymph leukocyte count oscillations under light-dark (LD) and dark-dark (DD) conditions; n = 3–37 mice, one-way ANOVA. (C and D) Remaining cellular numbers (in %) in lymph node (C) and lymph (D) over 24 hr after block of leukocyte homing. Lymph node: n = 3–10 mice; lymph: n = 3–6 mice, unpaired Student’s t test. (E) Lymph CD4⁺ and CD8⁺ T cell counts in T cell-specific Bmal1^−/− mice; n = 3–8 mice, one-way ANOVA. (F) Remaining cells (in %) in lymph nodes of adoptively transferred control and BMAL1-deficient CD4⁺ T cells 12 hr after transfer; n = 4–13, unpaired Student’s t test. (G) Mathematical model of leukocyte homing and egress. The model is expressed as a line based on the indicated data points. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001. All data are represented as mean ± SEM. See also Figures S2–S4.

Figure 5

Rhythmic Lymphocyte Egress Depends on Oscillatory S1pr1 Expression (A) Q-PCR analysis of LN S1pr1 over 24h. n = 3–5 mice, one-way ANOVA. (B) Lymph counts after blockade of S1P-receptor function using FTY720 at the indicated times; n = 3–33 mice, two-way ANOVA. (C) FTY720 titration and respective lymph counts at two time points. n = 3–5 mice. (D) Q-PCR analysis of CD4⁺ T cell S1pr1 over 24 hr in control and T cell-specific Bmal1^−/− mice; n = 4–9 mice, one-way and two-way ANOVA. (E) Normalized activity (in %) of Gaussia Luciferase driven by murine S1pr1 promoter (S1pr1-GLuc) after co-transfection with various doses of Clock and Bmal1 plasmids in HEK293 cells (n = 6). Data shown are pooled from two independent experiments, one-way ANOVA with Tukey’s multiple comparisons test. (F) LN CD4⁺ and CD8⁺ T cell counts in control and T cell-specific S1pr1 heterozygous mice; n = 3–6 mice, one-way and two-way ANOVA. (G) Lymph CD4⁺ and CD8⁺ T cell counts in control and T cell-specific S1pr1 heterozygous mice; n = 3–12 mice, one-way and two-way ANOVA. (H) Mass spectrometric analysis of sphingosine-1-phosphate (S1P) in lymph and blood plasma; n = 9–11 mice. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001. All data are represented as mean ± SEM. See also Figure S5.

Figure 6

T Cell Clock Function Regulates Disease Severity in EAE (A) Oscillations of CD69⁺ T cell numbers in lymph node; n = 3–5 mice, one-way ANOVA. (B) Oscillations of migratory dendritic cells (DCs) in lymph node; n = 6–12 mice, one-way ANOVA. (C) EAE disease scores of mice immunized at ZT8 or ZT20. Disease score EC₅₀ comparisons show accelerated symptom progression in ZT8-immunized mice; n = 5 mice, two-way ANOVA (left panel) and unpaired Student’s t test (right panel). (D) Quantification of demyelination in lumbar spinal cord sections; n = 5 mice, unpaired Student’s t test. (E) Luxol Fast Blue staining of lumbar spinal cord sections of mice immunized at ZT8 (top) or ZT20 (bottom) at the peak of the disease showing demyelinated areas (arrows), scale bar represents 200 μm (left images), 100 μm (right images). (F) LN IL-2 mRNA after EAE induction; n = 3–5 mice, unpaired Student’s t test. (G) LN counts of IL-17⁺ and VLA-4⁺ CD4⁺ T cells after EAE induction; n = 5 mice, unpaired Student’s t test. (H–J) Diurnal profiles of inguinal lymph node counts of total CD4⁺ T cells (H), naive CD4⁺ T cells (I), and activated CD4⁺ T cells (J) on day 2 after EAE induction; n = 4 mice, two-way ANOVA with Bonferroni’s post hoc test. (K) EAE disease scores and EC₅₀ values in T cell-specific Bmal1^−/− mice immunized at ZT8 or ZT20; n = 5 mice. (L) Inguinal lymph node counts of CD4⁺ T cells, and CD8⁺ T cells in T cell-specific Bmal1^−/− mice at ZT15 on day 2 after EAE induction; n = 4–5 mice, unpaired Student’s t test. (M) Schematic diagram of circadian lymphocyte migration through lymph nodes. At night onset, increased homing due to higher CCR7 amounts leads to enhanced lymphocyte counts in the lymph node. During the day, higher S1pr1 expression induces the egression of lymphocytes into efferent lymph. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001. All data are represented as mean ± SEM. See also Figure S6 and Movie S1.