Lymph nodes as barriers to T-cell rejuvenation in aging mice and nonhuman primates

Abstract

In youth, thymic involution curtails production of new naïve T cells, placing the onus of T-cell maintenance upon secondary lymphoid organs (SLO). This peripheral maintenance preserves the size of the T-cell pool for much of the lifespan, but wanes in the last third of life, leading to a dearth of naïve T cells in blood and SLO, and contributing to suboptimal immune defense. Both keratinocyte growth factor (KGF) and sex steroid ablation (SSA) have been shown to transiently increase the size and cellularity of the old thymus. It is less clear whether this increase can improve protection of old animals from infectious challenge. Here, we directly measured the extent to which thymic rejuvenation benefits the peripheral T-cell compartment of old mice and nonhuman primates. Following treatment of old animals with either KGF or SSA, we observed robust rejuvenation of thymic size and cellularity, and, in a reporter mouse model, an increase in recent thymic emigrants (RTE) in the blood. However, few RTE were found in the spleen and even fewer in the lymph nodes, and SSA-treated mice showed no improvement in immune defense against West Nile virus. In parallel, we found increased disorganization and fibrosis in old LN of both mice and nonhuman primates. These results suggest that SLO defects with aging can negate the effects of successful thymic rejuvenation in immune defense.

Figure 1

Thymic rejuvenation increases thymocyte populations in old mice. (a) Representation of naïve T cells among total CD4 (gray) or total CD8 (black) pool from mice across the lifespan is shown. Cells were gated on CD4+ or CD8+ cells, and then on CD62^HI CD44^LO. The slopes denote a decline from youth, and are significantly different from zero, p < 0.0001 for CD8 and p = 0.0003 for CD4. Data represent n = 81 mice of indicated ages across the timeline. (b–f) Mice were treated with either degarelix (D) or KGF and analyzed 42 days or 1 month post‐treatment, respectively. (b) Thymus from an untreated old mouse (left) and a degarelix‐treated old mouse (right). (c) Thymocyte numbers from adult, old, and old mice treated with degarelix. (d) Double‐positive thymocytes from adult, old, and old mice treated with degarelix. (e) Thymocyte numbers from adult, old, and old mice treated with KGF. (f) Numbers of double‐positive thymocytes from adult, old, and old mice treated with KGF. For degarelix experiments, n = 7 to 13 mice per group pooled from two independent experiments. For KGF experiments, n = 3–7 mice per group. Means + SEM are shown (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Figure 2

Thymic rejuvenation does not improve the frequency of naïve CD8 in the blood. (a) Percentage of CD8 T cells with naïve phenotype (CD62^HI CD44^LO) in the blood of adult, old, and old mice treated with degarelix (D) for 42 days. (b) Percentage of CD8 T cells with naïve phenotype in the blood of adult, old, and old mice treated with KGF at 1 month post‐treatment. (c) Percentage of CD4 T cells with naïve phenotype (CD62^HI CD44^LO) in the blood of adult, old, and old mice treated with degarelix for 42 days. (d) Percentage of CD4 T cells with naïve phenotype in the blood of adult, old, and old mice treated with KGF at 1 month post‐treatment. (e) Percentage of naïve T cells (CD95^LO, CD28^MOD) in the CD8 T‐cell pool of RM adult, old, and KGF‐treated old at 21 days post‐treatment. (f) Percentage of naïve T cells (CD95^LO, CD28^MOD) in the CD4 T‐cell pool of RM adult, old, and KGF‐treated old at 21 days post‐treatment. For RM experiments, n = 3 adults, n = 7 old, and n = 7 treated old from three independent experiments. For degarelix experiments, n = 7 to 13 mice per group pooled from two independent experiments. Bar graphs means + SEM are shown (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Absolute numbers provided in the text

Figure 3

Thymic rejuvenation is insufficient to protect against lethal WNV infection. (a) Survival of adult, old, and old mice treated with degarelix for 42 days then challenged with WNV n = 20–50 mice per group pooled from two independent experiments. (b) Percent of H‐2D^b/NS4b₂₄₈₈ tetramer cells in the blood of adult, old, old treated with degarelix‐treated mice at day 7 following infection n = 5 mice per group, one experiment showed representative of two experiments. (c) WNV‐specific IgG at day 7 following infection, n = 10 mice per group, one experiment showed representative of two experiments. For bar graphs, means + SEM are shown (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Figure 4

Secondary lymphoid compartments are not restored by recent thymic emigrants following thymic rejuvenation. (a) Frequency of total T cells that are recent thymic emigrants in the blood of adult, old, and old mice treated with degarelix (D). (b) CD4 (gray) and CD8 (black) T cells present in spleens of adult, old, and old mice treated with degarelix (D). (c) CD4 (gray) and CD8 (black) T cells present in the peripheral lymph nodes (pool of one of each inguinal, popliteal, and brachial). (d) Naïve T cells in the spleen of adult, old, and old mice treated with degarelix. (e) CD4 (gray) and CD8 (black) naïve T cells in the peripheral lymph nodes of adult, old, and old mice treated with degarelix (D). (f) CD4 (gray) and CD8 (black) recent thymic emigrants present in the spleen of adult, old, and old mice treated with degarelix (D). (g) CD4 (gray) and CD8 (black) recent thymic emigrants in the peripheral lymph node pool of adult, old, and old + degarelix (D). n = 7 to 13 mice per group, results are pooled from two independent experiments. Statistics were done on pooled CD4+ and CD8+ for each population shown. Means + SEM are shown (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Statistics were done on the total bar

Figure 5

Thymic influence on naïve T‐cell pool in SLO is altered with age. (a) Double‐positive thymocytes compared to the number of CD3+ naïve T cells per ul of blood in old mice, old mice treated with degarelix, adult mice, and adult mice treated with degarelix, one curve fits both data sets. (b) Double‐positive thymocytes compared to the number of CD3+ naïve T cells per spleen, one curve fits both data sets. (c) Double‐positive thymocytes compared to CD3+ naïve T cells in peripheral lymph node pool, a different curve set for each adult compared to the old lymph node data set (p = 0.0142). (d) Number of blood CD3 RTE determined by Rag2pGFP reporter compared to the number of double‐positive cells in the thymus, one curve fits both data sets. (e) Number of spleen CD3 RTE compared to double‐positive thymocytes, different curve set is needed for old compared to adult data sets (p = 0.0011). (f) Number of lymph node CD3 RTE compared to double‐positive thymocytes, a different curve is needed to fit each data set (p = 0.0427). n = 19 mice per group, results are pooled from two independent experiments

Figure 6

Old LN exhibit increased fibrosis in both mice and nonhuman primates. Photomicrographs show lymph node sections stained for collagen with Picrosirius red under bright field (left) or polarization contrast (right) (Leica, DMI6000). Representative images (one animal of four analyzed, similar changes observed in others) showing collagen content in (a). Total IL7 protein (pg/ml) as determined by ELISA on total LN homogenate for adult (A), old (O, 19–21 months), and old (O, 22–24 months) mice. (b) Adult rhesus macaque (axillary LN, 8 years old—scale bar, 100 μm); (c) old rhesus macaque (axillary LN, 25 years old—scale bar, 100 μm); (d) adult mice (inguinal LN, 4 months of age—scale bar, 100 μm); (e) old mice (inguinal LN, 23 months of age—scale bar, 100 μm). Fibrosis in mouse LNs was quantified using ImageJ software and presented as (f) capsule thickness and (g) percentage of area positive for collagen inside the capsule. Mean + SEM are shown (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001), n = 4–6 mouse/group)