Aging of lymphoid stromal architecture impacts immune responses

Abstract

The secondary lymphoid organs (SLOs) undergo structural changes with age, which correlates with diminishing immune responses against infectious disease. A growing body of research suggests that the aged tissue microenvironment can contribute to decreased immune function, independent of intrinsic changes to hematopoietic cells with age. Stromal cells impart structural integrity, facilitate fluid transport, and provide chemokine and cytokine signals that are essential for immune homeostasis. Mechanisms that drive SLO development have been described, but their roles in SLO maintenance with advanced age are unknown. Disorganization of the fibroblasts of the T cell and B cell zones may reduce the maintenance of naïve lymphocytes and delay immune activation. Reduced lymphatic transport efficiency with age can also delay the onset of the adaptive immune response. This review focuses on recent studies that describe age-associated changes to the stroma of the lymph nodes and spleen. We also review recent investigations into stromal cell biology, which include high-dimensional analysis of the stromal cell transcriptome and viscoelastic testing of lymph node mechanical properties, as they constitute an important framework for understanding aging of the lymphoid tissues.

1. Introduction

The immune system is a collection of cells and tissues that work in concert to protect the entire organism. Timely response to pathogenic agents is critical to avoid catastrophic damage to life-sustaining functions. Thus, immune responses are expedited by the activity of organized lymphoid structures strategically located throughout the body. The secondary lymphoid organs (SLOs) increase the efficiency of immune responses by coordinating distinct subsets of immune cells and physically bringing innate and adaptive immune cells together. In addition to immune cells of hematopoietic origin, lymphoid tissues are also composed of heterogenous stromal cells, which have roles in both organ structural support and immune regulation. Fibroblastic stromal cells have mechanical properties to provide structural integrity, whereas blood and lymphatic vascular endothelial cells facilitate fluid transport. In addition to their contributions to SLO architecture, stromal cells have prominent roles in supporting immune homeostasis and function. Appreciation of the importance of stromal cells to immune regulation has been facilitated by technological advances and engineering approaches, which have enabled in-depth analysis of lymphoid cells and their structural properties.

Biological aging is associated with dramatic changes to the tissue integrity of all organs, with the SLOs being no exception. Age-associated decline of immune function has been correlated to structural changes in aging lymphoid tissues, and investigations into how aging impacts lymphoid architecture and function are ongoing. Here, advanced age in humans is roughly defined as being greater than 55 years-old, which is approximated in laboratory mice and rats that are 18-24 months of age. [1,2] Aging has been strongly associated with increased baseline levels of inflammatory signaling markers[3,4] and the accumulation of senescent cells,[5-7] the contributions of which to immune regulation are an active area of study. In this review, we will focus on the impact of aging on the non-hematopoietic stroma of the SLOs, with emphasis on the lymph nodes and spleen, of which age-related changes have been most explored. Age-associated changes to the primary lymphoid organs, the bone marrow and thymus, have also been described and have been reviewed by us and others.[8-10]

2. Influence of tissue microenvironment on aged immune outcomes

Changes in the homeostasis and function of immune cells with age, particularly T cells and B cells, have been well documented.[11-13] It has been consistently observed that the frequency and numbers of naïve T cells (CD45RA⁺CD62L⁺CD95⁻) decreased sharply with age in human blood, though CD95⁺ memory T cell numbers remains constant.[14] The decline in naïve T cells is also apparent in laboratory mice, with a gradual decline of CD44^loCD62L^hi cells in the blood in adulthood from 3-16 months, and a sharper decrease in frequencies thereafter.[15] For both humans and mice, circulating naïve T cell decline is more dramatic in CD8⁺ T cells.[14,15] Naïve T cell attrition is significant, as it has been associated with diminished protection against infectious disease.[16] However, it has been difficult to uncouple age-associated T and B cell defects that are due to intrinsic changes in the lymphocytes from those driven by the aging tissue microenvironment. Despite these challenges, there is growing evidence that the age of lymphoid tissues impacts the maintenance and response of T and B cells beyond intrinsic age-related impairments.

Several lines of study indicate that the mechanisms of homeostatic maintenance and activation of naïve T cells decline with age. Experiments in mice suggest that impaired recruitment to and persistence of naïve T cells is exacerbated within the aged tissue microenvironment. Both CD4⁺ and CD8⁺ naive T cells, isolated from young mice (2-3 months-old), were adoptively transferred into sub-lethally irradiated mice, and had diminished homeostatic proliferation within aged hosts (16-22 months-old).[17,18] When equivalent numbers of young naïve T cells, either polyclonal[18] or T cell receptor (TCR) transgenic,[19] were adoptively-transferred into young or aged mice, reduced frequencies and numbers of donor-derived cells were recovered from the SLOs of aged hosts. In other experiments, the recovery of young CD44^lo naïve and CD44^hiCD49d^hi virtual-memory T cells two months after transfer into aged mice was significantly diminished as compared to transfer into young hosts.[20] The complementary scenario, in which either young or aged naïve T cells were transferred into young hosts, revealed comparable homeostatic turnover between the naïve T cell ages.[17] Furthermore, aged TCR transgenic CD8⁺ naïve T cells that were transferred into young hosts were able to expand robustly in response to Listeria monocytogenes (Lm) infection.[21] The aged microenvironment had reduced induction of CD8⁺ T effector transcriptional and metabolic programs upon acute infection, defects that could be rescued with exogenous addition of interleukins-12 and −18 (IL-12, IL-18) in the aged mice.[21] These experiments suggest that naïve T cells have impaired ability to traffic to and survive within the aged SLOs, and that aged T cells may in fact retain much of their function when presented with an intact microenvironment. The influence of the lymphoid microenvironment is likely pertinent to humans as well, as the capacity for CD45RA^hi naïve T cells to maintain their naïve phenotype was dependent on the 3-D structure of human lymphoid tissue FRC organoids.[22]

Experiments using parabiosis, the surgical conjoining of the blood circulation of a pair of mice, have revealed whether immune alterations are transferrable via circulating hematopoietic cells, or are determined by the resident stroma of the host. Studies that conjoined mice of different ages resulted in the seeding of both ages of T cells to the lymph nodes, yet the cellularity of the aged mouse lymph nodes remained significantly reduced compared to that of its young partner, indicating that lymph node capacity was determined by the hosts’ resident cells.[23] Parabiosis experiments have also recently demonstrated that the failure to differentiate B cells during peptide immunization was specific to the aged host, despite circulating young lymphocytes from the conjoined young partner.[24] Thus, aged lymphoid tissues can reduce the maintenance and activation of lymphocytes, even for transferred lymphocytes isolated from young organisms, but the investigation into the mechanisms by which aged tissues limit homeostasis and function are still ongoing.

3. Lymphoid architecture organization and maintenance with age

The lymph nodes and splenic white pulp are organized into compartments (Figure 1). Separate regions are dedicated to the support and function of T and B lymphocytes, and passages facilitate the access and transport of lymph and blood. Like a scaffold, stromal cells form a meshlike network within the lymphoid compartments, thus facilitating the migration of adaptive and innate immune cells through the lymphoid tissues.[25] Stromal cells form only about 1% of SLO cellularity,[26] and can be differentiated based on their lack of hematopoietic marker CD45, with subsets broadly distinguished based on expression levels of the surface markers podoplanin (PDPN; gp38) and CD31 (PECAM).[27] These subsets consist of fibroblastic reticular cells (FRCs; PDPN⁺CD31⁻), lymphatic endothelial cells (LECs; PDPN⁺CD31⁺), blood endothelial cells (BECs, PDPN⁻CD31⁺), and double-negative progenitor cells (DNs; PDPN⁻CD31⁻). Though the investigation of stroma from the human SLOs is challenging, studies have indicated that they are structurally and transcriptionally similar to those in murine SLOs, which are easier to harvest and study.[27,28] Single-cell RNA sequencing (scRNAseq) of mouse lymph nodes have revealed as many as nine transcriptionally-distinct clusters of FRCs[29-31] and four groups of LECs[32], which enables the functional specialization of the lymphoid compartments. Correlates for the FRC subsets identified within mice were found by scRNAseq analysis of human FRCs, obtained from resected lymph nodes.[30] FRC subsets have also been analyzed by scRNAseq of the murine spleen, which were correlated to human splenic FRCs by microscopy.[33] These transcriptionally distinct stromal subsets form specialized neighborhoods to support immune cell function. At the time of this review, there have not been published datasets of aged stromal cell scRNAseq, though recently Bennett and colleagues have RNA sequenced ex vivo cultured lymph node stromal cells obtained from aged mice.[34] They found that gene expression from aged stromal cells stimulated with either recombinant interferon-alpha (IFNα) or West Nile virus particles was similar to that of young stromal cells, and in fact had upregulated expression of immediate early response genes upon stimuli.[34] scRNAseq of aged stromal cells is certainly to be expected within the next couple years and would further elucidate how aging affects the subset distribution of stromal cell subsets.

Figure 1. Age-associated changes to lymph node architecture.

The lymph node is organized into compartments that express CCL19 and CCL21 (orange) in the T cell zone or CXCL13 (purple) in the B cell follicles. In adulthood (young- left panel), CCL19/21 and CXCL13 expression organizes the T cell and B cell zones. With advanced age (aged- right panel) the lymph node is atrophied, and chemokine expression is reduced and less responsive to immune activation. Contraction of lymphatic vessels deliver solutes and cells via the lymph, but lymphatic vessels with age become more permeable and generate less contractile force. Within the cellular niches of the T cell zone, B cell follicle, and subcapsular sinus (insets) specialized stroma create structures to coordinate hematopoietic cell function. The aged T cell zone is smaller, with increased fibrosis and/or lipomatosis. The aged B cell follicle loses its definition and harbors less follicular dendritic cells (FDCs) during the germinal center response. Lymphatics drain into the subcapsular sinus to allow antigen sampling from the lymph. With age, collagen of the capsule is thickened, gaps between stromal cells widen, though macrophage numbers remain constant. Stroma elements and hematopoietic cells are color-coded in the top right legend.

The SLOs arise during embryonic development through the interactions of hematopoietic lymphoid tissue inducer (LTi) cells and mesenchymal lymphoid tissue organizer (LTo) cells, the latter of which gives rise to heterogenous stromal cells (reviewed in [35-37]). Commitment of LTo cells to the fibroblast and endothelial cell fates is driven by constitutive signals received through the lymphotoxin-beta receptor (LTβR) by lymphotoxin ligands that are expressed by LTi cells and T and B lymphocytes.[38] The interactions between hematopoietic cells and stroma sustains constitutive crosstalk via LTβR,[39] and drives a transcriptional program within stroma to further promote lymphocyte homing to the SLOs.[40] Signaling through LTγR is essential for SLO development, in that Lta^−/− and Ltbr^−/− mice fail to develop lymph nodes and have diminished spleens,[41] and smaller lymph nodes with defective antiviral immunity was also observed when Ltbr was deleted in a stromal cell-specific manner.[42] Inducible deletion of Ltbr in adult mice also resulted in reduced size and organization of lymph nodes and spleen, indicating a continued requirement of signaling via LTβR for postnatal SLO maintenance.[43] Disorganization can similarly be observed after infections that bear tropism to the SLOs, such as lymphocytic choriomeningitis virus (LCMV); furthermore, the ability for SLOs to recover stromal architecture post-infection is dependent on LTβR signaling, as demonstrated by LCMV-infected spleens treated with a LTβR-blocking fusion protein.[44]

Despite its well-investigated roles in development, the direct impact of LTβR signaling on the size and organization of aged SLOs is unknown. Our studies have indicated that Ltbr is reduced in bulk aged stroma from murine lymph nodes.[18] Along these lines, an overall reduction in lymphocyte cellularity in aged murine lymph nodes and spleen has been observed.[15] Furthermore, aging is associated with disorganization of the T and B cell compartments within the lymph nodes[18,45] and the splenic white pulp.[46] Though immune defects are studied in advanced age, there are indications that SLO changes begin earlier; by inducible labeling of recent thymic emigrants (RTEs) in mice, Sonar and colleagues have demonstrated the preferential loss of RTE retention within the SLOs as early as six months of age in the skin-draining lymph nodes, which coincided with stromal network disorganization and defective immunity against West Nile virus.[47] The molecular mechanisms by which hematopoietic and stromal cells regulate the size and organization of the SLOs in adulthood remain an active area of inquiry.

3.1. FRCs of the T cell zone support T cells

Non-hematopoietic stromal cells are sources of the survival cytokine interleukin-7 (IL-7) and thus play a fundamental role in T cell homeostasis.[48] Levels of IL-7 are tightly regulated, as both innate and adaptive hematopoietic cells express the IL-7 receptor and thus form a control loop modulating IL-7 availability.[49] T cell zone FRCs (TRCs) are major components of the stromal cell networks within the SLOs, being the major sources of IL-7 in the lymph node and the primary mediators of naïve T cell survival.[25,50] The close contacts between T cells and TRCs facilitate homeostatic signaling,[51] as IL-7 is immobilized to cell surfaces by glycosaminoglycans.[52] It should be noted that Il7 is also expressed by LECs,[53] though it is not clear whether they equally support T cell survival.[50] This is perhaps because LECs can lose their gene expression signature during in vitro manipulation.[44] Interestingly, transcript expression of Il7 is maintained with age in mouse lymphoid tissues[17,18] and IL-7 protein expression remains stable in aged mouse and human serum.[17] It has been suggested that despite its continued expression with age, IL-7 is not bioavailable in the aged microenvironment. Experiments in aged mice were able to restore homeostatic proliferation of naïve T cells by the administration of IL-7/M25 antibody complexes.[17,54] Notably, studies using FRC and LEC-specific Il7-knockout mice indicated that naïve T cells may be able to procure IL-7 from other sources in vivo, though central memory T cells remained sensitive to a loss of FRC-derived IL-7.[55] An in vitro study with splenic stromal cells demonstrated that their presence was required for the survival of LTi-like cells isolated from adult mice, but that IL-7 itself was not.[56] Further study is needed to determine whether age-associated changes to IL-7 access within the tissue microenvironment could potentially improve aging immune outcomes.

Stromal expression of the chemokine ligands CCL19 and CCL21 promotes the recruitment of CCR7⁺ immune cells from the blood and their positioning within the SLOs.[57] Homeostatic expression of CCL19 and CCL21 is maintained by non-canonical NFκB signaling, downstream of LTβR.[39] Though often discussed together, CCL19 and CCL21 have different tissue expression patterns and signaling through CCR7.[58] Surface-immobilized CCL21 induces integrin activation and cell adhesion, which promotes robust and sustained T cell motility.[58] In contrast, binding of CCL19 causes desensitization and internalization of CCR7, which can attenuate T cell migration.[58] The roles of CCL19 and CCL21 are likely not redundant, as Ccl19^−/− mice had lymph nodes with distinguished T and B cell zones yet decreased maintenance of T cells; this is in contrast to plt/plt mice, which lack both CCL19 and CCL21 and as a result have disorganized lymph nodes and minimal T cells.[50] Emphasizing their prominent role, diphtheria toxin-induced depletion of FRCs significantly reduced expression of both CCL19 and CCL21, as well as the retention of naïve T cells within the lymph nodes of mice, though this effect was less pronounced in the spleen.[59] Studies have indicated that aged murine lymph nodes have decreased homeostatic expression of either Ccl19[17] or Ccl21[18] transcripts. Inconsistencies in reported results of chemokine expression may be due to differences in stromal cell isolation techniques, which rely on different digestive enzymes such as collagenase or dispase that can cleave certain surface molecules, variability in mechanical stress applied to disrupt the lymphoid issues, or in the choice of lymph node sites that are pooled.[50,60] Nonetheless, both studies determined that reduced Ccl19 or Ccl21 expression by lymph node stromal cells was associated with decreased naïve T cell maintenance within aged mice.[17,18] During acute infection, immune activation further increases CCL19 and CCL21 levels in the SLOs, to promote naïve T cell recruitment. However, upon immunization, protein levels of CCL21 were dramatically decreased in aged as compared to young murine lymph nodes.[19,61] As discussed earlier, SLOs have been reported to decrease in size with age, though the relative changes to the different compartments are likely not equivalent; immunofluorescence imaging revealed smaller T cell zones with decreased overall cellularity within aged murine lymph nodes.[45] In contrast, the murine spleen has been either described as having similar total cellularity yet smaller T cell zones with age,[62] or as having increased T cell zone occupancy, which drove a larger white pulp area with age.[63] Changes to chemokine distribution may be a mechanism for disorganization of T and B cell compartments with age, but more evidence is needed to establish this link.

Not only do FRCs express IL-7, CCL19, and CCL21 to promote immune homeostasis, but they also regulate the magnitude of lymphocyte expansion during immune activation. Mouse studies have revealed that FRCs have receptors for pro-inflammatory ligands including interferon-gamma (IFNγ) and tumor necrosis factor-alpha (TNFα), which when sensed will trigger the production of nitric oxide (NO) and dampen T cell proliferation.[64] NO-mediated control of T cell proliferation depended on strong TCR signaling, but FRCs were able to regulate both weakly and strongly stimulated T cells in vitro by expressing prostaglandin E2 (PGE2), the lipid mediator of fever, pain, and swelling during inflammation.[65] PGE2 is generated by cyclooxygenase (COX) enzymes from arachidonic acid, and constitutive activation of COX-2 enables FRCs to generate PGE2 that signal the prostaglandin EP4 receptor on T cells.[65] T cell regulation by murine FRCs has been confirmed with in vitro culture of human T cells with human tonsil tissues, with T cell activation being restored when indoleamine 2,3-dioxygenase (IDO), adenosine, PGE2, and transforming growth factor-beta receptor (TGFβR)-signaling pathways were blocked.[66] Lymphocyte expansion was notably reduced upon infection in aged murine lymph nodes,[61] but the roles of NO or PGE2 in suppressing T cell expansion with age have not been explored. Interestingly, upregulation of COX-2 expression has been investigated as a driver of aging[67] and in chronic diseases, including cancer.[68]

As introduced above, high-dimensional analysis of lymph node stroma from adult mice has revealed that fibroblast subsets carry dynamic transcriptional signatures. For example, Ccl19^hi TRCs facilitate immune homeostasis, but can adopt an activated, Cxcl9⁺ TRC signature upon acute inflammation.[29,31] There are also indications that FRCs from different SLOs will be specialized to their anatomical niches, as careful quantification has shown that mesenteric lymph nodes have less FRCs than skin-draining sites.[60] The development of FRCs appears to be site-specific, as the unique trajectories of FRCs in the mesenteries[69] and Peyer’s Patches[70] have been recently explored. Our initial studies have indicated that aging also impacted FRCs in a site-specific manner, as we found that the frequency of PDPN^hi FRCs was significantly diminished in aged murine lymph nodes of the mesenteries, though not of the skin-draining sites, whereas DNs and BECs frequencies were increased.[18] We further found that putative FRC progenitors, identified by lower expression levels of the adhesion molecule VCAM-1,[38] had decreased surface expression of LTBR and Ly6A/SCA-1,[18] a fibroblast activation marker.[31] Given the essential role of TRCs in antiviral immunity,[31,42] further work is needed in studying immune activation of TRCs from different tissue sites within aged organisms.

Not only is the crosstalk between FRCs and T cells important, but the interplay among FRCs, T cells, and dendritic cells (DCs) is critical for not only T cell activity, but in the maintenance of the SLOs. Like naïve T cells, activated DCs express CCR7, which enables them to home to the SLOs to traffic upon CCL19- and CCL21-expressing stroma.[25] By migrating along the stromal network within the T cell zone, DCs can present antigens to activate T cells.[51] Evidence supports a model in which stroma are not merely a passive scaffold for DCs searching for T cells, but that they actively signal with DCs to promote stromal activation. In vitro culture of lymph node stroma with activated DCs and TNFα stimulated stromal cell proliferation, an effect that was dependent on expression of the surface marker SIRPα by DCs.[71] SIRPα⁺ DCs were similarly shown to be important for FRC maintenance in the splenic white pulp.[72] In addition to their responses to stromal CCL19 and CCL21, DC expression of the receptor CLEC-2 has been identified as an axis by which FRCs influence the motility of DCs.[73] Studies have shown that Clec1b-deficient DCs from the skin have reduced migratory capacity to the lymph nodes, and it was further shown, using 3-D cell cultures, that CLEC-2 engagement of PDPN stimulated actin polymerization, thus enabling DCs to extend protrusions and crawl along the FRC network.[73] The engagement of aged DCs with aged stroma has not been addressed, though age-associated changes to the DC compartment have been reported.[74,75] In naïve aged mice, numbers of DC subsets were constant, though their frequencies within the spleen were decreased.[75] When aged mice were challenged with Lm, CD8α⁺ DCs had impaired expansion and exhibited maturation defects, reflected in poor upregulation of MHC-II, CD86, and CD40.[74] Changes to DCs with age and their ability to engage the stromal cell network may have repercussions on lymph node remodeling during immune activation.

3.2. FRCs of the follicles support B cells

Distinct from TRCs, FRCs in the B cell follicles (BRCs) express CXCL13 and CXCL12,[76,77] serve to support B cell maintenance, antigen sampling, and the generation of germinal centers. Mature B cells expressing CXCR5 are recruited to the follicles in response to the CXCL13 chemokine gradient,[78] and are furthermore maintained by stroma expressing B-cell activating factor (BAFF).[79] Immunofluorescence detection and protein quantification has reported that CXCL13 coverage and abundance in aged mice is reduced in the lymph nodes[45] and spleen,[19,80] though some studies concluded instead that CXCL13 was increased when analyzed by immunofluorescence staining, mRNA transcript expression,[17] and protein quantification.[80] Immunization of aged mice indicated that protein expression of CXCL13 failed to increase in aged murine lymph nodes to the same extent as in young mice, which was correlated to impaired development of humoral responses.[61] B cells compete for BAFF to ensure survival,[11] though aging is associated with an expansion of a B cell phenotype that does not require BAFF signaling for survival.[81] It has not been determined whether stromal expression of BAFF in the SLOs is impacted by aging.

In addition to TRCs and BRCs, some FRCs express intermediate levels of CCL19 or CXCL13, delineating a boundary between T and B cell zones.[29] These interfollicular FRCs serve important roles facilitating interactions between the two compartments.[31] Notably, during immune activation, a subset of activated CD4⁺ T cells will upregulate CXCR5 in order to migrate to the CXCL13-expressing B cell follicle as they differentiate into T follicular helper (Tfh) cells, and interact with activated B cells to initiate the germinal center response.[82] Live-cell 2-photon microscopy has revealed that CD4⁺ T cell:B cell interactions occur in the interfollicular regions within 1 to 3 days post-immunization.[83] With age, the boundary between the B cell follicles and T cell zone becomes increasingly irregular in both the murine lymph nodes[45] and the spleen,[63] with the B cell follicles becoming less defined and more diffuse. Experiments in which Ccl19-expressing FRCs were conditionally-depleted resulted in reduced B cells, loss of follicular boundaries, mixing of T and B cell zones, and ultimately reduced germinal center formation.[84] Further studies are needed to determine whether age-associated loss of follicular compartmentalization is related to changes to stromal organization.

It has been well established that germinal centers, organized tissue structures that develop within the follicles during immune activation,[85,86] are reduced in numbers and performance in both mice[87,88] and humans.[89,90] Germinal centers are divided into two histologically distinct regions, the light the dark zones, which contain both lymphocytes and specific BRC subsets. Supporting the notion that B-cell extrinsic influences diminish germinal center responses, young antigen-specific B cells, transferred into young or aged mice and then immunized with their cognate antigen, had reduced positive selection in aged hosts, as read out by c-Myc expression.[91] On the other hand, antigen-specific B cells, isolated from young and aged mice, had comparably robust germinal center expansion within young hosts.[91] Much effort has focused on age-associated defects to lymphocytes, particularly CD4⁺ Tfh cells, in germinal center initiation and maintenance,[19,87,92] but recent progress has also been made in determining how BRCs impair germinal centers.

The CXCL13⁺ BRC subset of follicular dendritic cells (FDCs) have been implicated in diminished germinal centers with age. FDCs are distinguished by their expression of surface molecules CD35 and CD21,[93] and are distributed sparsely throughout the follicle at steady state. Upon immunization, they become concentrated into foci within the follicles to serve their predominant role in promoting germinal center responses.[94] Their localization to the light zone of the germinal center and ability to retain antigen complexes enable them to effectively drive the selection of germinal center B cells.[85] Study of aged murine lymph nodes had indicated that FDCs covered a smaller follicular space with age, despite the follicles retaining comparable total area.[45,91] Corresponding to their decreased follicular coverage, aged FDCs had decreased capacity for the uptake of fluorescently-labeled immune complexes.[45] Opposite of FDCs in the light zone, a subset of Cxcl12-expressing BRCs in the dark zone have been identified as supporting germinal center cycling by attracting CXCR4⁺ germinal center B cells.[77,95] Without cell surface-immobilized CXCL12, transgenic mice have disorganized germinal centers and impaired antibody affinity maturation.[96] Recently, Silva-Cayetano and colleagues have found that CXCR4 expression was increased CD4⁺ Tfh cells in aged mice.[91] Expression of CXCR4 drove Tfh mislocalization to the dark zone, and restoring light zone-positioning of Tfh promoted FDC expansion and germinal center outputs.[91] It is unclear if CXCL12⁺ BRCs themselves change with age, as immunofluorescence of aged murine germinal centers showed comparable CXCL12 expression patterns.[91]

Interstitial fluids drain to the lymph nodes and accumulate as lymph in a cavity just beneath the lymph node capsule, known as the subcapsular sinus (SCS). In a similar fashion, blood is filtered through the marginal sinus of the spleen. For the SLOs, transit of fluids through these sinuses is necessary to screen for lymph- or blood-borne antigens. The sinus floors are formed from a layer of LECs or BECs, respectively, and the uptake of antigens is carried out by specialized CD169⁺ SCS macrophages via cellular protrusions extended through the endothelial barrier.[97] A type of FRC known as the marginal reticular cell (MRC) forms a reticular cell niche lining the lymph node SCS or splenic marginal sinus.[93] MRCs are distinguished among FRCs by expression of the adhesion molecule MAdCAM-1, increased expression of CXCL13, even at steady state, and lack of CCL21 expression.[93] MRCs can respond to LTβ and TNFα signaling in order to give rise to FDCs,[94] which support the germinal center responses as described above. Despite increased frequencies and numbers of SCS macrophages, MRCs are decreased in the aging murine lymph nodes.[45] The clear boundary of MAdCAM-1⁺ MRCs along the marginal zone of the murine spleen is similarly disrupted with age.[46] Denton and colleagues recently established the contribution of stroma to impaired humoral responses, demonstrating that aged mice had significantly diminished MRC and FDC compartments, which failed to proliferate upon immunization.[24] Administration of Toll-like receptor-4 (TLR4)-stimulating adjuvant was able to restore MRC and germinal center B cell expansion in immunized aged mice; however, antibody responses were not restored,[24] suggesting other factors yet contribute to diminished antibody generation with age.

3.3. Tissue integrity

The lymph nodes must be able to swell up to ten-times their original size within one week after immunization to accommodate the rapid proliferation of lymphocyte effectors.[98,99] SLO architecture is comprised of a strong yet deformable conduit network of FRCs surrounding extracellular matrix (ECM) fiber bundles, creating a reticular network that is highly connected.[100] As the conduits are formed by the bodies of FRCs wrapped around collagen fibers, the FRCs are thus positioned to make direct contact with lymphocytes and can therefore rapidly respond upon lymphocyte activation.[101] In vitro studies demonstrated that FRCs can be induced to lay down ECM to generate the reticular network when stimulated by TNFα and LTβ ligands provided by immune cells.[102] Inflammation-induced expansion of the FRCs network leads to local fissures in the conduits, but the intricate channels provide enough redundancy to maintain connectivity of the network.[103] The FRC network has been modeled using graph theory as a small-world network,[101] which similarly captures the idea that FRCs, modeled as network nodes, are highly interconnected and can thus maintain structural integrity even when perturbed. In this context, where lymph node architecture is treated as a reticular network, it was demonstrated that FRCs in the T cell zone of aged murine lymph nodes responded to immunization by maintaining the same degree of branching and network length as those in young lymph nodes.[104] Rather, the study concluded that it was the organization of the aged B cell follicle stromal network and the interfollicular zones that was disrupted upon immunization, unlike what was observed in the T cell zone.[104] This suggests that the capacity for the FRC network to interact with T cells during lymph node expansion is not diminished with age; what is left is whether the quality of the FRC:T cell interaction is also maintained with age.

Network expansion is thus cued by lymphocyte activation, which provides cytokine signals as well as mechanical stress cues.[98] In peptide-immunized mice, the first wave of proliferation is by LECs, followed closely by FRC expansion.[105] Though stroma in the developing spleen emerge from perivascular precursors,[106] mosaic-labelling of sparse TRCs reveal clusters of progeny cells proliferating in place in response to local activating signals.[107] In aged mice, quantification of lymph node stromal subsets indicated that magnitude of expansion for FRCs and LECs was diminished and delayed.[104] Recently, the lymph node has been analyzed as a viscoelastic system, in which the stress-strain responses of the FRC network was determined. The network was held under tension, even as the density of T cells packed within the spaces between network fibers increased.[107,108] The mechanical forces generated by the increased tension were relieved by engagement of PDPN on FRCs by CLEC-2-expressing DCs, which downregulated the activities of actin-tethering proteins and enabled FRC elongation.[108] In addition to network stretching, increased structural stiffness was sensed via PDPN to induce FRC proliferation.[107,108] As FRCs receive mechanical feedback to proliferate until the network is large enough to accommodate the lymphocytes therein, yet it appears that aged lymph nodes do not expand to the same size as young lymph nodes,[104] it would be interesting to determine whether this mechanical stress feedback loop is perturbed in aging.

The lymph nodes of aged mice have capsule thickening and accumulation of fibrosis within the parenchyma,[15,18] suggesting that the integrity of the stromal network could be changing with age. Our live-cell 2-photon microscopy analysis of murine slice explants revealed that the motility of young, adoptively-transferred naïve T cells was diminished in aged lymph nodes when in proximity to collagen deposition.[18] Lymph node fibrosis can result from chronic disease, such as human or simian immunodeficiency virus infection,[109-111] lymphedema caused by impaired lymphatic flow,[112] or during transplant rejection,[113,114] but it is unclear if the etiology of fibrosis seen in aging lymph nodes is analogous to these pathologies. When exploring the role of Hippo signaling in FRC maturation, Choi and colleagues demonstrated that genetic hyperactivation of Hippo pathway mediators YAP and TAZ drove a fibrotic lymph node architecture.[115] The authors demonstrated that YAP and TAZ were hyperactivated in a FRC-specific, Ltbr-deficient mouse, resulting in a fibrotic lymph node and thus demonstrating a link between Hippo and LTβR signaling.[115] While there are reports that human lymph nodes can become fibrotic with age,[89,116] or among populations in developing countries with endemic diseases,[117] lipid accumulation also appears to be a predominant phenotype observed.[118] A recent study by Bekkhus and colleagues found that, with age, stromal cells of the lymph node medulla co-stained for both fibroblast and adipocyte markers; an associated reduction in the expression of LTβ, necessary for commitment to the fibroblast lineage,[38] suggested a model in which loss of LTβR signaling could drive the differentiation of mesenchymal precursors away from the fibroblast and towards the adipocyte fate.[118] Unlike the fibrotic lymph node observed with hyperactivated YAP and TAZ, Choi and colleagues had also found that an adipogenic lymph node fate was promoted by genetic cessation of YAP and TAZ expression.[115] Thus, changes to lymph node stroma may also play a role in SLO aging, where aberrant YAP and TAZ activity, either hyperactivation or abrogation, potentially increases fibrotic deposition in the SLOs or transforms FRC precursors to adipocytes, respectively.

3.4. Lymphatic transport

The SLOs are hubs in a circuit composed of lymphatic and blood vasculature, which essentially forms a body-wide fluid transport network. The lymphatic vasculature is a unidirectional fluid transport system that carries interstitial fluid—containing cellular products, solutes, and antigenic material—from the organs and body barrier tissues to eventually empty into the blood circulation (reviewed in [119]). The lymphatics serve a critical link between the innate and adaptive immune responses, as antigens taken up at the barrier surfaces by activated CCR7⁺ DCs will be guided by CCL21 expression on LECs of the lymphatic capillaries,[120] where they will drain to the nearest lymph node and begin screening for antigen-specific T cells. Thus, effective lymphatic transport is integral to the generation of adaptive immune responses, as had been demonstrated in transgenic mice lacking lymphatic capillaries.[121]

The permeability and contractility of lymphatic vessels govern the efficiency of lymphatic transport. Fluid transport must be driven by the lymphatic vessels themselves, as there is no pump like the heart in blood circulation. These properties are regulated by both biochemical and mechanical stress cues. Vessels are surrounded by a glycocalyx, a structure composed of carbohydrates and proteins that also plays a significant role in regulating vascular permeability, and smooth muscle that contracts and drives fluid in one direction through one-way valves. In addition to its immune regulatory role, NO promotes vascular permeability and impacts contractile pumping.[122] Studies in aged rats indicated that NO was dysregulated, impairing lymphatic flow.[123] Thus with age, the lymphatics are more permeable and less contractile,[124] which has significant implications on the efficacy of immune responses. Analysis of lymphatic vessels from aged rats using electron microscopy revealed declining structure with reduced ECM, which corresponded to defects in the glycocalyx and reduced integrity of tight junctions.[125] In addition to signs of increased permeability, structural analysis of aged rat lymphatic vessels revealed a reduction in the expression of proteins associated with smooth muscle contraction, as well as inefficient in vivo pumping.[125] Given their structural and signaling roles in the lymphatic vascular apparatus, further analyses into the properties of aging LECs of the SLOs and the lymphatic vasculature are needed in order to determine their impacts on immune cell trafficking and activation.

4. Conclusions

The last decade has seen increased research activity in the field of lymphoid stromal cell immunology, capitalizing on new analytical technologies. Furthermore, the study of immunobiology has become infused with engineering concepts, as we now understand that both biochemical and mechanical forces are inputs for immune control loops. In this review we provided a basic understanding of how the immune functions of the SLOs are dictated by their structure and form. This knowledge is important, as the organization and structural properties of the SLOs may be altered by the biological processes of aging, thus impairing their efficient performance. We have focused on the known subsets of SLO fibroblastic and endothelial stromal cells, but our review is incomplete, as analyses of these subsets with age is yet ongoing. As technology will enable more sophisticated ways to describe the SLOs as interconnected systems, we expect that therapies will also be developed that address tissue-specific changes elicited by aging. Immune function is context-dependent, and so targeting SLO stromal cells to restore the immune microenvironment is a potential strategy for improving immune outcomes for older individuals.

Highlights.

• Reduced immune outputs with age coincide with secondary lymphoid organ degradation.

• Crosstalk of lymphocytes and stroma is necessary for lymphoid tissue maintenance.

• Aged stromal cells have impaired expansion during immune activation and reduced chemokine expression.