Interplay Of Immune And Kidney Resident Cells in The Formation Of Tertiary Lymphoid Structures in Lupus Nephritis

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Simin Jamaly et al

ABSTRACT

Kidney involvement confers significant morbidity and mortality in patients with systemic lupus erythematosus (SLE). The pathogenesis of lupus nephritis (LN) involves diverse mechanisms instigated by elements of the autoimmune response which alter the biology of kidney resident cells. Processes in the glomeruli and the interstitium may proceed independently albeit crosstalk between the two is inevitable. Podocytes, mesangial cells, tubular epithelial cells, kidney resident macrophages, and stromal cells with input from cytokines and autoantibodies present in the circulation alter the expression of enzymes, produce cytokines and chemokines which lead to their injury and damage of the kidney. Several of these molecules can be targeted independently to prevent and reverse kidney failure. Tertiary lymphoid structures with true germinal centers are present in the kidneys of patients with lupus nephritis and have been increasingly recognized to associate with poorer renal outcomes. Stromal cells, tubular epithelial cells, high endothelial vessels, and lymphatic venule cells produce chemokines which enable the formation of structures composed of a T-cell-rich zone with mature dendritic cells next to a B-cell follicle with the characteristics of a germinal center surrounded by plasma cells. Following an overview on the interaction of the immune cells with kidney resident cells, we discuss the cellular and molecular events which lead to the formation of tertiary lymphoid structures in the interstitium of the kidneys of mice and patients with lupus nephritis. In parallel, molecules and processes that can be targeted therapeutically are presented.

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Introduction

Systemic lupus erythematosus (SLE) is an autoimmune disease with impressive clinical heterogeneity which presents with manifestations from multiple organs. A multitude of pathogenic pathways has been identified originating from genetic, epigenetic, hormonal, environmental, and immunoregulatory factors and all converge at causing inflammation of tissues and organ damage [1,2]. Every aspect of the innate and adaptive immune response has been reported to be involved in patients with SLE and they contribute to the expression of the disease in distinct subsets of patients. The presence of a plethora of autoantibodies has typified the disease while the production of those directed against nuclear antigens, small nuclear ribonucleoproteins, double-stranded DNA (dsDNA), and nucleosomes represent the hallmark of the disease [1,3]. Autoantibodies from soluble immune complexes (IC) with autoantigens (such as nucleosomes) released in abundance in the circulation of patients with SLE, may deposit at basal membranes at various organs including the kidney, and initiate inflammation. Autoantibodies may bind directly to kidneys antigens and form in situ IC as is typified by cationic anti-dsDNA antibodies which bind to the glomerular basement membrane [4–6]. In parallel, the excessive production of cytokines including type I interferon (IFN), interleukin (IL)-17, and IL-23 further advance immune cell abnormalities or act directly on kidney resident cells to cause damage [7]. Last, but not least, autoreactive T cells infiltrate the kidney where they may form tertiary lymphoid structure (TLS) and cause organ damage.

Autoantibody or IC deposition within the kidney, along with the action of cytokines and the infiltration of immune cells contribute to the development of kidney inflammation in patients with SLE which manifests as lupus nephritis (LN) with significant morbidity and mortality [8,9]. After an update on the interaction of resident and immune kidney cells, we will discuss in detail the formation of TLS in the renal interstitium and its effect on kidney function.

Kidney resident cells

2.1. Podocytes

Podocytes are specialized cells on the visceral side of the Bowman's capsule that surround glomerular capillaries. They are part of the glomerular filtration machinery and are critical for maintaining renal function [10]. They express unique proteins including synaptopodin, nephrin [11], podocin [12], and Wilms' tumor protein [13], all of which are essential in the maintenance of their structure and function [14]. Genetic or acquired defects in the expression of key podocyte molecules lead invariably to their detachment and the development of renal failure [15]. Podocyte injury is notable in people with LN and accounts for the development of proteinuria and glomerular damage [16,17].

Podocytes are known to produce and express components of the complement pathway which along with the deposition and activation of complement from the circulation contributes to podocyte injury. Inhibition of the complement pathway has been entertained in clinical trials to treat people with LN [18]. Further, podocytes express all Toll-like receptors (TLR) and Nod-like receptor protein-3 (NLRP3) and caspase 1 [19]. Homocysteine activates NLRP3 inflammasomes in the podocytes of lupus-prone mice and patients with LN [20] and its suppression decreases proteinuria, histologic renal lesions, and podocyte foot process effacement [16] suggesting that NLRP3 can be targeted therapeutically.

Podocytes from lupus-prone mice and people with LN express increased levels of major histocompatibility molecules along with the costimulatory molecules CD80 and CD86 which are considered markers of cell injury but simultaneously they may activate passerby lymphocytes and contribute to their accumulation in the renal parenchyma. Reversely, breaches in Bowman's capsule, in human crescentic glomerulonephritis may allow CD8+ T cells to reach the glomerular tuft and podocytes and cause their destruction [21].

Podocytes express the neonatal Fc receptor (FcRn)which enables the transfer of IgG from the capillary to the urine space. IgG from patients with LN enters podocytes using FcRn and causes upregulation of calcium/calmodulin-dependent protein kinase IV (CaMK4) which phosphorylates 14–3-3 β, the scaffold protein of synaptopodin, which upon its release is degraded. Synaptopodin is important in the maintenance of podocyte structure [22]. In parallel, CaMK4 activates NFkB which suppresses the expression of nephrin, an important protein of the split diaphragm, by promoting the function of the transcriptional repressor SNAIL [23]. IgG from patients with active LN causes the upregulation of CaMK4 under being under-galactosylated [23]. Global deletion of Camk4 in MRLlpr lupus-prone mice effectively suppresses LN [24]. More importantly, podocyte-targeted delivery of a CaMK4 inhibitor suppresses all elements of LN and obviates the deposition of IC [22], suggesting that maintenance of the structure and function of podocytes by suppressing the activity of CaMK4 IC are not deposited. This line of information bespeaks the importance of local factors in the development of organ damage and the value of cell/organ-specific delivery of drugs to limit organ damage in autoimmunity.

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2.2. Mesangial cells

Mesangial cells and mesangial matrix make the renal corpuscle's vascular pole and are important in removing aggregated proteins and small IC from the basement membrane [19]. They are involved in the pathogenesis of LN as mesangial cell proliferation and mesangial matrix are present invariably in the LN kidneys [25]. Mesangial cells express Toll-like receptors (TLRs) [25,26], and when stimulated with a TLR3 ligand (dsRNA) they produce type I IFN [25] – a cytokine that is claimed to be important in the pathogenesis of SLE [25,27].

Antibodies to dsDNA bind to mesangial cells and activate inflammatory and fibrotic pathways, particularly those which involve the mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways, leading to the production of proinflammatory cytokines [27,28]. Mesangial cells secrete interleukin (IL)-6, which on its own can drive the development of glomerulonephritis [29]. CaMK4 is requisite for the proliferation of mesangial cells and the production of cytokines. Specifically, mesangial cells from the lupus-prone MRLlpr mice which lack genetically CaMK4, do not proliferate in response to platelet-derived growth factors and do not produce IL-6 [30].

2.3. Renal tubular epithelial cells

Renal tubular epithelial cells are involved in the pathophysiology of LN. They secrete pathogenic cytokines, including type I IFN [31] and B cell-activating factor (BAFF) [32], both of which have significant roles in the development of SLE (Fig. 1). Furthermore, renal tubular epithelial cells from LN patients express the costimulatory molecule B7-H4, suggesting that they can activate T cells. The addition of anti-dsDNA antibodies to renal tubular epithelial cells in culture leads to the sequential upregulation of tumor necrosis factor (TNF) α, IL-1β, and IL-6 [33], which suggests the cells contribute to the inflammatory processes in the tubulointerstitium in LN [34]. Kidney tubular epithelial cells express apoptotic endonucleases [35] which apparently when activated through yet unknown mechanisms, can cause cell death [36]. More recently, it was shown that tubular epithelial cells can produce CXCL12 in response to IL-23 to promote interstitial and its genetic deletion only in these cells limited glomerulonephritis in lupus-prone mice [7].

BAFF, is a well-established B cell growth and differentiation factor which helps auto-reactive B cells to survive and escape peripheral tolerance [37,38]. BAFF blockade with an antibody (Benlysta) has been approved to treat with SLE [39] and LN [40]. BAFF is also expressed by tubular epithelial cells of people with proliferative LN and the levels of expression correlate with the histopathology-defined activity index [32]. BAFF may promote further differentiation of B cells which are present in the interstitial space of kidneys from patients with LN [41]. Further, BAFF has been claimed to promote the formation of TLS in the kidney by increasing the number of T cells positioned inside the glomeruli and increasing inflammation in mice [42] which may explain the therapeutic effect of Benlysta in patients with LN [40]. Benlysta targets B cells maturation and signaling by inhibiting B cells survival, and reducing differentiation to Ig-producing plasma cells in patients with LN [43].

2.4. Mesenchymal stem cell

Mesenchymal stem cells (MSCs) are multipotent progenitor immunomodulatory cells present in all tissues [44]. They seem to have a role in dendritic and T-cell suppression [45]. Previous studies have shown that when concentrations of proinflammatory cytokines are low, MSCs may have immunostimulatory potential [45,46]. MSCs are detectable within the pelvis wall and TLS in the kidneys of lupus-prone mice [47]. Stimulation of MSCs with inflammatory cytokines leads to the expression of TNF-α, IL-1β, CCL19, and ICAM [47]. Although unclear, MSCs seem to have a role similar to that of lymphoid tissue organizer (LTo) cells and that resident tissue-specific MSCs function like lymphoid tissue inducer (LTi) cells. They can reprogram and start an early inflammatory cascade by interacting with T cells [47]. MSC differentiation and immune cell accumulation cause an expansion of lymphatic vessels and therefore the formation of TLS [48].

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2.5. Macrophages

Resident macrophages in the kidney normally are seen in the interstitium surrounding the glomeruli [49]. Peripheral monocytes after entry to kidney tissue and differentiation to macrophages act as main players in inflammation, injury, and fibrosis in acute and chronic kidney disorders [50]. CD16+ or CD14+ macrophages are recruited to injured kidneys in the presence of cytokines and chemokines [50]. Several subtypes of macrophages (M1, M2a-c) [51] have been recorded present in LN tissues with unknown origin and function [49,50,52]. In general, it seems that if resident macrophages are exposed to endosomal TLR ligands and damage-associated molecular pattern molecules (DAMPs) [53], they transit from a resolution phase to an inflammatory phase. During the inflammation phase, macrophages switch their phenotype to M1 and express Ly6C/Gr1 and secret pro-inflammatory cytokines [54,55]. In contrast, during the repair or resolution phase, they polarize into the M2 phenotype [56,57]. Therefore, it seems that macrophages have dual functionality and display high plasticity during the course of kidney disease.

3. Tertiary lymphoid structures

The term 'tertiary lymphoid' was introduced by Picker and Butcher [58] to explain extra-lymphoid sites in non-lymphoid tissues. TLS has been referred to in many ways, including tertiary lymphoid organs, tertiary lymphoid tissues, and ectopic lymphoid structures. The accumulation of lymphocytes in peripheral non-lymphoid tissues, and the degree to which they become organized, varies according to the type and duration of antigenic inflammatory stimuli [59]. As a result, lymphoid aggregates range from loose collections, comprising a few T or B cells, to organized tissues displaying the hallmarks of TLS [60–63].

TLS is composed of a T-cell-rich zone with mature DCs next to a Bcell follicle with the characteristics of a germinal center surrounded by plasma cells. The minimal attributes needed to form functional TLS are not known, but TLS is defined as a lymphoid aggregate with organized stromal components consisting of follicular dendritic cells (FDCs) and fibroblastic reticular cells (FRCs), and characteristically, with highendothelial venules (HEVs) and lymphatic vessels (LVs) [64,65]. A definition based on these criteria will exclude aggregates of B or T cells in response to inflammation lacking differentiated stromal compartments (Box 1).

TLS develop in various kidneys pathologies, including IgA nephropathy [66], early-stage IgG4-related tubulointerstitial nephritis [67], acute kidney injury [68,69], cancer [70], pyelonephritis [71], transplantation and LN [41,72,73]. In lupus-prone mice, TLS are found close to the pelvic wall, next to large arteries and veins [74]. In autoimmune diseases such as rheumatoid arthritis, Sjogren's syndrome, multiple sclerosis, diabetes, Hashimoto's thyroiditis, primary sclerosing cholangitis and primary biliary cirrhosis, and myasthenia gravis, TLS may enable the in situ generations of autoreactive T and B cells and the production of autoantibodies that perpetuate the pathogenic process [63,70,75,76].

3.1. Crosstalk of kidney's immune cells with tertiary lymphoid structures

T cells maintain immune homeostasis under physiologic conditions and promote tolerance against self-antigens. In autoimmune kidney disorders malfunction of T-cell tolerance to autoantigens can lead to the generation of autoantibodies, inflammation, immune cell infiltration, and development of different types of nephritis [77,78].

T cells may infiltrate the kidney tissue either because they have been activated in the periphery and express adhesion molecules or they may be naïve and become activated after they enter the kidney parenchyma by podocytes or tubular epithelial cells as discussed above. Activated cells express adhesion molecules such as CD44 which, when associated with phosphorylated ESRIN/redesign/moesin [79], binds to its ligand hyaluronic acid, the synthesis of which is increased in the kidneys of lupus-prone mice [80]. Since esrin/redesign/moesin is phosphorylated by Rho-kinase, inhibition of its activity limits entry of T cells into the kidney [81]. Similarly, inhibition of hyaluronic acid synthesis decreases the entry of T cells into the kidneys of lupus-prone mice [80] Interestingly, the numbers of CD3+CD44+ cells in the peripheral blood of people with SLE correlate with kidney disease activity [82].

The majority of the cells in TLS are CD3+ T cells [74] and they include cytotoxic granule-expressing CD8+ T cells and CD4+ T cells that display a TH1 cell phenotype and CD4+ Treg cells [83–86]. It is assumed that mature dendritic cells (DCs) present antigen to CD4+ T cells in the T-cell zone of TLS [87], but DC-LAMP+ DCs have also been detected in the germinal centers, suggesting they have a role in antigen presentation to B cells [88]. B cells organize into germinal centers with plasma cells. B-cell areas contain CD21+ FDCs whereas T cell areas contain MIDC-8+ DCs [74].

Double-negative (DN) T cells are defined by the presence of T-cell receptor (TCR) αβ+ and the absence of CD4 and CD8 molecules. They are expanded in the peripheral blood of patients with SLE, provide help to B cells to produce autoantibody [89], and produce IL-17 [90]. It seems that they derive from CD8+ T cells [91,92] in response to stimulation with autoantigen and the presence of IL-23 [93]. Mechanistically, the CD8 locus is shut off through epigenetic modifications imposed by the repressor cAMP response-element modulator α (CREMα) [94]. More interestingly, DN T cells are present in the kidneys of patients with LN and they produce IL-17 [90] pointing to their direct contribution to kidney inflammation.

The TH17 subset of αβ T cell is defined by the expression of lineage determining transcription factor RORγt. They promote an autoimmune response in humans and mice by producing granulocyte-macrophage colony-stimulating factor (GM-CSF), IFNγ, and IL-17, − 21 and − 22 [95,96]. TH17 cells express C–C motif chemokine receptor type 6 (CCR6) and are recruited to the kidney by C–C motif chemokine 20 (CCL20), which is produced by mesangial cells after stimulation by IL-17 (also produced by neutrophils or γδT cells) [97] and by TEC exposed to IL-23 [7]. TH17 cells present in the kidney secret IL-17 and promote inflammation by generating TLS [98], promoting B-cell activation and loss of tolerance [99,100].

Follicular T-helper (TFH) cells are CD4+ T cells that express the transcription factor BCL6 and C-X-C motif chemokine receptor type 5 (CXCR5). These cells migrate into germinal centers in response to C-X-C motif chemokine 13 (CXCL13). They also express three surfaces receptors, including the inducible T-cell costimulatory, CD40L, PD-1, and produce IL-21 to advance B-cell activation and differentiation of B cells into memory B cells and plasmablasts [101–103].

Levels of circulating TFH cells are increased in people with autoimmune diseases including SLE and studies in lupus-prone mice have confirmed their pathogenic role [102]. In SLE patients, the frequency of a subset of these cells – extrafollicular TH cells – correlates with levels of anti-dsDNA antibody and the quantity of plasmablast B cells. Extrafollicular TH cells define a CCR6+ subset, which expresses CXCR5 but not BCL6 and can secrete IL-17 and facilitate immunoglobulin production by B cells [103,104].

Treg cells are TCRαβ+Foxp3+CD4+ T cells that develop in the thymus or the periphery. They display suppressive activity and control most immune responses through various mechanisms [105]. One of their best-known roles in the kidney is the secretion of the anti-inflammatory cytokine IL-10 [106]. Some of the molecular events that lead to the dysfunction of Treg cells have been elucidated. The molecules involved include protein phosphatase 2A (PP2A), mammalian target of rapamycin complex 1 (mTORC1), phosphatidylinositol 3,4,5-trisphosphate 3- phosphatase, dual-specificity protein phosphatase (PTEN), and calcium/calmodulin-dependent protein kinase IV (CaMK4). Targeting these may rescue or suppress Treg cell function and modulate kidney inflammation [107].

Analysis of T-cell clonotypes from various tissues of LN mice, as well as from the peripheral blood or kidneys of patients with SLE, has revealed expansion of a restricted subset of TCR repertoire, indicating a response to the defined number of autoantigens [93]. This repertoire remains stable for months or years [108,109]. In SLE patients, the clones in the peripheral blood are different from those in the kidney [110], suggesting that naive T cells are activated independently in the periphery and the kidney.

Local production of antibodies is critical in TLS formation. Interestingly, the kidney immune system-related TLS gene profile in lupus-prone mice is similar to that in lymph nodes during active stages of LN [74]. TLS provides the T- and B-cell survival factors IL-7 and BAFF which recruit lymphocytes and favor the interaction between T and B cells in a confined environment [111]. Local B-cell activation has been demonstrated in TLS by the expression of Activation Induced Cytidine Deaminase (AICDA), an enzyme responsible for class switch recombination and somatic hypermutation [112] and active proliferation. Local differentiation of autoreactive plasma cells has also been observed [113].

In a model of pristane-induced murine SLE, B cells proliferate and class-switch within the TLS, and Sm/RNA antibody-producing plasma cells and plasmablasts produce locally autoantibodies [114,115]. Moreover, kidneys from LN patients contain germinal center-like structures containing FDCs. These centers may have a role in active local tissue-specific immune responses [41]. Identifying the events that initiate TLS formation should advance our understanding of kidney damage in patients with LN. BAFF and serum autoantibody levels correlate with TLS formation in the kidneys [42]. Reducing the levels of BAFF reduced T-cell numbers in the glomeruli and prevented LN and the formation or maintenance of TLS [42].

Furthermore, the presence of FDCs, or the binding of IgG-ICs to FcγRIIB, may provide a source of intact antigen [41,116,117] for expansion and activation of B cells, and the production of lymphotoxinα1β2, which may further promote the development of TLS [118,119]. Alternatively, CD11b+ myeloid cells secrete a high level of BAFF and enhance the chemotactic ability of B cells by modulating chemokine-induced signaling [120], thus leading to cell aggregation and compartmentalization of TLS. Since increased levels of BAFF amplify local T-cell activation [121], they may promote the activity of TFH and prolong in situ germinal center responses of kidney TLS. Further, in patients with LN, only cell-to-cell interactions between TFH cells and B cells induce high levels of Bcl-6 and IL-21 in the interstitium [122]. This may represent the effect of BAFF which promotes the expression of ICOSL on activated B cells [123] and induces the formation of TFH cells [124,125]. Thus, renal TLS may form in LN due to hematopoietic cell infiltration into the kidney, but high BAFF levels are required to form or maintain correctly compartmentalized TLS. This suggests that the high level or prolonged production of BAFF may be a key event in the formation of compartmentalized TLS.

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3.2. Molecular cues for the formation of tertiary lymphoid structures

CXCL13 which is produced by fibroblastic stromal cells is a crucial chemokine for B and lymphoid-tissue inducer (LTi) cells. Mice lacking CXCL13 do not form lymph nodes except for the facial, cervical, and mesenteric ones [126]. Induction of TLS formation [127] in mice can be accomplished by overexpressing CXCL13 driven by the rat insulin promoter (RIP), which is active in the pancreas and the kidney. This leads to the formation of TLS that is characterized by segregated B- and T-cell zones, the presence of conventional DCs, and a dense network of stromal cells and high endothelial venules (HEV)-type blood vessels [128]. CXCL12 (or stromal cell-derived factor 1 (SDF1)) is expressed by stromal cells of the bone marrow and is critical in bone-marrow hematopoiesis and B-cell development [129]. CXCL12 is expressed by HEVs in secondary lymphoid organs (SLO) and acts as an essential B-cell recruitment chemokine, while the T cells are mostly unresponsive [130] (Table 1).

Tubular epithelial cells from lupus-prone mice can express IL-23 receptors and produce the chemokine CCL20 which can attract lymphocytes to the kidney interstitium (Fig. 1) [7]. Further, IL-23 acting on tubular epithelial cells can suppress the production of arginase 1 which catabolizes arginine and therefore leads to increased concentrations of arginine which are necessary for the local growth of lymphocytes [7]. Through the first mechanism, tubular epithelial cells are capable to produce proinflammatory chemokines to attract lymphocytes which can be activated locally. Through the second mechanism, tubular epithelial cells can display immunosuppressive capacities which can be obviated in the presence of IL-23 and possibly other stimulants.

CCL19 and CCL21 are expressed by HEVs and some stromal cells. They are the ligands for CCR7 present on T cells, DCs, and LTi cells. Plt mice that lack the CCL19 gene and CCL21 expressed by lymphoid tissue in lymphatic vessels revealed a critical role for CCR7 and CCL19/CCL21 in T-cell homing. In the RIP-overexpression model, CCL21 proved to be more effective than CCL19 in inducing TLS formation [131,132]. However, even with CCL21 overexpression, there is no clear formation of B-cell follicles [131]. CCL28 has a role in the recruitment and homing of B and T cells and promotes adaptive immune responses [133–135]. Signals from the interaction between CCL28 and CCR3/CCR10 drive these processes and attract various immune cells from the local neighborhood [135,136]. Recently, the recruitment of Treg cells by CCL28 has been observed, demonstrating that it has a role in the modulation of the immune system, maintaining tolerance to self-antigens, and preventing the development of autoimmune diseases [137,138] (Table 1).

table 1

Members of the TNF superfamily (TNFSF), namely TNFα, lymphotoxin (LT) α, and β, and their signaling receptors TNFRI/II and LTβR, were suggested to promote the formation of TLS. Also, ectopic expression of TNFα or LTα, but not LTβ, under the control of RIP led to the formation of TLS [139,140]. The most substantial effect was seen when LTα and LTβ were co-expressed, resulting in invasive leukocyte accumulation in the pancreatic islets, and significantly larger TLS than those formed in LTα transgenic mice [139]. TNFR-I is the fundamental regulator of lymphoid tissue organogenesis and germinal-center formation, rather than TNFR-II [141], and it mediates LTα-induced pancreatic TLS [142]. Activation of TNFR-I and LTβR has also been implicated in aortic TLS, in which aberration of LTβR signaling leads to the suppression of CCL21 and CXCL13 expression, with the consequence of reduced HEV formation and disrupted TLS development [143,144] (Fig. 2).

While an effect of LTα, alone or with LTβ, appears clear, the role of TNFα is disputed. In some inflammatory diseases, including those involving TLS, TNFα has anti-inflammatory activity [144]; insulitis in NOD mice and lupus in New Zealand mice improve after injection of TNFα [144,145].

Transgenic overexpression of IL-6 and IL-6R leads to perivascular accumulation of B cells and mature plasma B cells [146]. IL-1β produced by MSCs is overexpressed in lupus-prone mice and may contribute to TLS formation [47]. Stimulation of T cells with IL-4 or IL-7 induced expression of LTαβ; IL-7 was most potent for CD4+ T cells [131]. The IL-17 gene family is vital in defense against pathogens and has been implicated in various chronic inflammatory scenarios. Like members of the TNFRSF, IL-17 receptor signals through NF-κB and IL-17 T cells are induced by IL-6, TGFβ, and IL-23, but inhibited by IL-27. IL-17 is, therefore, an essential mediator for lipopolysaccharide-induced about [147]. IL-7R is expressed by LTi cells and along with CXCR5) IL-7 promotes their formation in SLOs [126].

BAFF may promote tissue injury by affecting the quality and quantity of T-cell-driven cytokines such as IL-17, IL-4, and IFNγ. Increased levels of BAFF in the kidneys may prompt glomerular damage by invading T cells inside the glomeruli, or by inducing the formation of TH17 cells. It is unclear whether the position of the T cells is a parallel or codependent process that promotes glomerulonephritis and tubulointerstitial nephritis. It has been shown that blocking T-cell co-stimulation [148] or neutralizing IFNγ and IL-4 [149,150], leads to an improvement or delay in renal pathology. Comparably, T-cell infiltration and aggregation have been found in kidney biopsies from SLE patients [151]. Immune-cell infiltration into tubulointerstitial areas in SLE is associated with LN [41], suggesting that the position of T cells within the kidney is vital in the disease.

3.3. Vessels in tertiary lymphoid structures

TLS is similar to lymph nodes in terms of structure, vasculature, cellular composition, and chemokine profile. Immune cells include Tand B-cell zones and antigen-presenting cells, including FDCs and mature DCs. The vessels in TLS mainly divide into lymphatic and blood vessels (Fig. 3).

Renal lymphatic vessels (LVs) are considered part of the interstitium because they do not have a basement membrane, and they are blind-ended and lack pericytes [152]. Lymphatic capillaries express PROX-1, LYVE-1, CCL21, podoplanin, VEGFR-2, and VEGFR-3 [153]. The lymphatic vessels of TLS express lymphatic markers such as LYVE-1, PROX-1, podoplanin (in both mice and humans), and D2–40 (in humans) [154], as reported by various studies of chronic kidney rejection [155,156], cardiac allografts [157], transgenic mouse models [158] and a mouse model of age-related primary Sjogren's-like ¨ disease [159]. Nevertheless, there is still much to be elucidated.

It is not known whether TLS vessels carry out the same functions as those in lymph nodes. It seems that they contribute to fluid drainage, but this has not been fully explored. It is also not known whether LVs carry antigen and cells within the TLS and cells away from TLS, like afferent and efferent vessels in lymph nodes. That TLS LVs frequently contain cells [159,160], suggests they have a role as transporters through the expression of CCL21, which interacts with CCR7-expressing cells. However, LVs in some TLSs accumulate cells, suggesting that they do not facilitate cellular drainage and have impaired efferent function.

Lymph node resident cells express sphingosine-1 phosphate (S1P) and its interaction with the S1P1 receptor on lymphocytes is important for their egression from the lymph nodes. FTY720 (fingolimod) is an S1P1 agonist that causes its internalization and accumulation of lymphocytes in lymph nodes [161], thus functioning as an immunosuppressant. When NOD mice with pancreatic TLS are treated with FTY720, they do not go on to develop islet destruction and diabetes [162]. FTY720 inhibits disease progression only at the time that the mice exhibit TLS [163]. Their pancreatic TLS were associated with high insulitis scores after FTY720 treatment, indicating that cells are trapped within them. Islet destruction and diabetes occurred within days of stopping FTY720 treatment [162,164]. Thus, it seems that the S1P gradient affects lymphocyte trafficking in TLS LVs. Fingolimod may force TLS to dissolve in patients and mice with lupus.

LVs transport soluble or cell-associated antigens into lymph nodes. Plasmalemma vesicle-associated protein (PLVAP) is expressed by blood vessels lymphatic endothelial cells in the lymphatic sinus in lymph nodes. PLVAP-positive lymphatic endothelial cells contribute to the sieving of lymphocytes and high-molecular-weight antigens entering the lymph nodes [165]. Since TLS contain a conduit system [166], it is reasonable to question whether LVs in TLS and lymph nodes function similarly. Antigen transport may be less critical than in SLOs because the antigen is an actual component of TLS. However, since antigen-presenting cells are usually present in TLS, this is debatable.

As noted above, LVs in lymph nodes present self-antigens [167–169] either directly through the expression of major histocompatibility complex (MHC) molecules or antigen on ‘classical’ antigen-presenting cells. Presentation of self-antigen by LVs [167] may facilitate induction of either tolerance or T-cell activation in lymph nodes or TLS. Studies investigating the ability of TLS LVs to present antigen and induce either of these outcomes have not been conducted.

HEVs are specialized peripheral-node addressin (PNAd)-positive blood vessels with a distinct structure. HEVs appear to have a role in the transport of blood-borne lymphocytes into TLS. This phenomenon is a kind of specialized infiltration that mainly memory T cells with a low expression of L-selectin (possibly due to the expression of PNAd) can enter the kidney [139]. An experimental study in mice deficient in either LTα or LTβLN found that the development of PNAd-expressing HEVs is stunted, leading to a reduction in the size and cellularity of lymphoid infiltrates [139]. Thus, LTβR signaling may be required for organized lymphoid aggregation and HEV formation.

4. Conclusions and open questions

Although immune responses generated in SLOs can generate protection against pathogens, auto-immune responses in TLS may be destructive. The germinal centers in TLS have similar characteristics to germinal centers in SLOs and provide a ground for immune cell clonal expansion and somatic hypermutation [41]. Although the presence of immune complexes has been considered important in the formation of TLS, more recent evidence suggests that under the influence of cytokines tubular epithelial cells can produce cytokines able to attract T cells [7].

B cells present in TLS have been shown to have undergone somatic hypermutation [41] and therefore the local production of autoantibodies and the possible formation of in situ ICs is certain. Th17 cells are present in the kidneys of people and mice with lupus indicating the direct contribution of these cells in the inflammatory response and kidney damage [101,170–172]. The fact that the TCR repertoire of kidney infiltrating cells in mice and people with lupus is restricted [93] indicates that kidney-specific antigens, still at large, are being recognized. Th17 cells are vital in TLS formation in the propagation of inflammation in the central nervous system and the neonatal lungs [173–175]. A similar role can be projected for the cells in the establishment and maintenance of inflammation in LN.

The presence of Treg cells in the kidney TLS and their possible function is unknown. It is possible that they are excluded through unknown mechanisms or if present they become bereft of their expected function. It is known that Treg cells in the presence of an inflammatory environment lose their regulatory function [78].

Although it has been claimed that the intensity of the interstitial inflammation represents an ominous sign of renal function it is still unknown how TLS contributes to kidney damage. T cells may destroy kidney resident cells like it has been shown for podocytes [21], through direct cytotoxicity or by compromising the function of kidney cells through the action of cytokines as was shown for IL-23 [7] and BAFF [123].

Completely unchartered is the field of the contribution of TLS to the development of kidney fibrosis which is irreversible and defines the end of the function. Cytokines produced by the infiltrating cells along with the contribution of other factors produced by kidney resident cells may promote collagen production by fibroblasts.

Upcoming technologies including single-cell RNA sequencing [176] and spatial transcriptomics will enable the characterization of the interactions between cells comprising the TLS and kidney resident cells. They may also allow the characterization of subsets among patients with LN, as it is certain that LN is clinically and pathogenetically heterogeneous. The efforts to reverse kidney pathology by delivering drugs to kidney resident cells (podocytes [22], tubular epithelial cells [7]) should allow more effective restoration of the function of kidney cells while side effects resulting from the systemic administration are obviated.