Although an ever-increasing number of cell types have been shown to be capable of presenting antigen to immune cells, the classical professional antigen-presenting cell is the conventional dendritic cell (23).

Their involvement in various types of TLO has been demonstrated by a number of studies. In a model of viral lung infection, Halle et al. (2) showed that early infiltration of CD11c⁺ cells into the perivascular and peribronchiolar space (4 days post infection) precipitated recruitment of lymphocytes to the infected tissue, with subsequent development of organized inducible bronchus-associated lymphoid tissue (iBALT) structures. Within these highly developed structures, DCs resided primarily within the T cell area, as in SLOs. When CD11c⁺ cells were selectively depleted at various time points using a diphtheria toxin receptor (DTR) transgenic model, the size, but not frequency, of iBALT was reduced, suggesting an important role for DCs, and possibly alveolar macrophages, in maintaining TLO integrity (2). A concurrent study, investigating induction of iBALT in a model of influenza infection, also demonstrated a key role for CD11c⁺ cells in maintenance of these lymphoid structures. Again, using a DTR-transgenic model, this study showed that selective depletion of CD11c⁺ cells from lungs with mature iBALT led to disintegration of the TLO and gradual dispersal of lymphocytes from the lung (3). Notably, influenza-specific plasma cells were found to be undetectable soon after DT-induced depletion of CD11c⁺ cells, while total B cells and peanut agglutinin (PNA)⁺ GC B cells were also substantially reduced. The level of class-switched immunoglobulin, specifically IgA, was also significantly reduced in bronchoalveolar lavage fluid. These results indicate a prominent role for DCs in the function and maintenance of iBALT following influenza infection, as well as suggesting an important role for the TLO in local production of class-switched antibodies. Somewhat surprisingly, depletion of CD11c⁺ cells also led to a significant reduction in the level of systemic hemagglutinin-specific antibody present, indicating a potential role for TLO GCs in generation of long-lived plasma cells that home to the bone marrow (BM). To investigate the role of antigen presentation by DCs in this tissue, lung DCs were isolated from animals challenged with influenza virus expressing the MHC-II OVA_323–339 epitope, at days 4 and 17 post infection. While these DCs were able to activate OVA-specific CD4⁺ T cells (OT-II) at day 4, this was no longer the case at the later time point. However, they retained antigen-presenting ability, as demonstrated by DC-mediated activation of OT-II cells after addition of pre-processed OVA peptide. The authors suggest that the primary role of the DC population in maintenance of the iBALT is production of LTβ, which in turn induces high levels of CXCL13, an important chemokine in B cell migration and retention. Finally, the study also demonstrated that adoptive transfer of granulocyte-macrophage colony-stimulating factor (GM-CSF)-cultured BM-derived cells (a mix of conventional DCs and monocyte-derived macrophages) intratracheally into the lungs of naïve mice leads to iBALT development (3).

In a model of thyroid TLO development, where high levels of CCL21 were artificially induced in the thyroid, CD3⁺CD4⁺ T cells from an adoptively transferred mixed splenocyte population were found to be the initiating cell type in development of ectopic lymphoid tissue. Subsequent recruitment of host DCs and DC/T cell interactions were found to be important for the formation of peripheral-node addressin-positive (PNAd⁺) HEVs in the developing TLO, in a LTα-/LTβR-dependent manner (16). A subsequent paper by the same group confirmed that depletion of CD11c⁺ DCs led to reduced lymphangiogenesis in the thyroid (24).

LTβR is known to have an important role in the maintenance of HEVs within LNs (25), again indicating that these structures are bona fide lymphoid organs. Interestingly, in a model of insulin-dependent diabetes mellitus induced by adoptive transfer of specific antigen-expressing DCs, only mice that showed early infiltration of leukocytes and formation of islet-associated organized lymphoid structures in the pancreatic parenchyma went on to develop diabetes, suggesting a link between antigen presentation by DCs to T cells, TLO formation, and development of autoimmunity (26). More recently, the presence of mature DCs in tumor TLOs was highly associated with a favorable clinical outcome in patients with lung cancers (27, 28); however, to date, there is no direct demonstration that APCs in TLOs permit efficient local T-cell priming against tumor-associated antigens.

Macrophages are some of the earliest immune cells to encounter antigen at sites of infection or injury. The response to antigenic stimulus is context-dependent but production of inflammatory cytokines is a key function of these cells in the early stages of inflammation. In the case of atherosclerosis, macrophages that infiltrate the early plaque take up oxidized low-density lipoprotein (ox-LDL) particles and are activated, including upregulation of antigen-presentation genes and increased production of inflammatory cytokines (29). Recently, work from Guedj et al. has proposed a role for pro-inflammatory macrophages as a kind of lymphoid tissue inducer (LTi) cell in the development of artery tertiary lymphoid organs (ATLOs) during atherosclerosis. In this study, BM-derived macrophages were incubated with both LPS and IFNγ to yield a “pro-inflammatory” phenotype or with IL-4 to generate “alternatively activated” macrophages. Vascular smooth muscle cells (VSMCs) incubated with LPS/IFNγ-stimulated macrophages, which produced TNFα and LTα, developed a lymphoid tissue organizer (LTo) phenotype, while those incubated with IL-4-stimulated macrophages did not (20). This activity did not require LTβR signaling but was dependent on TNF receptor involvement. In addition, Jupelli et al. (4) have reported that iNOS-expressing macrophage involvement in early stages of bacterial lung infection precedes development of iBALT in the lungs of infected mice in their model. Intratracheal transfer of “pro-inflammatory” macrophages (generated from BM-derived macrophages cultured with IFNγ) into infected lungs leads to increased lung inflammation and iBALT formation. It should be also noted that, although the recruitment of CD11c⁺ DCs is clearly a crucial step in the development of iBALT in the viral infection model from Halle et al. (2), the earliest infiltrate recorded was that of alveolar macrophages, within 5–7 h of infection, with DC accumulation described from 4 days post infection. As TLOs, by their nature, form during inflammatory events, and particularly during sustained inflammation, it is logical to assume that macrophage production of inflammatory cytokines following antigen encounter is a necessary, but probably not sufficient, primary event in TLO formation. As described for ATLO formation, a possible role for macrophages as a type of inducible LTi remains to be demonstrated for other types of ectopic lymphoid tissues.

The main role of B cells in an immune response is production of antibodies. B cell presentation of antigen to T cells is an integral aspect of this function. These interactions allow B cells to receive survival signals and direct them appropriately to generate high affinity antibody specific to the antigen encountered (30). Well-established, highly organized TLOs contain secondary B cell follicles, which form following antigen encounter and activation of B cells (31–33). The GCs of these follicles are structurally and functionally similar to those within SLOs, with FDC development described within a number of ectopic lymphoid tissues. This lends credence to the hypothesis that TLOs provide a venue for local production of antibody proximal to the site of inflammation, with either beneficial (e.g., during infection, cancer, or atherosclerosis) or deleterious (e.g., autoimmunity) effects depending on the context in which the TLO forms.

B cells are involved in FDC development (34) in a LT- and TNF-dependent manner, with B cell aggregates shown to induce FDC through LTα1β2 production in SLOs (35, 36). LTα1β2 expression by naïve B cells is induced by CXCL13 (also known as B-lymphocyte chemoattractant), which is itself induced by LTα1β2 in a positive feedback loop (37), with FDCs likely the major source of CXCL13 in the follicle (36). In mice with B cells lacking LTβ, FDC structures in the spleen were disrupted, though not wholly absent (13). Similarly, LTα is not fully required for formation of iBALT, as lymphocytic aggregates form in influenza-infected Lta^−/− mice and lymphoid chemokines CXCL13 and CCL21 are detectable. However, these structures lack the level of development and organization of the TLO observed in mice expressing LTα (17). A similar role in promoting FDC formation has been suggested for B cells in TLOs that arise in the salivary glands of Sjögrens syndrome patients (38).

Follicular dendritic cells are cells of the immune system found in B cell follicles. FDCs are integral to the function of the follicle, presenting antigen in the form of immune complexes bound to their surface (39, 40). FDCs are believed to provide a uniquely long-lasting “depot” of antigen that can be accessed by B cells well beyond clearance of the initial infection or injury from which the antigen was acquired. They are thought to be important in the affinity maturation of the B-cell receptor (BCR). Only B cells expressing a receptor of high enough affinity will be successful in acquiring sufficient antigen from the FDC to in turn present the antigen to their cognate T cell and receive survival signals (36). A recent study has shown that disruption of the FDC network in a model of arthritis led to reduced GC formation in lymphoid follicles, impaired recruitment of follicular helper T (Tfh) cells into B cell areas, diminished autoantibody production, and attenuation of disease (41).

Although there is some debate over how B cells within a TLO might perceive antigen due to the likelihood of increased availability of local antigen compared to SLOs, a number of studies have identified FDCs within ectopic lymphoid structures (5, 42–45). The source of these cells within TLOs is unclear, but, as discussed above, various studies indicate that B cell production of LTα1β2 is important for differentiation of FDCs within ectopic lymphoid organs (1, 35, 38), even though follicle formation in BALT has been reported also in the absence of differentiated FDCs (46). In addition to providing a platform for antigen presentation, FDCs are also known to produce a variety of cytokines and chemokines involved in B cell migration survival and proliferation, as well as recruitment of Tfh cells into B cell areas, such as CXCL13, BAFF, IL-15, and IL-6 (45, 47). Therefore, a similarly multi-faceted role for these cells in mature TLOs is anticipated.

As reviewed by Kambayashi and Laufer recently, a number of cells not traditionally considered “professional” APC may nonetheless under specific circumstances be induced to express MHC-II on their surface and have been shown to interact with T cells in an antigen-specific manner (23, 48). In Sjögrens syndrome, salivary gland epithelial cells (SGECs) may play an important role in the presentation of self-antigen. Numerous lines of evidence point to this ability, including expression of co-stimulatory molecules, such as CD80, CD86, and CD40 (49, 50), the ability to express adhesion molecules and human leukocyte antigen (HLA)-DR (51), and the ability to activate antigen-specific T cells (48). Ishimaru et al. also suggest expression of IFNγ by SGECs may be involved in increased expression of MHC-II by these cells (48). Self-antigen presentation by thyroid epithelial cells – indicated by MHC-II expression and an ability to induce T cell activation – was described more than 30 years ago, with the authors suggesting that the cells might preferentially present self-antigen (52). Other non-hematopoietic cells have also been implicated in presentation of self-antigen. In 2010, Cohen and colleagues described a role for lymphatic endothelial cells (LECs) in the induction of peripheral tolerance through autoimmune regulator (AIRE)-independent presentation of self-antigen (53). Additionally, extrathymic AIRE-expressing cells (eTACs) have been identified in pancreatic TLO of non-obese diabetic (NOD) mice (54). The ability of eTACs to induce peripheral tolerance in TLOs is yet to be demonstrated, but expression of AIRE in these cells has been linked to non-canonical NF-κB activation, which contributes to peripheral tolerance induction (55). Finally, fibroblastic reticular cells (FRCs) express and present peripheral tissue-restricted antigen to T cells as part of the peripheral tolerance mechanism, and their ability to stimulate T cells is altered depending on the inflammatory state of the tissue (56). These cell types have also been detected in TLOs, again pointing to roles in directing the immune response that unfolds within (5, 45, 57).

While the presence in TLOs of each of the APCs described thus far has been robustly reported in the literature and across a variety of TLOs, in the vast majority of cases there has been limited or no direct investigation of actual antigen presentation in these tissues. What mechanisms exist to allow TLO-associated APCs access to antigen? Who are the main APCs presenting antigen, what are the nature of the antigens, and what are the ultimate immunological consequences of antigen presentation in TLOs?