Information flow in the spatiotemporal organization of immune responses

Summary

Immune responses must be rapid, tightly orchestrated, and tailored to the encountered stimulus. Lymphatic vessels facilitate this process by continuously collecting immunological information (i.e., antigens, immune cells, and soluble mediators) about the current state of peripheral tissues, and transporting these via the lymph across the lymphatic system. Lymph nodes (LNs), which are critical meeting points for innate and adaptive immune cells, are strategically located along the lymphatic network to intercept this information. Within LNs, immune cells are spatially organized, allowing them to efficiently respond to information delivered by the lymph, and to either promote immune homeostasis or mount protective immune responses. These responses involve the activation and functional cooperation of multiple distinct cell types, and are tailored to the specific inflammatory conditions. The natural patterns of lymph flow can also generate spatial gradients of antigens and agonists within draining LNs, which can in turn further regulate innate cell function and localization, as well as the downstream generation of adaptive immunity. In this review, we explore how information transmitted by the lymph shapes the spatiotemporal organization of innate and adaptive immune responses in LNs, with particular focus on steady state and Type-I vs. Type-II inflammation.

Role of lymph and lymph nodes in the immune response

The primary function of the vertebrate cardiovascular system is the regulation of body homeostasis. Blood vasculature supplies oxygen and nutrients to cells and tissues as well as removes waste products for elimination, while the lymphatic system maintains tissue balance by returning interstitial fluids via lymph back into the circulation¹. In addition to these essential functions, the cardiovascular system is also integrally connected to the organization and function of the immune system^2,3. Most adaptive lymphocytes and many innate cell types continuously recirculate via the blood, and this promotes rapid trafficking to lymphoid and non-lymphoid organs for surveillance and host defense. The lymphatics, on the other hand, carry information pertaining to the state of peripheral tissues, such as antigens, immune cells, inflammatory mediators, and other soluble molecules^2–7. The lymphatic vasculature originates within the tissues as a network of blind-ended, thin-walled lymphatic capillaries, which are highly permeable and specialized in fluid uptake^1,3,8. The lymphatic capillaries within the tissues coalesce into larger collecting lymphatic vessels that facilitate lymph transport, and these vessels eventually converge onto the thoracic duct, which empties the lymph into the subclavian veins to return this fluid back into the blood circulation¹.

Located along the path of lymph return are specialized lymphoid organs, called lymph nodes (LNs), which coordinate the information derived from adjacent peripheral tissues via the lymphatics, as well as promote surveillance of this information by the recirculating lymphocytes. Indeed, the formation of LNs themselves is intimately tied to both the blood and lymphatic vascular networks, as blood vessels supply the first lymphoid tissue inducer cells to drive early LN anlagen, while interstitial flow within the lymphatic vessels provides the spatial and mechanical cues to form the subcapsular sinus and promote LN expansion⁹. Critically, LNs are strategically positioned along the lymphatic network to intercept information transported by the lymph, in order to contain pathogens and limit dissemination, as well as to present this antigenic information to the recirculating T and B cells¹⁰. Lymph-borne proteins are further concentrated within LNs via the reticular conduit network, which transports fluids and small molecules back into the blood circulation, while retaining larger particulate proteins or pathogens for sampling by the LN innate sentinel cells^11–14. These innate cells are localized in a highly organized manner within LNs, and this allows for an extraordinary level of efficiency in sampling of antigens, detection of dangerous insults and pathogens, transmission of signals among responding innate immune cells and cognate lymphocytes, and the generation of robust antigen-specific adaptive responses specifically tailored to the nature of the stimulus^15–17 (Figure 1). Together, this intersection of information gathering via the lymphatic system, adaptive lymphocyte recirculation via blood vessels, and local cellular organization makes LNs ideal hubs for the induction and regulation of immunity, based on the specific organismal needs.

Figure 1. Initiation and organization of immune responses within lymph nodes.

Diagram depicts the preferential localization of different immune populations within steady state LNs. Biased spatial positioning is seen for both innate and adaptive cell subsets. (i) At both steady state and during inflammation, information about the state of peripheral tissues is delivered into the nearest LNs via the lymphatics either through direct antigen drainage or active transport by migratory cDCs. (ii & iii) During Type-I inflammation, macrophages and resident cDCs located directly within or proximally to the lymphatic sinuses readily capture antigens and inflammatory agonists. (ii) Innate and adaptive immune cells positioned near the subcapsular sinus initiate early inflammatory responses to promote pathogen containment and clearance. (iii) Resident cDCs also undergo maturation and relocalize into the T cell zone, while inflammatory monocytes infiltrate the LN from the blood. Together, these two cell types functionally cooperate to drive early T cell activation and effector differentiation. Migratory cDC1s can also support Th1 differentiation. (iv) Antigen-bearing migratory cDC2s preferentially position along the T-B border, where they interact with responding CD4 T cells to promote TfH responses. During Type-II inflammation, these cDCs are also the dominant drivers of Th2 differentiation (also see Figure 2). Yellow arrows indicate direction of lymph flow.

Abbreviations: CD8 TCM, central memory CD8 T cell; HEV, high endothelial venule; LC, Langerhans cell; Mϕ, macrophage; Med, medullary; Mig, migratory; MO, monocyte; Res, resident; SCS, subcapsular sinus

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Lymph node organization and function at steady state

At both steady state and during inflammation, lymph formed in upstream tissues enters LNs via the afferent lymphatic vessels, passes through the subcapsular and cortical lymphatic sinuses, and then exits via the efferent lymphatic vessels located in the LN medulla to eventually return to the blood circulation. Most LN macrophages, including subcapsular sinus macrophages lining the B cell follicles as well as medullary macrophages, are localized directly within the lymphatic sinuses (Figure 1). This enables efficient surveillance of draining lymph, and promotes rapid capture of pathogens that arrive via the lymphatics^18,19. Strategically prepositioned close to the subcapsular sinus macrophages are multiple innate lymphoid cells, including NK cells, gamma-delta T cells, NKT cells, and innate-like CD8 T cells^20–22. These cells are poised to immediately respond to macrophage-derived signals upon inflammation and help initiate early immune responses²¹. Central memory CD8 T cells are also localized nearby, and survey neighboring innate cells for cognate antigens presented on MHC molecules^23–25. Together, these cells serve to patrol the outer periphery of the LN and defend the site against invading pathogens that disseminate from the lymphatics¹⁰.

In addition, our quantitative 2-dimensional (2D) and 3D imaging studies have revealed that LN resident conventional dendritic cells (cDCs), and in particular resident cDC2s, also preferentially localize near or directly within the lymphatic sinuses^26,27. This enables robust sampling of draining antigens and allows these cells to induce T cell responses during inflammation^13,27,28. While some resident cDC1s also reside within these lymphatic sinus regions, most cDC1s are found more evenly distributed throughout the T cell zone and in close association with LN blood vessels^26,29. Although this reduces their efficiency in sampling of lymph-borne antigens^13,27, more centralized positioning may enhance information exchange with migratory cDCs, which localize there, and allow the interception of potentially infected or dying cells during viral infections and cancer^30–32. Close associations of resident cDC1s with blood vessels may also promote homeostatic maintenance of high endothelial venules to support normal lymphocyte recirculation^29,33. These differences in cDC subset positioning have been recapitulated in human LNs, thus revealing the conservation of cDC subset patterning across species and suggesting likely functional importance for maintenance of homeostasis or host defense³⁴.

cDCs residing directly within peripheral tissues also constitutively sample local antigens. In the steady state, tonic signaling within tissues induces a fraction of these cells to undergo homeostatic maturation^35–38, and this induces their migration via the lymphatics into the nearest LNs in a CCR7-dependent manner^6,39–41. Within LNs, different migratory cDC subsets segregate into distinct spatial compartments, and this is observed both at steady state and during inflammation¹⁶. Specifically, work by our group and others on cutaneous LNs (the focus of this review) has demonstrated that migratory cDC1s and Langerhans cells (ontogenically closely related to macrophages) preferentially migrate into the deep T cell zone, while migratory cDC2s generally localize to the interfollicular regions and outer LN paracortex^26,29,42,43. Further segregation of migratory cDC2 subpopulations is seen based on CD301b and SIPRα marker expression. CD301b+ cDC2s are primarily found at the upper paracortex bordering the B cell follicles, while SIRPα- migratory cDC2s (also described as “triple negative” cDC2s) predominantly accumulate in the lower cortical ridge bordering the medulla^29,44–46.

At steady state, lymph supplies a large repertoire of tissue-derived self-antigens, including byproducts from apoptotic cells and catabolized extracellular matrix components^4,7. The dominant role of these steady state cDC populations in LNs is to present captured peripheral self-antigens on MHC molecules to T cells in a non-immunogenic fashion. This is essential for inducing peripheral tolerance to potential self-reactive T cells, which may have escaped central thymic selection and entered the circulation^35,37,38,47. While cDCs are critical for this process, we have found that they can also aberrantly activate self-reactive T cells^48,49. In these instances, local regulatory T cells help maintain immune quiescence by forming discrete clusters around the autoreactive cells and quickly constraining self-reactive responses^48–52.

Stromal cells in the organization of lymph node innate immune cells

Such intricate compartmentalization of immune cells in LNs is thought to be at least in part mediated by the signals provided by the underlying stroma. Recent single-cell RNA sequencing studies have revealed the existence of multiple subsets of LN stromal cells with divergent properties, such as expression of distinct chemokines and adhesion molecules, as well as preferential production of survival factors to support immune cell homeostasis^53–59. In brief, lymphatic endothelial cells (LECs) and marginal reticular cells line the subcapsular sinus and promote the homeostatic maintenance of subcapsular sinus macrophages^60,61, as well as express chemokine scavenging receptors to establish CCL21 chemokine gradients that help guide migratory cDCs from the sinuses into the LN parenchyma⁶². Follicular dendritic cells, located within the B cell follicles, secrete abundant amounts of CXCL13 to recruit and position CXCR5+ naïve B cells, T follicular helper (TfH) cells, as well as CXCR5-expressing migratory cDCs in certain inflammatory settings^58,63–66. In the paracortex, multiple populations of T-zone reticular cells (TRCs) can be found, which secrete the chemokines CCL19 and CCL21 to promote homing of CCR7-expressing T cells and activated cDCs to the deep T cell zone^63,67–70. A unique population of CCL19-low TRCs are found along the T-B border and the interfollicular regions, and these stromal cells preferentially generate the chemotactic ligands for Ebi2 receptor-expressing immune cells⁵⁴.

With regard to innate cell positioning, Ebi2 is preferentially expressed on resident and migratory cDC2 populations and is thought to promote their localization and maintenance within the marginal zone bridging channels of the spleen, as well as within the outer paracortical regions of the T cell zone^71–73. Nevertheless, our quantitative imaging studies have demonstrated that disruption of the Ebi2 chemotactic axis has minimal effects on cDC distribution within cutaneous LNs⁷⁴, suggesting that additional mechanisms can contribute⁷². Our group’s findings on the preferential localization of resident cDC1s, and to a lesser degree cDC2s, near LN blood vessels also suggest existence of additional guidance cues emanating either directly from the blood endothelial cells or the surrounding pericytes²⁹. Of note, the discrete spatial patterning of resident cDC1 and cDC2 populations results in preferential coating of distinct vascular trees, with little intermixing between subsets, and this could be related to the substantial heterogeneity seen in the LN blood endothelial cells^75,76. Alternatively, preferential cDC coating of vasculature may also result from local recruitment of pre-cDC1 vs. pre-cDC2 precursors via distinct blood vessels and their spatially segregated proliferation to repopulate the mature cDC subsets^77–79. In this regard, a recently identified subset of Grem1+ TRCs has been shown to localize within the outer LN paracortex, a region highly enriched in high endothelial venules^57,80. This stromal cell population expresses several genes related to cDC recruitment and survival, and plays a crucial role in resident cDC homeostasis. It must also be noted that reciprocal crosstalk of cDCs with both LN fibroblastic reticular cells and blood endothelial cells via lymphotoxin beta and Clec2a signaling has been shown to be critical for promoting the survival and function of LN stromal cells themselves, during both steady state and inflammation^33,81–85. Thus, while the exact mechanisms remain to be worked out, the extensive stromal cell heterogeneity within LNs is thought to establish the appropriate spatial patterning and survival of various cDC subsets, and in return these cDCs support normal stromal cell homeostasis.

Generation of immune responses - Type-I Inflammation

In contrast to steady state conditions, inflammation of peripheral tissues, and specifically for this review, the murine skin, induces large-scale activation of immune cells both within the affected tissues and the draining LNs to support critical immunological functions. Many microbial infections of barrier surfaces or injected vaccines result in rapid release of pathogens, pathogen or danger associated molecular patterns (PAMPs, DAMPs), foreign antigens, alarmins, and other inflammatory mediators into the tissue parenchyma and subsequently the lymph³. Within minutes, these agonists flood the draining node, and the innate immune cells positioned within the lymphatic sinuses provide the first line of defense to contain the incoming threats as well as to trigger the initial wave of inflammation (Figure 1). Macrophages lining the lymphatic sinuses serve as local “flypaper,” and readily capture antigens, immune complexes, and pathogens as they enter through the afferent lymph. Medullary macrophages are highly phagocytic and rapidly degrade the captured microbes¹⁸. In contrast, subcapsular sinus macrophages are less degradative, and instead deliver the sequestered antigens to adjacent B cells to drive humoral immune responses^86–89, as well as secrete type-I interferon (IFN-I) to alert and protect neighboring cells from further pathogen spread^90–92.

In addition to direct containment, inflammasome activation and locally released cytokines from subcapsular macrophages elicits robust antigen-independent production of Type-II interferon (IFN gamma, IFNγ) by nearby innate lymphoid cells (NK cells, gamma-delta T cells, NK T cells, and innate-like CD8 T cells). This rapid wave of IFNγ in turn enhances the phagocytic activity of macrophages to amplify host defense²¹. Inflammasome activation also stimulates the influx of additional innate effector cells from the blood^21,93, including neutrophils, which provide further support for pathogen containment^21,94–96. Memory CD8 T cells close to the subcapsular sinus similarly secrete IFNγ upon detection of cognate antigen, inducing local CXCL9 production by macrophages and stromal cells. This leads to the repositioning of additional CXCR3-expressing memory T cells towards the site of infection, and these bystander-activated CD8 T cells rapidly mediate pathogen clearance^23,24,97. CXCL9 can also be displayed on the lumen of high endothelial venules, enabling additional recruitment of central and effector memory CD8 T cells from the circulation⁹⁸. Thus, within just a few hours of inflammation, various innate effector and adaptive memory cells that are pre-positioned in or rapidly recruited to the subcapsular sinus niche create a barrier to pathogen entry and prevent dissemination into the blood circulation as well as the deeper parenchymal regions of the LN.

Also enriched near the lymphatic sinuses are LN resident cDCs, and as shown by our group as well as by others, this allows them to efficiently intercept and present draining antigens^{27,28,36,99,100}. The peripheral positioning of resident cDCs near the lymphatic sinuses, however, minimizes their chances of interacting with cognate naïve T cells, which are primarily localized within the T cell zone. In contrast to macrophages, which recruit immune cells locally, we demonstrated that activated resident cDCs instead undergo rapid maturation to express the chemotactic receptor CCR7, and use the CCL19/CCL21 chemotactic gradient within the LN parenchyma to relocalize from the lymphatic-rich peripheral regions into the T cell zone²⁸. As these resident cDCs mature, they also increase surface expression of MHC and costimulatory molecules, thus facilitating enhanced antigen presentation and subsequent scanning by and activation of cognate T cells²⁸. Further efficiency in reducing the search time for cognate cDC–T cell interactions is achieved directly within the T cell zone. As we and others have shown, resident and migratory cDC subsets segregate here, with activated cDC2s preferentially localizing in the outer paracortex, and cDC1s predominantly positioned within the deeper T cell zone, and this is conserved across most, but not all, Type-I inflammatory conditions^16,28,72,73. This segregation of innate cells is also mirrored by the non-random distribution of naïve CD4 and CD8 T cells. CD4 T cells express higher levels of Ebi2 and also preferentially localize within the outer paracortex, thus paralleling the distribution of Ebi2-expressing cDC2s^74,101. Given the enhanced abilities of cDC2s in MHC-II antigen presentation, and the converse specialized ability of cDC1s to cross-present antigens on MHC-I, we found that this spatial coordination between the cDC and T cell subsets further reduces the search space necessary for the T cells to locate their MHC-presenting cognate antigens, and ensures rapid generation of T cell responses in the event of an inflammatory insult⁷⁴.

Our group as well as others have also demonstrated that as the resident cDCs migrate towards the T cell zone to elicit these cognate responses, inflammatory monocytes within the blood circulation also begin to infiltrate the draining LNs in large numbers via the high endothelial venules^28,102–104. Upon entering the LN parenchyma, the recruited monocytes acquire markers of activation and migrate into the T cell zone, eventually colocalizing with the co-clustered resident cDCs and early activated T cells²⁸. In comparison with resident DCs, monocyte maturation and entry into the T cell zone is relatively delayed, and monocytes do not appear to play a significant role in early antigen presentation and T cell activation in these settings. However, monocytes do arrive at the optimal time to provide an ample source of IL-12 to support the differentiation of recently activated T cells, and thus cooperate with the cDCs to enhance T cell effector programming^28,102,104. Therefore, we suggest that the spatiotemporal dynamics of resident cDC and monocyte migration, maturation, and function represent critical features of T cell activation and differentiation in LNs during Type-I inflammation.

Following these very early T cell activation events, additional involvement of the CXCL9/10–CXCR3 chemokine axis has been shown to help reinforce subsequent effector T cell programming¹⁰⁵. Expression of CXCL9 and CXCL10 chemokines by stromal and myeloid cells in the peripheral regions of inflamed LNs, such as the interfollicular zones and along the T-B boundary, elicits the recruitment of early differentiated effector T cells expressing CXCR3^{103,106–110}. Multiple cellular sources likely contribute to the generation of this chemotactic gradient, including monocytes which are significant producers of CXCL9 and CXCL10 following skin immunization^{103,106–109}. Migration of early effector T cells towards these microenvironments exposes these cells to additional sources of antigen and inflammation, and thus further enhances their differentiation^103,107,108. Of note, early activated CD4 T cells expressing Tbet and CXCR3 can also utilize this chemotactic axis to migrate to the T-B border and promote humoral immunity¹¹⁰. In contrast, less-differentiated CD8 T cells are predominantly retained within the deep T cell zone, which further enhances the bifurcation of T cell responses¹⁰⁷.

Additional innate support for the generation of T cell responses during Type-I inflammation has been shown for NK cells and plasmacytoid DCs (pDCs). IFNγ secreted by NK cells within the subcapsular sinus regions can enhance the activation of neighboring myeloid cells. Consistent with this, depletion of NK cells or blockade of IFNγ in certain inflammatory settings can lead to reduced cDC and monocyte responses, including diminished expression of IL-12 and CXCL9^{106,111–113}. Reciprocally, IL-12 or IL-18 cytokines released by the activated cDCs or monocytes may amplify NK cell-mediated IFNγ production to create a positive feed-forward inflammatory cascade^{109,112,114,115}. pDCs have also been shown to facilitate adaptive responses. During viral infection, pDCs can undergo CCR5-mediated intranodal migration within LNs to colocalize with responding CD8 T cells and cDC1s in order to optimize cDC1 maturation and downstream T cell priming via IFN-I signaling¹¹⁶. The CCR5 chemotactic axis has also been previously shown to enhance CD4 T cell help to CD8 T cell responses, by promoting more efficient scanning of cognate antigens by naïve CD8 T cells and for generating productive memory responses^{30,31,117–121}.

In addition, migratory cDCs residing directly within inflamed tissues also integrate pathogen-derived signals and inflammatory cues, undergo full maturation, and traffic to the draining LNs in a CCR7-dependent manner to facilitate the generation of adaptive immunity^36,41,122. The relative contributions of migratory vs. resident cDCs in T cell priming appear to be condition specific. As we and other groups have demonstrated, in settings of robust antigen and inflammatory agonist drainage via the lymph, with the exception of TfH responses, migratory cDCs appear mostly dispensable for early T cell responses^{27,28,99,100,123}. However, during highly tropic or slowly replicating microbial infections, such as HSV infection, foreign antigens and stimuli remain largely trapped in the periphery and do not enter the lymph in sufficient quantities to elicit responses by LN-resident innate cells. In these settings, antigen-bearing migratory cDCs are indispensable for the initiation of T cell immunity^31,124,125. In particular, migratory cDC1s can produce copious amounts of IL-12 and directly support the induction of Th1 and CD8 effector responses^124,126,127. Dermal cDC2s can also undergo functional maturation to support Th1, Th2 (below) or Th17 polarization, based on the specific types of encountered stimuli^122,128,129. In addition to direct induction of T cell responses, in certain infection or tumor models, migratory cDCs can transfer antigens to resident cDCs to further potentiate T cell activation^30–32,130. It has also been shown that lymph-borne antigens can be stored within LN LECs for extended periods of time, and then slowly released to the transmigrating migratory cDCs, leading to continued induction of T cell immunity^131–133.

In sum, Type-I inflammation induces a large-scale, orchestrated activation, recruitment, and crosstalk of multiple innate and adaptive cell types within draining LNs, which together promote rapid pathogen containment, as well as facilitate optimal generation of adaptive immunity.

Generation of immune responses - Type-II Inflammation

In contrast to Type-I responses discussed above, Type-II inflammation of cutaneous tissues does not appear to elicit strong LN resident innate responses. As we and others have shown, this is characterized by the absence of resident cDC activation, lack of IFNγ production by innate cells, and minimal monocyte influx and IL-12 expression^28,106,109. In fact, absence of such stimuli (i.e., IFNγ and IL-12) may be essential for promoting Th2 immunity, as these signals can readily override the program of Th2 differentiation¹³⁴. Consistent with this, migratory cDC1s, which constitutively secrete IL-12 within LNs, can inhibit Th2 responses, while genetic ablation of cDC1s can enhance Th2 immunity and promote control of nematode infection of the intestine¹³⁵, or increase house dust mite induced airway inflammation¹³⁶.

Nevertheless, Type-II inflammation still requires critical innate–adaptive immune cell crosstalk and the generation of specialized microenvironments within LNs to support Th2 responses (Figure 2). In these settings, peripheral tissue-derived migratory cDC2s, and in particular the IRF4/Klf4-dependent and CD301b+ subsets, appear to play a dominant role in Th2 induction^{45,109,137–141}. Exposure to inflammatory stimuli and alarmins following barrier tissue damage from proteases or helminths leads to a specific program of maturation in local dermal cDC2s, and their migration via the lymphatics to the LN subcapsular sinus⁴⁴. Here, additional CCL8 secretion by activated subcapsular sinus macrophages can synergize with CCR7 chemotactic signaling to promote CD301b+ cDC2 entry into the LN parenchyma¹⁴². Once in the LN, these CD301b+ cDC2s appear to predominantly localize at the T-B border^45,142. Given that we and others find similar spatial patterns in steady state LNs^29,45, this indicates that such localization is largely intrinsic to the cDC subsets, rather than elicited by the specific inflammatory conditions. In accordance with migratory cDC2 localization, Th2 cells also appear to accumulate along the T-B border^65,143,144, suggesting that cellular crosstalk between cDC2s and CD4 T cells within this LN microenvironment may be necessary for optimal Th2 generation. Indeed, in addition to Ebi2, preferential expression of CXCR5 by migratory cDC2s has been in part attributed to their enrichment along the T-B boundary, and this positioning was found critical for induction of Th2 responses during nematode intestinal infection^65,145.

Figure 2. Distinct immunological programs involved in the initiation of Type-I vs. Type-II immunity.

Type-II responses (left) are elicited in response to multiple skin-derived stimuli generated due to tissue damage and cell death. Various epithelial-derived alarmins, as well as mast cell-, neuronal-, and ILC-derived signals are integrated by the local dermal cDC2s, which next migrate into LNs to induce Th2 cell differentiation at the T/B border. In contrast, Type-I inflammatory settings (right) are elicited in response to innate sensing of microbe-associated molecular patterns and downstream inflammatory cytokines. In settings of ample antigen and agonist drainage to LNs, T cell responses are primarily induced through cooperation between LN-resident cDCs and blood-derived monocytes, as well as through participation of additional innate cell types (also see Figure 1). During highly tropic infections, peripheral tissue-derived dermal cDC1 and cDC2s more dominantly promote the generation of responses. Location of early activated Th1 and Th2 T cells depends on the positioning of specific antigen-presenting cDC subsets, as well as the chemokine receptors expressed on the responding cells.

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Specific signals provided by migratory cDC2s to induce Th2 differentiation remain enigmatic^122,141. Expression of OX40L and Notch ligands on cDCs have been shown important, but whether these molecules selectively promote Th2 responses or more broadly enhance the generation of multiple CD4 T cell helper subsets remains to be demonstrated^146–149. IL-4 signaling has been long known to drive Gata3 expression and promote Th2 differentiation in vitro¹⁴⁹. While the in vivo role of IL-4 in Th2 programming and localization has been less clear due to the confounding contributions of IL-4-producing TfH cells^150,151, a more recent study has clarified that IL-4 is indeed critical for generating early Th2 responses in vivo¹⁵². Nevertheless, the very early cellular source of IL-4 to initiate the Th2 differentiation cascade in the LN has not been definitively established, and migratory cDC2s do not appear to express this cytokine. Instead, basophil recruitment to reactive LNs or local NKT cell activation within the interfollicular regions have been proposed, albeit the precise roles of these innate cells in Th2 differentiation have come under scrutiny^153–157. It has recently been suggested that cDC-derived IL-10 signals can also elicit Gata3 expression and Th2 responses, indicating that multiple cytokines may mediate this process¹⁵⁸. Given that several cell types can express IL-10 during inflammation, additional studies to explore the precise role of cDC-derived IL-10 in Th2 priming are necessary. Finally, TCR signal strength has also been shown to influence Th2 polarization both in vitro and in vivo, although how this contributes to polyclonal T cell responses in more complex in vivo settings of infection or protease-mediated tissue damage remains to be explored^{141,159–161}. Thus, a combination of multiple stimuli provided by migratory cDC2s, and possibly other cells, as well as the complete absence of Th1 polarizing stimuli, within the peri-follicular microenvironments in LNs mediate the generation of Th2 responses.

It is important to highlight that in addition to Th2 differentiation, migratory cDC2s can also participate in the generation of multiple additional CD4 T helper cell subsets (Th1, Th17 and TfH), based on the specific nature of encountered agonist^109,128. This indicates that migratory cDC2s are relatively plastic and can drive divergent T cell helper subtypes though distinct inducible transcriptional programs imparted on them by the signals encountered within the peripheral tissues, rather by their intrinsic propensities to promote one or another form of immunity. In particular, migratory cDC2s have been closely linked with the generation of TfH responses^{141,162–164}. During infection, antigen-bearing migratory cDC2s accumulate at the T-B border^45,65,163, and express the high affinity IL-2 receptor alpha chain (CD25). This helps augment Tfh differentiation by quenching T cell-derived IL-2 signals, which would otherwise restrict BCL6 expression in responding lymphocytes¹⁶². Similarly, during intestinal nematode infection, localization of cDC2s near B cell follicles via CXCR5 chemotactic signaling is critical for the induction of both Th2 and TfH responses⁶⁵. This suggests that the close spatial juxtaposition of migratory cDC2-driven T cell responses with nearby B cell follicles may promote downstream interactions between activated T and B cells and thus elicit TfH programming⁶⁴. In addition, during certain viral infections, early exposure to IFN-I leads to IL-6 production by migratory cDC2s, and this can help promote Tfh polarization¹⁶⁴. Surprisingly, while most experimental models have demonstrated the combined loss of Th2 and TfH responses after broader blockade of cDC2 migration using conditional IRF4 deficiency models^138,163, selective depletion of CD301b+ cDC2s resulted in enhanced generation of TfH responses. This suggests the potential for reciprocal cross-regulation of T cell differentiation by the different migratory cDC2 subsets¹⁶⁵. Together these data support the model that migratory cDC2s are potent sensors and integrators of information within peripheral tissues and promote the generation of adaptive responses based on the corresponding organismal needs¹⁰⁹.

Finally, additional migratory antigen presenting cells are also involved in generation of other helper cell responses in certain cutaneous infection settings. In particular, during Candida albicans fungal infection, Langerhans cells migrate to draining LNs and secrete polarizing cytokines, such as IL-1β, IL-6, and IL-23 to promote Th17 differentiation^124,129. Given the preferential localization of these cells within the deeper regions of the T cell zone, as well as the unique cytokine profiles after fungal exposure, the LN microenvironments associated with Type-III inflammation are also likely to be different from other inflammatory settings.

Differences between Type-I and Type-II responses

These studies make it clear that divergent programs of T cell immunity (e.g., Type-I vs. -II) are driven by the distinct participation and activation states of specific innate immune cell subsets in the reactive LNs and the site of initial inflammation. It is therefore critical to consider how such innate responses are elicited in the different inflammatory conditions. In this regard, the mechanisms leading to the induction of innate Type-I vs. -II responses are quite distinct (Figure 2). As noted above, Type-I inflammation is initiated by the recognition of microbial and vaccine-derived PAMPs detected by conserved pattern recognition receptors on both innate and stromal populations^{36,122,141,166,167}. This elicits a potent multicellular cascade of inflammatory signaling events, that together drive adaptive immunity. We propose that inflammatory signaling within both barrier sites and LNs likely contribute to functional adaptive responses, and the relative contribution of these is dictated by pathogen tropism, speed of replication, as well as the amount of cellular damage and local inflammation caused by the infection. In the case of vaccination, the formulation of antigen, type/size/charge of adjuvant, as well as the site of immunization will dictate the relative participation of tissue vs. LN -derived innate responses in driving T cell immunity^168,169.

In contrast, Type-II responses are thought to be initiated by the recognition of “patterns of pathogenesis” in the form of allergens, venoms, adjuvants, cell death, tissue stress and destruction, and the release of non-specific alarm signals from barrier tissues, that together act on a variety of tissue-localized immune cell types¹⁷⁰. Protease activity is likely one major upstream trigger of Type-II immunity, and most clinically relevant allergens have protease activity which can cause local activation and release of alarmins from the epithelial cells, as well as cleave epithelial tight junctions in affected barrier surfaces^171–174. Helminth parasites, which drive potent Type-II responses, also employ proteases for migration through host tissues^175,176, thereby causing widespread tissue damage. Other forms of cell death, such as migration-induced cell shattering of mononuclear phagocytes, have also been recently associated with Th2 differentiation¹⁷⁷. Thus, the initiation of adaptive immunity during Type-II settings is likely to be critically dependent on innate cell sensing of local signals induced by tissue damage directly within the affected site.

Several immune cell populations reside in the skin, which together form complex cellular networks that function to maintain barrier integrity as well as promote immune responses. cDCs within these networks physically interact with and integrate signals from multiple distinct cell types before migrating into the nearest LN to instruct T cell responses^122,178,179. With regards to Th2 generation, barrier tissue damage elicits the release of specific inflammatory mediators (i.e., IL-33, IL-18, IL-25, and TSLP), and these signals can act directly on locally pre-positioned cDCs, as well as several other nearby cell types, to induce their activation^149,171,180. In particular, TSLP released by damaged epithelial cells activates cDCs within the skin, and this can drive the upregulation of OX40L and other costimulatory molecules, as well as promote their accumulation within draining LNs to induce Th2 responses in certain settings^{46,147,181,182}. Similarly, IL-33 can also lead to the maturation and upregulation of co-stimulatory molecules in cDCs, suppress IL-12 expression, as well as mediate their migration to LNs^183–186.

In addition, other responding immune cell types also potentiate cDC2 activation in the skin. Mast cells have been shown to directly interact with cDCs within the dermis¹⁸⁷, and can release inflammatory mediators to induce the maturation and migration of neighboring cDCs to the skin-draining LNs^{179,187–189}. Activated ILC2s can further amplify cDC responses through the production of the Type-II cytokine, IL-13^190,191. In many tissues, including the skin, ILC2s predominantly reside within the adventitial cuffs, sites highly enriched with small blood vessels, lymphatics, peripheral nerves, as well as other key components of immunity^192,193. Notably, CD301b+ cDC2s can colocalize with ILC2s within these niches¹⁹², suggesting that crosstalk between these two cell types may preferentially take place within this unique tissue microenvironment. More recently, a role for neuronal signaling has also been demonstrated in Type-II responses. Within the dermis, CD301b+ cDC2s can be found closely associated with the sensory neurons. TRPV1+ sensory neurons in particular can respond to allergens, and upon activation, locally release the neuropeptide Substance P to promote CD301b+ cDC2 migration into draining LNs and subsequent generation of Th2 responses¹⁹⁴. Finally, an additional role for IFN-I signaling in cDC2-mediated Type-II immunity has also been noted^44,195.

Thus, in contrast to anti-microbial Type-I responses which are characterized by broad-scale induction of innate cell activation through conserved microbial pattern recognition receptors and cytokine signaling in both peripheral tissues and draining LNs, Type-II responses appear highly dependent on the activation of multiple cell lineages within peripheral tissues in response to local tissue damage. Combined action of these cells appears to impart a unique differentiation trajectory for the locally distributed dermal cDC2 cells, which in turn migrate to draining LNs to elicit the corresponding Th2 responses. Together, this supports the model that the induction of Type-I vs. -II signatures of adaptive immunity are initiated as evolutionarily conserved mechanisms allowing either protection against microbial pathogenesis vs. wound repair and healing of tissue damage, respectively¹⁷².

Information gradients in the generation of immune responses

Additional considerations to how immune responses are propagated within reactive LNs are the spatial distribution and kinetics of lymph-borne antigens, inflammatory agonists, and the incoming migratory cDCs (Figure 3). Imaging studies by us and others have demonstrated that drainage of antigens and other soluble mediators from the inoculation site to LNs can happen within several minutes^{13,14,99,196–198}. Upon reaching the lymphatic sinuses, proteins and particles are physically separated based on molecular weight, with smaller antigens (<70kDa) and fluids entering the LN conduit network distributed throughout the interfollicular regions and the T cell zone^{197,199–202}. These conduits are also directly connected to the nearby blood vessels, and within a few hours, fluid-borne antigens, cytokines, and chemokines that entered the conduits are rapidly cleared out into the circulation. This can promote deposition of chemokines and inflammatory mediators on the lumen of high endothelial venules and lead to enhanced trafficking of immune cells into the reactive LNs^200,203,204. In contrast, large antigens and pathogens are retained within the lymphatic sinuses, and instead can directly cross the floor of the lymphatic sinuses into the parenchyma either by passive diffusion via local fenestrations or by active transcytosis by the LECs^{13,14,196,205}. We have previously shown that this results in enhanced accumulation of certain antigens within the lymphatic sinuses and the sinus-proximal parenchymal space, and the generation of steep antigen gradients across the LN. Significantly greater amounts of antigen are deposited near the lymphatic sinuses, while reduced quantities are found within the deeper T cell zone¹³. Given the preferential distribution of macrophages and resident cDC2s near the lymphatics, this leads to increased uptake of antigens by these populations^13,14. In contrast, lower abundance of antigens within the deeper T cell zone leads to reduced antigen capture by the centrally localized cDC1s. As a consequence, subunit immunizations with limiting doses of antigen result in enhanced CD4 T cell activation as compared to CD8 T cells¹³. This suggests that the spatial positioning of cDC subsets within the LN, coupled with the generation of antigen gradients across the tissue, directly impacts which classes of cellular immunity are generated. Such spatial regulation of T cell responses may reflect the distinct roles of these T cell subsets in immunity. Preferential CD4 T cell responses to lymph-borne soluble antigens may promote an appropriate balance of activation against extracellular draining antigens. Early and efficient activation of CD4 helper T cells may also be necessary in order for these cells to provide timely help for CD8 T cell and humoral responses^{30,31,64,118,206}. This is in contrast with intracellular infections, which are less likely to result in copious amounts of soluble antigens. In these settings, the more centrally localized cDC1s may be optimally positioned to acquire and cross-present antigen derived from infected host cells or antigen-bearing migratory cDCs, thus promoting robust CD8 T cell responses during conditions when the effector functions of cytotoxic CD8 T cells would be most utilized^30–32.

Figure 3. Generation of information gradients within lymph nodes.

(A) Confocal microscopy image of a draining LN demonstrating gradient formation 2 hours after subcutaneous injection of EαGFP (antigen, yellow) fluorescent protein. cDCs and stromal networks are also revealed with CD11c and Collagen-IV staining, respectively. (B) Schematic showing the radial and lateral gradients of antigen and agonist which are generated within the draining LNs (top). Influence of radial gradients is depicted in the zoom insets (i & ii). (i) Resident cDC2s are enriched near the lymphatic sinuses, and robustly acquire draining antigens. (ii) Lower abundance of antigen is observed within the T cell zone, therefore limiting antigen capture by the more centrally localized cDC1s. Bottom panel depicts the influence of lateral gradients on downstream myeloid cell localization and function.

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We have recently demonstrated that in addition to the radial axis of antigen dispersal across the lymphatic and T cell zone regions, lateral dispersal gradients also likely contribute to the generation of immune responses (Figure 3). LNs contain multiple incoming afferent lymphatic vessels, each draining a unique peripheral tissue, which are distributed in distinct sites across the subcapsular sinus. Accordingly, during infection or immunization of a given tissue, the draining node receives incoming stimuli via one dominant lymphatic vessel. This in turn results in markedly greater deposition of antigens and agonists in regions most proximal to the involved afferent vessel, and leads to establishment of lateral gradients across the LN^13,28. Concentration of antigen and inflammatory signals on one side of the LN is likely to influence the activation, localization, and function of multiple innate cell types. In this regard, we found highly polarized infiltration of inflammatory monocytes during Type-I inflammation, leading to varied abundance of innate cell subsets across the tissue²⁸. Similar considerations likely apply to migratory cDCs. As various migratory cDC subsets accumulate within the draining LN^{31,43,207,208}, they similarly enter from the specific afferent lymphatic vessel draining the inflamed tissue, and this likely reinforces the polarized distribution of antigen-bearing cDCs across the LN parenchyma. Consistent with this idea, in a variety of infection settings, more robust migratory cDC-driven T cell priming can be observed on one side of the draining LN^{31,107,209,210}. Together, this lateral axis of innate cell composition and activity sets up the generation of multiple distinct microenvironments within a single LN, in which T cells experience divergent conditions during early activation dependent on their localization²⁸. In turn, this imparts distinct differentiation properties to the responding T cells. As we demonstrated in settings of Type-I inflammation, increased infiltration of IL-12-producing activated monocytes into one side of the LN after immunization results in the localized generation of highly differentiated effector T cells. Conversely, less differentiated T cells expressing markers of memory precursor cells are preferentially observed in regions with fewer inflammatory monocytes and a greater abundance of cDCs²⁸. We thus propose that the fundamental rules of lymphatic flow and information dispersal across the LN can directly regulate the heterogeneity of the resultant T cell response.

CONCLUSIONS

Overall, these studies reveal several key features of how immunity is generated and provide important considerations for rational vaccine design. In particular, our past observations of reduced antigen capture by resident cDC1s and less efficient CD8 T cell activation during subunit immunization may have critical implications for vaccine outcomes. In line with this, while subunit vaccines have been shown to generate robust CD4 T cell and antibody responses in humans, they do not appear to induce robust CD8 T cell responses, even when delivered with potent adjuvants^211,212. Thus, strategies to better retain antigens within the draining LN, while also promoting higher diffusive properties, may promote more potent CD8 T cell responses during vaccination^168,169. In this regard, vaccine formulations that are designed to promote long-term antigen persistence in draining LNs have been shown to augment CD8 T cell responses^213–215. Work by many groups, including ours, also highlight the notion that functional cooperation between diverse innate cell types is intimately involved in regulating the magnitude and quality of both cell-mediated and humoral immunity. Therefore, vaccine strategies that simultaneously target multiple innate cell populations should be explored. Finally, it should be noted that different routes of immunization can induce distinct kinetics and biodistribution of vaccine derived materials. This can influence myeloid cell maturation and function as well as downstream adaptive immunity, which can in turn further shape vaccine outcomes^216,217.

In sum, the lymphatics are an information-rich superhighway that enables efficient crosstalk between peripheral tissues and the draining LNs. Such transmission of information is necessary for promoting immune quiescence at steady state, as well as for mounting productive immune responses during inflammation. During microbial infection or vaccination, the spatial organization of innate cells within LNs permits effective sampling of information and threat detection, as well as facilitates highly coordinated crosstalk among different immune cells to drive the generation of functional adaptive responses. During Type-II inflammation of barrier surfaces, information on local tissue damage is potently integrated by peripheral tissue cDCs, and is delivered into draining LNs to promote alternate forms of T cell activation geared to this type of insult. At the same time, the drainage patterns of antigens and agonists, coupled with localized innate cell positioning, generates discrete immune microenvironments which control the magnitude, quality, and heterogeneity of downstream adaptive immunity. Together, these large-scale and spatially orchestrated crosstalk events across tissues and among immune cell populations represent the mechanisms responsible for the appropriate generation of immune responses to distinct classes of stimuli.