Salivary gland macrophages and tissue-resident CD8⁺ T cells cooperate for homeostatic organ surveillance

Systemic viral infection leads to the establishment of T_RM in salivary glands

We used a viral infection model for a comparative analysis of CD8⁺ T cell populations in lymphoid tissue and SMG (fig. S1A). Systemic infection with lymphocytic choriomeningitis virus (LCMV)–ovalbumin (OVA), a replication-competent, attenuated LCMV mutant expressing OVA as model antigen (21), led to transient and low viral titers in spleens on day 3 postinfection (p.i.) that remained below the detection limit in peripheral lymph nodes (PLNs) and SMGs (fig. S1B). Adoptively transferred green fluorescent protein (GFP)⁺ OT-I (OVA-TCR-1) CD8⁺ T cell receptor (TCR) transgenic (tg) T cells (which recognize the OVA_257–264 peptide in the context of H2-K^b) (22) underwent a prototypic expansion-contraction kinetic in the spleen and PLN over the course of 30 days (fig. S1, C and D). Despite the lack of detectable viral titers, OT-I T cells accumulated in SMGs from day 6 p.i. onward, with a stable population maintained until at least day 30 p.i. (fig. S1, C and D). By day 30 p.i., OT-I T cells isolated from SMGs but not PLN or spleen showed increased expression of CD103 and CD69, while losing the KLRG1⁺ (killer cell lectin-like receptor subfamily G member 1) population present on day 6 p.i. (fig. S1, E and F). These data suggest that most SMG CD8⁺ T cells had developed into bona fide T_RM at day 30 p.i. (23). Memory OT-I CD8⁺ T cells isolated from PLN were about 65% CD62L⁺ CD44^high T_CM, with the remaining population being CD62L⁻ CD44^high memory T cells. To take this heterogeneity into account, we refer to memory T cells isolated from PLN as T_PLN-M.

Homeostatic SMG T_RM migration is characterized by dynamic cell shape changes

We next determined the localization of T_PLN-M and T_RM in their target organs during the memory phase after LCMV-OVA infection (Fig. 1A). Immunofluorescence sections showed that most GFP⁺ OT-I T cells in PLN and SMG were dispersed evenly throughout tissue at day 30 p.i. (Fig. 1B). In PLN, OT-I T_PLN-M cells localized mainly with smooth muscle actin (SMA)⁺ fibroblastic reticular cells, whereas most OT-I T_RM in SMG were within or adjacent to EpCAM⁺ (epithelial cell adhesion molecule) acini and ducts (Fig. 1B). We developed a sequential surgery method to visualize homeostatic tissue surveillance of T_PLN-M and T_RM in PLN and SMG, respectively, in the same host by 2PM (24). T_PLN-M displayed characteristic amoeboid shapes and moved with speeds similar to values reported for T_N (11.8 ± 4.0 μm/min, median ± SD) (Fig. 1, C and F to H, and movie S1). Compared with T_PLN-M, SMG T_RM displayed more pronounced shape changes, with several protrusions probing the microenvironment during migration, at times with thin and elongated cell bodies (Fig. 1, D to G, and movie S2). Although T_PLN-M and T_RM covered large distances throughout the observation period of intravital imaging sequences (20 to 60 min), both populations differed in their speed and arrest coefficients, i.e., percentage of track segments with speeds <2.5 μm/min. Thus, SMG T_RM moved slower than T_PLN-M (Fig. 1H) and had higher arrest coefficients (Fig. 1I). Nonetheless, SMG T_RM retained a relatively high motility coefficient, which is a proxy of a cell’s ability to scan the environment during random migration, of >15 μm²/min (Fig. 1J). Accordingly, their median speed of 6.8 ± 3.4 μm/min was notably higher than values reported for epidermal T_RM (1 to 2 μm/min) (10), with some cells achieving speeds of >12 μm/min. Both T_PLN-M and SMG T_RM retained a fast response to antigenic stimulation, as systemic administration of cognate peptide resulted in immediate arrest and secretion of interferon-γ (IFN-γ) (Fig. 1, J and K).

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To examine a distinct CD8⁺ T cell population, we transferred GFP⁺ P14 CD8⁺ TCR tg T cells (which recognize the LCMV epitope glycoprotein (gp)_33–41 in the context of H2-D^b) (25) and performed 2PM of PLN and SMG at >30 days p.i. with the LCMV Armstrong strain. We measured similar speeds and meandering indices for GFP⁺ P14 T_PLN-M and T_RM as with OT-I CD8⁺ T cells, both before and after cognate peptide administration (Fig. 1, L to N). Furthermore, GFP⁺ OT-I T cells patrolled the structurally comparable lacrimal gland (LG) in the same speed range as in SMG (7.6 ± 4.3 μm/min, median ± SD; Fig. 1O). Collectively, these data suggest that migration parameters of T_RM patrolling exocrine glands during homeostasis are independent of TCR specificity and may reflect tissue properties.

T_RM colocalize with tissue macrophages in salivary and LGs

To explore the microenvironmental context of exocrine gland T_RM migration, we used a CD11c–yellow fluorescent protein (YFP) reporter strain that preferentially labels CD64⁺ SMG macrophages (fig. S2A), as previously described (18). In tissue sections, CD11c-YFP⁺ cells were also positive for the macrophage marker Iba-1, whereas some Iba-1⁺ cells were CD11c-YFP^low/negative, indicating that most but not all tissue macrophages were labeled in CD11c-YFP mice (fig. S2B). Confocal analysis of thick tissue sections showed that CD11c-YFP⁺ tissue macrophages extended numerous protrusions from their cell bodies throughout the SMG tissue and were located within EpCAM⁺ ducts and acini, as well as SMA⁺ perivascular structures of the interstitium (fig. S2C). We further investigated the spatial arrangement of macrophage protrusions with regard to epithelial BM markers. The laminin ligand CD49f (α6) was prominent on the basal side of acini and, to a lesser extent, on ducts, which were identified by the presence of the tight junction protein ZO-1 on the luminal side. In some cases, tissue macrophage protrusions appeared to project between adjacent acini and ducts (fig. S2D).

To assess the spatial relationship between tissue macrophages and T_RM, we transferred GFP⁺ OT-I T cells into CD11c-YFP recipients 1 day before infection with LCMV-OVA and analyzed tissue sections by confocal microscopy in memory phase (≥day 30 p.i.). We observed a notable spatial proximity of T_RM and tissue macrophages in SMGs, with about 70% of OT-I T cells directly in contact with CD11c-YFP⁺ cells (Fig. 2, A and B, and movie S3). A comparable association of T_RM and tissue macrophages was observed in LGs after LCMV-OVA infection (Fig. 2C). The close spatial association between SMG tissue macrophages and T_RM was confirmed by correlative light and electron microscopy imaging, with both cell membranes adjacent to each other (Fig. 2D). Electron microscopy images also highlighted the compact tissue structure of acinar and ductal epithelium linked by tight junctions and surrounded by a dense ECM (Fig. 2E). Occasionally, we observed small T_RM clusters around tissue macrophages (Fig. 2F). CXCR3^−/− OT-I T_RM failed to accumulate at tissue macrophage clusters, suggesting the existence of local CXCL9/CXCL10 “hotspots” at these sites (Fig. 2F). Similarly, CD3⁺ T cells colocalized with CD68⁺ macrophages in human parotid gland sections, both as dispersed individual cells and in clusters (Fig. 2, G and H). Together, T_RM displayed a prominent colocalization with tissue macrophages in noninflamed salivary glands across species.

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Migrating T_RM follow tissue macrophage topology during SMG surveillance

The spatial proximity of T_RM to SMG macrophages in tissue sections raised the question whether patrolling T_RM migrate alongside macrophages. 2PM imaging of GFP⁺ T_RM in LCMV-OVA memory phase CD11c-YFP recipients confirmed that T_RM crawled along CD11c-YFP⁺ macrophages during most of the observation period, with T_RM shapes often closely matching the underlying macrophage topology. This was particularly evident along thin macrophage protrusions, which T_RM often followed (Fig. 3A and Movie 1). At the same time, T_RM protrusions occasionally detached from macrophages, apparently scanning the surrounding environment. Accordingly, we identified occasional T_RM track segments that were not associated with tissue macrophages, with a minor reduction in T_RM speeds (7.0 ± 5.3 μm/min with macrophages versus 6.1 ± 4.8 μm/min without macrophages; P < 0.001).

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Depletion of tissue macrophages disrupts T_RM patrolling within and between acini and ducts

Our observations prompted us to examine T_RM motility in the absence of tissue macrophages. To this end, we generated bone marrow chimera by reconstituting C57BL/6 or Ubi-GFP mice with control CD11c-YFP or CD11c-DTR (diptheria toxin receptor) bone marrow. At 6 weeks of reconstitution, we adoptively transferred GFP⁺ or DsRed⁺ OT-I T cells, followed by LCMV-OVA infection. In some experiments, we directly transferred OT-I T cells into CD11c-DTR mice and infected mice with LCMV-OVA. Both approaches allowed us to deplete CD11c⁺ macrophages by diphtheria toxin (DTx) treatment in the memory phase without affecting the unfolding of the adaptive immune response.

2PM imaging in DTx-treated mice revealed that SMG T_RM patrolling behavior was disrupted when macrophages were depleted (Fig. 3B). T_RM motility was decreased, reflected by less displacement (Fig. 3C) and slower speeds (Fig. 3D). In addition, we observed cells that returned and migrated back the same path within acini and ducts after macrophage depletion (Fig. 3B and movie S4). To quantify this behavior, we developed a method to specifically retrieve U-turns from track parameters (Fig. 3E). This analysis confirmed that the percentage of T cell tracks showing U-turns was doubled in DTx-treated CD11c-DTR SMG from 8.1 to 16.7% of tracks (Fig. 3F). We observed a similar impact of macrophage depletion on T_RM speeds in LG (7.6 ± 4.3 and 5.5 ± 3.2 μm/min in control and macrophage-depleted LG; P < 0.001), with a 2.5-fold increase in U-turns (fig. S3, A and B).

We next asked how impaired motility affects organ surveillance. We generated tracks in silico from the datasets obtained by 2PM imaging of DTx- and control-treated SMG and assessed the average T_RM dwell time in a sphere with a diameter of 80 μm as a surrogate epithelial structure (Fig. 3E). This analysis uncovered a nearly threefold increased sphere dwell time from 24 ± 1.8 min for control SMG to 69 ± 6.5 min (median ± SEM) for DTx-treated CD11c-DTR SMG (Fig. 3G). A similar finding was made for control and macrophage-depleted LG (fig. S3C). Together, macrophage depletion disrupted T_RM patrolling and increased the propensity of T_RM to make U-turns.

Absence of macrophages reduces T_RM movements into and out of epithelium

We investigated whether lack of macrophages may also affect T_RM transitions into and out of the epithelium as part of the impaired motility pattern, as suggested by movie S4. To address this point, we reconstituted irradiated Ubi-GFP mice expressing GFPs in all cells with CD11c-YFP bone marrow before transfer of DsRed⁺ OT-I T cells and systemic LCMV-OVA infection. We found that in these chimera, acini and ducts of surgically prepared SMGs were GFP^bright and readily identifiable by their glandular shapes, whereas connective tissue was GFP^low. Using case-by-case 3D rendering of 2PM image sequences in memory phase (≥30 days p.i. with LCMV-OVA), we observed that DsRed⁺ T_RM were not restricted to individual epithelial structures but occasionally crossed between adjacent acini or between epithelial and connective tissue compartments in a bidirectional manner along macrophage protrusions (Fig. 4A and movie S5). We confirmed this observation in a mouse model expressing membrane tomato and CD11c-YFP (Fig. 4B). In total, 75% of T_RM transits (n = 42) into and out of epithelial structures occurred along macrophage protrusions (Fig. 4C). Given that not all tissue macrophages are YFP⁺ (fig. S2B), the actual percentage of macrophage-assisted transitions may still be higher. DTx treatment of CD11c-DTR SMGs reduced, but did not abolish, T_RM transit into or out of acini and ducts. In total, we observed 55 T_RM crossing events into or out of acini in CD11c-YFP versus 12 events in CD11c-DTR chimera SMGs. These data corresponded to a 77% fewer crossing events per hour track duration (Fig. 4D). Because this defect is more pronounced than the relatively mild decrease in T_RM speeds after macrophage depletion (Fig. 3D), it is unlikely to be fully explained by decreased motility. Accordingly, when we normalized T_RM transitions to migrated distance, we continued to observe impaired epithelial crossing in the absence of macrophages (Fig. 4E). Reduced T_RM crossing into and out of epithelial structures was also observed when we prolonged DTx treatment for 5 days (movie S6).

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Clustering of SMG T_RM is impaired after macrophage depletion

We set out to examine the impact of macrophage-T_RM cooperativity in a local recall response. We infected CD11c-YFP or CD11c-DTR mice with LCMV-OVA 1 day after transfer of GFP⁺ OT-I T cells and depleted macrophages after 4 weeks using DTx (fig. S4A). One day later, we rechallenged mice with an mCherry- and OVA-expressing mouse cytomegalovirus (MCMV-OVAmCherry) (26, 27) administered locally via retrograde WD injection. After 48 hours, we analyzed SMG tissue sections for the presence and intensity of viral foci identified by mCherry⁺ signal. This analysis confirmed the strictly local reinfection because no mCherry signal was observed in the adjacent sublingual gland (fig. S4B). We observed close proximity of OT-I T cells and CD11c-YFP⁺ cells with MCMV-infected cells after SMGs infection (fig. S4C), with macrophages occasionally engulfing infected cells (fig. S4D). This observation is in line with the well-described macrophage core function of engulfing apoptotic cells (efferocytosis) (28–31). Accordingly, we observed massively increased numbers of infected cell foci in macrophage-depleted SMGs after WD infection with MCMV-OVAmCherry as compared with SMGs containing tissue macrophages, irrespective of the presence of T_RM (fig. S4E). In the absence of tissue macrophages, T_RM partially suppressed viral replication as assessed by decreased mCherry intensity in viral foci (fig. S4F). Given the increased viral load after DTx treatment, tissue macrophage depletion did not permit the direct assessment of the functional impact of reduced T_RM migration patterns in the presence and absence of these cells.

We therefore designed an experiment to examine T_RM cluster formation in response to exogenously added inflammatory chemokine as a prerequisite for efficient elimination of infected cells (15). We treated LCMV-OVA–immunized CD11c-YFP and CD11c-DTR BM chimera mice with DTx, followed 1 day later by local injection of the CXCR3 ligand CXCL10 and quantum dots into SMG (Fig. 4F). We also administered anti-α4 and LFA-1 blocking monoclonal antibodies (mAbs), which inhibit recruitment of circulating T cells to SMG (32). At 4 hours after CXCL10 administration, we isolated SMG and quantified T_RM enrichment in thick confocal SMG sections according to the area marked by the coinjected fluorescent quantum dots. CXCR3^−/− OT-I T cells did not show accumulation in CXCL10 injection sites, supporting the specificity of chemokine-triggered clustering (49 to 63 cells/cm² > 500 μm versus 60 to 68 cells/cm² < 500 μm from injection site; range from two SMG). Wild-type (WT) OT-I T_RM were twofold enriched at CXCL10 injection sites, suggesting that these cells had followed a CXCL10 gradient or became retained during their surveillance path (Fig. 4G). In contrast, local accumulation of T_RM was lost when macrophages had been depleted, although T_RM numbers outside the site of chemokine injection remained comparable with macrophage-containing SMGs (Fig. 4G). These data suggested that presence of SMG macrophages permitted T_RM clustering in response to local inflammatory chemokines.

Macrophages show discontinuous attachment to neighboring cells within SMG

We examined macrophage embedding within salivary gland tissue in more detail. In immunofluorescent sections, most macrophage protrusions contained phosphotyrosine-positive patches (Fig. 5, A and B), suggesting the presence of focal adhesion–like structures at these sites. We investigated how these local signals correlated with macrophage attachment in situ. We applied the super-resolution shadow imaging microscopy (SUSHI) technique, which was originally developed to visualize the complex topology of the extracellular space (ECS) in living brain slices (33). SUSHI can also be used to follow dynamic changes in ECS in response to a hyperosmotic challenge, which leads to cell shrinkage and ECS widening in brain tissue. We adapted SUSHI imaging to acutely sliced SMG sections, which were superfused with the cell-impermeable fluorescent dye calcein to examine macrophage anchorage to adjacent epithelium (Fig. 5C). Steady-state imaging revealed that the interstitium contained more ECS as compared with the tightly packed epithelium (Fig. 5, D and E). We reasoned that SUSHI in combination with hyperosmotic challenge could be applied to explore attachment between neighboring cells. Performing time-lapse ECS imaging, we gradually increased the osmolarity to induce cell shrinkage, which led to a strong increase in ECS in the interstitium (movie S7). In turn, interepithelial junctions remained relatively stable and only mildly increased their spacing under osmotic challenge, reflecting the presence of adherens and tight junctions known to link epithelial cells (Fig. 5F). In contrast, hyperosmolarity induced intraepithelial CD11c-YFP⁺ macrophages detachment from the adjacent epithelium (Fig. 5, G and H). This finding confirms previous observations that tissue macrophages do not form continuous adhesive contacts with the epithelium, unlike the extensive cell-to-cell contacts between acinar epithelial cells (34).

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For an examination of macrophage protrusions and epithelial BM, we analyzed laminin-stained tissue sections from immunized CD11c-YFP mice. We observed, in some cases, macrophage protrusions penetrating between adjacent acini, or between epithelium and connective tissue, thus bridging adjacent compartments separated by BM (Fig. 5I and movie S8). Using correlative confocal and transmission electron microscopy (TEM), we validated that some macrophage protrusions transversed BM (Fig. 5J). Together, our data uncover discontinuous attachment of tissue macrophages to neighboring cells and occasional incursions of macrophage protrusions across the epithelial BM, creating paths of least resistance for migrating T_RM. These observations offer an explanation for T_RM migration in accordance with tissue macrophage topography.

In vivo motility of SMG T_RM remains largely intact upon integrin and chemokine receptor blockade

Our data established a correlation between macrophage topography and T_RM migration patterns in noninflamed salivary and LGs. Next, we set out to dissect the molecular mechanisms that drive homeostatic T_RM motility in SMG, focusing on well-described canonical chemoattractant- and integrin-signaling pathways. Given the expression of promigratory chemokines and adhesion receptors including intercellular adhesion molecule–1 (ICAM-1) on tissue macrophages (35), we examined their influence on T_RM migration parameters. Integrins mediate attachment and force transmission through engagement of their ligands expressed by many cell types including macrophages. SMG T_RM express α1, α4, αE, αL, β1, β2, and β7 integrins and low levels of αV (figs. S1 and S5A). To assess their involvement in SMG T_RM immune surveillance, we administered a mix of integrin-blocking mAbs against the major lymphocyte integrin αL (CD11a/CD18 and LFA-1), the E-cadherin receptor αE (CD103), and α4 (VLA-4 and α4β7) to LCMV-OVA memory phase mice containing T_PLN-M and T_RM (Fig. 6A). We confirmed that mAbs were saturating surface integrins at the time point analyzed (fig. S5B). We then followed OT-I T cell motility in PLN and SMG on ≥day 30 p.i., using dual surgery 2PM of the same recipient mouse as above. Integrin blockade lowered T_PLN-M speeds from 11.7 to 8.8 μm/min (fig. S5C), similar to the decreased cell speeds of CD18-deficient T_N in lymphoid stroma (36). In contrast, T_RM speeds and crawling along tissue macrophages remained unaltered by this treatment (Fig. 6, B and C, and movie S9). We did not detect Mac-1 (CD11b/CD18) expression on SMG T_RM by flow cytometry, and addition of anti-Mac1 mAb to the integrin-blocking mix did not decrease T_RM speeds or guidance by tissue macrophages (7.1 ± 2.9 μm/min). Similarly, inclusion of blocking mAbs against α1 and αV, together with αL, α4, and αE, had no impact on T_RM speeds or association with macrophages (6.5 ± 2.7 μm/min).

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Poor surface saturation of blocking anti-β1 mAbs on OT-I T cells preempted our assessment of the role of β1 integrins for T_RM motility by this approach. As an alternative, we directly administered the β1-blocking peptide RGD or the control peptide RAD through the WD into SMG and followed its impact on T_RM motility parameters by 2PM. The WD channels saliva from SMG into the oral cavity and can be used to administer reagents or pathogens through retrograde duct cannulation (37). Control experiments using WD administration of OVA_257–264 peptide led to instantaneous arrest of T_RM similar to systemic injection, suggesting efficient peptide permeation of SMG by this route. WD injection of either peptide slightly lowered T_RM speeds; yet, we did not observe an impact on RGD administration on T_RM motility parameters as compared with control peptide (Fig. 6D). This reflected low β1 integrin levels in the interface between macrophages and T_RM (fig. S5D). Furthermore, E-cadherin levels on macrophages and T_RM were barely detectable in SMG tissue sections, arguing against a role for this cadherin in mediating close spatial association with tissue macrophages (fig. S5E).

Cytokine-driven chemoattractant production plays a key role for T cell trafficking. Because the CXCR3 ligands CXCL9 and CXCL10 play a role in T_RM clustering in SMG (Fig. 2F), we cotransferred WT and CXCR3^−/− OT-I T cells 1 day before LCMV-OVA infection. Consistent with a recent report (32), we found that the absence of CXCR3 did not impair T_RM formation in SMG after viral infection. Nonclustered CXCR3^−/− OT-I T_RM showed no differences in speeds as compared with WT T_RM (Fig. 6E), and lack of CXCR3 did not prevent T_RM patrolling along tissue macrophages (Fig. 2F). These data argue against a role for CXCR3 in mediating baseline homeostatic motility of SMG T_RM, despite its function in facilitating local clustering in response to proinflammatory CXCR3 ligands (Fig. 4F).

To comprehensively assess a function for potential chemoattractants, we inhibited Gαi signaling by systemic pertussis toxin (PTx) treatment (38) and performed 2PM analysis of OT-I T cell motility parameters on ≥day 30 after LCMV-OVA infection. To control for inhibitor efficacy, we took advantage of the dual surgery of PLN and SMG in the same recipient. Systemic PTx administration slowed T_PLN-M down from 11.3 μm/min in control to 8.6 μm/min in PTx-treated recipients (fig. S5F), resembling observations made with PTx-treated TN in PLN (39). Speeds were also decreased in SMG T_RM (from 6.6 to 5.5 μm/min) by PTx treatment (Fig. 6F), suggesting a role for chemoattractants in mediating high T_RM speeds. Nonetheless, we observed a robust residual motility and continued T_RM crawling along tissue macrophages in the presence of PTx (Fig. 6G and movie S10). Furthermore, PTx treatment had essentially no impact on U-turn frequency (1.15-fold increase as compared with PTx_mut), in contrast to the absence of macrophages (Fig. 3F). These data suggest that although Gαi-coupled receptors contribute to SMG T_RM motility, they are not required for T_RM association with tissue macrophages. Last, we interfered with matrix metalloproteinase (MMP) activity using the broad MMP-9, MMP-1, MMP-2, MMP-14, and MMP-7 inhibitor marimastat as previously described (40). MMP inhibition did not reduce T_RM migration speeds (6.9 ± 3.0 μm/min). Together, with the exception of a minor effect by PTx, the in vivo inhibitor treatments examined here did not alter T_RM motility and close spatial proximity to tissue macrophages.

To directly assess intercellular adhesion, we coincubated freshly isolated tissue macrophages and T_RM ex vivo and analyzed cluster formation by flow cytometry (Fig. 6H). As a positive control, we preincubated macrophages with cognate OVA_257–264 peptide. Although addition of OVA_257–264 to tissue macrophages induced detectable binding to T_RM, the baseline association between both populations remained low (Fig. 6, H and I). In sum, within the technical limitations of our experimental approach, our in vivo and in vitro observations did not identify specific molecules that provide strong adhesion of resting SMG T_RM to tissue macrophages. Our data rather suggested that T_RM association to macrophages occurred preferentially in the context of the SMG microanatomy. Our data do not exclude the presence of unidentified adhesion receptors mediating T_RM association to tissue macrophages in vivo.

SMG T_RM motility is induced by confinement and can be tuned by extrinsic factors

To address the molecular mechanisms underlying homeostatic T_RM motility, we used under-agarose assays that allow one to precisely control environmental factors and provide confinement required for T cell motility (Fig. 7A) (41). To benchmark our system, we transferred T_N on CCL21- and ICAM-1–coated plates as surrogate lymphoid tissue microenvironment. We observed high chemokinetic T_N motility with similar speeds as measured in vivo (13.3 ± 5.9 μm/min) (Fig. 7, B and C, and movie S11) (36, 41). Similarly, T_RM showed a high motility (11.4 ± 3.0 μm/min) when migrating on CXCL10 + CXCL12– and ICAM-1–coated plates (Fig. 7C and movie S12). These observations show that SMG T_RM respond to inflammatory chemokines and adhesion molecules with high speeds.

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Because we found that in vivo motility of resting T_RM was largely refractory to inhibition by PTx and integrins, we examined T cell displacement on plates coated with fatty acid–free human serum albumin (HSA) and thus free of chemoattractants and specific adhesion ligands. In line with previous findings (41), T_N and T_PLN-M remained essentially immobile throughout the observation period (Fig. 7, D to F, and movies S13 and S14). Under these conditions, only 12% of T_N and 31% of T_PLN-M migrated faster than 3 μm/min and showed low directionality (Fig. 7, G and H). Most SMG T_RM showed robust intrinsic motility on HSA-coated plates despite the absence of chemoattractants and adhesion molecules (Fig. 7, D to F, and movies S13 and S14). Almost 70% of SMG T_RM migrated faster than 3 μm/min with high directionality, with their median speed of 5.5 μm/min approaching values observed in vivo (Fig. 7, G and H). High temporal resolution imaging revealed that migratory T_RM often formed several protrusions along the leading edge that appeared to probe the environment, followed by rapid displacement of the cell body along one of the protrusions (movie S15).

Next, we performed under-agarose assays in the presence of SMG tissue macrophages to assess their influence on T_RM motility. On the few occasions when motile T_RM contacted coplated tissue macrophages, these contacts were mostly transient (Fig. 7, I and J). Furthermore, T_RM did not crawl along macrophage protrusions as observed in vivo (movie S16). These observations support the notion that T_RM-macrophage association occurred preferentially in the SMG microenvironment.

We examined whether spontaneous motility was a common feature of all T_RM populations. We isolated epidermal OT-I T cells from >30-day LCMV-OVA–infected mice, which had been recruited to DNFB (dinitrofluorobenzene) + OVA_257–264–treated skin during the expansion phase. In line with the reported sensitivity to in vivo PTx treatment (13), epidermal T_RM did not show spontaneous motility in the absence of chemokine (Fig. 7, K and L, and movie S17), although these cells remained responsive to exogenous chemokines (5.6 ± 4.2 μm/min in the presence of CXCL10 and CXCL12). Thus, confinement alone was sufficient to induce spontaneous SMG T_RM migration, a behavior that, to the best of our knowledge, had not been previously reported for resting T cells. Their speeds were increased in the presence of chemokines and adhesion molecules, suggesting that extrinsic promigratory factors tune intrinsic cell motility.

Friction mediates SMG T_RM migration in the absence of chemokines and ICAM-1

We set out to characterize the requirements for autonomous SMG T_RM motility using under-agarose assays. Reflecting the absence of chemoattractants and integrin ligands, PTx treatment or addition of the β1-blocking peptide RGD did not affect T_RM speeds in this setting (Fig. 7M). Although Mac-1 binds weakly to serum albumin (42), addition of anti–Mac-1 mAb did not cause a reduction in T_RM speeds (Fig. 7M). These observations suggested a friction-based migration mechanism (43). Friction is the resisting force when two elements slide against each other and may be composed of a number of fundamental forces. Although the nature of the weak interactions between T_RM and migratory surface causing friction are not defined, we hypothesized that these might, in part, involve bivalent cations. Chelation of bivalent cations by EDTA caused a strong decline of T_RM speeds under agarose (Fig. 7M). High temporal resolution imaging showed that despite the lack of translocation in the presence of EDTA, SMG T_RM continued to probe the environment via transient protrusion formation, essentially “running on the spot” (Fig. 7, N and O, and movie S18, left). This behavior precipitated a loss in the motility coefficient (Fig. 7P). In sum, our data suggest that bivalent cation-dependent friction between SMG T_RM and the confining 2D surfaces generated sufficient traction for translocation in the absence of considerable surface binding.

SMG T_RM insert protrusions between adjacent structures for translocation

In addition to friction-based migration, protrusion insertion has emerged in recent years as a complementary mechanism to allow cell migration without specific adhesions (43). The continuous probing of T_RM in the presence of EDTA (Fig. 7N) provided an opportunity to test whether topographic features of the environment such as narrow intercellular spaces may rescue cell motility by permitting insertion of pseudopods as mechanical “footholds” (44, 45). As a surrogate approach to reintroducing a “2.5D” environmental geometry in under-agarose assays, we cotransferred a surplus of T_N together with T_RM and performed time-lapse imaging in the presence of EDTA and in the absence of chemoattractants and adhesion molecules (Fig. 8A). T_RM localized within T_N clusters frequently showed lateral displacement despite the presence of EDTA (movies S18, right, and S19). Under these conditions, T_RM displacement occurred through insertion of protrusions between adjacent T_N and subsequent translocation of the cell body accompanied by dynamic cell shape changes (Fig. 8B). T_RM within T_N clusters were faster than isolated T_RM (5.8 ± 3.0 and 2.3 ± 1.7 μm/min, respectively), displayed higher directionality, and resembled in cell shape and speeds T_RM migrating in vivo (Fig. 8, C to E). Once T_RM had traversed T_N clusters, they returned to their probing behavior without efficient translocation, indicating a close interdependence on physical contact and motility (movie S18, right).

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We then examined whether potential residual molecular interactions between T_N and T_RM might act as drivers of migration. We therefore transferred uncoated polystyrene beads with T_RM in under-agarose assays. These beads replaced T_N as surrogate 2.5D structures and allowed us to examine protrusion insertion in the absence of potential adhesive interactions. In this setting, T_RM recapitulated the behavior observed within T_N clusters, showing effective cell displacement only when in contact with clusters of beads for protrusion insertion (Fig. 8F and movie S20). T_RM speeds increased to 6.4 ± 1.9 μm/min and became more directional when in contact with beads, whereas isolated T_RM showed no displacement (Fig. 8, G and H). This was also seen when beads were passivated with pluronic acid, which prevents any unspecific residual adhesion. We further observed that T_RM moved around dense bead areas, in line with a search for permissive gaps for locomotion (movie S20). In sum, our data uncover the remarkable ability of resting SMG T_RM to migrate by adapting to topographic features of the environment through protrusion insertion and shape deformation, even in the absence of considerable friction, chemoattractants, and adhesion receptors (Fig. 8I). At the same time, these cells retain responsiveness to chemokine signals, follow macrophage topology to short-cut SMG epithelial barriers, and accumulate at local inflammatory hot spots (Fig. 8J).