# Macrophages Induce Long-Term Trapping of γδ T Cells with Innate-like Properties within Secondary Lymphoid Organs in the Steady State

Alexandra Audemard-Verger, Matthieu Rivière, Aurélie Durand, Elisa Peranzoni, Vincent Guichard, Pauline Hamon, Nelly Bonilla, Thomas Guilbert, Alexandre Boissonnas, Cédric Auffray, et al.

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## To cite this version:

Alexandra Audemard-Verger, Matthieu Rivière, Aurélie Durand, Elisa Peranzoni, Vincent Guichard, et al. Macrophages Induce Long-Term Trapping of γδ T Cells with Innate-like Properties within Secondary Lymphoid Organs in the Steady State. Journal of Immunology, 2017, 199 (6), pp.1998-2007. 10.4049/jimmunol.1700430. hal-03863963

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Macrophages induce long-term trapping of γδ T cells with innate-like
properties within secondary lymphoid organs in the steady state

By

Alexandra Audemard-Verger¹*, Matthieu Rivière¹*, Aurélie Durand¹*, Elisa Peranzoni¹,
Vincent Guichard¹,², Pauline Hamon³, Nelly Bonilla¹, Thomas Guilbert¹, Alexandre
Boissonnas³, Cédric Auffray¹, Gérard Eberl⁴, Bruno Lucas¹** and Bruno Martin¹**

¹ Paris Descartes Université, Institut Cochin, CNRS UMR8104, INSERM U1016, 75014 Paris, France; ² Paris Diderot Université; ³ Sorbonne Universités, UPMC Université Paris 06, INSERM U1135, CNRS ERL8255, Centre d'Immunologie et des Maladies Infectieuses, 91 Boulevard de l'Hôpital, 75013 Paris, France; ⁴ Unité Microenvironment & Immunity, Institut Pasteur, 75724 Paris, France, INSERM U1224, 75724 Paris, France.

* Alexandra Audemard-Verger, Matthieu Rivière and Aurélie Durand contributed equally to this paper.

** Bruno Lucas and Bruno Martin contributed equally to this paper.

**Corresponding author:** Dr. Bruno Martin (bruno.martin@inserm.fr), Cochin Institute, 75014 Paris, France. Phone: 33-1-40516589; Fax: 33-1-40516535

**Running title:** γδ T-cell residency in secondary lymphoid organs

**Keywords:** Gamma delta T cells; Secondary lymphoid organs; T-cell trafficking; Steady state
28

ABSTRACT

29 So far, peripheral T cells are mostly described to circulate between blood, secondary
30 lymphoid organs and lymph in the steady state. This nomadic existence would allow
31 them to accomplish their surveying task for both foreign antigens and survival signals.

32 Although it is now well established that γδ T cells can be rapidly recruited to
33 inflammatory sites or in certain tumor microenvironments, the trafficking properties of
34 peripheral γδ T cells have been poorly studied in the steady state. In the present paper,
35 we highlight the existence of resident γδ T cells in the secondary lymphoid organs of
36 specific pathogen free mice. Indeed, using several experimental approaches such as the
37 injection of integrin-neutralizing antibodies which inhibit the entry of circulating
38 lymphocytes into lymph nodes and long-term parabiosis experiments, we have found
39 that, contrary to Ly6C⁺/CD44lo and Ly-6C⁺ CD44hi γδ T cells, a significant proportion
40 of Ly-6C⁺ CD44hi γδ T cells are trapped for long periods of time within lymph nodes and
41 the spleen in the steady state. Specific in vivo cell-depletion strategies have allowed us to
42 demonstrate that macrophages are the main actors involved in this long-term retention
43 of Ly-6C⁺ CD44hi γδ T cells in secondary lymphoid organs.
44 INTRODUCTION

γδ T cells are unique and distinct from other lymphocyte subsets, such as NK cells, B cells, and αβ T cells, in that they combine adaptive features with rapid, innate-like responses that allow them to play an important role in all phases of an immune response. γδ T cells are crucially involved in host immune defense against infections (1) but are also known to have a strong clinical association with various autoimmune diseases such as inflammatory bowel disease (2, 3), rheumatoid arthritis (or collagen-induced arthritis, the murine model of rheumatoid arthritis) (4, 5), and multiple sclerosis (or experimental autoimmune encephalomyelitis, the murine model of multiple sclerosis) (6, 7).

In addition, there is compelling evidence to indicate that γδ T cells play an important role in immunity to cancer by sensing and reacting to cellular stress. This has been clearly demonstrated in murine models of spontaneous (8), chemically induced (9), transgenic (10), and transplantable tumors (11, 12). However, it seems that the activity of γδ T cells in response to tumors can differ radically according to the tumor type or environment (13, 14). These data may actually reflect the high diversity of the γδ T cell compartment.

In this regard, we have recently showed that within murine secondary lymphoid organs (SLOs), γδ T cells can be subdivided into four subsets according to CD44 and Ly-6C expression. More precisely, we have characterized the functional heterogeneity of the peripheral γδ T cell compartment that comprises Ly-6C⁻ CD44ʰʰ γδ T cells exhibiting innate-like features and naive and memory adaptive-like γδ T cells (Ly6C⁺/ CD44¹º γδ T cells and Ly-6C⁺ CD44ʰʰ γδ T cells respectively; (15)).

This phenotypic and functional heterogeneity of the peripheral γδ T-cell compartment may be also associated with differential T-cell trafficking dynamic properties. So far, peripheral T
cells are mostly described to circulate between blood, SLOs and lymph in the steady state.
This nomadic existence would allow them to accomplish their surveying task for both foreign
antigens and survival signals (16, 17). In line with this concept, Mandl et al., have showed in
a recent study that the mean transit time for CD4⁺ and CD8⁺ αβ T cells in peripheral lymph
nodes (pLNs) were of 12 and 18 hours respectively (18). Concerning γδ T cells, it is now well
established that γδ T cells can be rapidly recruited to inflammatory sites or to certain tumor
microenvironments (19, 20). These γδ T-cell infiltrations are notably orchestrated by
inflammatory cytokines such as IL-1β or IL-23 or chemokine axes such as CCL2/CCR2 (12,
21, 22, 23). However, the T-cell trafficking dynamics of peripheral γδ T-cell subsets in the
steady state have still been poorly studied.
Here, using several experimental approaches such as the injection of integrin-neutralizing
antibodies which inhibit the entry of circulating lymphocytes into lymph nodes (LN) and
long-term parabiosis experiments, we have found that contrary to Ly-6C⁺ CD44⁺ᵢᵢ and CD44⁺ᵢ
γδ T-cell subsets which are circulating cells, an important proportion of Ly-6C⁺ CD44⁺ᵢᵢ γδ T
cells is residing within SLOs in the steady state. Moreover, specific in vivo cell-depletion
strategies have allowed us to demonstrate that macrophages are the main actors involved in
this long-term trapping of Ly-6C⁻ CD44⁺ᵢᵢ γδ T cells in SLOs in the steady state.
# MATERIALS AND METHODS

## Mice

C57BL/6 CD45.1 mice, C57BL/6 CD45.2 mice, C57BL/6 µMT mice and C57BL/6 CD11c.DTR mice (24), were maintained in our own animal facilities, under specific pathogen-free conditions. C57BL/6 RORγt-GFP mice were initially obtained from Dr. Gerard Eberl, Institut Pasteur, France (25). 6- to 12-week-old mice were used for experiments. Animal housing, care and research were carried out in accordance with the guidelines of the French Veterinary Department. All procedures were approved by the French animal experimentation and ethics committee and validated by “Service Protection et Santé Animales, Environnement” with the numbers C-75-562 and A-75-1315. Sample sizes were chosen to assure reproducibility of the experiments and in accordance with the 3R of animal ethics regulation.

## Cell suspensions

Peripheral lymph nodes (pooled cervical, axillary, brachial and inguinal lymph nodes; pLN), mesenteric LNs (mLN), cervical LNs (cLN), and spleen were homogenized and passed through a nylon cell strainer (BD Falcon) in RPMI 1640 Glutamax (Gibco) supplemented with 10% fetal calf serum (FCS; Biochrom) for adoptive transfer and cell culture (LN only), or in 5% FCS, 0.1% NaN3 (Sigma-Aldrich) in phosphate saline buffer saline (PBS), for flow cytometry.

To verify the efficiency of DCs or macrophages *in vivo* depletion, LNs and Spleens were disrupted in RPMI medium containing 5% FCS, 1 mg Collagenase from Clostridium histolyticum type II and type IV (Sigma) and 0.5 mg DNase I (Sigma) and incubated for 20 min at 37 °C.

## Fluorescence staining and flow cytometry
111 Cell suspensions were collected and dispensed into 96-well round-bottom microtiter plates (Greiner Bioscience; 6x10⁶ cells/well). Surface staining was performed as previously described (26, 27). Briefly, cells were incubated on ice, for 15 minutes per step, with 112 antibodies (Abs) in 5% FCS (Biochrom), 0.1% NaN₃ (Sigma-Aldrich) PBS. Each cell-staining 113 reaction was preceded by a 15-minute incubation with a purified anti-mouse CD16/32 Ab 114 (FcγRII/III block; 2.4G2) obtained from hybridoma supernatants. For determination of 115 intracellular cytokine production, cells were stimulated with 0.5 µg/ml PMA (Sigma- 116 Aldrich), 0.5 µg/ml ionomycin (Sigma-Aldrich), and 10 µg/ml BrefeldinA (Sigma-Aldrich) 117 for 2 hours at 37°C. Cells were then stained for surface markers, fixed in 2% 118 paraformaldehyde in PBS, and permeabilized with 0.5% saponin, followed by labeling with 119 specific cytokine Abs. 120

122 S1PR1 was detected with a rat monoclonal antibody (R&D Systems). Briefly, cells were first 123 incubated for 90 min with the anti-S1PR1 rat mAb (50µg/ml) in PBS containing 0.5% FCS, 124 1mM EDTA, 0.05% azide and 2% normal mouse serum. After washing, cells were then 125 incubated with donkey biotinylated anti-rat IgG polyclonal Abs (Jackson ImmunoReaserch) 126 for 30 min in the above medium followed by streptavidin-APC (BioLegend). 127

127 Multicolor immunofluorescence was analyzed using BD LSR2 and BD Fortessa cytometers 128 (BD Biosciences). List-mode data files were analyzed using Diva software (BD Biosciences). 129 Data acquisition was performed on the Cochin Cytometry and Immunobiology (CIBIO) 130 facility. 131

Parabiosis
---

132 Female host parabionts were generated with 8-12 week-old C57BL/6 CD45.1 mice and 133 C57BL/6 CD45.2 mice. The percentage of host cells in a given T-cell subset corresponds to 134 the percentage of CD45.1⁺ cells for the CD45.1 parabiont and the percentage of CD45.2⁺ cells 135 for the CD45.2 parabiont. 136
136 **Blocking T-cell entry into LNs**

137 Purified anti-VLA-4 (α4) (clone PS/2) and anti-LFA-1 (αL) (clone M17/4) Abs were
138 obtained from Bio-XCell. For short-term treatment, mice were injected intraperitoneally one
139 time with 200µg of both Abs and then analyzed 48 hours later. For long-term treatment, mice
140 were injected intraperitoneally every 2 days during 6 days (4 injections) with 200µg of both
141 Abs and analyzed 24 hours later.

142 *In vivo cell depletions*

143 For dendritic cell depletion, C57BL/6 CD11c.DTR mice (referred as CD11c DOG mice) were
144 injected intraperitoneally every 2 days during 6 days (4 injections) with 250ng of Diphtheria
145 Toxin (DT).

146 The CSF1R inhibitor PLX3397 incorporated into rodent chow at 290 p.p.m. (delivering daily
147 doses of approximately 45 mg kg⁻¹) was provided along with control chow from Plexxikon
148 Inc. (Berkeley, CA). For macrophage *in vivo* depletion, C57BL/6 mice were fed with
149 PLX3397 or control chow during 7 days.

150 **FTY720 treatment**

151 Mice were injected intraperitoneally daily with FTY720 (Cayman Chemical) in saline at a
dose of ~1µg/g during 7 days.
152

153 **Immunofluorescence microscopy**

154 Inguinal, axillary, brachial and maxillary mouse LNs were initially fixed overnight in 0.1M
155 phosphate buffer containing 75mM L-Lysine, 10mM NaIO4 and 1% PFA (PLP fixative
156 buffer). These organs were then embedded in 5% low-gelling-temperature agarose (type VII-
157 A; Sigma-Aldrich) prepared in PBS. 230-µm LN-slices were cut with a vibratome (VT
158 1000S; Leica) in a bath of ice-cold PBS. Slices were then incubated at 37°C, for 20 minutes
159 per step, with Abs in PBS. Stained slices were photographed on an upright Leica stand with a
160 confocal spinning disk Yokogawa head with a 25x 0.95 Leica objective. Acquisitions were
made with Flash 4 LT HAMAMATSU camera. Analysis has been performed using Image J
software. Data acquisition was performed on the Cochin imaging photonic (IMAG'IC)
facility.

164 *In vivo* LPS challenge

165 C57BL/6 RORγt-GFP mice were injected intravenously with 100µg of LPS (from Escherichia
166 Coli 0111:B4; Sigma). Three hours after LPS challenge, cells recovered from pLNsm, mLNsm
167 and the spleen were incubated with 10 µg/ml BrefeldinA (Sigma-Aldrich) for 2 hours at 37°C
168 for determination of intracellular cytokine production.

169 **Statistics**

170 Data are expressed as mean ± SEM, and the significance of differences between two series of
171 results was assessed using the student's paired or unpaired t test. Values of p < 0.05 were
172 considered significant. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).
173 **RESULTS**

174 **An important proportion of Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T lymphocytes are trapped in LNs in the**
175 **steady state**

176 According to Ly-6C and CD44 expression, we have recently characterized the heterogeneity
177 of the peripheral γδ T cell compartment by showing that it comprises Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T
cells exhibiting innate-like features and naive and memory adaptive-like γδ T cells (Ly6C<sup>-/+</sup>
178 CD44<sup>lo</sup> γδ T cells and Ly-6C<sup>+</sup> CD44<sup>hi</sup> γδ T cells respectively (15)). To go further, we have
179 now decided to study the T-cell trafficking dynamics of these subsets. We first inhibited the
180 entry of circulating lymphocytes into LNs through the injection of integrin-neutralizing
181 antibodies (18, 28, 29). More precisely, C57BL/6 mice were injected i.p. with anti-LFA-1
182 (αL) and anti-VLA-4 (α4) antibodies (Fig. 1A). We first verified that all γδ T cells from
183 peripheral LNs, mesenteric LNs and the spleen were expressing both LFA-1 and VLA-4
184 integrins (Fig. S1). The phenotype of γδ T cells remaining within LNs was analyzed 48 hours
185 after initiating the treatment (Fig. 1A). Interestingly, at that time-point, Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T
cells were greatly enriched within the γδ T cells remaining within peripheral LNs (pLN; Fig.
186 1B-C). This resulted from the fact that the great majority of Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells (~75%)
187 remained trapped within pLN whereas almost all the other γδ T cells have egressed from
188 them (Fig. 1D). Such a phenomenon could also be observed, to a lower extent, in mesenteric
189 LNs (mLN) as about 15% of the Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells were still present within this SLO
190 48 hours after the injection of integrin-neutralizing antibodies (Fig. 1C-D). Of note, in this
191 model, we observed a strong increase in the absolute numbers of all the different γδ T-cell
192 subsets in the spleen (Fig. 1D). This phenomenon could result from the accumulation in the
193 spleen of T lymphocytes egressing from LNs.
194
195 Narayan et al. (30) have previously showed that distinct cell types in the γδ T-cell lineage
196
197 could be identified on the basis of their TCR repertoire usage. Therefore, we then analyzed
198 the Vγ-chain repertoire of Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells remaining within pLN s and mLN s after
199 the injection of integrin-neutralizing antibodies (Fig. 1E). Although we noticed a decreased
200 proportion of Vγ1.1-expressing cells associated with an enrichment of Vγ2-expressing cells
201 within pLN s following treatment, such a discrepancy was not observed within mLN s,
202 indicating that Vγ-chain usage does not allow to precisely identify LN-trapped γδ T
203 lymphocytes.

204 Consistent with the fact that Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T lymphocytes are the only γδ T cells able to
205 produce IL-17 in response to stimulation (15), we observed, after treatment, within both
206 peripheral and mesenteric LNs, a clear and significant increase in the proportion of IL-17-
207 producing γδ T cells correlated with a diminished ability of the remaining γδ T cells to
208 produce IFNγ (Fig. 1F-G).

209 Because circulatory dynamics could differ between γδ T-cell subsets (i.e. Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T
210 cells could need more time to exit LNs than Ly-6C<sup>+</sup> CD44<sup>hi</sup> and CD44<sup>lo</sup> γδ T cells), we then
211 decided to extend to 7 days the duration of the treatment blocking T-cell entry into LNs from
212 the blood (Fig. 1H). In this setting, we still observed that whereas almost all Ly-6C<sup>+</sup> CD44<sup>hi</sup>
213 and CD44<sup>lo</sup> γδ T cells have left LNs, the great majority of Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells were still
214 trapped in pLN s and to a lesser extent in mLN s (Fig. 1I). Taken together, our data suggest that
215 contrary to the other γδ T-cell subsets, Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T lymphocytes from LNs are in part
216 non-circulating cells.

217 A significant proportion of Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T lymphocytes is long-term SLO-resident
218 cells in the steady state

219 To explain the results presented above, one may argue that Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells could
220 enter into LNs in a LFA-1- and VLA-4-independent manner. To address this issue, we then
examined the cell exchange rate of the various γδ T-cell subsets from SLOs (using CD45.1 and CD45.2 congenic markers) that occurred between parabiotic pairs (Fig. 2A). Four weeks following surgery, although both Ly-6C⁺ CD44⁺¹⁾ and CD44<sup>lo</sup> γδ T cells have achieved full chimerism in all studied SLOs, Ly-6C⁻ CD44<sup>hi</sup> lymphocytes showed only limited exchange between the parabionts (Fig. 2B). In line with a weak circulatory dynamic of Ly-6C⁻ CD44<sup>hi</sup> γδ T cells, we also observed a limited exchange of splenic IL-17-producing γδ T cells between parabiotic mice (Fig. 2C). To examine whether these results were stable over time, we performed longer-term parabiosis experiments (Fig. 2D). Eight weeks post-surgery, in all studied SLOs, Ly-6C⁻ CD44<sup>hi</sup> γδ T lymphocytes still showed limited exchange between the paired mice (Fig. 2E). Altogether, these findings suggest strongly that an important proportion of Ly-6C⁻ CD44<sup>hi</sup> γδ T lymphocytes corresponds to long-term SLO-resident cells in the steady state.

**Macrophages are responsible for the long-term trapping of Ly-6C⁻ CD44<sup>hi</sup> γδ T lymphocytes within SLOs in the steady state**

We then attempted to identify the cellular actors involved in the retention of Ly-6C⁻ CD44⁺¹⁾ γδ T cells within SLOs. We first studied the potential involvement of B cells in this process. B-cell-deficient or -proficient C57BL/6 mice were treated or not with integrin-neutralizing antibodies and analyzed 48 hours later (Fig. 3A). The retention rate (recovery) of Ly-6C⁻ CD44⁺¹⁾ γδ T cells in pLN s and mLN s was comparable in both mouse strains, 48 hours after blocking T-cell entry (Fig 3B). Thus, B lymphocytes do not seem to be involved in the trapping of Ly-6C⁻ CD44⁺¹⁾ γδ T cells within LNs.

We next assessed whether dendritic cells (DCs) played a role in the retention of this γδ T-cell subset in LNs. For this purpose, C57BL/6 CD11c.DTR mice (designated CD11c.DOG), that allow, following Diphtheria Toxin (DT) injections, a continuous *in vivo* depletion of DCs,
245 were injected or not with anti-αL and anti-α4 antibodies (Fig. 3C). We first assessed the 246 depletion efficiency of such a protocol (Fig. S2A-B). Depletion was quite efficient for 247 plasmacytoid DCs in all LNs and better for the other DC subsets in mLNs than in pLN 248 recovery of Ly-6C⁻CD44⁺⁻γδT cells in both pLN and mLNs 48 hours after blocking T-cell 249 entry into LNs was not diminished by DC depletion (Fig. 3D) suggesting that these latter cells 250 are not crucially involved in the retention of Ly-6C⁻CD44⁺⁻γδT cells within SLOs. 251

Finally, we examined whether macrophages could contribute to Ly-6C⁻CD44⁺⁻γδT-cell 252 retention within SLOs. To test this hypothesis, C57BL/6 mice were fed with mouse chow 253 containing an inhibitor of CSF1R (PLX3397, provided by Plexxikon Inc.) for 7 days (Fig. 254 3E). We first assessed the depletion efficiency of such a protocol (Fig. S1C). This protocol led 255 to the depletion of the majority of subcapsular (F4/80⁻CD169⁺) and medullary (F4/80⁺ 256 CD169⁺) sinus macrophages in both pLN and mLNs whereas F4/80⁺CD169⁻ macrophages 257 were only poorly affected especially in pLN. We found that the absolute numbers of Ly-6C⁻ 258 CD44⁺⁻γδT cells in pLN, mLNs and the spleen dropped dramatically in mice fed 7 days 259 with PLX3397 chow (Fig. 3F). Although the absolute numbers of the other γδT-cell subsets 260 were also affected, the observed decreases were far less pronounced (Fig. 3F). 261

We hypothesized that macrophages would be required for Ly-6C⁻CD44⁺⁻γδT-cell retention 262 within SLOs. In such a model, macrophage depletion would lead to the egress of Ly-6C⁻ 263 CD44⁺⁻γδT cells from SLOs and that would account for the significant drop in absolute 264 numbers observed in mice treated 7 days with PLX3397. The membrane receptor S1PR1, by 265 allowing T cells to respond to the sphingolipid S1P present within efferent lymph, is the 266 central mediator of lymphocyte egress from SLOs (31, 32). First, we studied S1PR1 267 expression by γδT-cell subsets recovered from pLN, mLNs and the spleen (Fig. S3). We 268 found that Ly-6C⁻CD44⁺⁻γδT cells from peripheral and mesenteric LNs exhibited higher
269 S1PR1 surface levels than the other γδ T-cell subsets. Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells should be thus
270 highly sensitive to S1P gradients. Then, C57BL/6 mice were fed with PLX3397 chow and
271 daily injected or not for 7 days with FTY720 (Fig. 3G). Interestingly, FTY720 injections fully
272 abrogated the strong decrease in Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T-cell absolute numbers provoked by the
273 PLX3397 treatment in both pLNsm and mLNsm (Fig. 3H). These results suggest that CSF1R
274 inhibitor administration is not toxic to γδ T cells but rather allow them to egress from LNs
275 through a S1PR1 dependent mechanism. Of note, FTY720 administration did not abolish the
276 effect of the PLX3397 treatment in the spleen but rather accentuated the observed decrease of
277 the absolute number of Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T-cells (Fig. 3G). That could be explained by the
278 duration of the S1PR1 agonist treatment. More precisely, it has already been described that
279 whereas a single injection of FTY720 induces T-cell sequestration in the spleen (33, 34), a
280 prolonged FTY720 treatment rather leads, in this SLO, to a decrease in the absolute numbers
281 of T lymphocytes (34, 35, 36).

282 Taken together, these last results suggest that macrophages play a crucial role in the long-term
283 trapping of Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T lymphocytes within SLOs.

284 **Membrane blebs reveal the close intimacy between Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T lymphocytes and**
285 **macrophages in SLOs**

286 It has recently been shown that an important fraction of IL-17-committed T lymphocytes from
287 pLNsm could acquire membrane blebs from subcapsular sinus macrophages, reflecting the
288 intimacy of the crosstalk between these 2 cell types (37). We thus examined the proportion of
289 Ly-6C<sup>-</sup> CD44<sup>hi</sup>, Ly-6C<sup>+</sup> CD44<sup>hi</sup> and CD44<sup>lo</sup> γδ T lymphocytes expressing macrophage markers
290 such as CD11b, F4/80 and CD169 (Fig. 4A-B). Expression of these 3 molecules was solely
291 detected on the cell surface of Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells. Interestingly, the surface expression
292 of CD11b, F4/80 and CD169 by Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T lymphocytes seemed to vary depending
293 on the location of these cells (pLNsvs mLNs vs the spleen; Fig. 4B) suggesting the ability of
294 Ly-6C⁻CD44⁺γδTlymphocytes to initiate intimate crosstalk with various macrophage
295 subsets such as subcapsular sinus macrophages (CD11b⁺F4/80⁻CD169⁺), medullary sinus
296 macrophages (CD11b⁺F4/80⁺CD169⁺) or spleen red pulp macrophages (CD11b⁻F4/80⁺
297 CD169⁻)(38, 39, 40). More precisely, the observed proportions of Ly-6C⁻CD44⁺γδT cells
298 acquiring CD11b, F4/80 and CD169 expression suggest that these cells interact preferentially
299 with subcapsular and medullary sinus macrophages within pLNs (30% of Ly-6C⁻CD44⁺γδT
300 cells from pLNsexpressed CD11b and CD169 whereas only 15% expressed F4/80), with
301 medullary sinus macrophages in mLNs [equal proportions (15% ) of cells from mLNs
302 expressing CD11b, F4/80 and CD169] and with red pulp macrophages in the spleen (Ly-6C⁻
303 CD44⁺γδTcells only expressed F4/80) (Fig. 4B and Fig. S4).

304 Using the PLX3397⁺ feeding strategy, we then studied the consequences of macrophage
depletion on the expression of macrophage markers by Ly-6C⁻CD44⁺γδTlymphocytes (Fig.
305 4C). Following PLX3397 treatment, there was a gradual decrease with time in the proportion
306 of SLO Ly-6C⁻CD44⁺γδTcells expressing CD11b, F4/80 or CD169 (Fig. 4D). These latter
307 results confirm that the expression of CD11b, F4/80 and CD169 by this γδT-cell subset
308 results from the transfer of membrane blebs from macrophages to γδTcells.
309

310 Altogether, our results suggest that Ly-6C⁻CD44⁺γδTlymphocytes initiate strong adhesive
311 interactions with macrophages that may lead to their long-term retention within SLOs.

312 RORγt expression allows to determine the localization of resident γδ T cells within pLNs
313 We have described above a clear enrichment in IL-17-producing cells among γδ T cells in
314 LNs, 48 hours after blocking T-cell entry into these SLOs (Fig. 1E-F). We thus hypothesized
315 that the expression of the transcription factor RORγt may allow a precise identification of
316 resident γδ T cells in SLOs. We first evaluated the proportion of Ly-6C⁻CD44⁺, Ly-6C⁺
317 CD44<sup>hi</sup> and CD44<sup>lo</sup> γδ T lymphocytes expressing RORγt in SLOs by using RORγt-GFP
318 transgenic mice (Fig. 5A-B). In agreement with the fact that only Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T
319 lymphocytes are able to produce IL-17 (15), we found that RORγt expression was restricted to
320 these cells. Surprisingly, the proportion of RORγt<sup>+</sup> cells within Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells
321 differed considerably depending on the organ studied. Indeed, whereas the vast majority
322 (~80%) of Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T lymphocytes recovered from pLNs expressed RORγt, this
323 proportion was quite lower in mLNs and the spleen (Fig. 5B). We then examined the
324 expression of RORγt by LN-resident γδ T lymphocytes 48 hours after blocking T-cell entry
325 (Fig. 5C-E). In pLNs, after treatment, the proportion of Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells expressing
326 RORγt was significantly higher than in pLNs from control mice (Fig. 5D). Accordingly, 48
327 hours after blocking T-cell entry, the recovery of RORγt<sup>+</sup> Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells was far
328 better than the recovery observed for their RORγt<sup>-</sup> cell counterparts (Fig. 5E). This was not
329 the case for mLNs as in this SLO, RORγt<sup>-</sup> Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells were more efficiently
330 retained after treatment than RORγt<sup>+</sup> Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells (Fig. 5D-E).

331 Altogether, these results suggest that the expression of RORγt can be considered as a reliable
332 marker to identify resident γδ T cells within pLNs (but not in mLNs). Thus, in order to study
333 the location of resident γδ T cells in pLNs, we imaged pLN slices recovered from RORγt-GFP
334 mice by immunofluorescence microscopy (Fig. 5F-H). In line with the capacity of Ly-6C-
335 CD44<sup>hi</sup> γδ T lymphocytes to acquire membrane blebs from subcapsular and medullary sinus
336 macrophages, we clearly observed that RORγt<sup>+</sup> γδ T cells (the majority of which are resident
337 cells within pLNs) were localized close to CD169<sup>+</sup> sinus macrophages (Fig. 5F-G).
338 Quantification of the distance between RORγt<sup>-</sup> or RORγt<sup>+</sup> γδ T cells and CD169<sup>+</sup> cells
339 confirmed that RORγt<sup>+</sup> γδ T cells were very significantly far closer to CD169<sup>+</sup> cells than their
340 RORγt<sup>-</sup> cell counterparts (Fig. 5H). Such a close vicinity may help explaining the capacity of
341 Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells to acquire macrophage-related surface markers and such interactions
342 may lead to the long-term retention of these cells within SLOs.

343 **Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T lymphocytes from SLOs respond rapidly in vivo to LPS challenge**

344 As described above, our results suggest that Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T lymphocytes initiate strong
345 adhesive interactions with macrophages that may lead to their long-term retention within
346 SLOs. In addition, we observed that resident RORγt<sup>+</sup> γδ T cells were localized close to
347 CD169<sup>+</sup> sinus macrophages within pLN s. We thus hypothesized that SLO resident γδ T cells
348 could represent sentinels surveying the pathogen-exposed surface of SLOs.

349 To address this issue, RORγt-GFP transgenic mice were injected i.v. with LPS. IL-17 and
350 IFNγ productions by Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells recovered from pLN s, mLN s and the spleen
351 were then analyzed 3 hours later (Fig. 5I). We observed that, in all SLOs, around 20% of Ly-
352 6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells were already producing IL-17 in response to LPS challenge at this early
353 time-point (Fig. 5J). In addition, we noticed that IL-17 production was restricted to RORγt
354 expressing cells (Fig. 5K). Of note, in the same setting, we detected a small proportion of
355 IFNγ-producing Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells in the spleen and this IFNγ production was
356 restricted to RORγt<sup>-</sup> cells (Fig. 5J-K). The capacity of Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells to rapidly
357 produce IL-17 upon LPS challenge strongly suggest that these SLO resident γδ T cells could
358 play a crucial surveying role to protect these organs from infections.
359

DISCUSSION

360

361

To achieve their surveying task for foreign antigens and survival signals, peripheral T cells have been described as having a nomadic existence between blood, SLOs and lymph in the steady state (16, 17). Transit times within SLOs should be (a) not too long as T cells must continuously scan antigen-presenting cells (APCs) from different LNs in order to find where the next pathogen will cross the epithelial barrier, but also (b) not too rapid to avoid missing the rare APCs presenting their specific antigen in SLOs in the early phases of an infection.

362

363

364

365

366

367

368

369

370

371

372

However, in parallel to that model, recent findings highlight the existence of T cells residing for long periods of time within non-lymphoid tissues (41). More precisely, subsets of CD8⁺ and CD4⁺ αβ T cells but also subsets of NK cells, NKT cells and γδ T cells have been shown to reside within non-lymphoid tissues such as the skin, gut, lungs or adipose tissue providing a rapid and efficient protection against reinfection by tissue-tropic pathogens but also promoting the repair of damaged tissues (42, 43, 44, 45, 46, 47).

373

374

375

376

377

378

379

380

381

382

383

384

In the present paper, using injection of integrin-neutralizing antibodies and parabiosis strategies, we highlight the existence of γδ T cells residing in the steady state in the SLOs of specific pathogen-free mice. In line with our results, Zhang et al. have recently shown that innate-like T cells defined as expressing high surface levels of CD127 and CCR6, among which about one fourth corresponds to γδ T cells, accumulated near the subcapsular sinus in pLN s (32). Here, we confirm and extend these observations. First, while Zhang et al. examined the amount of cell exchange 2 weeks after surgery in parabiotic mice or 2 days after T-cell photoconversion in photoconvertible fluorescence protein Kaede transgenic mice, our parabiosis experiments clearly showed that full chimerism between the 2 parabionts was still not reached in pLN s for Ly-6C⁺CD44⁺hi γδ T cells at least up to 2 months after surgery (Fig. 2). Thus, we here demonstrate the long-term residency of these cells within pLN s. Second, we showed that the long-term retention of Ly-6C⁺CD44⁺hi γδ T cells was not a peculiar feature of
385 pLNs as it also applies to mLNs and the spleen. Third, while we confirmed that in pLN5, 386 innate-like Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T lymphocytes were located close to subcapsular sinus 387 macrophages and were able to acquire, from this vicinity, the apparent expression of CD11b 388 and CD169, our results strongly suggest that other subsets of macrophages are involved in the 389 retention of these cells within SLOs (see below). Finally, we have also studied the behavior of 390 the other γδ T-cell subsets (i.e. Ly-6C<sup>+</sup> CD44<sup>hi</sup> and Ly-6C<sup>-/+</sup> CD44<sup>low</sup> γδ T cells) in SLOs and 391 showed that whatever their naïve-like or memory-like phenotype, they corresponded to 392 circulating cells. 393

393 In addition to the CD11b and CD169 markers, about half of CD11b-expressing innate-like γδ
394 T cells in pLN5 and all of them in mLNs were also acquiring F4/80 expression (Fig. 4B). Like
395 for CD11b and CD169, F4/80 surface expression by γδ T cells decreased gradually following
396 *in vivo* macrophage depletion suggesting that innate-like γδ T cells are not only interacting
397 with subcapsular (CD11b<sup>+</sup> F4/80<sup>-</sup> CD169<sup>+</sup>) sinus macrophages but also with medullary sinus
398 (CD11b<sup>+</sup> F4/80<sup>+</sup> CD169<sup>+</sup>) macrophages. Finally, our parabiosis experiments show that a
399 fraction of Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells are also residing for long periods of time in the spleen. In
400 this SLO, only F4/80<sup>-</sup> was detected at the cell surface of Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells. This latter
401 result suggests that resident γδ T lymphocytes are interacting with splenic red pulp (CD11b<sup>-</sup>
402 F4/80<sup>+</sup> CD169<sup>-</sup>) macrophages. Such a hypothesis would be in agreement with previous studies
403 that have observed a preferential localization of human and mice γδ T lymphocytes within the
404 red pulp of the spleen (48, 49). Interactions between tissue resident γδ T cells and F4/80<sup>+</sup>
405 macrophages have been reported in the lungs in mice (50). Indeed, it has been shown that
406 pulmonary resident γδ T cells had an intrinsic preference to interact with F4/80<sup>+</sup> macrophages
407 (51). These continuous interactions would be important for their protective role as sentinels of
408 airways and lung tissues.
Altogether, our data suggest that the precise "nature" of resident γδ T cells but also of the macrophages involved in their retention varies according to the secondary lymphoid organs analyzed (pLNs vs mLNs vs the spleen). Indeed, whereas in pLNs, the residence of Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells was mostly restricted to RORγt<sup>+</sup> cells, RORγt<sup>-</sup> cells were more efficiently trapped in mLNs than their RORγt<sup>+</sup> cell counterparts. Furthermore, whereas both subcapsular and medullary sinus macrophages would be involved in the retention of Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells in pLNs, the role of subcapsular sinus macrophages would be less crucial in mLNs and the spleen. Indeed, medullary sinus and red pulp macrophages appear as the main actors implied in the retention of γδ T cells in mLNs and the spleen respectively. Sinus and red pulp macrophages have a propensity to capture particulate material arriving in the lymph and the blood (52-54). Thus, we hypothesize that resident Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells could play a critical surveillance role in protecting SLOs from infections. In our previous study, we have showed that Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells were possessing innate-like properties such as the ability to respond directly to TLR ligands *in vitro* (15). Here, we have found that Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells from SLOs were rapidly able to produce IL-17 *in vivo* in response to LPS challenge. In this setting, LPS may activate them directly or indirectly through the induction of IL-23 and IL-1beta production by neighboring macrophages as it has been described that RORγt<sup>+</sup> γδ T cells express IL-23R and produce IL-17 in response to IL-1beta and IL-23, without T-cell receptor engagement (6). We are thus proposing that resident γδ T cells and macrophages could have complementary roles and may represent a major barrier blocking the systemic spread of pathogens within SLOs by providing rapid innate responses.
430 ACKNOWLEDGMENTS

431 We are grateful to Plexxikon Inc. for providing the PLX3397 inhibitor. We thank the Cochin

432 Cytometry and Immunobiology (CIBIO), Cochin imaging photonic (IMAG'IC) and Cochin

433 Animal Core facilities.

434
435

## FOOTNOTES

436

This work was supported by grants from la Ligue contre le cancer, the Association pour la

437

Recherche contre le Cancer, and the Fondation pour la Recherche Médicale. M.R. was

438

supported by a fellowship from Fondation pour la Recherche Médical. A. A. V. and P. H.

439

were supported by a PhD fellowship from the “Ligue contre le Cancer” and from INSERM.

440

V. G. was supported by a PhD fellowship from the French Ministry of National Education,

441

Research, and Technology. E.P. was supported by a fellowship from Fondation de France.
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594 **Figure 1: An important proportion of Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ<sup>+</sup> T lymphocytes is trapped in**
595 **LNs in the steady state**

596 C57BL/6 mice have been injected i.p. with integrin-neutralizing anti-LFA-1 (αL) and anti-
597 VLA-4 (α4) antibodies. Two days after treatment, pLNsmLNsspleen (Spl.) were
598 recovered separately. (A) Diagram illustrating the experimental model. (B) CD44/Ly-6C dot-
599 plots for gated TCRγδ<sup>+</sup> cells recovered from pLNs of representative control and treated mice.
600 Numbers on FACS dot-plots indicate the percentage of Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ<sup>+</sup> T cells among the
601 γδ<sup>+</sup> T lymphocyte compartment (C) Pie charts illustrating the proportions of γδ<sup>+</sup> T-cell subsets
602 recovered from pLNsmLNsspleen ( Spl.) of control (Ctrl.) and treated mice. (D)
603 Percentage of γδ<sup>+</sup> T cell-subset recovery within pLNsmLNsspleen ( Spl.) of anti-αL and
604 anti-α4 treated mice. The percentage of recovery was calculated by dividing the absolute
605 numbers in treated mice by the mean absolute number obtained in untreated animals. (E)
606 Percentage of Vγ1.1<sup>+</sup> and Vγ2<sup>+</sup> cells among Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ<sup>+</sup> T cells recovered from pLNsm
607 and mLNs of control (Ctrl.) and anti-αL and anti-α4 treated mice. (F) IL-17/IFNγ dot-plots
608 for gated TCRγδ<sup>+</sup> cells recovered from pLNs of representative control and treated mice. (G)
609 Proportion of IL-17- and IFNγ-producing TCRγδ<sup>+</sup> cells recovered from peripheral and
610 mesenteric LNs of control and treated mice. (H-I) C57BL/6 mice have been injected i.p. every
611 2 days during 6 days with 200μg of both integrin-neutralizing Abs and analyzed 24 hours
612 later. (H) Diagram illustrating the experimental model. (I) Percentage of γδ<sup>+</sup> T-cell subset
613 recovery within pLNsmLNsspleen ( Spl.) of 6 days treated mice.

614 (C, D, E, G, I) Results are shown as means±s.e.m. for at least two mice per group per
615 experiment, from at least three independent experiments and each point indicates an
616 individual mouse. Significance of differences between two series of results was assessed
617

using a two-tailed paired (D, H) or unpaired (C, F) Student's t-test. Values of P<0.05 were considered as statistically significant (*P<0.05; **P<0.01; ***P<0.001; and ****P<0.0001).

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**Figure 2: Ly-6C<sup>hi</sup> CD44<sup>hi</sup> γδ T lymphocytes are largely long-term SLO-resident cells in the steady state**

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Parabiotic mice were established between CD45.1 and CD45.2 C57BL/6 mice. Four weeks after surgery, parabiotic pairs were analyzed. (A) Diagram illustrating the experimental model. (B) Proportions of host cells, CD45.1⁺ for the CD45.1 parabiont and CD45.2⁺ for the CD45.2 parabiont, among the indicated γδ T-cell subsets recovered from pLN s, mLN s and spleen ( Spl.) from parabiotic pairs. (C) IL-17 and IFNγ dot-plots for gated TCRγδ⁺ cells recovered from spleen of representative parabiotic mice and percentage of host cells among these IL-17- and IFNγ-producing γδ T cells. (D-E) Parabiotic mice were established between CD45.1 and CD45.2 C57BL/6 mice and analyzed 8 weeks after surgery. (D) Diagram illustrating the experimental model. (E) Proportions of host cells among the indicated γδ T-cell subsets recovered from pLN s, mLN s and spleen ( Spl.) from parabiotic pairs. Results are shown as means±s.e.m. for at least three mice per group per experiment, from at least three independent experiments.

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**Figure 3: Macrophages are responsible for the long-term trapping of Ly-6C<sup>hi</sup> CD44<sup>hi</sup> γδ T lymphocytes within SLOs in the steady state**

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(A-B) C57BL/6 and C57BL/6 µMT mice have been treated or not with integrin-neutralizing antibodies and analyzed 48 hours later. (A) Diagram illustrating the experimental model. (B) Percentage of γδ T cell-subset recovery (calculated as in figure 1D) within pLN s and mLN s of anti-αL and anti-α4 treated C57BL/6 and C57BL/6 µMT mice. (C-D) C57BL/6 CD11c.DTR

638

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640
mice (designated CD11c.DOG) have been injected or not with integrin-neutralizing
antibodies and Diphtheria Toxin (DT). (C) Diagram illustrating the experimental model. (D)
Percentage of γδ T cell-subset recovery within pLNs and mLNs of anti-αL and anti-α4 treated
control (Ctrl.) and DT injected mice. (E-F) C57BL/6 mice were fed with mouse chow
containing or not an inhibitor of CSF1R (PLX3397). Seven days after PLX3397 feeding,
pLNs, mLNs and spleen were recovered separately. (E) Diagram illustrating the experimental
model. (F) Percentage of γδ T-cell subset recovery within pLNs, mLNs and spleen ( Spl.) of
PLX3397 fed mice. (G-H) C57BL/6 mice were fed with mouse chow containing or not
PLX3397 and daily injected or not with FTY720. Seven days after PLX3397 and FTY
treatments, pLNs, mLNs and spleen were recovered separately. (G) Diagram illustrating the
experimental model. (H) Absolute numbers of Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T cells recovered from
pLNs, mLNs and spleen of control (Ctrl.), PLX3397 and PLX3397+FTY720 treated mice. (B,
D, F, H) Results are shown as means±s.e.m. for at least three mice per group per experiment,
from at least two independent experiments.

655

<caption>656 Figure 4: Membrane blebs reveal the close intimacy between Ly-6C<sup>-</sup> CD44<sup>hi</sup> γδ T
lymphocytes and macrophage subsets in SLOs
657</caption>

(A) CD11b, F4/80 and CD169 fluorescence histograms of γδ T-cell subsets recovered from
pLNs are shown for a representative C57BL/6 mouse. Numbers in each histogram represent
the percentage of CD11b+, F4/80+ or CD169+ cells among γδ T-cell subsets. (B) The
proportions of CD11b+, F4/80+ or CD169+ cells among γδ T-cell subsets recovered from
pLNs, mLNs and spleen ( Spl.) of C57BL/6 mice were calculated. (C-D) C57BL/6 mice were
fed with mouse chow containing or not an inhibitor of CSF1R (PLX3397). Two, four and
seven days after PLX3397 feeding, pLNs, mLNs and spleen were recovered separately. (C)
Diagram illustrating the experimental model. (D) The proportions of CD11b+, F4/80+ or
666 CD169⁺ cells among Ly-6C⁻ CD44⁺ᵢᵢ γδ T cells recovered from pLN s, mLN s and spleen ( Spl.)
667 of C57BL/6 mice were calculated. Results are shown as means±s.e.m. for at least three mice
668 per group per experiment, from at least three independent experiments.
669

670 **Figure 5: RORγt expression allows to determine the location of resident γδ T cells**
671 **within pLN s**

672 (A) RORγt/TCRγδ dot-plots for gated γδ T-cell subsets recovered from pLN s of a
673 representative C57BL/6 RORγt-GFP mouse. (B) The proportions of RORγt⁺ cells among γδ
674 T-cell subsets recovered from pLN s, mLN s and spleen ( Spl.) of C57BL/6 RORγt-GFP mice
675 were calculated. (C-E) C57BL/6 RORγt-GFP mice have been injected i.p. with integrin-
676 neutralizing anti-LFA-1 (αL) and anti-VLA-4 (α4) antibodies. Two days after treatment,
677 pLN s and mLN s were recovered separately. (C) Diagram illustrating the experimental model.
678 (D) The proportions of RORγt⁺ cells among Ly-6C⁻ CD44⁺ᵢᵢ γδ T cells recovered from pLN s
679 and mLN s of treated or untreated mice were calculated. (E) Percentage of RORγt⁺ and RORγt⁻
680 Ly-6C⁻ CD44⁺ᵢᵢ γδ T-cell recovery (calculated as in figure 1) within pLN s and mLN s of anti-
681 αL and anti-α4 treated mice. Results are shown as means±s.e.m. for at least three mice per
group per experiment, from at least three independent experiments.
682

683 (F-H) Location of RORγt⁻ and RORγt⁺ γδ T cells within pLN s from C57BL/6 RORγt-GFP
684 mouse has been examined. (F) Representative immunofluorescence TCRγδ, CD169 and
685 RORγt-GFP staining of fixed pLN s slices (maxillary LN is shown here). RORγt⁺ γδ T cells
686 appear in yellow. Squares indicate zoomed areas presented on the figure. (G) Location
687 modeling of RORγt⁻ and RORγt⁺ γδ T lymphocytes within pLN s (maxillary LN is shown
688 here). Coordinates of all RORγt⁻ and RORγt⁺ γδ T cells were calculated from
689 immunofluorescence staining shown in (F) and were then re-applied on CD169 stained LN
slice. (H) Distance between RORγt⁻ and RORγt⁺ γδ T lymphocytes and CD169⁺ cells were

calculated. (I, J, K) C57BL/6 RORγt-GFP mice were injected i.v. with LPS. Three hours later,

cells recovered from pLN~, mLNs and spleen were incubated during 2 hours with Brefeldin A

for determination of intracellular cytokine production. (I) Diagram illustrating the

experimental model. (J) Percentage of IL-17- and IFNγ-producing cells among Ly-6C⁺CD44⁺

γδ T cells recovered from pLN~, mLNs and spleen of control (Ctrl.) and LPS treated mice.

(K) Percentage of IL-17- and IFNγ-producing cells among RORγt⁻ and RORγt⁺ Ly-6C⁺CD44⁺

γδ T cells recovered from pLN~, mLNs and spleen of LPS treated mice. Results are shown as

means±s.e.m. for at least three different LNs slices from three different mice.

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<figure><img src="image_3.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_11.png" /></figure>

Figure 1

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<figure><img src="image_12.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_16.png" /></figure>

Figure 2

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A

Ab injection

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

B

<figure><img src="image_19.png" /></figure>

E

<figure><img src="image_20.png" /></figure>

F

<figure><img src="image_21.png" /></figure>

C

<figure><img src="image_22.png" /></figure>

D

<figure><img src="image_23.png" /></figure>

G

<figure><img src="image_24.png" /></figure>

H

<figure><img src="image_25.png" /><figcaption>Figure 3</figcaption></figure>

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711
<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /></figure>

Figure 4

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<figure><img src="image_30.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_32.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_33.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_34.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_35.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_36.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_37.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_38.png" /><figcaption>J</figcaption></figure>

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /><figcaption>K</figcaption></figure>

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /><figcaption>Figure 5</figcaption></figure>