The transcription factor SpiB regulates the fibroblastic reticular cell network and CD8+ T‐cell responses in lymph nodes

Fibroblastic reticular cells (FRCs) construct microanatomical niches that support lymph node (LN) homeostasis and coordination of immune responses. Transcription factors regulating the functionality of FRCs remain poorly understood. Here, we investigated the role of the transcription factor SpiB that is expressed in LN FRCs. Conditional ablation of SpiB in FRCs impaired the FRC network in the T‐cell zone of LNs, leading to reduced numbers of FRCs and altered homeostatic functions including reduced CCL21 and interleukin‐7 expression. The size and cellularity of LNs remained intact in the absence of SpiB but the space between the reticular network increased, indicating that although FRCs were reduced in number they stretched to maintain network integrity. Following virus infection, antiviral CD8+ T‐cell responses were impaired, suggesting a role for SpiB expression in FRCs in orchestrating immune responses. Together, our findings reveal a new role for SpiB as an important regulator of FRC functions and immunity in LNs.


INTRODUCTION
Lymph nodes (LNs) are secondary lymphoid organs that form a network of tissues designed as a filtration and surveillance system.The microarchitecture of LNs is organized into distinct compartments with the primary goal of capturing and presenting antigens from peripheral tissues to cells of the immune system, generating immune responses. 1Several types of nonhematopoietic stromal cells support the lymphoid architecture and function by constructing networks and defining compartments.CD31 + lymphatic and blood endothelial cells build the vasculature of LNs required for the entry and exit of immune cells.Contractile pericytes expressing the adhesion molecule CD146 are also found in association with blood vessels.Fibroblastic reticular cells (FRCs) are the most prominent stromal cell population in LNs that form an interconnected cellular network that supports immune cell migration. 2In addition, FRCs create a conduit system composed of extracellular matrix components and reticular fibers that facilitates the transport of lymph-derived antigens and signaling molecules, assisting in the induction of immune responses. 3Inflammation transcriptionally reprograms FRCs toward immune-related pathways that further support ongoing immune responses, [4][5][6] yet the role of FRC-specific transcription factors in controlling their function is unexplored.
Fibroblastic reticular cells comprise several subsets based on their intranodal location, markers and functions that all express podoplanin (PDPN).This heterogeneity in FRCs creates anatomical niches that support the homeostasis and support of immune responses in LNs. 7][10] B-cell zone reticular cells (BRCs) include follicular dendritic cells (FDCs) and other CXCL13expressing reticular cells that define this compartment and support humoral responses. 11FDCs can arise from the differentiation of MRCs. 12Within the T-cell zone, reticular cells [i.e.T-zone reticular cells (TRCs)] express the chemokines CCL19 and CCL21 that promote the attraction and retention of T cells and DCs that express CCR7 and produce cytokines such as interleukin (IL)-7 that are critical for T-cell survival. 13,14In addition, MRCs, BRCs and TRCs are all characterized by the high expression of the bone marrow stromal cell antigen-1, or CD157.Conversely, in the medulla, CD157 low FRCs (medRC) were recently shown to support plasma cells by providing IL-6. 7,15Finally, adventitial reticular cells (ARCs) that express CD34 and Ly6C form a specific niche by surrounding blood vessels and may function as precursors of adult FRCs. 16,17N FRCs develop from fibroblast activation protein-a + lymphoid tissue organizer cells of mesenchymal origin. 18heir development requires sequential differentiation and maturation steps that are not fully understood.0][21][22] Deficiency in these pathways results in a reduction in the cellularity of FRCs and these immature FRCs harbor lower expression of the homeostatic chemokines CCL19, CCL21 and CXCL13 as well as reduction in IL-7.Interestingly, immature FRCs still produce reticular fibers and a functional conduit network in LNs, but the overall size and cellularity of the tissue are decreased. 19,20These defects in the FRC network also resulted in impaired CD8 + T-cell responses during viral infections, indicating that the generation of optimal immune responses require a functional and healthy FRC network. 19,20Surprisingly, despite the diversity of signaling pathways that regulate FRC function, little is known about how transcription factors engage in controlling FRC biology.
We previously identified the transcription factor SpiB as a regulator of FRC maturation in the spleen. 17,23Here, we investigated the role of SpiB in LN homeostasis and immune responses.We found that SpiB expression is conserved in LN FRCs and conditional ablation of SpiB expression impacted the cellularity and functionality of the FRC network in the T-cell zone at steady state.Deletion of SpiB in FRCs did not affect LN tissue size or homeostasis of immune cells but impacted T-cell priming following viral infection.These data indicate conserved function of the transcription factor SpiB in lymphoid organ FRCs for the induction of pathogen-specific T-cell responses.

RESULTS
The transcription factor SpiB is expressed by lymph node FRCs We previously identified a role for SpiB in FRCs in the spleen. 17We first sought to determine whether SpiB expression was conserved in LNs.Integration of single cell RNA-sequencing data sets analyzing FRCs from both spleen and LNs revealed expression of Spib messenger RNA in FRCs from LN B and T-cell zones as well as medullary reticular cells (medRCs) but low to no expression in LN ARCs or pericytes (Supplementary figure 1a).BRCs had higher expression of Spib compared with TRCs.We then used SpiB-tdTomato reporter mice to examine expression in LN stromal cells by flow cytometry and observed that FRCs and lymphatic endothelial cells had the highest levels of tdTomato while blood endothelial cells and pericytes had low expression (Figure 1a, b).We then used a new gating strategy to further define FRC subsets in LNs.We identified CD21/35 + FDC, MadCAM1 + MRCs, CD157 + TRC, Ly6C + CD157 À ARCs and Ly6C À CD157 À medRCs (Figure 1c). 15,17,24Flow cytometry analysis revealed that FDCs and MRCs had the highest expression of tdTomato followed by TRC, while ARC and medRC had low levels of tdTomato (Figure 1d).

The transcription factor SpiB supports FRC homeostasis
To investigate a role for SpiB in LN FRCs, we used Ccl19-Cre/SpiB flox/flox mice (SpiB DCCL19 ) as previously described 17 and confirmed the absence of Spib in sorted populations of TRCs, ARCs and medRCs by qPCR (Supplementary figure 1b).We observed a significant reduction in the numbers of FRCs, lymphatic endothelial cells and blood endothelial cells but not of pericytes in the LN of SpiB DCCL19 mice (Figure 2a).The reduction in lymphatic endothelial cells and blood endothelial cells may reflect a bystander effect because endothelial cells are not targeted in CCL19-Cre mice. 20,25Among FRC subsets, TRCs and MRCs were reduced in cellularity in SpiB DCCL19 mice, but FDCs, ARCs and medRCs were not changed (Figure 2b).Thus, expression of SpiB was required for maintenance of the stromal cell networks that support defined areas of LNs.
We previously showed that in the absence of SpiB, splenic TRCs had increased expression of genes expressed by ARCs, notably the stem marker CD34, and decreased expression of mature TRC markers, including Il7, Ccl19 and Grem1, which indicated a role for SpiB in supporting the differentiation of spleen TRCs from adventitial precursor cells. 17We did not observe changes in Ccl19 expression in SpiB-deficient LN TRCs (Supplementary figure 1c); however, the expression of the homeostatic cytokine Il7 was significantly reduced in FRC subsets and Grem1 expression was reduced in TRCs (Figure 2c).Expression of Cd34 remained low in TRCs and expression of the immature genes Cspg4 and Mfge8 were not changed in the absence of SpiB (Supplementary figure 1c).This suggests that SpiB likely regulates distinct aspects of the differentiation of FRCs in LNs.We then asked whether SpiB regulates the expression of other canonical markers in FRCs, including the proteins PDPN, CD157, VCAM-1 and CD140a (PDGFRa).We observed small changes in expression of these markers in MRCs in the absence of SpiB and no differences among other LN FRC subsets except for CD140a that was also slightly reduced in LN TRCs (Supplementary figure 1d).In addition, SpiB DCCL19 FRCs demonstrated normal expression of the chemokines Ccl2, Ccl7, Cxcl9, Cxcl10, Cxcl12, Cxcl13 and the alarmin Il33, except for a reduction of Ccl7 expression in TRCs and Cxcl13 in medRC (Supplementary figure 1e).We observed a significant reduction in intracellular CCL21 expression in SpiB DCCL19 FRCs, particularly within TRCs, and no changes in ARCs and medRCs (Figure 2d; Supplementary figure 1f).The absence of SpiB also did not affect CCL21 expression in endothelial cells or pericytes (Figure 2d).Histological examination showed no difference in CCL21 deposition in the T-cell zone of SpiB DCCL19 mice (Figure 2e), possibly because of accumulation of the chemokine on the reticular network via heparan sulfate binding. 26LN FRCs that surround high endothelial venules and lymphatic endothelial cells also predominantly express the leptin receptor (LepR), which might promote FRC survival and functions. 27We found that Lepr was reduced in TRCs in the absence of SpiB and confirmed the decrease of LepR expression by flow cytometry in SpiB-deficient TRCs (Figure 2f, g).Overall, these data show that SpiB regulates the maintenance of TRCs and MRCs in LNs and regulates discreet components of FRC function.

Normal lymph node architecture in mice lacking SpiB in FRCs
As FRCs play crucial roles in regulating LN homeostasis, we investigated whether the reduction in numbers of TRCs and altered functionality impacted immune cell cellularity and the LN architecture.The loss of SpiB in FRCs resulted in a small but nonsignificant decrease in total LN cellularity, composed of small but nonsignificant changes in the numbers of B cells, CD4 + and CD8 + T cells (Figure 3a; Supplementary figure 2a).We observed a small but nonsignificant reduction in CD44 À CD62L + na€ ıve and CD44 + CD62L + central memory CD8 + T cells in the LN of SpiB DCCL19 mice (Supplementary figure 2a, b).We also found a nonsignificant decrease in na€ ıve CD44 À CD62L + CD4 + T-cell cellularity but not CD44 + CD62L À activated CD4 + T cells, corresponding to a small but significant reduction in the percentage of na€ ıve CD4 + T cells and an increase in activated CD4 + T cells in SpiB DCCL19 mice (Supplementary figure 2a, b).Among B cells, IgM + and IgD + subsets were unchanged in SpiB DCCL19 mice (Supplementary figure 2a, b).Other immune cells including NK and NK-T cells, monocytes, neutrophils and dendritic cell subsets, apart from plasmacytoid dendritic cells, were not altered in the LNs of SpiB DCCL19 mice (Figure 3a; Supplementary figure 2a, c).
The gross architecture and total surface area of LNs remained unchanged in SpiB DCCL19 mice (Figure 3b, c; Supplementary figure 2d).The absence of SpiB expression in FRCs also did not affect the organization or size of the LN T-cell zone, B-cell follicles or medulla (Figure 3c).However, a closer examination of the PDPN + TRC network in the T-cell zone revealed that the TRC network was less dense in SpiB DCCL19 mice (Figure 3d).Quantification of the spacing between FRCs in the T-cell zone network using gap analysis confirmed that the space between the reticular network fibers was increased in the absence of SpiB (Figure 3e).The reduction in FRCs and lymphocyte cellularity resulted in maintenance of the ratio of T cells to TRCs (Figure 3f).Thus, increased spacing between TRCs in LNs from SpiB DCCL19 mice occurred in the absence of a reduction in tissue size, suggesting that the FRC network stretched to maintain T-cell homeostasis.

SpiB expression enables FRC to regulate T-cell immunity
Having identified a role for SpiB in regulating the TRC function and network properties at homeostasis, we then investigated whether SpiB expression in FRCs supports immune responses.For this, we labeled with CellTrace Violet gBT-I CD8 + T cells specific for a herpes simplex virus (HSV) glycoprotein B epitope and transferred them into SpiB DCCL19 and control mice followed by subcutaneous HSV-1 KOS infection (Figure 4a).We tracked CD45.1 + gBT-I CD8 + T cells in the draining popliteal LNs of infected mice (Figure 4b).Proliferation of gBT-I CD8 + T cells 3 days after infection was diminished in SpiB DCCL19 mice, reflected by a lower average number of divisions (proliferation index) and reduced fold expansion (replication index) among dividing cells (Figure 4b, c).Accumulation of divided gBT-I CD8 + T cells required SpiB expression in FRCs (Figure 4d), yet upregulation of the activation markers CD69 and CD25 by gBT-I cells was unaffected, suggesting normal differentiation of the CD8 + T cells that entered division (Figure 4e).
We then explored whether the reduced early proliferation of gBT-I cells would further impact their effector functions.For this, we similarly tracked and analyzed the differentiation of gBT-I cells into short-lived effector cells and memory precursor effector cells based on the expression of KLRG1 and the IL-7R, respectively, in the draining popliteal LNs of infected mice 8 days after infection (Figure 4f, g).Although total numbers of CD8 + T, CD4 + T and B cells were similar in LNs of SpiB DCCL19 mice when compared with littermate control mice, we observed a significant reduction in numbers of virus-specific gBT-I CD8 + T cells (Figure 4h).The differentiation of gBT-I cells into short-lived effector cells and memory precursor effector cells was not affected (Figure 4i), and induction of GL7 + CD38 À germinal center B cells, CD138 + antibody-secreting cells and CXCR5 + PD-I + T FH cells was not altered in the LNs of HSV-1-infected SpiB DCCL19 mice (Supplementary figure 3a, b).This suggested that SpiB expression in FRCs preferentially supported CD8 + T-cell responses.To confirm this, we infected mice with lymphocytic choriomeningitis virus and examined cellularity in inguinal LNs.Expansion of endogenous CD4 + and CD8 + T cells and virus-specific transgenic P14 CD8 + T cells was reduced in SpiB DCCL19 mice, but differentiation of effector CD8 + T cells was not altered (Supplementary figure 2c, d).
Finally, we observed significantly fewer TRCs in SpiB DCCL19 mice during the course of HSV-1 infection that resulted in a significant increase in the ratio of total T cells to TRCs in LNs (Figure 4j; Supplementary figure 3e).The decrease in the TRC network was likely not because of a defect in their proliferation as we did not observe changes in Ki-67 expression in SpiB DCCL19 mice, nor an increase in apoptosis because FRCs had high expression of the prosurvival protein BCL-2 (Supplementary figure 3f, g).The expansion and organization of the LN T-cell zone, B-cell follicles or medulla were also not affected by the absence of SpiB (Supplementary figure 3h, i).However, analysis of the PDPN + TRC network revealed that the increase of space between the reticular network was maintained in the absence of SpiB following HSV infection (Figure 4k, l).These results suggest that SpiB expression in FRCs is critical for the expansion of the FRC network that parallels and regulates the early priming and proliferation of CD8 + T cells during viral infection.Together, our findings expand our understanding of the role of SpiB in regulating the LN FRC network and a crucial role in supporting the induction of CD8 + T-cell responses.

DISCUSSION
This study identified the transcription factor SpiB as a regulator of FRC homeostasis and functionality in LNs.We found that in the absence of SpiB, FRCs were decreased in cellularity and key homeostatic factors expressed by FRCs such as CCL21 and Il7 were reduced.However, we found that the size and architecture of LNs were not affected.In addition, we found that the absence of SpiB expression in FRCs did not affect the expression of canonical FRC markers such as PDPN, CD140a, BST1 and VCAM1, or the expression of other chemokines, including Ccl19, suggesting normal differentiation of FRCs from precursor cells.0][21] In these models, homeostatic chemokine expression such as CCL21 and CCL19 was also strongly reduced in FRCs that impacted the recruitment of immune cells, leading to smaller LN size and deformed architecture, notably the delineation of the T-and B-cell compartments.We found that even though CCL21 expression was reduced in FRCs in the absence of SpiB, our histological examination of LNs showed normal CCL21 accumulation on the reticular network, suggesting that reduced chemokine expression in FRCs might be compensated from other sources such as endothelial cells, which are a source of the chemokine in lymphoid tissues. 5,14In addition, intact expression of Ccl19 in our model might support the recruitment and positioning of lymphocytes within LNs and its normal compartmentalization.
Despite normal compartmentalization of the LN, we observed that the space between the reticular network fibers within the T-cell zone of LNs was increased in the absence of SpiB expression.This implies that the FRC network has remodeled to maintain the size of the LN and ratio with T cells in the steady state.Under basal conditions, FRCs maintain tension via PDPN expression that facilitates the contraction of the actomyosin cytoskeleton 28,29 and this contraction of the FRC network regulates the stiffness of the LN as well as their size.During the first few days of an immune response, CLEC-2 expression from mature dendritic cells engages and blocks PDPN functions and downstream signaling to relax the actomyosin cytoskeleton and induce the stretching of FRCs. 28,291][32] We did not find that SpiB regulates PDPN expression in FRCs, suggesting that additional molecules involved in FRC contractility should be investigated in our model.A recent report identified that the PDPN-binding partner surface proteins CD44 and CD9 suppress PDPN functions and the contractility of FRCs. 33Whether SpiB regulates expression of both CD44 and CD9 or regulates the tension of the TRC network remains to be addressed.
Our data identified that SpiB expression in FRCs is required for the optimal activation of na€ ıve CD8 + T cells during viral infection.In the absence of SpiB, the early proliferation of antiviral CD8 + T cells was delayed.This corroborates our previous findings where we identified a role for SpiB expression in spleen FRCs in regulating CD8 T-cell responses to acute and chronic systemic viral infection. 17Previous studies also identified a defect in CD8 + T-cell responses when the FRC network was impaired in the absence of the lymphotoxin beta signaling pathway or the expression of YAP/TAZ in FRCs. 19,20In these two studies, the decrease of FRC as well as reduction in homeostatic chemokine expression resulted in a paucity of immune cells in LNs, and disorganized T/B segregation that likely contributed to the reduced T-cell response.Similarly, gradual depletion of the CCL19 + FRC network revealed that the topology of the network can accommodate up to 50% decrease before affecting immune cell recruitment to LNs, intranodal cell motility or priming of CD8 + T cells. 34Here we found that deletion of SpiB expression in FRCs led to an approximate 50% reduction in TRC numbers and altered spacing of the TRC network at steady state.This disadvantage persisted during virus infection, resulting in an increased ratio of T cells to TRCs, suggesting that T cells might make fewer contacts with FRCs as the LN expands.This could, at least in part, explain impaired T-cell priming following virus infection of SpiB DCCL19 mice.However, we do not rule out additional functional programming of TRCs by SpiB.In support of this, we found reduced expression of CCL21, Il7 and LepR in SpiBdeficient TRCs.Two recent studies found that FRC numbers were reduced in LNs from mice lacking LepR. 27,35uture studies would need to uncouple the role of SpiB in regulating FRC cellularity and FRC cellular physiology during inflammation to determine factors that influence immune responses beyond providing the framework required for optimal T-DC interactions.Interestingly, SpiB is also expressed in human FDCs and TRCs, suggesting potential conserved functions across species. 36In summary, our study establishes a role for the transcription factor SpiB in regulating distinct LN FRC functions and provides new insights into how the FRC network supports immune responses.

METHODS Mice
C57BL/6, CCL19-Cre, SpiB flox/flox , gBT-I3B6.SJL-PtprcaPep3b/ BoyJ (gBT-I.CD45.1)and P143B6.SJL-PtprcaPep3b/BoyJ (P14.CD45.1)mice were bred in the Doherty Institute.SpiB-tdTomato were bred at the Walter and Eliza Hall Institute of Medical Research.SpiB DCCL19 mice were generated by crossing CCL19-Cre and SpiB flox/flox mice.Animal experiments were approved by the University of Melbourne Animal Ethics Committee.Mice were maintained under specific pathogen-free conditions and housed in individually ventilated cages.All mice were sex-and age-matched, and both female and male mice were used between 8 and 14 weeks of age.

Figure 1 .
Figure 1.LN FRCs express the transcription factor SpiB. (a) Gating strategy to identify LN stromal cells by flow cytometry.Hematopoietic cells were excluded, and stromal cells were identified with the markers PDPN, CD31 and CD146.(b) Flow cytometry of LN stromal cell subsets in SpiB-TdTomato mice.Representative histograms of SpiB expression (left) and pooled data (means AE standard error of the mean) from eight mice combined from three independent experiments.Dotted line represents baseline fluorescence from WT control.(c) Gating strategy to identify LN FRC subsets by flow cytometry.(d) Flow cytometry of LN stromal cell subsets in SpiB-TdTomato mice.Representative histograms of SpiB expression (left) and pooled data (means AE standard error of the mean) from eight mice combined from three independent experiments.Dotted line represents baseline fluorescence from WT control.*P < 0.05, **P < 0.01, ****P < 0.0001.ns, nonsignificant, by analysis of variance with Tukey's multiple comparisons test (b, d).ARC, adventitial reticular cell; BEC, blood endothelial cell; FDC, follicular dendritic cell; FRC, fibroblastic reticular cell; LEC, lymphatic endothelial cell; LN, lymph node; MedRC, medullary reticular cell; MRC, marginal reticular cell; PDPN, podoplanin; TRC, T-zone reticular cell; WT, wild type.

Figure 2 .
Figure 2. The transcription factor SpiB controls the T-zone reticular cell network and functionality.(a, b) Enumeration of LN stromal cells and FRC subsets from control SpiB flox/flox and SpiB DCCL19 mice by flow cytometry.Graphs show pooled data (means AE standard error of the mean) from two independent experiments with eight mice per group.(c) Analysis of Il7 and Grem1 expression in LN-sorted TRCs, ARC and medRC of control SpiB flox/flox and SpiB DCCL19 mice by qPCR.n = 6 mice from three independent sorts.(d) Flow cytometry analysis and representative histograms (left) of intracellular CCL21 expression in FRCs from control SpiB flox/flox and SpiB DCCL19 mice.Fluorescence minus one staining is shown by the histogram with a dotted line and used to discriminate CCL21 + from CCL21 À cells.Percentage (right) of CCL21 + stromal cell subsets and TRCs in the LNs of control SpiB flox/flox and SpiB DCCL19 mice.Graphs show pooled data (means AE standard error of the mean) from two independent experiments with seven mice per group.(e) Skin-draining LN sections from control SpiB flox/flox and SpiB DCCL19 mice were stained for PDPN and CCL21 and analyzed by confocal microscopy, and the area of CCL21 was quantified in the T-cell zone of LN.Graph shows pooled data (means AE standard error of the mean) from two independent experiments with five and six mice per group.Scale bar, 50 lm.(f) Analysis of Lepr expression in LN-sorted TRCs, ARC and medRC of control SpiB flox/flox and SpiB DCCL19 mice by qPCR.n = 6 mice from three independent sorts.(g) Flow cytometry analysis of LepR expression in TRCs from control SpiB flox/flox and SpiB DCCL19 mice.Left: representative histograms of LepR staining with fluorescence minus one staining shown as dotted line and used to discriminate LepR + from LepR À cells.Right: graphs show the percentage of LepR + TRCs in LNs and the mean fluorescence intensity (GeoMean) of LepR in TRCs.Data are representative of one experiment out of two, with three mice per group.*P < 0.05, **P < 0.01, ns, nonsignificant, by the unpaired two-tailed t-test (a, b, g) and the Mann-Whitney U-test (c-f).ARC, adventitial reticular cell; BEC, blood endothelial cell; FRC, fibroblastic reticular cell; LEC, lymphatic endothelial cell; LN, lymph node; MRC, marginal reticular cell; PDPN, podoplanin; qPCR, quantitative polymerase chain reaction; TRC, T-zone reticular cell.

Figure 3 .
Figure 3. SpiB deletion in FRC does not impact immune cell homeostasis or LN architecture.(a) Enumeration of immune cells from control SpiB flox/flox and SpiB DCCL19 mice by flow cytometry.Graphs show pooled data (means AE standard error of the mean) from two independent experiments with seven and eight mice per group.(b) Skin-draining LN sections from control SpiB flox/flox and SpiB DCCL19 mice were stained for Lyve-1, CD31, PDPN, CD3 and B220 and analyzed by confocal microscopy.Left images display the merged staining and right images the merged staining of CD3 and B220 only.Scale bar, 500 lm.(c) Quantification of the total LN size area (left graph) and LN compartment areas (right graph).Graphs show pooled data (means AE standard error of the mean) from two independent experiments with five mice per group.(d, e) TRC gap analysis on LN sections from control SpiB flox/flox and SpiB DCCL19 mice.Sections were stained for PDPN (left) and converted to threshold images (middle) and overlayed with circles for gap analysis (right).Graph shows the quantification of circle radius.Each point represents a circle radius and data are pooled from two independent experiments with five mice per group.Scale bar, 50 lm.(f) Ratio analysis of total T cells versus TRCs in control SpiB flox/flox and SpiB DCCL19 mice.Graph shows pooled data (means AE standard error of the mean) from two independent experiments with eight mice per group *P < 0.05, ****P < 0.0001, ns, nonsignificant, by the Mann-Whitney U-test (a, c, e, f).cDC, conventional dendritic cell; FRC, fibroblastic reticular cell; LN, lymph node; NK, natural killer; pDC, plasmacytoid dendritic cell; PDPN, podoplanin; TRC, T-zone reticular cell.

Figure 4 .
Figure 4. SpiB expression in FRC regulates T-cell responses during viral infection.(a) Experimental schematic of HSV-1 infection for T-cell priming.Mice were injected with 1.5 9 10 6 CTV-labeled gBT-I cells and 24 h later infected subcutaneously in the footpad with HSV-1.Draining popliteal LNs were analyzed 3 days after infection.(b) Flow cytometry analysis of CD45.1 + CD8 + gBT-I cells and representative histograms of CTV-labeled gBT-I cells in the draining popliteal LN of control SpiB flox/flox and SpiB DCCL19 mice, 3 days after subcutaneous HSV-1 infection.Dotted histogram represents CTV staining on gBT-I cells from na€ ıve mice used to gate on divided gBT-I cells.(c-e) Quantification of gBT-I cell expansion and activation.(c) The left graph shows the replication index (calculated as the total number of divided cells/cells that went into division) and the right graph shows the proliferation index (calculated as the total number of divisions/cells that went into division) of gBT-I cells in the draining popliteal LN of control SpiB flox/flox and SpiB DCCL19 mice.(d) The graph shows the enumeration of divided gBT-I cells, 3 days after subcutaneous HSV-1 infection.(e) Quantification of the percentage of CD25 + and CD69 + gBT-I cells.Graphs show pooled data (means AE standard error of the mean) from two independent experiments with nine and eight mice per group.(f) Experimental schematic of HSV-1 infection.Mice were injected with 5 9 10 4 gBT-I cells and 24 h later infected subcutaneously in the footpad with HSV-1.Draining popliteal LNs were analyzed 8 days after infection.(g-i) Flow cytometry analysis of CD45.1 + gBT-I cells in the draining popliteal LN, 8 days after subcutaneous HSV-1 infection.(g) Shortlived effector cells (SLECs) and memory precursor effector cells (MPECs) gBT-I cells were identified as KLRG1 + and IL-7R + , respectively.Enumeration of (h) lymphocytes and gBT-I cells and (i) gBT-I cell differentiation in the draining popliteal LN of control SpiB flox/flox and SpiB DCCL19 mice, 8 days after subcutaneous HSV-1 infection.(j) Ratio analysis of total T cells versus TRCs in control SpiB flox/flox and SpiB DCCL19 mice in the draining popliteal LN, 8 days after subcutaneous HSV-1 infection.Graphs (h, j) show pooled data (means AE standard error of the mean) from two independent experiments with seven and nine mice per group.(k, l) TRC gap analysis on LN sections from control SpiB flox/flox and SpiB DCCL19 mice at day 8 post-HSV infection.The graph shows the quantification of circle radius.Each point represents a circle radius and data are pooled from two independent experiments with five mice per group.Scale bar, 50 lm.*P < 0.05, **P < 0.01, ****P < 0.0001, ns, nonsignificant, by the unpaired two-tailed t-test (c-e, l) or the Mann-Whitney U-test (h-j).CTV, CellTrace Violet; FRC, fibroblastic reticular cell; HSV, herpes simplex virus; IL, interleukin; LN, lymph node; MPEC, memory precursor effector cell; PDPN, podoplanin; pLN, popliteal lymph node; SLEC, shortlived effector cell; TRC, T-zone reticular cell.