Abstract
Tissue-resident γδ intraepithelial lymphocytes (IELs) orchestrate
innate and adaptive immune responses to maintain intestinal epithelial
barrier integrity. Epithelia-specific butyrophilin-like (Btnl)
molecules induce perinatal development of distinct Vγ TCR^+ IELs,
however, the mechanisms that control γδ IEL maintenance within discrete
intestinal segments are unclear. Here, we show that Btnl2 suppressed
homeostatic proliferation of γδ IELs preferentially in the ileum. High
throughput transcriptomic characterization of site-specific Btnl2-KO γδ
IELs reveals that Btnl2 regulated the antimicrobial response module of
ileal γδ IELs. Btnl2 deficiency shapes the TCR specificities and TCRγ/δ
repertoire diversity of ileal γδ IELs. During DSS-induced colitis,
Btnl2-KO mice exhibit increased inflammation and delayed mucosal repair
in the colon. Collectively, these data suggest that Btnl2 fine-tunes γδ
IEL frequencies and TCR specificities in response to site-specific
homeostatic and inflammatory cues. Hence, Btnl-mediated targeting of γδ
IEL development and maintenance may help dissect their immunological
functions in intestinal diseases with segment-specific manifestations.
Subject terms: Mucosal immunology, Innate immunity
__________________________________________________________________
Panea et al showed that epithelia-specific butyrophilinlike 2 (Btnl2)
suppressed homeostatic proliferation of γδ intraepithelial lymphocytes
(IELs) preferentially in the ileum and used high throughput
transcriptomic characterization of Btnl2-deficient γδ IELs to
demonstrate that Btnl2 impacts γδ TCR specificities and repertoire
diversity of ileal γδ IELs. In addition, they showed that
Btnl2-deficient mice exhibited increased inflammation and delayed
mucosal repair in the colon, suggesting that it plays a key
immunological function in intestinal diseases.
Introduction
Tissue-resident intraepithelial lymphocytes (IELs) represent a
heterogenous population of antigen-experienced immune cells in the
intestinal epithelium that are involved in the maintenance of gut
homeostasis^[58]1,[59]2. In particular, IELs expressing αβ T cell
receptors (TCRs) are poised for mounting pathogen-specific memory
responses, while those possessing γδ TCRs strengthen tight junctions
and orchestrate innate and adaptive immunity during homeostasis,
inflammation, and infection^[60]1–[61]6. Interactions between
intestinal epithelial cells (IECs) and γδ IELs influence IEL
development and function^[62]7,[63]8. Notably, recent studies
emphasized that anatomical segregation could drive gut segment-specific
immunity^[64]9–[65]11, including functionally distinct γδ IEL immune
responses to chemically-induced and pathogen-induced epithelial
injury^[66]5,[67]12–[68]14. However, the mechanisms that regulate γδ
IEL development and maintenance in response to the local antigenic
environment remain poorly understood.
Recent studies provided some evidence that IEC-specific
butyrophilin-like (Btnl) molecules induce perinatal expansion and
maturation of distinct Vγ TCR^+ IELs^[69]5,[70]7,[71]15–[72]17. Indeed,
intestinal γδ IELs predominantly express Vγ7 in mice and Vγ4 in humans
that persist throughout the life of the host^[73]5,[74]7. γδ IELs
continuously sample both self and bacterial antigens from the local
environment to customize their TCR specificities^[75]5,[76]16.
Moreover, the contributions of Btnl molecules to shaping γδ TCR
repertoire diversity and regulating the distribution and function of γδ
IEL subsets across intestinal compartments^[77]18,[78]19 remain to be
elucidated and may inform our understanding of the compartmentalized
immune responses observed in the intestine^[79]9,[80]11,[81]14.
Btn/Btnl proteins are members of B7 immunoglobulin-superfamily and
analogous to other costimulatory and coinhibitory molecules (e.g.,
CD80, CD86, PDL1, and PDL2) have been shown to modulate αβ T cell
immune functions, including inhibition of CD4^+ T and CD8^+ T cell
activation, proliferation and cytokine production, induction of
regulatory T (Treg) cells and blockade of antigen-specific
proinflammatory responses^[82]20–[83]27. Btnl2, a member of the Btnl
family, has been shown to induce Treg differentiation and suppress T
cell activation and proliferation in vitro^[84]25. Accordingly,
Btnl2-KO chimera mice displayed increased susceptibility in a mouse
model of experimental cerebral malaria and higher frequencies of
peripheral CD4^+ T and CD8^+ T cells indicating a potential role for
Btnl2 in dampening infection-elicited T cell immune responses in
vivo^[85]28. Btnl2 is highly enriched in villous IECs across different
intestinal compartments and its expression is reported to be altered by
inflammatory cues such as epithelial injury and tumor
burden^[86]21,[87]29–[88]32. In particular, Btnl2 mRNA levels were
increased in the colon of Mdr1a-KO colitic mice and decreased in human
colon tumors^[89]29,[90]32. Furthermore, truncating single nucleotide
polymorphisms (SNPs) of BTNL2 were associated with ulcerative colitis
(UC) and chronic sarcoidosis, independent of linkage disequilibrium
(LD) with HLA^[91]21,[92]29–[93]31. The role of Btnl2 in regulating
intestinal immune responses during homeostasis and inflammation and,
particularly, in the induction and maintenance of intestinal γδ IELs
has not yet been addressed.
Here, we report the generation and characterization of Btnl2 knockout
mice and identify a role for Btnl2 in regulating the frequencies and
phenotype of γδ IELs preferentially in the ileum at a steady state. We
found that γδ IELs derived from the ileum, but not duodenum, of
Btnl2-KO mice possess a dysregulated antibacterial response module. By
integrating RNA and single-cell TCR expression data we identified
distinct transcriptional signatures and greater TCR repertoire
diversity in ileal Btnl2-KO γδ IELs. Upon DSS challenge, Btnl2-KO mice
displayed enhanced colonic, but not ileal, intestinal inflammation, and
delayed mucosal repair. Collectively, our findings suggest that
context-dependent Btnl2 expression fine-tunes intestinal immune
responses to protect against epithelial injury.
Results
Btnl2 is preferentially expressed in small intestinal epithelial cells
To determine the expression pattern of Btnl2 in the intestine during
homeostasis, we generated Btnl2-LacZ knock-in mice henceforth referred
to as Btnl2-KO mice (Fig. [94]1a). Consistent with previous
observations^[95]21,[96]29, Btnl2 was predominantly expressed in
terminally differentiated IECs of the small intestine (Fig. [97]1b).
Importantly, unlike Btnl1, Btnl4, and Btnl6^[98]7, Btnl2 expression was
detected in duodenal Brunner’s glands and duodenal, jejunal, ileal, and
colonic crypts, suggesting potential divergent roles of different Btnls
dictated by their region-specific expression patterns (Fig. [99]1b). To
further validate our observations, we measured Btnl2 mRNA levels in
IECs derived from terminally differentiated enterocytes isolated from
the duodenum, jejunum, ileum, and distal colon. Btnl2 was highly
enriched in duodenal IECs with a descending proximal-to-distal
gradient, such that Btnl2 expression in IECs isolated from the colon
was ~5-fold lower than in the ileum of WT mice (Fig. [100]1c).
Similarly, BTNL2 transcripts were detected in the small intestine, but
not colon samples pooled from healthy human tissues^[101]30.
Fig. 1. Btnl2 is preferentially expressed in small intestinal epithelial
cells.
[102]Fig. 1
[103]Open in a new tab
a Schematic representation of the WT and targeted locus of Btnl2−/−
mice. hUb, human Ubiquitin promoter. b Beta-galactosidase and Neutral
Red counterstaining in cryosections of segments of the small intestine
(duodenum, jejunum, and ileum) and colon of 15-week-old Btnl2-KO mice.
Arrowheads indicate regions of duodenal glands and duodenal, jejunal,
ileal, and colonic crypts and villi with weak Btnl2-LacZ expression.
Magnification is 20×; scale bar is 50 μm. c mRNA expression of Btnl2 in
intestinal epithelial cells from different segments of small intestine
and colon of cohoused 7-week-old Btnl2-KO and WT mice (n = 5, each),
normalized to β2m. Error bars represent mean ± SEM. Significance is
measured using multiple unpaired t-tests assuming similar SD,
*p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001, significantly
different from WT mice.
Given the close proximity of the Btnl2 gene to the H2 locus and other
Btnls^[104]33, we investigated the expression levels of several
adjacent genes in the IEC fraction isolated from duodenum, jejunum, and
ileum of Btnl2-KO and WT mice by bulk RNA-sequencing. We did not
observe any significant changes in H2-Aa, H2-Ab1, H2-Eb1, Tap1/2,
[105]BC051142, Btnl4, Btnl5, Btnl6, Notch4, and Ppt2 gene expression
levels across different segments of the small intestine, albeit
Btnl2-KO mice displayed a trend towards decreased levels of Btnl1
(Supplementary Figure [106]1), indicating no significant coregulation
of Btnl2 with adjacent genes near the H2 locus. Altogether, our data
confirm preferential expression of Btnl2 in terminally differentiated
enterocytes across different segments of the small intestine suggesting
a compartment-specific function of Btnl2.
Btnl2-KO mice display increased frequencies of γδ IELs preferentially in the
ileum
Under homeostatic conditions, Btnl2-KO mice did not exhibit any adverse
intestinal pathology, as determined by body weight loss, increased
epithelial sloughing, and pro-inflammatory cytokines (Supplementary
Figure [107]2a, b). In addition, we did not observe any significant
changes in genes associated with differentiation and maturation of
IECs^[108]34–[109]41, suggesting that IEC development and maintenance
are not altered in unchallenged Btnl2-KO mice (Supplementary
Figure [110]2c).
In light of the developing paradigm implicating members of
Btnl/BTN/BTNL family in shaping the γδ T cell
compartment^[111]5,[112]7,[113]16,[114]17,[115]42,[116]43 and intrigued
by the selective expression pattern of Btnl2 in different segments of
the small intestine, we postulated Btnl2 deficiency might impact the
maintenance of γδ IEL subsets in different segments of the small
intestine. To this end, we isolated IELs from the duodenum, jejunum,
and ileum of adult Btnl2-KO and WT mice. Consistent with the previous
observations^[117]14,[118]18,[119]19, γδ IELs were found at ~3-fold
higher frequency in the duodenum compared to the ileum of WT mice
(30.7% vs. 8.62% in total cells, Fig. [120]2a-right panel), however we
observed that compared to WT littermates, Btnl2-KO mice displayed a
30–40% increase in the frequency of γδ IELs in the jejunum (27.1% vs.
20.1%) and ileum (12.8% vs. 8.6%), but not duodenum (29.4% vs. 30.7%)
(Fig. [121]2a). Notably, this increase was observed predominantly in
ileal CD8αα^+ γδ IELs suggesting that Btnl2 may suppress their
proliferative capacity in situ (Fig. [122]2a). As the number of Vγ7^+
IELs have been reported to plateau in 11–16-week-old young
adults^[123]7, we investigated whether the observed effect of Btnl2 on
the percentage of ileal γδ IELs changed as mice approached middle
adulthood. Previous work suggested that ileal γδ IEL frequencies,
including CD8αα^+ γδ IELs, remain steady past 6 months of age in WT
mice^[124]44. In contrast, we observed that γδ IEL frequency was
reduced by 50% at 6 months of age in WT mice in our facility (39.5% vs.
17.1% in total γδ IELs) (Fig. [125]2b, d). Conversely, αβ IEL frequency
remained relatively unchanged at 6 months of age suggesting that αβ
IELs actively maintain their levels, possibly through in situ expansion
(Fig. [126]2c). We found that γδ IELs were significantly increased in
the ileum of young adult (up to 4 months old) Btnl2-KO compared to WT
mice (36.9% vs. 22.3% in CD8αα^+ γδ IELs), while mature adult mice
displayed similar levels of γδ IELs (Fig. [127]2b, d). αβ IELs were not
significantly altered in Btnl2-KO mice during this time frame
(Fig. [128]2c). As such, the Btnl2 effect in young adult mice suggests
it plays a role in γδ IEL maintenance under homeostatic conditions.
Notably, γδ T cells were observed at comparable frequencies in the
lamina propria (LP) of the duodenum, jejunum, and ileum, mesenteric
lymph nodes (mLN), and Peyer’s Patches (PP) of Btnl2-KO and WT mice
indicating that Btnl2 exerts its function specifically on γδ CD8αα^+
IELs (Fig. [129]2e).
Fig. 2. Btnl2-KO mice display increased frequencies of γδ IELs in the ileum.
[130]Fig. 2
[131]Open in a new tab
a Different segments of the small intestine were collected from
cohoused 7–17-week-old Btnl2-KO and WT mice (n = 3–8, each) and
processed for flow cytometry. Left panel-representative flow cytometry
plots of γδ IELs in the duodenum, jejunum, and ileum of cohoused
7-week-old Btnl2-KO and WT littermates. Displayed plots are gated on
live TCRαβ- cells. Right panel-frequencies of γδ IELs and CD8αα + γδ
IELs from 7–17-week-old Btnl2-KO and WT littermates. b Frequencies of
ileal γδ IELs at different ages (n = 6–23 mice/group). Error bars
represent mean ± SEM. Significance is measured by 2-way ANOVA with
Sidak’s multiple comparison test, *p < 0.05, **p < 0.005. c Frequencies
of ileal αβ IELs at different ages (n = 6–23 mice/group). Error bars
represent mean ± SEM. Significance is measured by 2-way ANOVA. d Ileum
was collected from cohoused Btnl2-KO and WT littermates of different
ages and intestinal intraepithelial lymphocytes (IELs) were isolated
and processed for flow cytometry. Left panel-representative flow
cytometry plots of γδ IELs in the ileum of 12–17-week-old Btnl2-KO and
WT littermates. Right panel-frequencies of ileal γδ IELs from
12–17-week-old Btnl2-KO and WT littermates. Data are pooled from 3
independent experiments with 3–6 mice/group. Error bars represent
mean ± SEM. Significance is measured using unpaired t-tests assuming
similar SD, *p < 0.05, **p < 0.005, significantly different from WT
mice. e Frequencies of γδ T cells in lamina propria (LP), mesenteric
lymph nodes (MLN), spleen, and Peyer’s Patches of 7–17-week-old
Btnl2-KO and WT littermates (n = 3–6 mice/group).
Prior studies had shown that recombinant Btnl2 can inhibit mLN CD4^+ T
cell proliferation and promote Treg cell differentiation under certain
activation conditions in vitro^[132]21,[133]22,[134]25,[135]29.
Nevertheless, we observed similar frequencies of CD4^+ T cells and
FoxP3^+ Tregs in the ileal LP, mLNs, and PPs of Btnl2-KO mice. In
addition, Btnl2-KO mice exhibited comparable immune cell profiles
across different tissues emphasizing the specificity and localized
effect of Btnl2 effects in the intestine on jejunal and ileal γδ IELs
(Supplementary Figure [136]3a–c).
Btnl2 suppresses proliferation of jejunal/ileal γδ IELs
To investigate the effects that Btnl2 exerted on jejunal and ileal γδ
IELs, we revisited its ability to suppress T cell proliferation. As
Btnl2 inhibitory function is dependent on concurrent TCR stimulation
and ligation with the putative Btnl2 receptor on CD4^+ T cells in
vitro^[137]21,[138]22,[139]25,[140]29, we activated CFSE-labeled CD4^+
T cells in the presence of equimolar concentrations of plate-bound
Btnl2-mFc, Pdl1-mFc, Pdl2-mFc, or mFc (Supplementary Figure [141]4a).
After 72 h of culture, we found that recombinant Btnl2 potently
suppressed proliferation and activation of CD4^+ T cells similarly to
Pdl1 and Pdl2, as evidenced by CFSE dilution and greater than 40%
decrease in cytokine production (Supplementary Figure [142]4b, c). In
line with previous reports^[143]25, CD28 co-stimulation rescued
production of TNFα and IFNγ, but not IL-2 (Supplementary
Figure [144]4c), which also coincided with ~50% decrease in Btnl2
binding to its putative receptor on activated CD4^+ T cells
(Supplementary Figure [145]4d).
We next sought to determine whether Btnl2 suppresses the proliferation
of γδ IELs in vitro (Supplementary Figure [146]5a). To obtain
comparable numbers to those from the duodenum, we pooled IELs from the
jejunum and ileum. Interestingly, duodenal CD8αα^+ γδ IELs showed
greater proliferative capacity compared to their jejunal/ileal
counterparts indicating that duodenum and jejunal/ileal γδ IELs may
require different TCR and/or cytokine stimulation (Fig. [147]3a).
Recombinant Btnl2 and Pdl1 potently inhibited the proliferation of both
duodenal and jejunal/ileal CD8αα^+ γδ IELs (Fig. [148]3a, b). However,
Btnl2 suppressive effect was 2-fold higher on jejunal/ileal CD8αα^+ γδ
IEL proliferation compared to one observed for duodenal CD8αα^+ γδ IELs
(Fig. [149]3b). Importantly, recombinant Btnl2 failed to inhibit the
proliferation of duodenal or jejunal/ileal CD8αβ^+ αβ IELs, suggesting
that the Btnl2 putative receptor may not be present on these cells
(Fig. [150]3b). Contrary to previous reports suggesting stronger
responsiveness of Btnl1-KO γδ IELs to α-CD3 stimulation^[151]7, we
found that duodenal and jejunal/ileal Btnl2-KO γδ and αβ IELs exhibited
equal proliferative capacity compared to their WT counterparts,
indicating that Btnl2 deficiency did not impair the ability of IELs to
respond to TCR and cytokine stimulation (Supplementary Figure [152]5b).
Moreover, recombinant Btnl2 similarly inhibited the proliferation of
Btnl2-KO and WT jejunal/ileal γδ IELs in vitro (Fig. [153]3c). Btnl2-KO
and WT jejunal/ileal γδ IELs also showed comparable expression profiles
of coinhibitory receptors (e.g. PD1) and markers associated with tissue
residence, maturation, and activation (e.g. CD69, CD44, CD27, and
CD122)^[154]7,[155]45 (Supplementary Figure [156]5c). Altogether, these
data indicate that Btnl2 preferentially suppresses jejunal/ileal
CD8αα^+ γδ IEL proliferation.
Fig. 3. Btnl2 suppresses proliferation of jejunal/ileal γδ IELs.
[157]Fig. 3
[158]Open in a new tab
IELs were isolated from duodenum and jejunum/ileum of cohoused
12-week-old Btnl2-KO and WT mice (n = 4–5, each), labeled with CFSE and
stimulated with α-CD3 and different Fc fusions in the presence of rh
IL-2, rmIL7, rm IL15 for 84 h. Supernatants from the cell cultures were
collected and cells were processed for flow cytometry. a Representative
flow cytometry plots of duodenal and jejunal/ileal WT CD8αα + γδ IELs
following 84 h of culture in the presence of equimolar concentrations
of Btnl2-Fc and control mFc fusion proteins. b, c Suppression of
proliferation calculated as the percent difference between the
proliferation in the presence of a specific Fc fusion and no Fc fusion,
relative to the proliferation in the absence of Fc fusion. b
Suppression of proliferation of duodenal and jejunal/ileal WT
CD8αα + γδ IELs and CD8αβ + αβ IELs. c Suppression of proliferation of
duodenal and jejunal/ileal Btnl2-KO CD8αα + γδ IELs and CD8αβ + αβ
IELs. Error bars represent mean ± SEM. Significance is measured using
one-way ANOVA, *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001,
significantly different from mFc fusion protein control. d, e Cohoused
11-week-old Btnl2-KO and WT mice (n = 4–5, each) were given BrdU at
0.8 mg/mL ad libitum in drinking water for 3 days. BrdU incorporation
was measured by intranuclear staining of γδ IELs from different
segments of the small intestine over time. d Representative flow
cytometry plots of BrdU incorporation in ileal CD8αα + γδ IELs. e BrdU
incorporation in CD8αα + γδ IELs and CD8αβ + αβ IELs across different
segments. Error bars represent mean ± SEM. Significance is measured
using unpaired t-tests assuming similar SD, *p < 0.05, **p < 0.005,
***p < 0.0005, ****p < 0.0001, significantly different from WT.
To determine the proliferative capacity of Btnl2-KO γδ IELs in vivo, we
measured BrdU incorporation in γδ IELs. As previously reported^[159]46,
high BrdU incorporation was observed in WT CD8αα^+ γδ IELs by day 3
(Fig. [160]3d, e). Notably, ileal Btnl2-KO γδ IELs incorporated BrdU
~2-fold more than their WT counterparts (9.86
[MATH: ± :MATH]
1.52 vs. 5.87
[MATH: ± :MATH]
1.18), emphasizing the enhanced proliferative capacity of γδ IELs in
the absence of Btnl2 (Fig. [161]3d, e). Overall, these observations
indicate that Btnl2 may serve as a γδ immune checkpoint molecule by
limiting the expansion of mature γδ IELs in the ileum and, potentially,
regulate their effector responses during the normal epithelial
lifespan.
Ileal Btnl2-KO γδ IELs display a subdued antibacterial response module
To gain insights into the impact of Btnl2 deficiency on γδ IEL
cytolytic potential, we performed transcriptomic analysis of γδ IELs
enriched from duodenum, jejunum, and ileum of 11-week-old cohoused
Btnl2-KO and WT mice at steady-state. Over 200 genes were significantly
downregulated (FDR < 0.05 and fold change > 1.5) in ileal Btnl2-KO γδ
IELs compared to their WT counterparts (Fig. [162]4a), whereas only
Btnl2 was significantly decreased in duodenal and jejunal Btnl2-KO γδ
IELs (Fig. [163]4b). The top 50 downregulated genes included signaling
molecules (e.g. Raph1, Cyr61, Tspan8), transcriptional regulators of
cell proliferation and apoptosis (e.g. Id1, Pbx1, Nupr1, Hoxb7), growth
factors (e.g. Kitl, Wnt3, Fgfbp1), antimicrobial molecules (e.g.
Ifi27l2b, Ccl25, Gsdmc3/4) and different classes of metabolic molecules
(e.g. aminoacid-Mgst1, lipid-Fabp6, Pnliprp2, sulfur-Sult1c2, Cth,
carbonic anhydrases-Car8) (Fig. [164]4a, b). Collectively, these
observations hint at some decrease in the metabolic function of ileal
Btnl2-KO γδ IELs compared to ileal WT γδ IELs.
Fig. 4. Ileal Btnl2-KO γδ IELs display an altered antibacterial response
module compared to ileal WT γδ IELs.
[165]Fig. 4
[166]Open in a new tab
γδ IELs from duodenum, jejunum, and ileum of cohoused 11-week-old
Btnl2-KO and WT littermates (n = 3–4/genotype, each a pool of 2 mice)
were sort-purified as CD45 + TCRβ-TCRγδ + cells. RNA sequencing was
performed, and gene set enrichment analysis using NextBio. Gene
ontology (GO) was employed to identify GO biological processes
differentially enriched in Btnl2-KO and WT γδ IELs. a Volcano plot
displaying genes differentially regulated between ileal Btnl2-KO and WT
γδ IELs. The Horizontal dashed line indicates FDR = 0.05 and a vertical
dashed line indicates |Fold Change | = 1.5. b Hierarchical clustering
of top 50 differentially expressed genes between ileal Btnl2-KO and WT
γδ IELs; duodenal and jejunal Btnl2-KO and WT γδ IELs were also
included as a comparison. c Gene ontology enrichment analysis of most
significantly impaired biological processes in ileal Btnl2-KO γδ IELs
compared to ileal WT γδ IELs. d Hierarchical clustering of top
dysregulated antibacterial response genes in ileal Btnl2-KO and WT γδ
IELs within the combined top 3 GO processes; duodenal and jejunal
Btnl2-KO and WT γδ IELs were also included as a comparison.
In line with these findings, gene ontology enrichment analysis revealed
that the most significantly dysregulated biological processes centered
around bacterial tolerance and clearance, emphasizing that ileal
Btnl2-KO γδ IELs display an impaired ability to secrete antimicrobial
molecules at a steady-state (Fig. [167]4c). Interferon-induced
molecules and several members of the α-defensin antimicrobial peptide
family were found among the genes significantly downregulated in ileal
Btnl2-KO γδ IELs compared to their WT counterparts (Fig. [168]4d).
Hence, our findings indicate that γδ IELs in the ileum, but not
duodenum or jejunum, may be specialized in secreting antibacterial
molecules in response to local microbial antigens.
Single-cell TCR sequencing highlights greater repertoire diversity in
Btnl2-KO γδ IELs
To determine whether the downregulated antibacterial response module
observed in ileal Btnl2-KO γδ IELs related to an altered γ/δ TCR
repertoire, we performed unbiased single-cell TCR sequencing on
duodenal, jejunal and ileal γδ IELs from Btnl2-KO and WT mice. In
total, we sequenced 28,679 cells and reassembled 24,961 productive γ
chains and 24,515 productive δ chains. Among all sequenced cells,
17,260 cells (60.2%) had paired γ and δ chains. We found that TRGV and
TRDV gene usage was comparable between Btnl2-KO and WT γδ IELs in the
duodenum, jejunum, and ileum (Fig. [169]5a). TRGV7 gene usage averaged
50% of TCR γ chains, consistent with the previous reports^[170]7,
whereas TRDV2-2, TRDV5, TRDV6D-1, and TRDV6D-2 genes were equally
represented and their combined gene usage surpassed 80% of TCR δ
chains. Ileal Btnl2-KO γδ IELs used the TRGV7 gene less frequently than
their WT counterparts (51.0% vs. 53.2%), however, they employed the
less common TRGV4 gene more frequently (11.5% vs. 8.6%; Pearson’s
chi-squared test, p = 1.78 × 10^−7). Similarly, ileal Btnl2-KO γδ IELs
showed reduced usage of TRDV2-2 and TRDV6D-2 genes compared to their WT
counterparts (23.2% vs. 25.3%; 21.9% vs. 26.2%, respectively), whereas
usage of TRDV6D-1 gene (18.6% vs. 13.4%) and of the less employed
TRDV12 gene (1.52% vs. 0.87%) increased.
Fig. 5. Ileal Btnl2-KO γδ IELs exhibit more diverse TRGV repertoire compared
to ileal WT γδ IELs.
[171]Fig. 5
[172]Open in a new tab
γδ IELs from the duodenum, jejunum, and the ileum of cohoused
11-week-old Btnl2-KO and WT littermates (n = 3–4/genotype, a pool of 2
mice, each) were sort-purified as CD45 + TCRβ-TCRγδ + cells. Two-thirds
of each sample were processed for deep bulk RNA sequencing and
one-third of each sample was pooled per genotype per segment and used
for single-cell sorting and single-cell TCR sequencing. γδ IELs from
duodenum, jejunum, and ileum of cohoused 11-week-old Btnl2-KO and WT
littermates (n = 8 mice, each) were single-cell sorted and single-cell
TCR sequencing analysis of TCR Vγ and TCR Vδ chain usage and CDR3
aminoacid sequences was performed. a TRGV*J and TRDV*J gene usage in
Btnl2-KO and WT γδ IELs. ND not detected. b Top-Diversity estimates for
TRG only CDR3 aminoacid sequences in Btnl2-KO and WT γδ IELs. Shaded
areas indicate the 95% confidence interval by 50 bootstrap replicates.
Bottom-TRG diversity estimates at interpolation point 3500, where p
value is derived from t-test based on 50 bootstrap replicates. c
Diversity estimates for TRD only and paired TRG and TRD CDR3 aminoacid
sequences, respectively, in ileal Btnl2-KO and WT γδ IELs. Shaded areas
indicate the 95% confidence interval by 50 bootstrap replicates. d
Diversity 50 (D50) index represented as the number of top unique clones
that comprise 50% of the TRGV and TRDV repertoires, respectively,
normalized to the total number of unique clones of duodenal, jejunal
and ileal Btnl2-KO and WT γδ IELs.
To establish TCR γ chain and δ chain clonotypes, we identified TCR γ
and δ sequences encoded by the same V gene and J gene segments with
identical aminoacid sequences in the third complementarity determining
regions (CDR3). Using the R iNext package^[173]47, we computed two
metrics (Species richness and Shannon diversity) to estimate TCR
diversity for each sample. Overall, ileal Btnl2-KO γδ IELs had
consistently higher TCR γ chain diversity than WT γδ IELs by both
measurements in both interpolated and extrapolated data. For example,
we randomly sampled 3500 γ chains from each sample by 50 bootstrap
replications and observed that the mean Shannon diversity of ileal
Btnl2-KO γ chains was 255.6, significantly higher than 226.8 in WT
(p = 0.0019, T-test) (Fig. [174]5b). Duodenal and jejunal Btnl2-KO γδ
IELs also showed a trend towards higher diversity in TCR γ chain
compared to WT γδ IELs but the difference was marginal when contrasted
to their ileal counterparts (p = 0.0616 and 0.2429, respectively for
Shannon diversity when sampling 3500 γ chains, Fig. [175]5b). Although
ileal Btnl2-KO γδ IELs had increased diversity in TCR δ chains
(Fig. [176]5c), Btnl2-KO γδ IELs isolated from all three segments
displayed an increased frequency of unique clonotypes that comprised
50% of the TRDV repertoire (duodenum: 19.0% vs. 17.6 %; jejunum: 20.2%
vs. 18.6%; and ileum: 18.5% vs. 18%) (Fig. [177]5d). In contrast, the
overall repertoire diversity of paired γ/δ chains was marginally
altered in ileal Btnl2-KO γδ IELs (Fig. [178]5c). Collectively, these
results suggested that the ileal TCR γδ repertoire diversity may be
continually shaped by both host and microbial antigens and metabolites
such that fluctuations in the frequencies of ileal γδ IELs as well as
perturbations in the antimicrobial response module could lead to
significant clonal revisions.
Shared TCR clonotypes display different frequencies in ileal Btnl2-KO and WT
γδ IELs
In addition to unique Btnl2-KO CDR3γ clones, 19 of the top 20 ileal
Btnl2-KO CDR3γ clones were shared by ileal WT γδ IELs as contracted or
expanded clones, possibly contributing to the TRGV repertoire diversity
in Btnl2-KO compared to WT γδ IELs (Fig. [179]6a). Overall, ~40% of
CDR3γ clones were shared by Btnl2-KO and WT γδ IELs in each segment
(Supplementary Figure [180]6a, c). In TCRδ chains, each segment was
characterized by a large number of unique CDR3δ clones and fewer than
3% shared clones between Btnl2-KO and WT mice (Supplementary
Figure [181]6b, c). More CDR3γ and CDR3δ clones were shared by jejunal
and ileal γδ IELs (131 vs. 72 and 270 vs. 179, respectively) in
Btnl2-KO compared to WT mice, highlighting the jejunum as a
transitional segment in the small intestine (Supplementary
Figure [182]6d). Btnl2-KO and WT γδ IELs carrying one CDR3γ/δ pair
showed virtually no overlap (less than 0.4%) of their CDR3γ/δ clonal
repertoire, emphasizing that individual mice carry unique
γ-chain-δ-chain pairings (Supplementary Figure [183]7a, b).
Nevertheless, up to 13% of CDR3γ/δ paired clones overlapped between two
or all three segments suggesting the presence of dominant CDR3γ/δ pairs
that populate all segments within individual mice (Supplementary
Figure [184]7c).
Fig. 6. Ileal γδ IEL transcriptome of shared Btnl2-KO and WT CDR3γ clones is
shaped by pairing with CDR3δ.
[185]Fig. 6
[186]Open in a new tab
Ileal γδ IELs from cohoused 11-week-old Btnl2-KO and WT littermates
(pool of 8 mice, each) were single-cell sorted, and single-cell TCR
sequencing and single-cell RNA sequencing were performed. a Top 20 TRG
clones (CDR3γ aminoacid sequences listed in order on the right) from
ileal Btnl2-KO γδ IELs, which are differentially enriched in ileal WT
γδ IELs. b Top largest CDR3γ clones identified during scTCRseq can be
found among individual ileal Btnl2-KO (n = 3) and ileal WT (n = 4)
samples, in which TCR sequences have been reconstructed from bulk
RNAseq. Shared clones are highlighted in bold. Each slice represents a
different sample and white slices mark the absence of the CDR3γ clone
from the individual sample. c Multiple Vγ7–J1 recombination events
converge to the same top 1 CDR3γ aminoacid sequence (CASWAGYSSGFHKVF)
in both ileal Btnl2-KO and ileal WT γδ IELs. d Distribution of top 1
CDR3γ chain (TRGV7*02/TRGJ1*01, CASWAGYSSGFHKVF; ~10% of total clones)
shared by ileal Btnl2-KO and ileal WT γδ IELs among different UMAP
clusters. e The pairing of top 1 CDR3γ (CASWAGYSSGFHKVF) with different
CDR3δ sequences, listed above the UMAP plot, shapes the transcriptome
of the γδ IELs. The number of clones per pair is denoted below the
CDR3δ sequences.
We next reconstructed CDR3γ sequences using bulk RNA-seq data from each
individual mouse. The most frequent ileal CDR3γ clones revealed by
single-cell TCR sequencing data were also found in different individual
mice, which suggested that the single-cell TCR repertoire was an
accurate representation of individual Btnl2-KO and WT CDR3γ diversities
(Fig. [187]6b). The top ileal Btnl2-KO CDR3γ clones also included
TRGV4, TRGV1, and TRGV7 genes, whereas ileal WT CDR3γ clones carried
TRGV7 almost exclusively (Fig. [188]6c). For the most frequent CDR3γ
chain (Vγ7-J1, CASWAGYSSGFHKVF), ~70% of IELs carrying this CDR3γ amino
acid sequences were translated from the same DNA sequence, which could
result from clonal expansion of one progenitor or from recurrent
independent recombinations that pair with distict Vδ sequences in each
clone, while the remainder of the IELs derived from smaller clones with
different DNA sequences (Fig. [189]6d). Similar convergent Vγ
recombination has been observed for common human Vγ9^+ clonotypes,
where their abundance has been proposed to be preconfigured since
birth^[190]48. Likewise, the most prevalent Vγ7^+ chain stemmed from
one major and several minor independent convergent recombination
events. These findings highlighted the presence of public TRGV
clonotypes of γδ IELs and suggested that the paired TRGV/TRDV
repertoire diversity may be driven by the CDR3δ sequence.
γδ IEL transcriptome of shared CDR3γ clones is shaped by pairing with CDR3δ
To further explore the relationship between TCR and γδ IEL
transcriptome, we performed scRNA-seq on the same duodenal, jejunal,
and ileal γδ IELs we have profiled for TCR sequencing. We identified
nine clusters in each sample (Supplementary Figure [191]8a) and the
transcriptome of single γδ IELs in clusters 0, 1, and 3 clearly
differentiated between duodenal and ileal origin with jejunal γδ IELs
exhibiting intermediate transcriptome profiles (Supplementary
Figure [192]8a, b).
Using the top 20 markers detected in each single cell cluster, in
conjunction with molecular signatures described in recent scRNA-seq and
bulk RNAseq reports^[193]5,[194]49, we propose γδ IEL attributes, such
as differentiation stage, maturation, and effector profile, to
distinguish among γδ IEL clusters (Supplementary Figure [195]8c). In
line with previous observations^[196]50, clusters 0 and 1 contain
mature and highly cytolytic IELs, clusters 2 and 3 include immature
IELs, whereas cluster 4 consists of newly activated IELs undergoing
transcriptional changes such as antigen-mediated differentiation
(Supplementary Figure [197]8c). The remaining IELs were subdivided into
smaller clusters with specialized effector profiles such as type I/III
interferon responses in cluster 5 (Isg15, Irf7, Stat1) and
subset-specific differentiation stage such as recently emigrated CD8β^+
IEL progenitors in cluster 6 (Klf2, Thy1, S1pr1, CD8b1, Sell)
(Supplementary Figure [198]8c)^[199]6,[200]7,[201]50. With respect to
TRGV distribution, TRGV7 gene usage was dominant in clusters 0-4, while
ileal Btnl2-KO γδ IELs had reduced frequencies of TRGV7 and higher
frequencies of TRGV1 and TRGV4 in cluster 1 compared to their WT
counterparts (40.4% vs. 45.9%, 15.2% vs. 10.3%, and 13.1% vs. 9.5%,
respectively; Supplementary Figure [202]8d).
We next examined the distribution of CDR3γ/δ pairings using the most
common ileal CDR3γ, encompassing ~10% of total CDR3γ clones across
different segments and genotypes (Vγ7-J1, CASWAGYSSGFHKVF). We found
that the top CDR3γ chain was preferentially enriched in cluster 0 of
ileal Btnl2-KO γδ IELs (51.3% vs. 37.2%), which is defined by the
largest number of maturation and cytolytic molecules (Supplementary
Figure [203]8c), and dominated cluster 1 in WT γδ IELs (47.4% vs 31.5%)
(Fig. [204]6c). The pairing of the top γ chain bearing the same
nucleotide sequence with distinct CDR3δ sequences shaped the
transcriptome of the pairs, as they are preferentially associated with
specific clusters (Fig. [205]6d).
Collectively, these RNA-seq and scTCR-seq observations indicate that
Btnl2 deficiency alters the transcriptome as well as the TRGV/TRDV
repertoire of ileal γδ IELs, such that their antigenic specificities
and antibacterial responses are changed. This report is the first to
describe intestinal γδ IEL transcriptome and TCR repertoire diversity
simultaneously at single-cell resolution, revealing a previously
uncharacterized heterogeneity in duodenal, jejunal and ileal γδ IELs
that may account for compartment-specific immune responses driven by
tissue-specific expression of immune-modulatory molecules.
Btnl2-KO mice exhibit more severe intestinal inflammation in chronic
DSS-induced colitis
Since BTNL2 SNPs have been associated with increased risk of UC and
Crohn’s disease (CD)^[206]31,[207]51–[208]53, we assessed the impact of
its deficiency on mucosal immune responses in the setting of
DSS-induced epithelial injury^[209]54,[210]55. Briefly, cohoused
Btnl2-KO and WT littermates were subjected to DSS-induced colitis by
administering DSS for 7 days followed by 8 days of water. While
Btnl2-KO and WT mice exhibited comparable intestinal damage in the
early phase of the disease (day 7), as demonstrated by comparable body
weight loss and increased myeloperoxidase activity (MPO) levels, a
biomarker of intestinal injury and neutrophilia^[211]56 (Fig. [212]7a,
d), we observed that Btnl2-KO mice exhibited a significant delay in
body weight recovery compared to WT littermates during the repair phase
of colitis (Fig. [213]7a). The observed delay in recovery was
accompanied by significantly shorter colons, increased granzyme A
levels, greater histopathological damage, and MPO activity in the colon
compared to WT littermates (Fig. [214]7b–d, f). Notably, DSS-treated
Btnl2-KO mice had ~2-fold higher levels of pro-inflammatory cytokines
such as IFNγ, IL-6, KC-GRO, TNFα, and IL-1β in the colon
(Fig. [215]7e). In contrast, MPO activity, a pro-inflammatory cytokine,
and granzyme A levels were not significantly altered in the ileum of
DSS-treated Btnl2-KO mice compared to WT littermates (Supplementary
Figure [216]9a–c). Corroborating these results, Btnl2 transcripts were
increased in the colon of DSS-treated WT mice, whereas the levels of
other family members, such as Btnl1 and Btnl6, were decreased with DSS
treatment (Fig. [217]7g). Btnl2 transcripts were unchanged in the ileum
of DSS-treated suggesting that Btnl2 expression in the colon may be
induced as a feedback regulatory mechanism at the site of injury to
attenuate DSS-triggered inflammation and facilitate the recovery
process (Supplementary Figure [218]9d).
Fig. 7. Btnl2-KO mice exhibit more severe intestinal inflammation in chronic
DSS-induced colitis.
[219]Fig. 7
[220]Open in a new tab
Cohoused 15-week-old Btnl2-KO (n = 11) and WT (n = 8) littermates were
subjected to 3% DSS-induced colitis for 7 days followed by water for 8
days. Control mice (n = 2–4) received water. a Body weight loss in
cohoused Btnl2-KO and WT littermates calculated as the percent
difference between the initial and actual body weight on the above
days. Error bars represent mean ± SEM. Significance is measured using
unpaired t-tests assuming similar SD, *p < 0.05, **p < 0.005,
***p < 0.0005, significantly different from DSS-treated WT mice. b
Colon length of water- and DSS-treated Btnl2-KO and WT mice on day 15.
c H&E histological sections and a pathological score of the colon from
water- and DSS-treated Btnl2-KO and WT mice. Scale bars are 200 μm
(WT/water), 250 μm (Btnl2-KO/water), 500 μm (WT/DSS and Btnl2-KO/DSS).
d Myeloperoxidase (MPO) activity in colon homogenates of water- and
DSS-treated Btnl2-KO and WT mice. e Levels of pro-inflammatory
cytokines in colon homogenates of water and DSS-treated Btnl2-KO and WT
mice. f Granzyme A mRNA levels in colon homogenates of water- and
DSS-treated Btnl2-KO and WT mice, normalized to β2m. Error bars
represent mean ± SEM. Significance is measured using unpaired t-tests
assuming similar SD, *p < 0.05. g Btnl1/2/6 mRNA levels in the colon of
water- and DSS-treated WT mice, normalized to β2m. Error bars represent
mean ± SEM. Significance is measured using one-way ANOVA, *p < 0.05,
**p < 0.005, ***p < 0.0005.
Discussion
Emerging research places the Btn/Btnl family of molecules at the heart
of γδ T cell development. Our studies shed light on Btnl2 as a
regulator of ileal γδ IEL maintenance. Specifically, we propose that
Btnl2 acts as a coinhibitory ligand to an unidentified receptor(s) on
γδ IELs and regulates both proliferation and segment-specific effector
profiles of ileal γδ IELs under homeostatic conditions.
Through our segment-focused approach, we found a temporal and spatial
window during which Btnl2 exerted its functions on intestinal γδ IELs.
ScTCRseq revealed that Vγ7^+ IELs dominated the small intestine of
11-week-old Btnl2-KO and WT mice suggesting that their development was
not affected. Although Btnl2 impacted γδ IEL proliferation
preferentially in the ileum, its deficiency reverberated throughout
distinct segments of the small intestine. Specifically, despite the
ability of Btnl2 to suppress both duodenal and jejunal/ileal γδ IEL
proliferation in vitro, this was confined in vivo only to ileal γδ IEL
expansion. However, at the molecular level, Btnl2 deficiency led to an
altered Vγ usage among Vγ7^− IELs and similarly altered Vδ usage across
all three segments of the small intestine suggesting overlapping as
well as unique roles for Btnl2 across the distinct segments of the
small intestine. This in turn was accompanied by dysregulated
antibacterial module in ileal Btnl2-KO γδ IELs, which may be relevant
for mucosal repair and clearance of segment-tropic pathogenic
microbes^[221]9.
Consistent with a region-specific effect, duodenal Btnl2-KO CDR3γ and
CDR3γ/δ clonal repertoires were not markedly different from those
identified in cohoused WT littermates underscoring the ileum as the
predominant site of Btnl2-mediated regulation at steady-state.
Importantly, most abundant Vγ clones in the ileal compartment of
individual Btnl2-KO mice included Vγ1^+ and Vγ4^+ clones, in contrast
to Vγ7^+ clones exclusively enriched in WT mice. Btnl2 may be important
for co-regulating ligands (i.e. Btnl1, Btnl6) of Vγ7^+ TCRs during
early adulthood. In support of this dynamic remodeling of the γδ TCRs,
Btnl2 deficiency led to different convergent recombination events, such
that pairing of the most common Vγ7^+ chain with distinct Vδ sequences
defined the transcriptome profiles of ileal γδ IELs. Based on the
previous studies^[222]5,[223]17, one possibility is that site-specific
metabolite levels and/or antigenic pressure led to multiple independent
in situ recombination events, suggesting an adaptive behavior of γδ
IELs towards local environmental antigens. Since ileal γδ IEL motility
along the villi-crypt axis is strictly dependent on the presence of
microbiota^[224]14, an impaired antibacterial profile could also be a
consequence of improper localization or ineffective surveillance of
ileal Btnl2-KO γδ IELs. Conversely, loss of epithelia-expressed Btnl2
could lead to alterations in the local microbiome, which would then
drive reshaping of the TCR repertoire and antibacterial response module
of ileal Btnl2-KO γδ IELs. Further studies are required to understand
how the reshaped Vγ-Vδ repertoire alongside the defective antibacterial
response module may affect the susceptibility of Btnl2-KO mice to small
intestinal infectious agents.
An acute reliance on Btnl expression at a predefined time has been
proposed for both murine Vγ7^+ IEL development and human Vγ4^+/Vδ1^+
IEL maintenance^[225]5,[226]7. Specifically, Btnl1 expression in adult
Btnl1-KO mice could not rescue Vγ7^+ development^[227]7, whereas
mucosal repair and Btnl8 expression restoration following adherence to
a gluten-free diet could not reconstitute Vγ4^+/Vδ1^+ IEL subsets in
patients with celiac disease^[228]5. In light of these observations, it
is tempting to speculate that Btnl molecules may regulate not only the
selective expansion of tissue-specific Vγ chains in neonates but also
their TCR specificities across distinct tissue compartments in young
adults. As such, segment-biased γδ TCR specificities may be determined
by the choice of dimerization partners among Btnl molecules and their
nuanced spatial and temporal expression in the intestine. While Btnl1
and Btnl6 jointly affect Vγ7 selection and maturation, there is no
known binding partner for Btnl2. In addition, no other Btnl molecules
were induced in the intestine to compensate for the loss of Btnl2
suggesting that their expression patterns were not co-regulated,
despite being encoded at the same locus. Of the various family members,
structurally, Btnl2 is unique in that it lacks the antigen-binding
B30.2 domain shared by most of the Btn/Btnl superfamily
members^[229]57, suggesting that the inhibitory effect of Btnl2 may
depend on the signaling pathways triggered downstream of engagement of
its putative receptor on γδ IELs. Btnl2 could either homodimerize or
heterodimerize with other intestine-specific Btnls through IgC
interactions independent of B30.2 domains^[230]42,[231]58. As a
heterodimer, Btnl2 interacting partner may contribute to the
B30.2-driven activation of the heterodimer and binding to the putative
receptor, whereby Btnl2 ligation would induce the downstream inhibition
of proliferation. Btnl1, Btnl4, and Btnl6 can be candidate binding
partners of Btnl2 due to their similar intestinal expression^[232]7.
Hence, despite higher expression on duodenal IECs, Btnl2 may exert more
profound inhibition on ileal γδ IELs due to increased regional
expression of its binding partner on IECs and putative receptor on γδ
IELs. As such, this region-specific interaction may be promoted by
local soluble antigens like bacterial metabolites. Btnl2 could also
function as a receptor antagonist prohibiting the binding of another
Btnl heterodimer to γδ TCR and suppressing γδ IEL proliferation.
Alternatively, as this suppression is only partial, Btnl2 may
indirectly target certain Vγ TCR(s) or TCR specificities via regulating
surface expression of Vγ ligands (i.e. Btnl6 for Vγ7
TCRs)^[233]16,[234]59. Further studies are required to address whether
Btnl2 can exert its inhibitory effects on γδ IELs across all intestinal
compartments during segment-specific inflammation.
Previous studies showed that γδ T cell depletion induces greater
colonic damage, reduced KGF secretion, increased IFNγ production by αβ
T cells and decreased IEC proliferation during DSS-induced colitis,
suggesting that γδ IELs can promote mucosal repair following epithelial
injury^[235]12,[236]60. Conversely, impaired IL-10 production by Tregs
leads to uncontrolled γδ IEL proliferation and spontaneous colitis in
Pdk1^f/f; CD4^cre mice, supporting a proinflammatory role for γδ IELs
in the colon^[237]61. Btnl2 expression is upregulated in the distal
colon during DSS-induced colitis and Btnl2-KO colitic mice exhibit a
delay in recovery during the mucosal repair phase of the disease,
potentially due to γδ IEL-dependent and -independent (i.e. Tregs,
proinflammatory helper T cells) mechanisms to controlling the damage
caused by epithelial injury. Interestingly, Btnl1 and Btnl6 transcripts
were downregulated in Btnl2-KO colitic mice, confirming previous
reports in which Btnl1/6 transcripts were shown to be significantly
reduced in the distal colon of Muc2-KO mice and BTNL8 expression was
diminished or lost in colonic and duodenal biopsies of patients with UC
and celiac disease, respectively^[238]5,[239]32. Hence, we speculate
that IECs upregulate Btnl2 expression in response to environmental
stress factors to limit the damage-induced expansion of γδ IELs and
induce the release of antibacterial molecules. As such, our findings
support the idea that close interaction between γδ IELs and
epithelium-specific Btnl molecules throughout the small and large
intestines drives their proliferation and function in homeostatic and
inflammatory settings^[240]14.
In conclusion, we have unveiled a novel role for Btnl2 in regulating
the expansion of ileal γδ IELs, sculpting of their Vγ and Vγ-Vδ TCR
specificities and altering their antibacterial response module. Our
scRNAseq and scTCRseq surveys revealed a highly dynamic γδ IEL
compartment finely adapted to environmental cues of each segment of the
small intestine during adulthood. Further studies are required to
establish whether the timing and choice of intestinal Btnl heteromers
drive the site-specific functions of γδ IELs in intestinal immune
disorders. Taken together, these studies suggest that Btnl-mediated
targeting of γδ IEL development and maintenance may help dissect their
immunological functions in intestinal diseases with gut
segment-specific manifestations.
Materials and methods
Mice
Eight- to twelve-week-old female C57BL/6 mice were obtained from
Jackson Laboratory. Btnl2-KO mice on a C57BL/6 background were
generated and maintained at Regeneron Pharmaceuticals Inc. using the
VelociGene technology^[241]62,[242]63. Briefly, a LacZ cassette was
inserted in-frame with the start codon followed by a selection cassette
that disrupted the transcription of the Btnl2 gene resulting in a null
allele. Heterozygous mice were interbred to produce homozygous KO and
WT littermates. Btnl2 expression pattern was confirmed by
β-galactosidase staining and Btnl2 targeted deletion was measured by
quantitative RT-PCR and RNA sequencing of the small intestine. Btnl2-KO
and WT female mice were used at 10-17 weeks of age for all the
experiments except when otherwise indicated. Female littermates were
cohoused after weaning for several weeks and assigned randomly to
experimental groups in disease settings. All animals were maintained
under pathogen-free conditions and experiments were performed according
to protocols approved by the Institutional Animal Care and Use
Committee at Regeneron Pharmaceuticals Inc.
Isolation of intestinal epithelial cells (IECs), intraepithelial lymphocytes
(IELs), and lamina propria lymphocytes (LPLs)
The small intestine was divided into three equal segments and
lymphocyte isolation proceeded as described previously^[243]64.
Briefly, to isolate IEC and IEL fractions, the small intestine was cut
into 2 cm pieces and incubated in HBSS containing 5 mM EDTA, 10 mM
HEPES and 2% fetal calf serum (FCS) twice for 15 min at 37 °C with
shaking at 150 rpm. After vigorous vortexing, the intestinal pieces
were washed over 100 μm cell strainer and centrifuged on a 40%/80%
Percoll gradient (GE Healthcare) at 2500 rpm for 20 min at 20 °C. The
top layer containing IECs was collected, washed, and resuspended in
Trizol for RNA extraction. IEL fraction was collected from the
interface, washed, and resuspended in Miltenyi MACS buffer. Following
IEL isolation, LPLs were isolated from intestinal pieces by incubation
in HBSS w/o Ca^2+/Mg^2+ supplemented with 50 U mL^−1 Collagenase D
(Roche), 0.25 mg mL^−1 DNase I (Sigma-Aldrich), 50 U mL^−1 Dispase
(Corning), and 5% FCS for two rounds of 25 min at 37 °C with shaking at
150 rpm. Cells were centrifuged on a 40%/80% Percoll gradient (GE
Healthcare) and LPLs were collected from the interface, washed, and
resuspended in MACS buffer for immediate surface cell staining.
Mesenteric lymph node and Peyer’s Patch immunophenotyping
Peyer’s Patches were collected from the whole small intestine, washed
with ice-cold DPBS, and incubated with 50 U mL^−1 Collagenase D
(Roche), 0.25 mg mL^−1 DNase I (Sigma-Aldrich), 50 U mL^−1 Dispase
(Corning), and 5% FCS for 25 min at 37 °C with shaking at 150 rpm.
Mesenteric lymph nodes were minced in HBSS with Ca^2+/Mg^2+containing
15 U mL^−1 Collagenase D (Roche) and 50 μg mL^−1 DNase I
(Sigma-Aldrich), and incubated for 20 min at 37 °C without shaking.
Cells were resuspended in MACS buffer for immediate surface staining.
Flow cytometry
Flow cytometry antibodies were purchased from Biolegend (US), BD
Biosciences (US), TONBO Biosciences (US), eBioscience (US) and
ThermoFischer (US). Dead cells were excluded using LIVE/DEAD fixable
blue dead cell stain (Thermo Fischer Scientific, Cat#[244]L23105). Fc
receptors were blocked using purified anti-mouse CD16/32 (BD Pharmigen,
Clone 2.4G2, Cat#553142) and 2% each of normal mouse serum (Jackson
ImmunoResearch, Cat#015-000-120), rat serum (Jackson ImmunoResearch,
Cat#012-000-120) and hamster serum (Jackson ImmunoResearch,
Cat#007-000-120). The following antibodies were used for the staining
according to manufacturer’s instructions: CD45-BV510 (Biolegend,
Clone#30-F11, Cat#103138), CD8α-AF700 (Biolegend, Clone#53-6.7,
Cat#100730), CD8β-PerCP/Cy5.5 (Biolegend, Clone#YTS156.7.7,
Cat#126610), TCRβ-BV711 (Biolegend, Clone#H57-597, Cat#109243),
TCRβ-APC/Cy7 (Biolegend, Clone#H57-597, Cat#109220), TCRγ/δ-PE/Cy7
(Biolegend, Clone#GL3, Cat#118124), CD11b-APC/Cy7 (Biolegend,
Clone#M1/70, Cat#101226), CD11c-APC/Cy7 (Biolegend, Clone#N418,
Cat#117324), CD11c-AF700 (Biolegend, Clone#N418, Cat#117320),
CD11c-PE/Cy7 (Biolegend, Clone#N418, Cat#117318), Gr1-APC/Cy7
(Biolegend, Clone#RB6-8C5, Cat#108424), B220-APC/Cy7 (Biolegend,
Clone#RA3-6B2, Cat#103224), B220-AF700 (Biolegend, Clone#RA3-6B2,
Cat#103232), B220-BV650 (Biolegend, Clone#RA3-6B2, Cat#103241),
NK1.1-APC/Cy7 (Biolegend, Clone#PK136, Cat#108724), MHCII-BV421
(Biolegend, Clone#M5/114.15.2, Cat#107632), Ly6C-PerCP/Cy5.5
(Biolegend, Clone#HK1.4, Cat#128012), CD64-PE (Biolegend,
Clone#X54-5/7.1, Cat#139304), CD103-FITC (Biolegend, Clone#2E7,
Cat#121420), CX3CR1-Biotin (Biolegend, Clone#SA011F11, Cat#149018),
Streptavidin-PE/Dazzle 594 (Biolegend, Cat#405248), NKp46-PE/Dazzle594
(Biolegend, Clone#29A1.4, Cat#137630), CD4-PerCP/Cy5.5 (Biolegend,
Clone#GK1.5, Cat#100434), CD4-VF450 (Tonbo Biosciences, Clone#GK1.5,
Cat#75-0041-U100), c-KIT-PE-Cy7 (Biolegend, Clone#ACK2, Cat#135112),
CD44-APC/Cy7 (Biolegend, Clone#IM7, Cat#103028), CD44-BV650 (Biolegend,
Clone#IM7, Cat#103049), RORγt-APC (eBiosciences, Clone#AFKJS-9,
Cat#17-6988-82), FoxP3-AF700 (eBioscience, Clone#FJK-16s,
Cat#56-5773-82), FoxP3-eF450 (eBioscience, Clone#FJK-16s,
Cat#48-5773-82), LegendScreen Mouse PE kit (Biolegend, Cat#700005). For
intranuclear staining, cells were incubated with fixable viability dye
and surface markers prior to fixation and permeabilization using the
FoxP3/Transcription factor fixation and permeabilization kit
(eBioscience) according to manufacturer’s instructions.
BrdU (Sigma-Aldrich) incorporation was assessed 3 days after continuous
administration in drinking water dissolved at 0.8 mg mL^−1 in 3%
sucrose. Briefly, IELs were fixed with BD Cytofix/Cytoperm™ for 20 min
at 20 °C, washed and incubated with 1x DPBS supplemented with
Ca^2+/Mg^2+, 10% FCS, and 10% DMSO for 10 min at 20 °C. Cells were
re-fixed with BD Cytofix/Cytoperm™ for 5 min at 20 °C, washed and
incubated with 0.5 mg mL^−1 DNAse I (Sigma-Aldrich) for 1 hr at 37 °C.
Cells were stained with BrdU-AF647 (MoBU-1) (Thermo Fisher Scientific,
Clone# MoBU-1, Cat#[245]B35133) at 20 °C, washed, and resuspended for
acquisition.
Flow cytometry was performed on the LSRFortessa X-20 instrument (BD
Biosciences), data were analyzed using FlowJo software (BD Biosciences)
and plotted using GraphPad Prism™ (GraphPad Software, Inc.).
Representative gating strategies for flow cytometry are provided in
Supplementary Figure [246]10.
In vitro IEL proliferation assay
Total 96-well flat-bottom plates were coated overnight with 1 μg mL^−1
purified anti-mouse CD3ε (Tonbo Biosciences, 145-2C11,
Cat#70-0031-M001) and 60 pmoles of mouse Btnl2-Fc, PDL1-Fc or mFc
(Adipogen Life Sciences) at 4 °C and washed twice with DPBS before
adding IELs to the cultures. Freshly isolated IELs were labeled with
CellTrace CFSE Cell Proliferation dye according to the manufacturer’s
instructions (Thermo Fischer Scientific, Cat#[247]C34554). CFSE-labeled
IELs were plated at 200,000 cells per well in RPMI 1640 supplemented
with 10% FCS, 1% Pen/Strep, 2% HEPES, 1% Glutamine, 1% nonessential
amino acids, 1% sodium pyruvate, 0.1% β-mercapto-ethanol (Gibco),
recombinant mouse IL-7 (10 ng mL^−1, R&D), recombinant mouse IL-15
(10 ng mL^−1, R&D) and recombinant human IL-2 (10 ng mL^−1, Peprotech).
Cells were incubated for 72–96 h at 37 °C in 5% CO[2] prior to
analysis.
In vitro CD4^+ T cell proliferation assay
Total 96-well flat-bottom plates were coated overnight with 1 μg mL^−1
purified anti-mouse CD3ε (Tonbo Biosciences, 145-2C11,
Cat#70-0031-M001), 1 μg mL^−1 purified anti-mouse CD28 (Tonbo
Biosciences, 37.51, Cat#70-0281-U500), and 60 pmoles of mouse Btnl2-Fc,
PDL1-Fc, PDL2-Fc or mFc (Adipogen Life Sciences) at 4 °C and washed
twice with DPBS before adding CD4^+ T cells to the cultures. CD4^+ T
cells were enriched from pooled spleen and lymph nodes using mouse CD4
(L3T4) microbeads (Miltenyi Biotec) and labeled with CellTrace CFSE
Cell Proliferation dye (Thermo Fischer Scientific, Cat#[248]C34554).
CFSE-labeled CD4^+ T cells were plated at 80,000–100,000 cells per well
in RPMI 1640 supplemented with 10% FCS, Pen/Strep, 2% HEPES, 1%
Glutamine, 1% nonessential amino acids, 1% sodium pyruvate and 0.1%
β-mercapto-ethanol (Gibco). Cells were incubated for 72 h at 37 °C in
5% CO[2] prior to analysis.
DSS-induced model of colitis
Fourteen-twenty-week-old cohoused female Btnl2-KO and WT mice with an
average body weight greater than 23 g were given 3% DSS (Sigma-Aldrich)
in drinking water for 6–7 days followed by distilled water for up to 10
days. Control group received only distilled water for the duration of
the study. Mice were weighed and monitored daily for clinical signs of
colitis (e.g. stool consistency, fecal blood). On day 15, mice were
euthanized, and colon length was measured.
Generation of colon and ileal homogenates and measurement of cytokines and
myeloperoxidase (MPO) activity
Total 6 mm pieces of distal colon or terminal ileum were placed in
T-per buffer (Thermo Fisher Scientific) containing 1× Halt Protease
Inhibitor Cocktail (Thermo Fisher Scientific), 0.5 M EDTA solution
(Thermo Fisher Scientific), and two 3 mm tungsten carbide beads
(Qiagen). Tissues were disrupted in TissueLyser II (Qiagen) for 10 min
at an oscillation frequency of 27.5 Hz. Generated tissue homogenates
were centrifuged at 15000 rcf for 10 min at 4 °C and the supernatants
were collected into deep 96-well plates. Protein assay dye (BioRad) was
used to quantify total protein content using Bradford protein assay
according to the manufacturer’s instructions. Cytokine concentrations
were measured using V-PLEX Plus Proinflammatory Panel 1 mouse kit
according to the manufacturer’s instructions (Meso Scale Diagnostics).
Absorbance was measured on the Meso SECTOR S600 instrument (Meso Scale
Diagnostics). Myeloperoxidase (MPO) activity was measured using a mouse
MPO ELISA kit according to the manufacturer’s instructions (Hycult
Biotech). Absorbance was measured on the SpectraMax i3x instrument
(Molecular Devices). Data analysis was performed using GraphPad Prism™
(GraphPad Software, Inc.). Cytokine and MPO levels were normalized to
total protein content.
Histology
Total 3 cm pieces of duodenum, jejunum, ileum, and colon were prepared
as swiss rolls, fixed in 10% buffered formalin, embedded in paraffin,
sectioned at 5 μm and H&E stained. Histology was performed by HistoWiz
Inc. (histowiz.com) using a Standard Operating Procedure and fully
automated workflow. After staining, sections were dehydrated and film
coverslipped using a TissueTek-Prisma and Coverslipper (Sakura). Whole
slide scanning (40x) was performed on an Aperio AT2 (Leica Biosystems).
Histopathological scoring was performed by an evaluator blinded to
genotype, group assignment, and experimental outcome. The following
features were evaluated for DSS-induced injury and scored based on
previous published criteria:^[249]65 degree of inflammation in lamina
propria, goblet cell loss, abnormal crypts, presence of crypt
abscesses, mucosal erosion, and ulceration, submucosal spread to
transmural involvement, number of neutrophils. Each parameter received
a score from 0 to 4 with a maximum cumulative score of 17. Mucosal
lesions in unchallenged mice were scored as described
previously:^[250]66 0, normal; 1, mild sloughing of epithelial cells;
2, moderate sloughing of epithelial cells; 3, severe mucosal edema; 4,
extensive mucosal injury. Data analysis was performed using GraphPad
Prism™.
Quantitative PCR
RNA was isolated from IECs derived from duodenum, jejunum, ileum, and
colon from cohoused unchallenged Btnl2-KO and WT mice. RNA was
extracted from distal colon and terminal ileum from cohoused, water-
and DSS-treated Btnl2-KO and WT mice. RNA was purified on Kingfisher
flex (Thermo Fisher Scientific) using the MagMAX-96 for Microarrays
Total RNA isolation kit (Thermo Fisher Scientific) with an additional
DNAse I (Sigma-Aldrich) step added between the first and second washes.
cDNA synthesis was performed using SuperScript® VILO™ Master mix
(Thermo Fisher Scientific) according to the manufacturer’s
instructions. qPCR was performed using MyTaq™ Mix (Bioline) and assay
mix (Thermo Fisher Scientific or LGC BioSearch). Probes for each gene
are listed in Table [251]1. qPCR was run on an ABI 7900HT Fast
Real-Time PCR System with a 384-well block module and automation
accessory (Thermo Fisher Scientific). Gene expression was normalized to
β2 m and differences were determined using the 2ΔC(t) calculation.
Table 1 .
RT-PCR Probes for select genes.
Gene Forward Reverse Probe
BTNL2 GGATTGCCCACGGTATAGTC AGGACCGACCACTCTGAAG TATCTGGCGTGGCTGCCTCCTT
BTNL1 GGTGCAGATGCCGGAATACAG GCCACACTTCCCATGTCAATG
CAGGACCCAGATGGTGAGACAAGC
BTNL6 GAGGCCATCTTGGAACTGAA CCACCGTCTTCTGGACCTTT TGGCAGCAATGGGCTCTGTCC
b2m GGGAAGCCGAACATACTGAACTG CCCGTTCTTCAGCATTTGGATTTC
ACGTAACACAGTTCCACCCGCCT
Gzma GGCGCTTTGATTGAAAAGAACTG TGTTCTGGCTCCTTATTGATTGAG
TGACTGCTGCCCACTGTAACGTGG
[252]Open in a new tab
Single-cell RNA sequencing of γδ IELs
Following IEL isolation, cells were stained with LIVE/DEAD fixable blue
dead cell stain as per the manufacturer’s instructions (Thermo Fisher
Scientific, Cat#[253]L23105). Fc receptors were blocked using purified
anti-mouse CD16/32 (BD Pharmigen, Clone 2.4G2, Cat#553142) and 2% each
of normal mouse serum (Jackson ImmunoResearch, Cat#015-000-120), rat
serum (Jackson ImmunoResearch, Cat#012-000-120) and hamster serum
(Jackson ImmunoResearch, Cat#007-000-120) and cells were stained using
the following antibodies: CD45-BV510 (Biolegend, Clone#30-F11,
Cat#103138), CD8α-AF700 (Biolegend, Clone#53-6.7, Cat#100730),
CD8β-PerCP/Cy5.5 (Biolegend, Clone#YTS156.7.7, Cat#126610), TCRβ-BV711
(Biolegend, Clone#H57-597, Cat#109243), TCRγδ-PE/Cy7 (Biolegend,
Clone#GL3, Cat#118124). Two mice were pooled per each sample. γδ IELs
were sorted from each sample using MoFlo Astrios EQ (Beckman Coulter).
Two-thirds of each sample were resuspended in RNA Lysis Buffer (Zymo
Research) and processed for bulk RNA sequencing. One-third of each
sample was pooled per segment of the small intestine per genotype,
resuspended in PBS with 0.04% BSA, and loaded on a Chromium Single Cell
Instrument (10X Genomics). RNAseq and V(D)J libraries were prepared
using Chromium Single Cell 5’ Library, Gel Beads & Multiplex Kit (10X
Genomics). After amplification, cDNA was divided into RNAseq and V(D)J
library aliquots. To enrich the V(D)J library aliquot for γδ TCRs, cDNA
was divided into two 10 ng aliquots and amplified in two rounds using
internally designed primers. In particular, the following primers were
used for the first round of amplification: MP147 for short R1
(ACACTCTTTCCCTACACGACGC), MP371 for mouse TRGC1-3
(/5Biosg/TTCCTGGGAGTCCAGGATRGTATTG), MP 372 for mouse TRGC4
(/5Biosg/CACCCTTATGACTTCAGGAAAGAACTTT), and MP369 for mouse TRDC
(/5Biosg/TTCCACAATCTTCTTGGATGATCTGAG). For the second round of
amplification, 20 ng aliquots from the first round were further
amplified using MP147 for short R1 (ACACTCTTTCCCTACACGACGC), MP373 a
nested R2 plus mouse
TRGC(GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTCCCAGYCTTATGGAGATTTGT), and
MP370 a nested R2 plus mouse TRDC
(GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTAGTCACCTCTTTAGGGTAGAAATCTT). V(D)J
libraries were prepared from 25 ng of each mTRGC and mTRDC amplified
cDNA. Paired-end sequencing was performed on Illumina NextSeq500 for
RNAseq libraries (Read 1 26 bp for a unique molecular identifier (UMI)
and cell barcode, 8 bp i7 sample index, 0 bp i5, and Read 2 55 bp
transcript read) and V(D)J libraries (Read 1 150 bp, 8 bp i7 sample
index, 0 bp i5, and Read 2 150 bp read). For RNAseq libraries, Cell
Ranger Single-Cell Software Suite (10X Genomics, v2.2.0) was used to
perform sample de-multiplexing, alignment, filtering, and UMI counting.
The mouse mm 10 genome assembly and RefSeq gene model for mouse were
used for the alignment. For V(D)J libraries, Cell Ranger Single-Cell
Software Suite (10X Genomics, v2.2.0) was used to perform sample
de-multiplexing, de novo assembly of read pairs into contigs, align and
annotate contigs against all the germline segment V(D)J reference
sequences from IMGT, label and locate CDR3 regions, group clonotypes.
Single-cell RNA sequencing data analysis
scRNAseq data were analyzed using Seurat R package^[254]67. Cells with
fewer than 500 genes or more than 10% of mitochondrial RNA content were
excluded during the quality control (QC) step. The remaining cells
underwent dimension reduction by PCA on the highly variable genes. Data
were further reduced to the 2D space on the first 20 PCs using uniform
manifold approximation and projection (UMAP). Cell clusters were
determined using a graph-based unbiased clustering approach implemented
in Seurat. Positive markers defining each cluster were identified using
the Wilcoxon rank-sum test. Six representative markers were selected
for each cluster to visualize in heatmaps.
Single-cell TCR sequencing data analysis
After V(D)J sequences were assembled and annotated, only productive γ
and δ TCR sequences were kept. Two TCR diversity metrics (i.e. species
richness and exponential of Shannon entropy) were estimated for each
sample using iNEXT R package^[255]47,[256]68. Species richness measured
total unique clone numbers, whereas the Shannon index computed the
uncertainty in predicting the identity of a sequence taken at random
from the dataset. Both interpolated and extrapolated diversities were
estimated, and a 95% confidence interval was based on 50 bootstraps.
TCR repertoires were visualized using the Treemap R package
([257]https://CRAN.R-project.org/package=treemap). Downstream TCR
analysis such as V(D)J usage, shared TCR, and integration of TCR and
RNA-seq was performed using customized R scripts (available upon
request).
Bulk RNA-sequencing and data analysis
cDNA was synthesized and amplified (16-cycle PCR) from 5 ng total RNA
using SMARTer® Ultra® Low RNA Kit (Clontech). Nextera XT library prep
kit (Illumina) was used to generate the final sequencing library (12
PCR cycles performed to amplify libraries) using 1 ng of cDNA as the
input. The amplified libraries were size-selected at 400 to 600 bp.
Sequencing was performed on Illumina HiSeq® 2500 (Illumina) by
multiplexed paired-read run with 2 × 100 cycles. The sequencing reads
were mapped to the customized mouse genome using ArrayStudio
(OmicSoft). Sense-strand exon reads were used to quantify the gene
expression level by RSEM algorithm implemented in ArrayStudio. Genes
were flagged as detectable with a minimum of 10 reads. Differentially
expressed gene analysis was performed using Deseq2^[258]69. Genes with
fold change | > 1.5 | and FDR < 0.05 were considered significantly
differentially expressed. The differentially expressed genes were
subjected to pathway enrichment analysis using the Running Fish exact
test in NextBio ([259]www.nextbio.com). TCR hypervariable-region
sequences were reconstructed using TRUST^[260]70.
Statistics and reproducibility
Statistical significance (p values) within the groups was determined by
using one of the following statistical tests: unpaired t-tests assuming
similar SD; one-way ANOVA with Tukey’s multiple comparison post-test;
or ordinary two-way ANOVA with Sidak’s multiple comparison post-test,
*p < 0.05, **p < 0.005, ***p < 0.0005. P values of < 0.05 were
considered significant. Statistical analyses were performed with
Graphpad Prism 8. Samples were defined as biological replicates and no
technical replicates were used to generate graphs. Each experiment was
repeated at least three times, sample sizes and numbers and the
statistical test used were indicated in each figure legend.
Reporting summary
Further information on research design is available in the [261]Nature
Research Reporting Summary linked to this article.
Supplementary information
[262]Supplementary Information^ (23.7MB, pdf)
[263]42003_2021_2438_MOESM2_ESM.pdf^ (76.7KB, pdf)
Description of Additional Supplementary Files
[264]Supplementary Data 1^ (71KB, xlsx)
[265]Reporting Summary^ (5MB, pdf)
Acknowledgements