The Establishment of Resident Memory B Cells in The Lung Requires Local Antigen Encounter

Articles
https://doi.org/10.1038/s41590-018-0260-6
The establishment of resident memory B cells in

the lung requires local antigen encounter
S. Rameeza Allie1, John E. Bradley1, Uma Mudunuru1, Michael D. Schultz2, Beth A. Graf2, Frances E. Lund2
and Troy D. Randall 1*
Memory B cells are found in lymphoid and non-lymphoid tissues, suggesting that some may be tissue-resident cells. Here we
show that pulmonary influenza infection elicited lung-resident memory B cells (BRM cells) that were phenotypically and func-
tionally distinct from their systemic counterparts. BRM cells were established in the lung early after infection, in part because
their placement required local antigen encounter. Lung BRM cells, but not systemic memory B cells, contributed to early plas-
mablast responses following challenge infection. Following secondary infection, antigen-specific BRM cells differentiated in
situ, whereas antigen-non-specific BRM cells were maintained as memory cells. These data demonstrate that BRM cells are an
important component of immunity to respiratory viruses such as influenza virus and suggest that vaccines designed to elicit
BRM cells must deliver antigen to the lungs.
D
urable humoral immunity is maintained by long-lived, anti- antibodies generated in the lymph node circulate systemically and
body-secreting plasma cells that home to the bone marrow1,2, efficiently protect the lung14, it is not clear whether BRM cells might
as well as resting memory B cells that are poised to respond be useful or if they even exist at all.
rapidly following a secondary encounter with antigen3. The genera- Here we used parabiosis to definitively demonstrate the presence
tion of both cell types requires help from T cells, typically in the ger- of BRM cells in the lung following influenza infection. We found
minal center (GC), where B cells rapidly proliferate and are selected that influenza-specific BRM cells, particularly IgM+ BRM cells,
for high-affinity antigen receptors4,5. As GC B cells differentiate into were established early after influenza infection and were phenotypi-
memory cells, they stop proliferating6,7 and, in some cases, home to cally distinct from their lymphoid counterparts. The formation of
mucosal surfaces8,9. For example, pulmonary infection with influ- BRM cells that colonized the lung was dependent on early CD40-
enza virus elicits memory B cells in the lung that express CXCR39, dependent interactions with T cells in lymphoid organs. However,
whereas memory B cells responding to intestinal infection with the placement of BRM cells in the lung also required encounter with
rotavirus express mucosal homing receptors, such as α4β7 integrin antigen in the lung itself. Importantly, the presence of BRM cells in
and CCR98. the lung led to a rapid secondary ASC response in the lung follow-
Early studies of memory B cells in the lung used limiting dilu- ing a challenge infection. This rapid secondary response was main-
tion and in vitro differentiation to indirectly enumerate memory tained when mice were treated with the sphingosine-1-phosphate
B cells by enzyme-linked immunospot (ELISPOT10) but did not receptor 1 (S1P1R) agonist FTY-720 to prevent lymphocyte recir-
directly characterize the memory B cells themselves. More recent culation, suggesting that BRM cells differentiated in situ rather than
studies use fluorescently labeled hemagglutinin (HA) to iden- in draining lymph nodes. Taken together, these data suggest that,
tify influenza-specific B cells in the lung and show that these cells along with resident memory T cells, BRM cells in the lung contrib-
have hallmarks of resident memory cells, such as the expression of ute to protection against secondary pulmonary infections.
CD699. Importantly, following adoptive transfer, memory B cells
from the lung respond more quickly to challenge infection than do Results
those from lymphoid organs9, suggesting that memory B cells in Identification of influenza-specific B cells. In order to identify
different locations may be functionally distinct. influenza-specific B cells, we expressed recombinant nucleoprotein
Although these studies imply that resident memory B (BRM) (NP) monomers with a biotinylation domain and a 6× his tag in
cells are generated following influenza infection, they do not test Escherichia coli and expressed recombinant HA from the A/PR8/34
whether potential BRM cells in the lung recirculate or whether they (PR8–H1N1) and A/X31 (X31–H3N2) viruses with a GNC4 trimer-
alter the properties of a secondary B cell response following infec- ization domain and either a 6× his tag or a biotinylation (Avi)
tion. In fact, it is not clear whether BRM cells in the lung would domain in 293 cells (Fig. 1a). Following purification over a nickel
benefit pulmonary immunity. For example, lung-resident mem- column (Fig. 1b), we enzymatically biotinylated the purified recom-
ory T (TRM) cells are a critical element of pulmonary immunity binant HA protein, and ‘tetramerized’ the recombinant HA and NP
because they must physically contact MHC-expressing target cells proteins with fluorochrome-conjugated streptavidin to generate
in a secondary response in order to exert their effector functions11. reagents that could be used in flow cytometry assays.
In contrast, memory B cells either differentiate into antibody- To determine whether our B cell tetramers could identify anti-
secreting cells (ASCs) or re-enter the GC and undergo additional gen-specific cells, we infected C57BL/6 mice with either PR8 or X31
rounds of proliferation and selection12,13. Both of these functions and determined the frequency of tetramer-binding cells in the GC
can be efficiently accomplished in draining lymph nodes. Given that population (complete gating strategy in Supplementary Fig. 1) from
Department of Medicine, Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, Birmingham, AL, USA. 2Department
1
of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA. *e-mail: randallt@uab.edu
Nature Immunology | VOL 20 | JANUARY 2019 | 97–108 | www.nature.com/natureimmunology 97

Articles NaTuRe IMMunoLogy
a b
Kd 1 2 3 4
NP Avi His
f mLN day 15 memory B cells
80
CD19 – APC Cy7
CD19 – APC Cy7

65 5 5 5
CD38 – AF700
10 10 10
CD5 HA-PR8 GCN4Zip His 14.8
. 9 6.1
4 4
infected
4
50 10 10 10
PR8
3 3
10
3 10 10
35 0 0
CD5 HA-PR8 GCN4Zip Avi 0
30
3 4 5 3 4 5 3 4 5
25 0 10 10 10 0 10 10 10 0 10 10 10
15 PNA – Pac. Blue NP – PE HA (PR8) – PE
c mLN day 15 GC B cells g mLN day 0 ISW memory B cells
CD95 (Fas) – FITC
CD19 – APC Cy7
CD19 – APC Cy7

5
6.3 10
5
9.4 10
5
9.6 5 5 5
HA (PR8) – PE
HA (PR8) – PE
10 10 10 10
IgM – Per 710

4 4
10 10
infected
4 4
10 4 4
Naive
10 10 10
PR8
3 3
10 10
3
10 2 2 10
3
3 3
10 10 10 10
0 0 0
40.6 0.5 0
2.3
0 0.0 0 0.5
3 4 5 2 3 4 5 2 3 4 5 3 4 5 3 4 5 3 4 5
0 10 10 10 0 10 10 10 10 0 10 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10
PNA – Pac. Blue NP – APC HA (X31) – APC IgD – eF450 NP – PE HA (PR8) – PE
d mLN day 15 GC B cells h mLN day 23 ISW memory B cells
CD95 (Fas) – FITC
CD19 – APC Cy7
CD19 – APC Cy7

10
5
4 10
5
43.5 10
5
0.2 5 5
HA (PR8) – PE
5 10 10
IgM – Per 710

10
4 4
10 10
NP – PE
infected
4 infected
10 4 4 4
10 10
PR8
10
X31
3 3
10 10
3
10 2 2 3 3 3
10 10 10 10 10
0 0 0
4 4.2 0 3.2
0 11.4 0 4.7
3 4 5 2 3 4 5 2 3 4 5 3 4 5 3 4 5 3 4 5
0 10 10 10 0 10 10 10 10 0 10 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10
PNA – Pac. Blue HA (X31) – APC HA (X31) – APC IgD – eF450 NP – PE HA (PR8) – PE
e mLN day 15 GC B cells i mLN 9 week ISW memory B cells
CD19 – APC Cy7
CD19 – APC Cy7

CD19 – APC Cy7
CD19 – APC Cy7
5 5 5
CD138 – BV605
5 5 5
10 0.3 10 10 10 10 10
IgM – Per710
20.1 6.2
S. mansoni
4 4
infected
infected
10 10 10 4 4 4
10 10 10
PR8
3 3 3
10 10 10 3 3 3
10 10 10
0 0 0.03
0 0
5.6 0 0 0.2
3 4 5 3 4 5 3 4 5 3 4 5 3 4 5 3 4 5
0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10
CD19 – APC Cy7 NP – PE HA (PR8) – PE IgD – eF450 NP – PE HA (PR8) – PE
Fig. 1 | Identification of influenza-specific B cells. a, Schematic of recombinant NP and HA proteins. b, Coomassie-stained sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS–PAGE) gel showing recombinant HA(PR8) (lane 2), HA(X31) (lane 3) and NP (lane 4). Image is representative
of 12 independent preparations of recombinant protein. c–f, Cells from the mLNs of day 15 influenza-infected mice were first gated on live, singlet CD19+
lymphocytes (Supplementary Fig. 1a) and then on PNA+CD95+ GC B cells (c–d), or first gated on live, singlet lymphocytes (Supplementary Fig. 1a) and
then on CD138+ plasmablasts (e), or first gated on live, singlet CD19+IgD– lymphocytes (Supplementary Fig. 1b) and then on PNAloCD38+ memory B cells
(f). Data are representative of four independent experiments with five mice. g–i, Cells from the mLNs of naïve mice (g), day 23 PR8-infected mice (h), and
week 9 S. mansoni–infected mice (i) were gated on live, singlet CD19+CD38hi lymphocytes (Supplementary Fig. 1c) and analyzed for NP-specific and
HA-specific isotype-switched (ISW) memory B cells. Data are representative of four experiments with five mice. eF450, eFluor 450; PE, phycoerythrin.
the mediastinal lymph nodes (mLNs). In mice infected with PR8 B cells in PR8-infected mice (Fig. 1h). Both IgM+ memory
for 15 days, we found that about 10% of the GC B cells bound the B cells and isotype-switched memory B cells could be found in
HA(PR8) tetramer and 40% bound the NP tetramer, but very few lungs, spleen and mLNs of PR8-infected mice, but not in those of
B cells bound the HA(X31) tetramer (Fig. 1c). Conversely, in mice naïve mice, on day 30 and day 70 after infection (Supplementary
infected with X31, about 4% of the GC B cells bound the HA(X31) Fig. 2). These results demonstrate the specificity of the B cell
tetramer and about 43% bound the NP tetramer, but very few bound tetramer reagents.
the HA(PR8) tetramer (Fig. 1d). We also observed both NP-specific
and HA(PR8)-specific cells in the CD138+ plasmablast population BRM cells in the lung and lymphoid tissues are distinct. To char-
and in the CD38+IgD–PNA– memory B cell population in the mLNs acterize non-circulating memory B cells in the mLNs, lung and
of PR8-infected mice (Fig. 1e,f). spleen, we infected mice with PR8 and, 56 days later, infused them
To further confirm that we were identifying antigen-specific with fluorochrome-conjugated anti-B220 5 min prior to euthanasia.
memory B cells rather than non-specific binding to sialic acid (for We subsequently gated on CD19+B220– (non-circulating) CD38hi
HA tetramers), we next gated on isotype-switched memory B cells in isotype-switched memory B cells and finally gated on NP-binding
the mLNs of naïve, PR8-infected and Schistosoma mansoni–infected cells (Fig. 2a–d). We found that the majority of NP-specific iso-
mice (Fig. 1g–i). We found essentially no binding of either NP or type-switched memory B cells in the mLNs and spleen expressed
HA(PR8) tetramers to isotype-switched memory B cells in either CD73 (Fig. 2e), a marker noted in previous memory B cell stud-
the naïve or S. mansoni–infected mice but observed about 11% ies15, whereas most isotype-switched memory B cells in the lung
NP-specific and 5% HA(PR8)-specific isotype-switched memory did not express CD73. NP-specific memory B cells in the lung also
98 Nature Immunology | VOL 20 | JANUARY 2019 | 97–108 | www.nature.com/natureimmunology

NaTuRe IMMunoLogy Articles
a b e d56 B220– (non-circulating) NP-specific ISW memory B cells
mLN Lung Spleen mLN Lung Spleen mLN Lung Spleen
105 105 105 41.2 51.9 5.8 32.4 31.2 61.2
94.4 66.8 98.5 80
**** ####
CD73 PD-L2 (%)

AF 647 – B220
CD73 – BV605
CD38 – AF700
104 104 104 60
+
103 103 10
3
40
34.3 8.0 24.6
+
2
10 102 0 20
0 0 1.7 5.2 12.6 49.1 5.2 2.3
0
3 4 5
0 103 104 105 0 3 4
10 10 10
5
0 10 10 10 mLN LG SP
CD19 – APCCy7 CD19 – APC Cy7 PD-L2 – FITC
c mLN Lung Spleen d mLN Lung Spleen f mLN Lung Spleen
CD80 – Pac. Orange

105 105 48.9 39.9 16.7 50.3 41.8 48.0
CD19 – APC Cy7

105
IgM – PE Cy7
CD83 PD-L2 (%)

104 104 19.5 24.3 6.5
4
40 **** ####
+
10 30
103 103
10
3 20
0 0 10
–
0
1.73 19.5 1.37 9.4 2.5 9.9 23.1 7.9 2.8
0
0 103 104 105 0 3 4
10 10 10
5
0 103 104 105 mLN LG SP
eF450 IgD NP – PE PD-L2 – FITC
g d121 B220– (non-circulating) NP-specific ISW memory B cells i d31 B220– (non-circulating) NP-specific ISW memory B cells
mLN Lung Spleen mLN Lung Spleen
105 89.9 100 *** * * ***
CD19 – APC Cy7

1.4 93.9 0.4 77.2 1.5
CD69 CD103 (%)
105 70.5 93 57.3 100

CD69 – BV605
4 90
CXCR3 (%)
–
10 80
80 104
3 60
+
10 3
70 10 40
+
0 60 0 20
8.6 0.0 5.8 0.0 21.3 0.0 50 0
103 10 105
4
0 103 10 105
4
0 mLN LG SP mLN LG SP
CD103 – APC CXCR3 – BV421
mLN Lung Spleen
h 105 77.9 13.4 92.6 1.5 72.7 6.1 50 j d44 B220– (non-circulating) NP-specific memory B cells
CD62L CD69 (%)
* **
CD69 – BV605
104 40 mLN Lung Spleen

+
30
103 6.1% IgA 7.1% IgA 10.2% IgA
20 20.4% IgG1 22.2% IgG1 19.4% IgG1
+
0 10 20.4% IgG2b 18.2% IgG2b 15.3% IgG2b

8.6 0.0 5.8 0.0 21.3 0.0 30.6% IgG2c 29.3% IgG2c 19.4% IgG2c
0 11.2% IgG3 9.1% IgG3 7.1% IgG3
103 10 105
4
0 mLN LG SP 11.2% IgM 14.1% IgM 28.6% IgM
CD62L – BV510
Fig. 2 | Phenotype of memory B cells in the lung and lymphoid organs. a–d, PR8-infected mice were infused with anti-B220 5 min prior to euthanasia, and
cells from the mLNs, lung and spleen were gated on live, singlet lymphocytes (Supplementary Fig. 1a) and then on B220– non-circulating B cells (a), CD38+
memory B cells (b), IgD–IgM– ISW cells (c) and NP-specific cells (d). e–i, Within the population of NP-specific memory cells described in d, we determined
the frequencies of CD73+PD–L2+ memory B cells (e), CD80–PD–L2+ memory B cells (f), CD69+CD103– memory B cells (g), CD62L+CD69+ memory B cells
(h) and CXCR3+ memory B cells (i). Data are representative of three independent experiments with five mice (a–d), two experiments with five mice
(g–h) or five experiments with five mice. (i). Graphs show individual data points as well as mean ± s.d. Data were analyzed by one-way ANOVA with Tukey’s
post-test for multiple comparison: ****P = 0.0001 and ####P = 0.0001 (e), ****P = 0.0001 and ****P = 0.0001 (f), ***P = 0.0002 and *P = 0.0105 (g),
*P = 0.0471 and **P =0.0042 (h), and *P = 0.0206 and ***P = 0.0008 (i). P < 0.05 is considered significant. j, Cells from the mLNs, lung and spleen of day
44 PR8-infected mice were gated on live, singlet lymphocytes (Supplementary Fig. 1a) and then gated on NP-specific CD19+CD38+IgD– memory B cells
(Supplementary Fig. 3c–e), and the frequency of IgA-, IgG1-, IgG2b-, IgG2c-, IgG3- and IgM-expressing cells was determined. Data are representative of
three independent experiments with five mice. AF647, Alexa Fluor 647; AF700, Alexa Fluor 700.
differed from those in the mLNs and spleen based on the expression resided in the lung, we infected C57BL/6 (CD45.2+) mice with influ-
of CD80 and PD-L2 (Fig. 2f). enza and, on day 44, surgically paired them with CD45.1+ partner
Resident memory T cells in the lung are often characterized using mice for an additional 15 days to allow recirculating cells to equili-
CD69 and CD10311. Although most NP-specific memory B cells in brate between the partners. To determine whether the pairs had
all three tissues expressed CD69, none of them expressed CD103 reached equilibrium, we enumerated both donor (CD45.2+) and
(Fig. 2g). Moreover, although approximately 12% of the BRM in partner (CD45.2–) naïve B cells in the mLNs and spleen. We found
mLNs and spleen expressed the LN homing receptor CD62L, almost that naïve B cells had equilibrated between donor mice and partner
none of the BRM cells in the lung expressed this marker (Fig. 2h). mice in the mLNs and spleens of both mice in each pair (Fig. 3a,b).
Finally, NP-specific memory B cells in the lung uniformly expressed We next gated on non-circulating IgM+ or isotype-switched memory
high amounts of CXCR3, whereas those in the mLNs and spleen had B cells in the lung and examined whether the NP-specific memory B cells
a substantial population of CXCR3– cells (Fig. 2i). These characteris- had equilibrated (Fig. 3c). When PR8-memory mice were paired with
tics were generally shared by NP-specific isotype-switched memory naïve mice, NP-specific IgM+ memory B cells as well as NP-specific iso-
B cells in the non-circulating (B220–) and total (B220+/–) populations type-switched memory B cells remained in the previously infected lung
(Supplementary Fig. 3a–b), although we should note that more than and did not migrate to the naïve lung (Fig. 3d,e). We also paired previously
95% of NP-specific isotype-switched memory B cells in the lung and infected CD45.2+ mice with CD45.1+ mice that had previously received
mLNs are in the B220– fraction. Despite the phenotypic differences intranasal lipopolysaccharide (LPS) to promote pulmonary inflamma-
between memory B cells in the lungs and lymphoid organs, the distri- tion. Again, we found that NP-specific IgM+ memory B cells as well as
bution of isotypes was remarkably similar, except for an increase in the NP-specific, isotype-switched memory B cells remained in the previ-
frequency of IgM+ BRM cells in the spleen (Fig. 2j and Supplementary ously infected lung and did not migrate to the LPS-treated lung (Fig. 3f,g).
Fig. 3c–e). These results demonstrate that BRM cells in the lung are Finally, we paired previously infected CD45.2+ mice with previously
phenotypically different from their lymphoid counterparts. infected CD45.1+ mice so that both lungs had been exposed to virus.
Again, we found that NP-specific IgM+ memory B cells as well as
BRM cells in the lung do not recirculate. To determine whether NP-specific, isotype-switched memory B cells remained in the lung of
the non-circulating, influenza-specific memory B cells permanently origin and did not migrate to the opposite lung (Fig. 3h,i). These data

CD45.1 CD45.2
a d 8 Naive PR8
CD45.1 CD45.2 Naive PR8 *
104
IgM NP (×103)
250,000 100 0.2 0 0.1 1.9 6
IgMNP – PE
3
200,000 53 47 49 51 10
CD45.2+(%)
80 4
SSC-A
150,000 60 102
100,000 40 2
101
50,000 20
0 0 0 0
0 103 104 105 CD45.1 CD45.2 0 101 102 103 104 Host Partner Host Partner
CD45.2 – FITC CD45.2 – FITC
b CD45.1 CD45.2
e Naive PR8 Naive PR8
CD45.1 CD45.2 40
250,000 104 0.1 0.7 0 ***
11.7
ISW NP (×103)
100
ISW NP – PE
200,000 52 47 45 54 103 30
CD45.2+(%)
80
SSC-A
150,000 60 102 20
100,000 40
101 10
50,000 20
0 0 0 0
0 103 104 105 CD45.1 CD45.2 0 101 102 103 104 Host Partner Host Partner
CD45.1 CD45.2
c f LPS PR8 LPS PR8
5 5 5
105 10 82.3 10 24 105 0 0.3 0.2 1.4 ****
CD38 – AF700
B220 – AF647
IgM – PE Cy7
IgM NP (×103)
4
IgMNP – PE
104 104
104 10 4
103
10 3 3
103 2
102 102 103
0 0 0 0
4.64 28 1
0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0
CD19 – APC Cy7 CD19 – APC Cy7 IgD – eF450 Host Partner Host Partner
CD45.2 – FITC
g LPS PR8 h PR8 PR8 i PR8 PR8

105 0 0 0.1 11.1 105 2.1 0.8 0.3 2.4 105 15 2.5 2.4 23.3
ISW NP – PE
IgM NP – PE
IgM NP – PE
4 104 104
10
103 103
103 0 0
0
0 103 104 105 0 103 104 105 0 103 104 105

CD45.2 – FITC CD45.2 – FITC CD45.2 – FITC
CD45.1 CD45.2 CD45.1 CD45.2 CD45.1 CD45.2

LPS PR8 6 PR8 PR8 PR8 PR8
25 40
**** **** ** **** **
ISW NP (×103)
20
IgM NP (×103)
IgM NP (×103)
30
4
15
20
10
2
5 10
0 0 0
Host Partner Host Partner Host Partner Host Partner Host Partner Host Partner
Fig. 3 | Identification of influenza-specific, non-circulating BRM cells in the lung. Mice were infected on day 0, surgically paired with partner
mice on day 44 and analyzed on day 59. a,b, Cells from the mLNs (a) or spleen (b) of each partner mouse were gated on live, singlet lymphocyte
CD19+CD38+IgD+IgMlo naïve B cells (Supplementary Fig. 1d), and the frequency of CD45.2+ cells was determined in each partner. Data are combined from
four independent experiments with three mice each. Graph shows individual points as well as mean ± s.d. c, Cells from the lung were gated on live, singlet
lymphocytes (Supplementary Fig. 1a) and subsequently gated on CD19+B220– (non-circulating), CD38+IgD–IgM+ (IgM) or CD38+IgD–IgM– (ISW) memory
B cells. d,e, The frequencies and numbers of NP-specific IgM+ (d) and ISW (e) memory B cells from host and partner mice were determined in the lungs
of naïve mice paired with PR8-infected mice. f,g, The frequencies and numbers of NP-specific IgM+ (f) and ISW (g) memory B cells from host and partner
mice were determined in the lungs of LPS-treated mice paired with PR8-infected mice. h,i, The frequencies and numbers of NP-specific IgM+ (h) and ISW
(i) memory B cells from host and partner mice were determined in the lungs of PR8-infected mice paired with PR8-infected mice. Data are representative
of three independent experiments, each with three pairs of mice (d,e), two experiments with three pairs of mice (f,g), or three experiments combined, with
a total of 11 pairs of mice (h,i). Graphs show individual data as well as mean ± s.d. Significance was determined using one-way ANOVA (paired) followed
by the Bonferroni–Sidak method for multiple comparison: *P = 0.0428 (d), ***P = 0.0001 (e), ****P = 0.0001 and **P = 0.0040 (f–h), and ****P = 0.0001
and **P = 0.0022 (i). P < 0.05 is considered significant. BV-421, Brilliant Violet 421; BV-510, Brilliant Violet 510; FITC, fluorescein isothiocyanate; SSC-A,
side scatter area.
suggest that antigen-specific memory cells in the lung are maintained as the IgM+ and isotype-switched fractions were strongly biased to
tissue resident cells (BRM cells) and do not migrate, even to previously remain in the lung of origin (Supplementary Fig. 4a–d). This bias
infected lungs. was less pronounced in the mLNs and was only significant in the
We also tested whether tissue residence was restricted to the isotype-switched memory B cells (Supplementary Fig. 4e–h). The
lung or also occurred in the mLNs and spleen. Using PR8-infected bias was even less evident in the spleen but did achieve signifi-
CD45.1+ mice paired with PR8-infected CD45.2+ mice, we found cance for NP-specific IgM+ and isotype-switched memory B cells
that both HA-specific and NP-specific memory B cells in both (Supplementary Fig. 4i–l).

a c
CD45.1 CD45.2
B220+Dead – AF488
105 105 105 9.8
250,000 100
CD38 – AF700
IgM – PE Cy7
56 42 48 50 104 4
200,000 80
10 104
CD45.2+(%)
103
SSC-A
150,000 60 103 83.9 103

102
100,000 40 0
0 0
50,000 20 5.9
15.8
0 0
3 4 5 0 103 104 105 0 103 104 105 0102 103 104 105
0 10 10 10 CD45.1 CD45.2
CD19 – APC Cy7 CD19 – APC Cy7 IgD – eF450
CD45.2 – FITC
b CD45.1 CD45.2 d CD45.1 CD45.2 CD45.1 CD45.2
250,000 105 1.1 0.2 0.1 0.5 8
55 43 56 42 100 * ***
IgM NP (×102)
200,000 80 104 6
CD45.2+(%)
NP – PE
SSC-A
150,000 60 103
4
100,000 102
40
0 2
50,000 20
0 0 0
Host Partner Host Partner
0 103 104 105 CD45.1 CD45.2 0 103 104 105
e CD45.1 CD45.2
f CD45.1 CD45.2
g CD45.1 CD45.2
105 18.5 1.8 5.0 16.9 105 2.0 0.4 0.3 1.3 105 7.2 0.8 1.0 5.3
HA(PR8) – PE
HA(PR8) – PE
10 4 10 4
104
NP – PE
103 103
103
0 102 102
0 0
0 103 104 105 0 103 104 105 0 103 104 105

CD45.2 – FITC CD45.2 – FITC CD45.2 – FITC
10 ** * 20 4 ** ##
* ***
ISW HA (×103)
IgM HA (×102)
ISW NP (×103)
8 15 3
6
10 2
4
5 1
2
0 0 0
Host Partner Host Partner Host Partner Host Partner Host Partner Host Partner
Fig. 4 | BRM cells in the lung are established early after infection. Mice were infected on day 0, surgically paired with partner mice on day 15 and analyzed
on day 30. a,b, Cells from the mLNs (a) or spleen (b) were gated on live, singlet lymphocyte CD19+IgD+IgMlo naïve B cells (Supplementary Fig. 1d), and
the frequency of CD45.2+ cells was determined in each partner. These data are combined from two independent experiments totalling eight pairs of mice.
Graphs show individual data points as well as mean ± s.d. c, Cells from the lung were gated on live, singlet (Supplementary Fig. 1a) and subsequently gated
on live, CD19+B220– (non–circulating), CD38+IgD–IgM+ (IgM) or CD38+IgD–IgM– (ISW) memory B cells. d,e, The frequencies of NP-specific IgM+ (d) and
ISW (e) memory B cells from the lungs of host and partner mice. f,g, The frequencies of HA(PR8)-specific IgM+ (f) and ISW (g) memory B cells from the
lungs of host and partner mice. Data in c–g are representative of three independent experiments, each with four pairs of mice. Graphs show individual data
as well as mean ± s.d. Significance was determined using one-way ANOVA (paired) followed by the Bonferroni–Sidak method for multiple comparison:
*P = 0.0457 and ***P = 0.0002 (d), **P = 0.0047 and *P = 0.0213 (e), *P = 0.0285 and ***P = 0.0002 (f), and **P = 0.0060 and ##P = 0.0080 (g).
P < 0.05 is considered significant. AF488, Alexa Fluor 488.
Memory B cells are established early after immunization7. To We found that GC B cells appeared in the mLNs as early as 7 days
determine whether BRM cells in the lung are also established early after infection, peaked around 15 days after infection and declined
after infection, we infected both CD45.1+ mice and CD45.2+ mice thereafter (Fig. 5a). In contrast, memory B cells in both the blood
with PR8, surgically paired them 15 days later and evaluated the (Fig. 5b) and lung (Fig. 5c) were barely detectable at day 7, but
presence of BRM cells 15 days after joining. We found that naïve peaked at day 15 and declined thereafter.
B cells in mLNs and spleen had equilibrated in the parabiotic pairs To test whether the appearance of BRM cells in the lung was
(Fig. 4a,b). However, after gating on non-circulating memory B cells dependent on CD40 signalling, we infected mice with PR8 and
(Fig. 4c), we found that NP-specific IgM+ memory B cells as well treated them at various intervals with a blocking antibody to CD40L
as NP-specific isotype-switched memory B cells remained mainly (MR1). We found that CD40L blockade for 2 weeks (regardless of
in the partner of origin (Fig. 4d,e). HA-specific IgM+ memory B when the treatment started) completely eliminated the GC B cell
cells as well as HA-specific, isotype-switched memory B cells also response in both the mLNs and the lung (Fig. 5d–g). Early CD40L
remained mainly in the partner of origin (Fig. 4f,g). Collectively, blockade prevented the placement of NP-specific IgM+ and iso-
these findings demonstrate that many BRM cells in the lung are type-switched BRM cells in the lung (Fig. 5h). Reductions in BRM
established very early after infection. cells were less pronounced when the blockade occurred between
day 10 and day 20 (Fig. 5i) or between day 20 and day 30 (Fig. 5j).
BRM cells are formed by early CD40-dependent mechanisms. However, BRM cells in the lung were not reduced when CD40L was
To establish the kinetics of GC formation relative to BRM cell gen- blocked between day 30 and day 40 (Fig. 5k), despite a complete loss
eration, we enumerated antigen-specific GC B cells in the mLNs, of GC B cells in the mLNs and lung. We obtained a similar result
as well as isotype-switched memory B cells in the blood and lung. when we examined HA-specific BRM cells (Supplementary Fig. 5),

a
Day 7 Day 15 Day 70 5
GC-NP
GC B cells (log10)
5 5 5
10 10 10
3 GC-HA
4 4
10 10
4 10
3
10
3
10
3 mLN
3
10 2 GC B cells
2
10
0
0 0 1
18.5 14.7 13.6 7 10 15 30 70
0 10
2
10
3 4
10 10
5
0 10
3
10
4
10
5
0 10
3
10
4
10
5
Days
b 100
10
5
10
5
10
5
ISW NP
CD19 – APC Cy7
ISW B cells (%)

4 4 ISW HA
10 10
4 10 10
3
10 3 10
3
Blood
10
2 1 ISW memory
10
0
0 0
1 6.7 2.3 0.1
0 10
2
10
3 4
10 10
5
0 10
3
10
4
10
5
0 10
3
10
4
10
5 7 10 15 30 70
c Days
10
5
10
5
10
5 4
ISW B cells (log10)

4 4
10 4 10
10 3
3
10 3
10
3 10 Lung
10
2 2 ISW NP ISW memory
0
0 0 ISW HA
0.8 24 22 1
0 10
2
10
3 4
10 10
5
0 10
3
10
4
10
5
0 10
3
10
4
10
5 7 10 15 30 70
Days
NP – PE
mLN GC B cells Lung GC B cells
d CT MR1 CT MR1
5 8 ##
2.0 **
5
10 10
GC B cells (×104)
GC B cells (106)
CD38 – APC
6
CD38 – APC
4 4
10 1.5 10
10
3
1.0 4
0 0
0.5 2
17.8 0.2 1.72 0.3
3 4 5
0.0 3 4 5
0
0 10 10 10 CT MR1 0 10 10 10 CT MR1
GL7 – PerCP GL7 – PerCP
e 6 *
**
5
10
5
1.5 10
GC B cells (×104)
GC B cells (106)
CD38 – APC
CD38 – APC
4
10
4
1.0
10 4
3 3
10 10
0.5 2
0 0
13.4 0.4 4.2 0.97
0.0 3 4 5
0
3 4 5
0 10 10 10 CT MR1 0 10 10 10 CT MR1
f ##
10
5 5 4
5 ** 10 GC B cells (×104)
CD38 – APC
GC B cells (105)
CD38 – APC
4
10
4
4 10 3
10
3
3 10
3
2
0
2
0 1
7.6 0.1 1
4.8 0.18
3 4 5
0 3 4 5
0
0 10 10 10 CT MR1 0 10 10 10 CT MR1
g #
10
5
10
5 5
*
GC B cells (×104)
CD38 – APC
CD38 – APC
3 4
GC B cells (105)
4 4
10 10
3
10
3 2 10
3
2
0 0
1 1
7.7 0.3 6.0 0.22
0
0 10
3
10
4
10
5
0 CT MR1 0 10
3
10
4
10
5
CT MR1
Fig. 5 | BRM cells in the lung are generated from early CD40-dependent precursors. a, Cells from the mLNs of PR8-infected mice were gated on live,
singlet lymphocyte, CD19+PNA+CD95+ GC B cells (Supplementary Fig. 1f), and NP-specific as well as HA-specific cells were enumerated. b,c, Cells from
the blood (b) and lungs (c) of PR8-infected mice were gated on live, singlet lymphocyte, CD19+CD38+IgM–IgD– ISW memory B cells (Supplementary
Fig. 1c), and the frequency (b) and number (c) of NP-specific as well as HA-specific cells were determined. Data are representative of three independent
experiments with five mice at each time point. The data points represent mean ± s.d. d–k, Mice were infected with PR8 and administered anti-CD40L
(MR1) or isotype-matched control antibody (CT) every other day for 10 days starting on day 5 (d, h), day 10 (e,i), day 20 (f,j) or day 30 (g,k). Cells from
the mLNs and lung were gated on live, singlet lymphocyte, CD19+CD138lo cells (Supplementary Fig. 1g) and subsequently gated on GL7+CD38lo GC B cells
(d–g). Cells from the lung were gated on live, singlet lymphocyte, CD19+CD138loCD38+IgM+IgD– IgM BRM cells or CD19+CD138loCD38+IgM–IgD– ISW
BRM cells (h–k) (gating in Supplementary Fig. 1g). Data in d–k are representative of three independent experiments with five mice per group per timepoint.
Graphs show mean ± s.d. as well as individual data points. Significance was determined using a Mann–Whitney U-test: **P = 0.0079 and ##P =0.0014 (d),
**P = 0.0079 and *P =0.0462 (e), **P = 0.0079 and ##P =0.0024 (f), *P = 0.0159 and #P= 0.0180 (g); or unpaired, two-tailed t-test: **P =0.0018 and
***P =0.0003 (h), and **P = 0.0011 (i). P < 0.05 is considered significant. PerCP, peridinin chlorophyll protein complex.

Lung: IgM memory Lung: ISW memory
h CT MR1 CT MR1
105 7.6 2.8 105 15 4.5
15 ** 3 ***
CD19 – APC Cy7
CD19 – APC Cy7
ISW NP (×104)
104
IgM NP (×103)
104
10 2
103 103
102 5 102 1
0 0
0 0
0 102 103 104 105 CT MR1 0102 103 104 105 CT MR1
NP – PE NP – PE
i ns
105 6 105 9.9
9.1 9.9 23.2 4 **
CD19 – APC Cy7
CD19 – APC Cy7

IgM NP (×103)
ISW NP (×104)
4 4
10 4 10 3
103 103 2
2
0 0 1
0 0
0 103 104 105 CT MR1 0 103 104 105 CT MR1
NP – PE NP – PE
j
105 5.3 4.3 105 20 21.9 8
ns
CD19 – APC Cy7
CD19 – APC Cy7

ns
ISW NP (×104)
IgM NP (×103)
4 6
10 4 104
103 3 103 4
2
0 0 2
1
0 0
0 103 104 105 CT MR1 0 103 104 105 CT MR1
NP – PE NP – PE
k ns
105 4.2 3.4 5 105 18.4 18.8 4
ns
CD19 – APC Cy7
CD19 – APC Cy7
ISW NP (×104)
IgM NP (×103)
104 4 104 3
3
103 103 2
2
102 102
0 1 0 1
0 0
0 103 104 105 CT MR1 0 103 104 105 CT MR1
NP – PE NP – PE
Fig. 5 | Continued.
although the IgM+ BRM cells were placed in the lung as early as specific (Fig. 6e) isotype-switched memory B cells in the lung. As
day 10, whereas the isotype–switched memory B cells required up expected, a pulmonary challenge with X31 elicited both NP-specific
to 30 days to fill the lung compartment. These data indicated that (Fig. 6d) and HA(X31)-specific (Fig. 6f) isotype-switched memory
the seeding of influenza-specific BRM cells in the lung is dependent B cells in the lung. Importantly, pulmonary challenge with X31 also
on early T cell interactions, possibly in the GC, and that IgM+ BRM recruited HA(PR8)-specific isotype-switched memory B cells to the
cells are seeded earlier than isotype-switched BRM cells. lung (Fig. 6e). We observed similar results on day 10 (Fig. 6d–f) and
day 45 (Fig. 6g–i) after the challenge infection. We also examined
BRM establishment in the lung is dependent on local antigen. To IgM+ memory B cells in the same experiment but found that so few
test whether local antigen was needed for the placement of BRM HA(PR8)-specific IgM+ memory B cells were recruited to the lungs
cells in the lung, we surgically paired naïve mice, waited 15 days that the result was not significant (Supplementary Fig. 6c,f). Taken
for them to attain equilibrium, infected one mouse with PR8 and together, these data indicate that BRM cells need to encounter anti-
the other with X31, and analyzed the influenza-specific BRM cells gen again for their placement in the lung.
in the lungs 10 days later. After gating on non-circulating isotype-
switched memory B cells (Fig. 6a), we found that PR8-infected BRM cells in the lung respond rapidly to infection. To directly
partners had many more HA(PR8)-specific BRM cells than the compare the ability of systemic or pulmonary infection to elicit
X31-infected partners had (Fig. 6b). Conversely, we found that the BRM cells in the lung, we infected (or mock infected) mice in
X31-infected partners had many more HA(X31)-specific BRM cells either the peritoneal cavity or the lung and, after 30 days, enumer-
than the PR8-infected partners had (Fig. 6b). Importantly, both ated NP-specific B cells. As expected, naïve mice lacked NP-specific
partners had similar numbers of NP-specific BRM cells (Fig. 6b). GC B cells (Fig. 7a) and isotype-switched memory B cells (Fig. 7b),
These data demonstrate that even in concurrently infected partner whereas the spleens of mice infected in the peritoneal cavity or the
mice, BRM cells tend to stay in the lungs that express the antigen to lung had numerous NP-specific GC and isotype-switched memory
which they respond. B cells. In contrast, only the lungs of mice intranasally infected with
We next tested whether antigen-non-specific pulmonary inflam- PR8 had NP-specific isotype-switched memory B cells (Fig. 7c). To
mation could recruit pre-existing systemic memory B cells to the test the effect of lung BRM cells on the outcome of a challenge infec-
lung. To do this, we infected mice with PR8 in the peritoneal cavity tion, we intraperitoneally, intranasally or mock infected mice with
(or not), challenged them (or not) 30 days later with X31, and exam- PR8, challenged all groups with X31 on day 30, measured weight
ined the placement of NP-specific, HA(X31)-specific and HA(PR8)- loss over 2 weeks and assayed viral titers on day 35. We found that
specific memory B cells (Fig. 6c). We found that peritoneal infection intranasally primed mice had the lowest drop in weight (Fig. 7d)
with PR8 did not elicit either NP-specific (Fig. 6d) or HA(PR8)- and the lowest viral titers (Fig. 7e).

Day 40 (day10 post-challenge with X31)
a d --- PR8 PR8 i.p. infection
X31 --- X31 i.n. infection
105 105 105 105 24 ****
CD38 – AF700
CD19 APC fire

B220 – AF488
IgM – Per 710

104
ISW NP (×103)
104 104 104 18
103
103 12
0 103 103
0.9 7.5 0.5 15.9
0 6
0 0
86.6 15.4
0
0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 --- PR8 PR8 i.p.
CD19 – APC Cy7 CD19 – APC Cy7 IgD – eF450 NP – PE X31 --- X31 i.n.
e
10
105
ISW PR8 (×103)

8
CD19 – APC fire

b PR8 infected X31infected
10 4
6 ***
105 13.8 0.5 4 103 4
**** 0.1 0.9 3.7
ISW PR8 (log10)

104 3 0 2
0
103 2
0 103 104 105 --- PR8 PR8 i.p.
0 1 HA (PR8) – PE X31 --- X31 i.n.
f
0 10
0 103 104 105 105
PR8 X31 **
ISW X31 (×103)

8
CD19 – APC fire

HA (PR8) – PE Infecting virus
6
4 104
105 0 11
* 4
ISW X31 (log10)
3 103
CD19 – APC Cy7
104 4.9 0.0 4.2 2

0
2 0
103
--- PR8 PR8 i.p.
0 1 0 103 104 105
X31 --- X31 i.n.
HA (X31) – PE
0
103 104 105 PR8 X31
0
Infecting virus
g Day 75 (day 45 post-challenge with X31)
HA (X31) – PE
--- PR8 PR8 i.p. infection
5 10.7 11.1
10 5 i.n. infection
ns X31 --- X31
ISW NP (log10)
104 4 6
105
CD19 – APC fire
ISW NP (×103)
103 4
2
104
0
1 **
2
0 103 19.2 0.0 11.3
0 103 104 105 PR8 X31 0
NP – PE 0
Infecting virus
103 104 105 --- PR8 PR8 i.p.
0
c NP – PE X31 --- X31 i.n.
105 105 78.2 105
h
2.0
B220 – AF488
105
IgM – Per710
CD38 – APC
ISW PR8 (×103)

104 104
CD19 – APC fire
4 1.5
10
103 0 104
2.9 1.0
103
0
0
33.3
103 0.5 0.0 1.1 0.5 **
0
0 103 104 105 0 103 104 105 0 103 104 105 0.0
CD19 – APC fire CD19 – APC fire IgD – eF450 0 103 104 105 --- PR8 PR8 i.p.
HA (PR8) – PE X31 --- X31 i.n.
i
2.0
105
CD19 – APC fire
ISW X31 (×103)
1.5
104
1.0
103 4.6 0 3.9 0.5 ***

0
0.0
0 103 104 105 --- PR8 PR8 i.p.
HA (X31) – PE X31 --- X31 i.n.
Fig. 6 | Establishment of BRM cells in the lung requires local antigen encounter. a,b, Naïve mice were surgically joined, and on day 15, one was infected
with PR8 and the other was infected with X31. BRM cells in the lung were analyzed on day 25. Cells from the lung were gated on live, singlet lymphocytes
(Supplementary Fig 1a) and subsequently gated on B220–CD19+CD38hiIgM–IgD– ISW BRM cells (a). The frequency and number of HA(PR8)-specific (top
row), HA(X31)-specific (middle row) and NP-specific (bottom row) BRM cells were determined in each partner (b). Data are representative of three
independent experiments with five pairs of mice. Significance was determined using one-tailed, paired t-test: ****P = 0.0001 and *P = 0.0477. d–i, Mice
were peritoneally infected with PR8 on day 0, intranasally challenged with X31 on day 30 and analyzed on day 40 (d–f) and day 75 (g–i). Cells from the
lung were gated on live, singlet lymphocytes (Supplementary Fig. 1a) and subsequently gated on CD19+B220–CD38+IgD–IgM– ISW memory B cells
(c). NP-specific (d,g), HA(PR8)-specific (e,h) and HA(X31)-specific (f,i) ISW BRM cells were enumerated on day 40 (d–f) and day 75 (g–i). These data
are representative of two independent experiments with five mice per timepoint. Data were analyzed with an unpaired t-test: ****P =0.0001 (d),
***P =0.0009 (e), **P = 0.0013 (f), **P =0.0047 (g), **P =0.0011 (h) and ***P =0.0004 (i). P < 0.05 is considered significant. i.n., intranasal.
Although intranasally infected mice have BRM cells, they also ASCs in their lungs before and after challenge, whereas intranasally
likely have resident memory T cells, which undoubtedly contribute to infected mice had some NP-specific ASCs in the lung prior to chal-
secondary immunity. To test whether BRM cells participate in pulmo- lenge and had significantly more ASCs 4 days later (Fig. 8a).
nary recall responses, we enumerated ASCs by ELISPOT in mice that We performed a similar experiment in which BLIMP-1–yel-
were intraperitoneally, intranasally or mock infected for 30 days and low fluorescent protein (YFP) reporter mice were intraperitone-
subsequently challenged with X31 (or not) for 4 days. We found that ally, intranasally or mock infected for 30 days and subsequently
naïve and peritoneally infected mice completely lacked NP-specific challenged with X31 (or not) for 3 days. Given that ASCs strongly

Spleen germinal center
Nv i.p. i.n.
a
105 250 **
104 200
GC NP (×103)
103 150
100
0
50
0 10.5 18.1
0
0 103 104 105 Nv i.p. i.n.
Spleen ISW memory

b
105 50 **
CD19 – APC-Cy7
ISW NP (×103)
10 40
103 30
20
0
10
0.2 2.2 4.9
0
0 103 104 105 Nv i.p. i.n.
Lung ISW memory

c
105 150
****
104
ISW NP (×103)
100
103
50
0
0 1.3 27
0
0 103 104 105 Nv i.p. i.n.
NP – PE
d 120
e 107 **
106
VFU per mL
100
Weight (%)
----, i.n. 105

i.p., i.n. 104
80
i.n., i.n. 103
60 102 LOD
101
0 2 4 6 8 10 12 14 ---- i.p. i.n. PR8 infection
Days post infection i.n. i.n. i.n. X31 infection
Fig. 7 | BRM cells are associated with protection from secondary infection. a–c, Mice were infected in the peritoneal cavity or in the lung and analyzed
on day 30. Cells were gated on live, singlet lymphocyte, CD19+B220–CD138–GL7+CD38lo GC B cells (Supplementary Fig. 1h), and NP-specific cells were
enumerated in the spleen (a). Cells were gated on live, singlet lymphocyte CD19+B220–CD38+IgD–IgM– isotype-switched memory B cells (Supplementary
Fig. 1e), and NP-specific cells were enumerated in the spleen (b) and lung (c). Data are representative of three independent experiments with three
mice in the naïve group, five mice in the intraperitoneally infected group and four mice in the intranasally infected group. Graphs show mean ± s.d. as well
as individual data points. Significance was determined with a one-way ANOVA with Tukey’s post-test for multiple comparisons: **P =0.0038 (a),
**P =0.0021 (b), and ****P =0.0001 (c). d, Weight loss after challenge infection. e, Viral titers on day 5 after challenge infection. Data in d,e are
representative of two experiments with five mice per group. Graphs show mean ± s.d. (d) or individual data points as well as the geometric mean ± s.d.
(e). Data were analyzed using the Mann–Whitney U-test: **P = 0.0079. P < 0.05 is considered significant.
express BLIMP-1, we gated on the reporter, YFP, to enumerate ASCs challenge with X31. We found that FTY720 did not reduce the
in the lung. We found that naïve and peritoneally infected mice NP-specific ASC response in the lung (Fig. 8d), demonstrating that
completely lacked NP-specific ASCs in their lungs before and after the observed spots were derived from BRM cells that differenti-
challenge, whereas intranasally infected mice had some NP-specific ated in situ. We also examined the fate of the BRM cells that reacted
ASCs in the lung prior to challenge and had significantly more with the challenge virus (NP specific) and the BRM cells that did
ASCs 3 days later (Fig. 8b). To test whether the ASCs remaining in not react with the challenge virus (HA(PR8) specific). We found
the lung following a primary infection declined over time, we per- that the NP-specific BRM cells declined after challenge (Fig. 8e),
formed the same experiment, but challenged the mice with X31 at whereas the HA(PR8)-specific BRM cells did not (Fig. 8f). Collectively,
day 60. We found a few remaining ASCs in the lungs of previously these findings demonstrate that BRM cells in the lung are important for
infected mice but a large increase in the pulmonary ASCs response rapid secondary ASC responses and that inflammatory signals alone
in mice with lung BRM cells, but not in mice with systemic memory are insufficient to trigger the differentiation of BRM cells in the lung16,17.
B cells (Fig. 8c).
To ensure that the ASCs that we observed in the lungs of chal- Discussion
lenged mice were due to responding BRM cells and not recirculat- Our data show that pulmonary infection with influenza virus
ing memory B cells, we treated PR8 memory mice with fingolimod elicits non-recirculating, lung-resident BRM cells. Unlike memory
(FTY720) to block lymphocyte recirculation just before and after B cells in lymphoid organs, BRM cells in the lung uniformly express

a PR8 infection i.p. i.n. i.p. i.n.

X31 infection i.n. i.n. i.n.
15 **
100,000 ##
NP ASCs (×103)
$$
25,000 10
Cells/well
6,250
5
1,563
391 0
i.p. i.n. i.p. i.n. PR8 infection
i.n. i.n. i.n. X31 infection
b PR8 infection i.p. i.n. i.p. i.n.
X31 infection i.n. i.n. i.n. ***
105 250,000 0.2 0.1 4.5 2.3 0.7 12.7 ****
Blimp-1 (YFP)
20 ####
NP YF P(×104)
4 10.3 200,000
10 15
SSC-A
150,000
103 100,000 10
0 50,000 5
0 0
0 103 104 105 0 103 104 105 i.p. i.n. i.p. i.n. PR8 infection
CD19 – APC-Fire NP – PE i.n. i.n. i.n. X31 infection
c PR8 infection i.p. i.n. i.p. i.n.

X31 infection i.n. i.n. i.n. ****
####
15
NP YFP (×104)
250,000 0.0 0.1 1.7 6.6 3.7 16.4
Blimp-1 (YFP)
5
10 $$$$
7.5 200,000
10
SSC-A
104 150,000
100,000 5
103
50,000
0 0
0 i.p. i.n. i.p. i.n. PR8 infection
0 103 104 105 0 103 104 105
i.n. i.n. i.n. X31 infection
CD19 – APC-Fire NP – PE
d e Lung ISW memory

CT FTY720 PR8 infection i.n. i.n.
100,000 X31 infection i.n. 60 *
30 105
ISW NP (103)
22.1 10.6
CD19 – APC
NP ASCs (×103)
25,000 40
Cells/well
104
20
fire
6,250 103
20
0
1,563 10
0
391 0 103 104 105 i.n. i.n. PR8 infection
0 NP – PE i.n. X31 infection
CT FTY720
f PR8 infection i.n. i.n.
X31 infection i.n. 60
10 5
3.6 3.9
CD19 – APC
(PR8) (103)
40
ISW HA
104
fire
103 20
0
0
0 103 104 105 i.n. i.n. PR8 infection
HA(PR8) – PE i.n. X31 infection
Fig. 8 | BRM cells are required for rapid secondary ASCs in the lung. a, NP-specific ELISPOTs in the lung 4 days after challenge infection. Graph shows
data points as well as mean ± s.d. Data are representative of five independent experiments with four mice per group. Data were analyzed with one-way
ANOVA and Tukey’s test for multiple comparisons: **P = 0.0037, ##P =0.002 and P = 0.003. b,c, BLIMP-1 reporter mice were infected with PR8 on day
0 and challenged with X31 on day 30 (b) or 60 (c), and ASCs were enumerated 3 days later. Cells in the lung were gated on live, singlet lymphocytes
(Supplementary Fig. 1a) and subsequently gated on NP-specific YFP+ cells. Data are representative of three independent experiments (b) or two
experiments (c) with three to five mice per group. Graphs show individual data points as well as mean ± s.d. Data are analyzed by one-way ANOVA with
Bonferroni–Sidak test for multiple comparisons: ***P =0.0003, ****P =0.0001, and ###P =0.0001 (b); or Tukey’s test for multiple comparisons:
****P = 0.0001, ####P =0.0001 and P =0.0001 (c). d, Mice were infected with PR8 on day 0, treated with FTY720 and challenged with X31 on day 30, and
ELISPOTs in the lung were analyzed 4 days later. Data are representative of two independent experiments with six mice per group (control) or four mice
per group (FTY720). Graph shows data points as well as mean ± s.d. Data were analyzed with a Mann–Whitney U-test. e,f, Mice were infected with PR8
on day 0, challenged with X31 on day 30 and analyzed on day 33. Cells from the lung were gated on live, singlet lymphocyte, CD19+B220–CD38+IgD–IgM–
ISW memory B cells (Supplementary Fig. 1e), and NP-specific (e) and HA(PR8)-specific (f) memory B cells were enumerated. Graphs show individual data
points as well as mean ± s.d. Data are representative of three independent experiments with three mice per group. Data were analyzed with Student’s t-test:
*P = 0.0488. P < 0.05 is considered significant.
the chemokine receptor CXCR3 and completely lack the lymph the first few weeks after infection, in part due to local antigen
node–homing receptor CD62L. Moreover, lung-resident BRM cells encounter. Finally, the presence of BRM cells in the lung leads to an
are evenly divided into CD73+ and CD73– populations, whereas accelerated ASC response in the lung following a challenge infec-
memory B cells in lymphoid organs are predominantly CD73+. tion. These data demonstrate that like TRM cells, BRM cells are an
Influenza-specific BRM cells are recruited mainly to the lung in instrumental component of pulmonary immunity to influenza.

The formation of BRM cells, like that of other memory B cells, situ from precursors already in the lung. This change in secondary
is dependent on CD40 signaling, most likely in GCs. Interestingly, response time may be simply due to location, with BRM cells in
GCs are found in both the mLNs and the lungs of influenza-infected the lung encountering antigen and inflammation earlier than their
mice, and some studies suggest that GCs in the lung select mainly lymphoid counterparts and accelerating their response accord-
for influenza-specific memory B cells in the lung18. However, we find ingly. Alternatively, BRM cells may be qualitatively different from
that the GC response in the mLNs develops very rapidly after infec- those in lymphoid organs, as their phenotype suggests, and may be
tion, whereas the GC response in the lung develops more slowly, poised to rapidly differentiate into ASCs with minimal stimulation.
likely because it takes time to form inducible bronchus-associated Consistent with this idea, memory B cells from the lung (presum-
lymphoid tissue (iBALT)19,20. Given our observation that BRM cells ably BRM cells) adoptively transferred to recipient mice provide
in the lung are placed very early after infection and that CD40L more efficient protection than that provided by memory B cells iso-
blockade at late times has a minimal effect on lung-resident BRM lated from secondary lymphoid organs9.
cells, despite eliminating GCs in both the mLNs and the lung, it In summary, our data demonstrate that influenza-specific
seems likely that most lung-resident BRM cells formed in response BRM cells are formed in the lung following pulmonary influenza
to a primary infection coming from GCs in the mLNs. infection and that these cells are necessary for an accelerated ASC
Interestingly, not all memory B cells transit through GCs, as mice response following challenge infection. BRM cells are formed early
lacking follicular helper T cells, and therefore GCs, still generate after infection and seed the lung in a process that depends on local
memory B cells21. Moreover, GC-independent memory B cells poorly encounter with antigen. These data suggest that vaccines designed
express CD7321. Given our observation that more than half of the to elicit highly effective, long-lived protection against influenza
BRM cells in the lung lack CD73, it is possible that some of these cells virus infection will need to deliver antigens to the respiratory tract.
were generated independently of the GC reaction either in the mLNs
or in the lung. These data are also consistent with our observation Online content
that many BRM cells, particularly IgM+ BRM cells, seed the lung very Any methods, additional references, Nature Research reporting
early after infection, and with previous studies showing that systemic summaries, source data, statements of data availability and asso-
memory B cells are also formed mainly early after immunization7. ciated accession codes are available at https://doi.org/10.1038/
Although memory B cells are generated mainly early after s41590-018-0260-6.
infection, we still observe them in the blood as late as 70 days after
infection, suggesting that recirculating memory B cells could be Received: 5 February 2018; Accepted: 15 October 2018;
recruited to the lungs for extended periods of time. In fact, we Published online: 3 December 2018
find that memory B cells established at systemic sites during a
primary response can be 'called' into the lung by a subsequent References
inflammatory response in the lung, such as a non-specific infec- 1. Halliley, J. L. et al. Long-lived plasma cells are contained within the
tion. However, during a primary response, the placement of newly CD19−CD38hiCD138+ subset in human bone marrow. Immunity 43,
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2. Dorner, T. & Radbruch, A. Antibodies and B cell memory in viral immunity.
a limited time after infection. Moreover, as shown by our experi- Immunity 27, 384–392 (2007).
ments with parabiotic mice concurrently infected with two differ- 3. Phan, T. G. & Tangye, S. G. Memory B cells: total recall. Curr. Opin.
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by inflammation, which is present in the lungs of both partners, 4. Bannard, O. & Cyster, J. G. Germinal centers: programmed for affinity
but by the availability of local antigen, suggesting that BRM pre- maturation and antibody diversification. Curr. Opin. Immunol. 45,
21–30 (2017).
cursors need to contact antigen in the lung in order to stay (or sur- 5. Shlomchik, M. J. & Weisel, F. Germinal center selection and the development
vive) as lung-resident BRM cells. These data are similar to those of memory B and plasma cells. Immunol. Rev. 247, 52–63 (2012).
showing that the placement of TRM cells in the skin and the lung 6. Schittek, B. & Rajewsky, K. Maintenance of B-cell memory by long-lived cells
requires contact with local antigen22,23. generated from proliferating precursors. Nature 346, 749–751 (1990).
Where might this encounter with antigen occur? One possibility 7. Weisel, F. J., Zuccarino-Catania, G. V., Chikina, M. & Shlomchik, M. J. A
temporal switch in the germinal center determines differential output of
is that BRM precursors encounter antigen in iBALT19,20. We know memory B and plasma cells. Immunity 44, 116–130 (2016).
that iBALT is formed in the lungs of influenza-infected mice and 8. Jaimes, M. C. et al. Maturation and trafficking markers on rotavirus-specific
that mice lacking conventional secondary lymphoid organs gener- B cells during acute infection and convalescence in children. J. Virol. 78,
ate and maintain long-lived B cell responses24. Moreover, GC-like 10967–10976 (2004).
B cells are observed in the lungs of infected mice19, even though 9. Onodera, T. et al. Memory B cells in the lung participate in protective
humoral immune responses to pulmonary influenza virus reinfection. Proc.
the existence of pulmonary follicular T cells remains controversial25, Natl. Acad. Sci. USA 109, 2485–2490 (2012).
possibly due to the altered phenotype of helper T cells in peripheral 10. Joo, H. M., He, Y., Sundararajan, A., Huan, L. & Sangster, M. Y. Quantitative
non-lymphoid tissues26. Interestingly, GCs in the lung seem to pref- analysis of influenza virus-specific B cell memory generated by different
erentially select for broadly reactive memory B cells18, although it is routes of inactivated virus vaccination. Vaccine 28, 2186–2194 (2010).
unclear whether the broad reactivity of BRM cells is due to site-spe- 11. Schenkel, J. M. & Masopust, D. Tissue-resident memory T cells. Immunity 41,
886–897 (2014).
cific properties of the GC response or to the early placement of less 12. Dogan, I. et al. Multiple layers of B cell memory with different effector
stringently selected (and perhaps more broadly reactive) BRM cells. functions. Nat. Immunol. 10, 1292–1299 (2009).
This latter idea is consistent with data showing that rapamycin treat- 13. Pape, K. A., Taylor, J. J., Maul, R. W., Gearhart, P. J. & Jenkins, M. K.
ment of influenza-infected mice leads to curtailed GC responses but Different B cell populations mediate early and late memory during an
endogenous immune response. Science 331, 1203–1207 (2011).
a more broadly reactive repertoire of memory B cells27.
14. Palladino, G., Mozdzanowska, K., Washko, G. & Gerhard, W. Virus-
Our data highlight another qualitative difference between BRM neutralizing antibodies of immunoglobulin G (IgG) but not of IgM or IgA
cells in the lung and memory B cells in lymphoid organs: the rapid- isotypes can cure influenza virus pneumonia in SCID mice. J. Virol. 69,
ity of secondary responses. Mice with NP-specific BRM cells in the 2075–2081 (1995).
lung generate secondary ASC responses in the lungs more rapidly 15. Tomayko, M. M., Steinel, N. C., Anderson, S. M. & Shlomchik, M. J. Cutting
edge: hierarchy of maturity of murine memory B cell subsets. J. Immunol.
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the accelerated secondary response in the lungs is maintained in 16. Bernasconi, N. L., Traggiai, E. & Lanzavecchia, A. Maintenance of serological
the presence of FTY-720, a functional antagonist of the chemotactic memory by polyclonal activation of human memory B cells. Science 298,
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17. Lee, F. E. et al. Circulating human antibody-secreting cells during Acknowledgements
vaccinations and respiratory viral infections are characterized by high The authors would like to thank U. Mudunuru and S. Simpler for animal husbandry
specificity and lack of bystander effect. J. Immunol. 186, 5514–5521 (2011). and the Rheumatic Diseases Core Center flow cytometry facility, which is supported
18. Adachi, Y. et al. Distinct germinal center selection at local sites shapes by AI078907. This work was supported by NIH grants HL69409, AI100127, AI097357,
memory B cell response to viral escape. J. Exp. Med. 212, 1709–1723 (2015). AI109962 to T.D.R. and AI120508 to S.R.A.
19. Moyron-Quiroz, J. E. et al. Role of inducible bronchus associated lymphoid
tissue (iBALT) in respiratory immunity. Nat. Med. 10, 927–934 (2004).
20. Hwang, J. Y., Randall, T. D. & Silva-Sanchez, A. Inducible bronchus- Author contributions
associated lymphoid tissue: taming inflammation in the lung. Front. Immunol. S.R.A., F.E.L. and T.D.R. designed the experiments. J.E.B., B.A.G. and T.D.R. designed
7, 258 (2016). the recombinant influenza proteins and J.E.B. and B.A.G. expressed, purified and
21. Taylor, J. J., Pape, K. A. & Jenkins, M. K. A germinal center-independent characterized the tetramers. U.M. and S.R.A performed the surgeries. M.D.S. performed
pathway generates unswitched memory B cells early in the primary response. the intratracheal infections. S.R.A performed and analyzed the experiments and
J. Exp. Med. 209, 597–606 (2012). generated the figures. S.R.A. and T.D.R. wrote the manuscript. All authors edited the
22. Zaid, A. et al. Persistence of skin-resident memory T cells within an manuscript.
epidermal niche. Proc. Natl. Acad. Sci. USA 111, 5307–5312 (2014).
23. McMaster, S. R. et al. Pulmonary antigen encounter regulates the
establishment of tissue-resident CD8 memory T cells in the lung airways and Competing interests
parenchyma. Mucosal Immunol. 11, 1071–1078 (2018). The authors declare no competing interests.
24. Moyron-Quiroz, J. E. et al. Persistence and responsiveness of immunologic memory
in the absence of secondary lymphoid organs. Immunity 25, 643–654 (2006).
25. Vu Van, D. et al. Local T/B cooperation in inflamed tissues is supported by Additional information
T follicular helper-like cells. Nat. Commun. 7, 10875 (2016). Supplementary information is available for this paper at https://doi.org/10.1038/
26. Rao, D. A. et al. Pathologically expanded peripheral T helper cell subset s41590-018-0260-6.
drives B cells in rheumatoid arthritis. Nature 542, 110–114 (2017). Reprints and permissions information is available at www.nature.com/reprints.
27. Keating, R. et al. The kinase mTOR modulates the antibody response to Correspondence and requests for materials should be addressed to T.D.R.
provide cross-protective immunity to lethal infection with influenza virus.
Nat. Immunol. 14, 1266–1276 (2013). Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in
28. Matloubian, M. et al. Lymphocyte egress from thymus and peripheral published maps and institutional affiliations.
lymphoid organs is dependent on S1P receptor 1. Nature 427, 355–360 (2004). © The Author(s), under exclusive licence to Springer Nature America, Inc. 2018

Methods number: P03438) were synthesized in frame with the human CD5 signal sequence
Mice and parabiosis. Male and female C57BL/6 J (CD45.2) and B6.SJL- upstream and the GCN4 isoleucine zipper trimerization domain downstream
PtprcaPepcb/BoyJ (CD45.1) mice were purchased from the Jackson Laboratory, and (GeneArt). These cDNAs were fused in frame with either a 6XHIS tag or an
BLIMP-1-YFP reporter mice29 were obtained from S. Crotty (La Jolla Institute for AviTag at the C-terminus and cloned into the pCXpoly( + ) mammalian expression
Immunology) and bred in the University of Alabama at Birmingham vivarium, as vector. Constructs encoding HA-6XHIS and HA-AviTag were cotransfected
noted in the Life Sciences Reporting Summary associated with this paper. Swiss using 293Fectin Transfection Reagent into FreeStyle 293–F Cells (ThermoFisher
Webster mice infected with Schistosoma mansoni (strain NMRI) were obtained Scientific) at a 2:1 ratio. Transfected cells were cultured in FreeStyle 293 Expression
from the Schistosomiasis Resource Center and distributed through BEI resources, Medium for 3 days, and the supernatant was recovered by centrifugation.
National Institute of Allergy and Infectious Diseases. Parabiosis surgery was Recombinant HA molecules were purified by fast protein liquid chromatography
performed as described24 using pairs of female mice that had been cohoused for (FPLC) using a HisTrap HP Column (GE Healthcare) and eluted with 250 mM of
at least 2 weeks prior to surgery. In brief, anesthetized mice were shaved and imidazole. The coding sequence of NP from PR8 was synthesized in frame with the
disinfected with betadine, and longitudinal incisions in the skin were made from coding sequence for a 15–amino acid biotinylation consensus site30 on the 3′ end
approximately 1 cm behind the ear to just past the hind limb without opening the (GeneArt). The modified NP sequence was cloned in frame to the 6X His tag
peritoneal cavity. The skin was loosened from the connective tissue, and the two in the pTRC–His2c expression vector (Invitrogen). NP protein was expressed in
mice were sutured together at the scapulae, flank and thigh. The dorsal edges of E. coli strain CVB101 (Avidity), purified by FPLC using a HisTrap HP Column (GE
the skin were joined using 9 mm stainless steel wound clips. Body temperature was Healthcare) and eluted with a 50–250 mM gradient of imidazole. Purified HA was
maintained using a heating pad during surgery and recovery. Mice were provided biotinylated by addition of biotin–protein ligase (Avidity). Biotinylated proteins
with buprenorphine (0.05 mg/kg) prior to surgery and carprofen (5 mg/kg) prior were then tetramerized with fluorochrome-labelled streptavidin (Prozyme).
to surgery and 24 h postsurgery. All animal procedures were approved by the Labelled tetramers were purified by size exclusion on a HiPrep 16/60 Sephacryl
University of Alabama Institutional Animal Care and Use Committee and were S–300 column (GE Healthcare).
performed according to guidelines outlined by the National Research Council.
ELISPOT. For ELISPOT assays, multiscreen HA plates (MAHAS4510, Millipore)
Infections and viral foci assay. Influenza A/PR8/34 (PR8) was used at 15,000 viral were coated with purified NP at 1 μg/ml in PBS overnight at 4 °C. Plates were
foci units (VFU) for intranasal infection and at 1 × 109 VFU for intraperitoneal washed with PBS and blocked with complete medium (RPMI supplemented with
(i.p.) infection. In Fig. 7a–d, the i.p. infections were repeated for 6 days at 1 × 108 10% fetal bovine serum (FBS), 0.5% Penicillin (100×), 0.5% streptomycin (100×),
VFU, 1 × 108 VFU, 1 × 109 VFU, 1 × 109 VFU, 1 × 108 VFU and 1 × 108 VFU. 1% l-glutamine (200 mM), 1% sodium pyruvate (100 mM), 1% HEPES pH7.4
Influenza X31 was used at 1.25 × 104 VFU for primary infection and 1.25 × 105 (1 M), 0.15% sodium bicarbonate, 1.2% amino acids (50×), 1.2% non-essential
VFU for secondary infections. Mice were anesthetized with vaporized isofluorane amino acids (100×), 1.2% vitamins (100×), 0.7% glucose, 0.1% 2-mercaptoethanol
and infected intranasally with 100 μl of inoculum or intratracheally with 75 μl (1000×, 55 mM). Single-cell suspensions were washed, diluted in complete medium
inoculum. Viral foci assays were performed by homogenizing lung samples in zero- and cultured on coated plates for 5 h. Cells were aspirated, and plates were washed
serum medium with 4 μg/ml trypsin and performing fivefold serial dilutions in with 0.2% Tween 20 in PBS. Bound IgG was detected using alkaline phosphatase–
the same medium. 100 μl of each sample was used to infect Madin–Darby Canine conjugated goat anti–mouse kappa (Southern Biotech) diluted (1:1000) in
Kidney (MDCK) cell monolayers. After 16 h of culture, MDCK cells were fixed 0.5% BSA, 0.05% Tween 20 in PBS for 1 h at 37 °C. Plates were washed with 0.2%
with 80% acetone, and infected cells were detected using an antibody to NP (Clone Tween 20 in PBS and developed with BCIP/NBT (Moss Substrates) substrate for
A3. Millipore–Sigma). Spots were revealed using alkaline phosphatase–conjugated 1 h. Spots were recorded using a CTL Immunospot S6 Macroplate Imager Reader
streptavidin (Life Technologies) and 5-bromo-4-chloro-3-indolyl phosphate/nitro (New Life Scientific) and counted manually.
blue tetrazolium (BCIP/NBT) substrate (Moss Substrates). Samples were plated in
duplicates and the mean foci count was used to calculate the VFU per ml of sample. Statistical analysis. GraphPad Prism software (Version 7.0) was used for data
analysis. Datasets that did not follow a Gaussian distribution were analyzed using
Antibody infusion, blocking antibodies and FTY-720 treatment. To identify non-parametric tests. Comparisons between two samples were performed with
circulating B cells, mice were intravenously administered 2.4 μg of fluorochrome- Student’s t-test or the Mann–Whitney U-test. Parabiosis experiments were analyzed
conjugated anti-B220 in 100 μl phosphate-buffered saline (PBS) 5 min prior using one-way analysis of variance (ANOVA) (paired) followed by the Bonferroni–
to euthanasia. CD40:CD40L interactions were blocked with 250 μg of MR1 Sidak method for multiple comparison. One-way ANOVA, followed by analysis-
(BioXCell). To prevent lymphocyte recirculation, the S1P1R agonist FTY-720 specific post-tests, was carried out when more than two variables were compared;
was dissolved at 10 mg/ml in dimethyl sulfoxide (DMSO), diluted to 0.5 mg/ml in P < 0.05 was considered statistically significant.
ethanol and mixed 50:50 with PBS prior to administration at 1 mg/kg.
Reporting Summary. Further information on research design is available in the
Tissue preparation and flow cytometry. Lungs were isolated, cut into small Nature Research Reporting Summary linked to this article.
fragments and digested for 30 min at 37 °C with 0.6 mg/ml collagenase A (Sigma)
and 30 μg/ml DNAse I (Sigma) in RPMI-1640 medium (GIBCO). Digested lungs, Data availability
mLNs or spleens were mechanically disrupted by passage through a wire mesh. The data supporting the findings of this study are available from the corresponding
Red blood cells were lysed with 150 mM NH4Cl, 10 mM KHCO3 and 0.1 mM author upon reasonable request.
ethylene diamine tetraacetic acid (EDTA). Fc receptors were blocked with
5 μg/ml anti-CD16/32 (BD Biosciences), followed by staining with fluorochrome-
conjugated antibodies. Analytic flow cytometry was performed on an LSR II (BD References
Biosciences) instrument available through the Comprehensive Flow Cytometry 29. Misulovin, Z., Yang, X. W., Yu, W., Heintz, N. & Meffre, E. A rapid method
Core at University of Alabama, Birmingham (UAB). for targeted modification and screening of recombinant bacterial artificial
chromosome. J. Immunol. Methods 257, 99–105 (2001).
Production of influenza proteins, immunoblot and tetramers. The coding 30. Beckett, D., Kovaleva, E. & Schatz, P. J. A minimal peptide substrate in biotin
sequences of PR8 HA16–523 (accession number: P03452) and X31 HA15–525 (accession holoenzyme synthetase-catalyzed biotinylation. Protein Sci. 8, 921–929 (1999).
Nature Immunology | www.nature.com/natureimmunology

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Sample size Sample size was determined to obtain a significant difference between groups, with a power of 80%, when the statistical significance was set
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were started with 8-10 pairs per group to account for the expected attrition, while all other experiments were with 5 animals per group.
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Antibodies
Antibodies used Specificity Clone Vendor fluorochrome Catalog number Lot #
B220 RA3-6B2 Invitrogen AF647, AF488 14-0452-81 A10235
CD95 Jo2 BD FITC 554257 4080693
April 2018
CD19 6D5 Biolegend APC-Cy7, APC Fire 115529, 115558 B253924, B243046
CD138 281-2 Biolegend BV605 142516 B240942
CD38 90 eBiosciences APC, AF700 17-0381-82, 56-0381-82 4314370, E108747
IgD 11-26 eBiosciences eFluor450 48-5993-82 4316984
IgM II/41 eBiosciences PerCP-eFluor710 46-5790-82 4321765
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IgG2b RMG2b-1 BD FITC 406705 B170450
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IgG3 R40-82 BD FITC 553403 M066986
CD45.2 102 BD Horizon V500 562129 5107958
GL7 GL7 Biolegend PerCP-Cy5.5 144610 B246315
PNA (lectin) N/A Sigma Pacific Blue L0881
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Laboratory animals Laboratory mice (mus musculus) strains C57BL/6, B6.SJL–PtprcaPepcb/BoyJ and BLIMP–1–YFP reporter mice were used
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Confirm that:
The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.
Methodology
Sample preparation Lymph nodes: mechanically disrupted through wire mesh, filtered and resuspended at 10e6/mL of PBS(10%FBS)
Spleen: mechanically disrupted through wire mesh, lysed RBCs, filtered and resuspended at 10e6/mL of PBS(10%FBS)
Lung: mechanically disrupted through wire mesh, lysed RBCs, filtered and resuspended at 10e6/mL of PBS(10%FBS)
Blood: 500uL of blood collected in heparin coated tubes. Lysed RBCs (2x), filtered and resuspended in 200uL of PBS(10%FBS)
Single cells suspensions were plated at 1-2x10e6 in 96-well v-bottom plates and stained with 50uL of the mix of tetramer,
antibodies and Fc block antibody in PBS (10% FBS) for 30min at 4 degrees centigrade. The cells were washed twice with PBS (10%
FBS) and fixed with 10% Formalin for 5min at room temperature and washed in PBS (10% FBS). The cells were resuspended in
PBS (10% FBS).
Instrument BD LSR II Flow cytometer
Software FLow cytometry data was collected using BD FACSDiva Software Version 8.0.1 from BD Biosciences
FlowJo 9.6: Analysis of flow cytometry data
Microsoft Excel: Organization of numerical data and calculation of numbers from proportions.
April 2018
GraphPad Prism 7: Graphing and statistical analysis.
Cell population abundance Sorted populations are not included in this study
Gating strategy All samples were initially gated on the lymphocytes in the FSC-A/SSC-A plot. The gate is placed above a threshold of 50,000 on
the FSC-A axis. Doublets are excluded, and live cells are selected using a dead cell dye. The following are sequential gates for
germinal center cells (GCs), antibody secreting cells (ASCs), Resident memory by parabiosis, resident memory by infusion and
phenotyping of resident memory cells (identified by infusion):
4
GGBs
CD138-CD19+CD38-GL7+NP+

CD138-CD19+CD38-GL7+PR8+
CD138-CD19+CD38-PNA+GL7++NP+
CD138-CD19+CD38-PNA+GL7++NP+
ASCs
C57BL/6 mice:
CD138+CD19+/lo
Blimp-YFP reporter mice:
YFP+CD19+/lo
YFP+CD19+/loCD138+
Resident memory (parabiosis)
Ex. Donor CD45.2+
aB220-CD19+CD38+IgM+IgD-CD45.2+NP+
aB220-CD19+CD38+IgM+IgD-CD45.2+HA+
aB220-CD19+CD38+IgM-IgD-CD45.2+NP+
aB220-CD19+CD38+IgM-IgD-CD45.2+HA+
Resident memory (infusion)
aB220-CD19+CD38+IgM+IgD-NP+
aB220-CD19+CD38+IgM+IgD-HA+
aB220-CD19+CD38+IgM-IgD-NP+
aB220-CD19+CD38+IgM-IgD-HA+
Infused BRM phenotyping, isotyping
aB220-CD19+CD38+IgD-NP+isotype+
aB220-CD19+CD38+IgD-PR8+ isotype+
Gating strategy (except lymphocyte gates, doublet exclusion and live/dead exclusion) is presented in most figures.
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.
Magnetic resonance imaging

Experimental design
Design type Indicate task or resting state; event-related or block design.
Design specifications Specify the number of blocks, trials or experimental units per session and/or subject, and specify the length of each trial
or block (if trials are blocked) and interval between trials.
Behavioral performance measures State number and/or type of variables recorded (e.g. correct button press, response time) and what statistics were used
to establish that the subjects were performing the task as expected (e.g. mean, range, and/or standard deviation across
subjects).
Acquisition
Imaging type(s) Specify: functional, structural, diffusion, perfusion.
Field strength Specify in Tesla
Sequence & imaging parameters Specify the pulse sequence type (gradient echo, spin echo, etc.), imaging type (EPI, spiral, etc.), field of view, matrix size,
slice thickness, orientation and TE/TR/flip angle.
Area of acquisition State whether a whole brain scan was used OR define the area of acquisition, describing how the region was determined.
Diffusion MRI Used Not used
Preprocessing
Preprocessing software Provide detail on software version and revision number and on specific parameters (model/functions, brain extraction,
segmentation, smoothing kernel size, etc.).
Normalization If data were normalized/standardized, describe the approach(es): specify linear or non-linear and define image types
used for transformation OR indicate that data were not normalized and explain rationale for lack of normalization.
Normalization template Describe the template used for normalization/transformation, specifying subject space or group standardized space (e.g.
April 2018
original Talairach, MNI305, ICBM152) OR indicate that the data were not normalized.
Noise and artifact removal Describe your procedure(s) for artifact and structured noise removal, specifying motion parameters, tissue signals and
physiological signals (heart rate, respiration).
Volume censoring Define your software and/or method and criteria for volume censoring, and state the extent of such censoring.
5
Statistical modeling & inference

Model type and settings Specify type (mass univariate, multivariate, RSA, predictive, etc.) and describe essential details of the model at the first
and second levels (e.g. fixed, random or mixed effects; drift or auto-correlation).
Effect(s) tested Define precise effect in terms of the task or stimulus conditions instead of psychological concepts and indicate whether
ANOVA or factorial designs were used.
Specify type of analysis: Whole brain ROI-based Both

Statistic type for inference Specify voxel-wise or cluster-wise and report all relevant parameters for cluster-wise methods.
(See Eklund et al. 2016)
Correction Describe the type of correction and how it is obtained for multiple comparisons (e.g. FWE, FDR, permutation or Monte
Carlo).
Models & analysis

n/a Involved in the study
Functional and/or effective connectivity
Graph analysis
Multivariate modeling or predictive analysis
Functional and/or effective connectivity Report the measures of dependence used and the model details (e.g. Pearson correlation, partial
correlation, mutual information).
Graph analysis Report the dependent variable and connectivity measure, specifying weighted graph or binarized graph,
subject- or group-level, and the global and/or node summaries used (e.g. clustering coefficient, efficiency,
etc.).
Multivariate modeling and predictive analysis Specify independent variables, features extraction and dimension reduction, model, training and evaluation
metrics.
April 2018

The Establishment of Resident Memory B Cells in The Lung Requires Local Antigen Encounter

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The Establishment of Resident Memory B Cells in The Lung Requires Local Antigen Encounter

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The establishment of resident memory B cells in

of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA. *e-mail: randallt@uab.edu

Nature Immunology | VOL 20 | JANUARY 2019 | 97–108 | www.nature.com/natureimmunology 97

CD19 – APC Cy7

CD19 – APC Cy7

CD19 – APC Cy7

CD19 – APC Cy7

IgM – Per 710

CD19 – APC Cy7

CD19 – APC Cy7

IgM – Per 710

e mLN day 15 GC B cells i mLN 9 week ISW memory B cells

CD19 – APC Cy7

CD19 – APC Cy7

98 Nature Immunology | VOL 20 | JANUARY 2019 | 97–108 | www.nature.com/natureimmunology

CD73 PD-L2 (%)

c mLN Lung Spleen d mLN Lung Spleen f mLN Lung Spleen

CD80 – Pac. Orange

CD19 – APC Cy7

CD83 PD-L2 (%)

CD19 – APC Cy7

105 70.5 93 57.3 100

104 40 mLN Lung Spleen

0 10 20.4% IgG2b 18.2% IgG2b 15.3% IgG2b

Nature Immunology | VOL 20 | JANUARY 2019 | 97–108 | www.nature.com/natureimmunology 99

g LPS PR8 h PR8 PR8 i PR8 PR8

0 103 104 105 0 103 104 105 0 103 104 105

CD45.1 CD45.2 CD45.1 CD45.2 CD45.1 CD45.2

100 Nature Immunology | VOL 20 | JANUARY 2019 | 97–108 | www.nature.com/natureimmunology

150,000 60 103 83.9 103

0 103 104 105 0 103 104 105 0 103 104 105

Nature Immunology | VOL 20 | JANUARY 2019 | 97–108 | www.nature.com/natureimmunology 101

ISW B cells (%)

ISW B cells (log10)

102 Nature Immunology | VOL 20 | JANUARY 2019 | 97–108 | www.nature.com/natureimmunology

CD19 – APC Cy7

CD19 – APC Cy7

CD19 – APC Cy7

CD19 – APC Cy7

Nature Immunology | VOL 20 | JANUARY 2019 | 97–108 | www.nature.com/natureimmunology 103

CD19 APC fire

IgM – Per 710

ISW PR8 (×103)

CD19 – APC fire

ISW PR8 (log10)

ISW X31 (×103)

CD19 – APC fire

104 4.9 0.0 4.2 2

ISW PR8 (×103)

ISW X31 (×103)

103 4.6 0 3.9 0.5 ***

104 Nature Immunology | VOL 20 | JANUARY 2019 | 97–108 | www.nature.com/natureimmunology

Spleen ISW memory

Lung ISW memory

----, i.n. 105

Nature Immunology | VOL 20 | JANUARY 2019 | 97–108 | www.nature.com/natureimmunology 105

a PR8 infection i.p. i.n. i.p. i.n.

c PR8 infection i.p. i.n. i.p. i.n.

d e Lung ISW memory

106 Nature Immunology | VOL 20 | JANUARY 2019 | 97–108 | www.nature.com/natureimmunology

Nature Immunology | VOL 20 | JANUARY 2019 | 97–108 | www.nature.com/natureimmunology 107

108 Nature Immunology | VOL 20 | JANUARY 2019 | 97–108 | www.nature.com/natureimmunology