Lymphotoxin (LT), a cytokine belonging to the TNF family, has established roles in the formation of secondary lymphoid structures and in the compartmentalization of T and B lymphocyte areas of the spleen. In this study, we examine the role of LT in directing the composition of intestinal lymphocytes. We report that mice deficient in LT have a normal composition of intestinal lamina propria (LP) T lymphocytes, and an absence of intestinal LP B lymphocytes. We further refine this observation to demonstrate that the interaction of LT with the LTβR is essential for the presence LP B lymphocytes. The LT/LTβR-dependent events relevant for the presence of LP B lymphocytes occur after birth, do not require the presence of Peyer’s patches, lymph nodes, or the spleen; and therefore, are distinct and independent from the previously identified roles of LT/LTβR. The LT-dependent signal relevant for the presence of LP B lymphocytes is optimally supplied by a LT-sufficient B lymphocyte, and requires a LTβR-sufficient radio-resistant, non-bone marrow-derived cell. Based upon the severity of the deficit of LP B lymphocytes we observed, these novel LT/LTβR-dependent events are of primary importance in directing the entry and residence of LP B lymphocytes.

Recent studies have established roles for lymphotoxin (LT),3 and the receptors, LTβR and TNFRI in the formation of secondary lymphoid structures and the normal compartmentalization of T and B lymphocytes in the spleen. LT, a TNF family member, exists in two forms, a membrane-bound heterotrimer, comprised of two β-chains and one α-chain (LTα1β2), which binds exclusively the LTβR, and a soluble homotrimer (LTα3), which has overlapping functions with TNF, binding both the TNFRI and TNFRII (1, 2). LTβR is expressed on nonlymphoid cells including monocytes. LT (LTα3 and LTα1β2) is expressed predominantly on T lymphocytes, B lymphocytes, and NK cells in adult mice (3). Ligation of the LTβR by LTα1β2 at defined points during embryogenesis is critical for the formation of organized lymphoid structures, as evidenced by the lack of lymph node (LN) and Peyer’s patch (PP) formation in mice deficient in LTα, LTβ, and LTβR (4, 5, 6); and mice in which LTβR signaling has been blocked at specific times during gestation (7). The relevant LTα1β2-expressing cell for the formation of PP and LN during embryogenesis is a non-T, non-B lymphocyte, as evidenced by the presence of LN in severe combined immunodeficient mice reconstituted as adults with LTα−/− bone marrow (8). LTα−/− mice have normal lymphatics, and LTα−/− B and T lymphocytes have normal function and can repopulate LN and PP when placed in an LT-sufficient environment (4, 9).

In addition to the role of LT in the formation of LN and PP, LT is also crucial for the normal compartmentalization of T and B lymphocyte areas of the spleen. Mice in which LTβR and/or TNFRI signal transduction have been blocked fail to develop follicular dendritic cells (FDC) and germinal centers (GC) (5, 10, 11, 12), and have diminished expression of secondary lymphoid tissue chemokine (SLC), B lymphocyte chemoattractant (BLC), and EBV-induced molecule 1 ligand chemokine by splenic stromal cells (13), all of which contribute to the normal segregation of B and T lymphocytes in the spleen.

The intestine, as a secondary lymphoid tissue, represents a unique challenge for the normal segregation of lymphocytes, as the major effector site, the lamina propria (LP), is distant from intestinal and nonintestinal inductive sites of the immune response. The relevant factors for the entry and residence of lymphocytes into intestinal effector sites are not well-known. Others have demonstrated these events are at least partially dependent upon the expression of the α4β7 and αEβ7 integrins by lymphocytes, allowing these lymphocytes to home to the LP and intraepithelial lymphocyte (IEL) compartments (14, 15, 16), and are dependent upon the expression of thymus-expressed chemokine by the small intestine epithelia and its corresponding receptor CCR9 by T lymphocytes (17, 18).

In addition to these observations, recent investigations have demonstrated a role for NFκβ-inducing kinase (NIK) in the migration of peritoneal B-1 B lymphocytes to the intestine (19). Alymphoplasia (aly) mice have a naturally occurring point mutation in the gene encoding NIK, and have an absence of LN and PP, disrupted splenic architecture, immunodeficiency, and lack LP B lymphocytes (19, 20, 21). Peritoneal B-1 B lymphocytes from aly/aly mice are unable to populate the intestine of wild-type mice with IgA producing plasma cells after adoptive transfer. This is presumed to result from impaired signal transduction downstream of the receptors for SLC, as evidenced by defective migration of aly/aly B and T lymphocytes in response to SLC. However, signal transduction downstream of the LTβR has also been shown to require NIK activity (22). Therefore, deficiencies in LTβR signal transduction in addition to the above defects in signal transduction downstream of SLC could play a role in the absence of LP B lymphocytes in the aly/aly mice.

LT is known to play a critical role in the formation of PP and LN, and the normal segregation of lymphocytes in the spleen. In this study, we examine the role of LTβR-dependent events in directing the entry and residence of lymphocytes into the intestine. We demonstrate a role for LTα1β2 interacting with the LTβR in directing B-2 B lymphocytes to the intestinal LP. The relevant LTα1β2 signal can be delivered in adulthood, does not require the presence of LN, PP, or the spleen, and can be blocked by the administration of LTβR antagonists after birth. Bone marrow-derived cell lineages may transmit the LT-dependent signals relevant for the presence of LP B lymphocytes; however, these signals are optimally transmitted by LT-sufficient B lymphocytes, and require a LTβR-sufficient, non-bone marrow-derived, radio-resistant cell population. Our observations define a novel role for LT/LTβR interactions in the intestine in directing the composition of the intestinal LP. This role is distinct and independent of the known roles of LT in the formation of PP, LN, and in the compartmentalization of the spleen. Based upon the severity of the deficit of LP B lymphocytes, these LTβR-dependent events are of greater hierarchal importance than previously recognized factors contributing to the entry and residence of LP B lymphocytes.

All mice used for this study were housed in a specific pathogen-free facility and fed routine chow diet. Animal procedures and protocols were conducted in accordance with the institutional review board at Washington University School of Medicine (St. Louis, MO). C57BL/6, TNFRII-deficient, recombination-activating gene (RAG)-1-deficient, and B cell-deficient JH−/− mice (23) on the C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, ME). TNFRI-deficient mice (10, 24) on the C57BL/6 background were a gift from Dr. J. J. Peschon (Immunex, Seattle, WA). LTβR-deficient mice (6) on the C57BL/6 background were a gift from Dr. K. Pfeffer (Technical University of Munich, Munich, Germany). LTα-deficient mice (4), a gift from Dr. D. Chaplin (University of Alabama, Birmingham, AL), were bred onto the C57BL/6 background for >10 generations before use in experiments. LTα−/− and C57BL/6 mice for flow cytometric analysis, Table I, were 7 wk of age. Fecal IgA levels were measured on feces from LTα−/− and C57BL/6 mice from 9 to 26 wk of age (Table I). Analysis of resident peritoneal cells, Table II, was performed on 14-wk-old LTα−/−, LTβR−/−, and C57BL/6 mice. TNFRI−/−, TNFRII−/−, and LTβR−/− mice for flow cytometric analysis, Table III, were 10–14 wk of age. Timed pregnant C57BL/6 female mice for use in experiments involving the injection of LTβR-Ig fusion protein, Table IV, were generated by matings with C57BL/6 male mice. Six to 10-wk-old LTα−/− and LTβR−/− mice were used as recipients for bone marrow transfers (Table V).

Table I.

Analysis of LP lymphocytes from C57BL/6 and LTα−/− mice

Fecal IgA (μg/m)aLP Supernatant IgA (ng/ml)aIgA SFC/106 LP CellsbCD45+ LP Cells/Intestine × 106% of CD45+ LP Cells Which Are TCRαβ+% of CD45+ LP Cells Which Are TCRγδ+% of CD45+ LP Cells Which Are CD4+% of CD45+ LP Cells Which Are CD8+% of CD45+ LP Cells Which Are CD19+% CD19+ LP Cells Which Are CD11b% of CD19+ LP Cells Which Are IgA+% of CD45+ Splenocytes Which Are CD19+
C57BL/6 176.7 ± 62.0 17,683 ± 3,374 22,433 ± 3,537 8.4 ± 4.3 39.1 ± 6.6b 3.5 ± 1.1 32.7 ± 9.1 7.7 ± 0.7 35.4 ± 11.9 87.9 ± 13.5 2.3 ± 0.3 46.9 ± 13.8 
 (n = 11) (n = 3) (n = 3) (n = 8) (n = 9) (n = 3) (n = 9) (n = 9) (n = 9) (n = 3) (n = 5) (n = 5) 
LTα−/− 3.8 ± 0.7c 0.6 ± 0.6c 256 ± 27.0c 9.1 ± 2.0 44.8 ± 4.9 4.8 ± 1.0 39.8 ± 4.5 14.5 ± 4.7c 1.5 ± 0.9c d d 64.3 ± 3.4c 
 (n = 11) (n = 3) (n = 3) (n = 5) (n = 6) (n = 3) (n = 6) (n = 6) (n = 6)   (n = 5) 
Fecal IgA (μg/m)aLP Supernatant IgA (ng/ml)aIgA SFC/106 LP CellsbCD45+ LP Cells/Intestine × 106% of CD45+ LP Cells Which Are TCRαβ+% of CD45+ LP Cells Which Are TCRγδ+% of CD45+ LP Cells Which Are CD4+% of CD45+ LP Cells Which Are CD8+% of CD45+ LP Cells Which Are CD19+% CD19+ LP Cells Which Are CD11b% of CD19+ LP Cells Which Are IgA+% of CD45+ Splenocytes Which Are CD19+
C57BL/6 176.7 ± 62.0 17,683 ± 3,374 22,433 ± 3,537 8.4 ± 4.3 39.1 ± 6.6b 3.5 ± 1.1 32.7 ± 9.1 7.7 ± 0.7 35.4 ± 11.9 87.9 ± 13.5 2.3 ± 0.3 46.9 ± 13.8 
 (n = 11) (n = 3) (n = 3) (n = 8) (n = 9) (n = 3) (n = 9) (n = 9) (n = 9) (n = 3) (n = 5) (n = 5) 
LTα−/− 3.8 ± 0.7c 0.6 ± 0.6c 256 ± 27.0c 9.1 ± 2.0 44.8 ± 4.9 4.8 ± 1.0 39.8 ± 4.5 14.5 ± 4.7c 1.5 ± 0.9c d d 64.3 ± 3.4c 
 (n = 11) (n = 3) (n = 3) (n = 5) (n = 6) (n = 3) (n = 6) (n = 6) (n = 6)   (n = 5) 
a

Fecal IgA levels and supernatant IgA levels displayed as mean ± SEM.

b

ELISPOT, cell counts, and flow cytometric analysis displayed as mean ± SD.

c

Values of p < 0.05 when compared to results from C57BL/6 mice.

d

Too few cells to analyze.

Table II.

Analysis of resident peritoneal cells from C57BL/6, LTα−/−, and LTβR−/− mice

Resident Peritoneal Cells/Mouse × 106a% of Peritoneal Cells Which Are CD19+% of CD19+ Cells Which Are CD11b% of CD19+ Cells Which Are IgA+
C57BL/6 1.4 ± 0.9 82.3 ± 3.8 38.0 ± 5.8 9.0 ± 9.6 
 (n = 9) (n = 10) (n = 5) (n = 10) 
LTα−/− 9.5 ± 2.1b 90.7 ± 3.1b 53.9 ± 6.4b 6.2 ± 3.0 
 (n = 5) (n = 5) (n = 5) (n = 5) 
LTβR−/− 3.5 ± 0.5b,c 83.1 ± 7.7 50.9 ± 5.8b 9.8 ± 1.5 
 (n = 4) (n = 4) (n = 4) (n = 4) 
Resident Peritoneal Cells/Mouse × 106a% of Peritoneal Cells Which Are CD19+% of CD19+ Cells Which Are CD11b% of CD19+ Cells Which Are IgA+
C57BL/6 1.4 ± 0.9 82.3 ± 3.8 38.0 ± 5.8 9.0 ± 9.6 
 (n = 9) (n = 10) (n = 5) (n = 10) 
LTα−/− 9.5 ± 2.1b 90.7 ± 3.1b 53.9 ± 6.4b 6.2 ± 3.0 
 (n = 5) (n = 5) (n = 5) (n = 5) 
LTβR−/− 3.5 ± 0.5b,c 83.1 ± 7.7 50.9 ± 5.8b 9.8 ± 1.5 
 (n = 4) (n = 4) (n = 4) (n = 4) 
a

Results displayed as mean ± SD for cell number and flow cytometric analysis.

b

Values of p < 0.05 when compared to results from C57BL/6 mice.

c

Values of p < 0.05 when compared to results from LTα−/− mice.

Table III.

Analysis of LP lymphocytes from TNFRI, TNFRII, and LTβR-deficient mice

MouseFecal IgA (μg/ml)aLP Supernatant IgA (ng/ml)IgA SFC/106 LP Cellsb% of CD45+ LP Cells Which Are CD19+% of CD19+ LP Cells Which Are IgA+% of CD19+ LP Cells Which Are CD11b% of CD45+ LP Cells Which Are TCRβ+% of CD45+ Splenocytes Which Are CD19+
TNFRI−/− 49.4 ± 12.3d,e 7,766 ± 884d,e 51,610 ± 6,787d,e 21.9 ± 5.5d 20.7 ± 1.5e 90.5 ± 3.1 46.6 ± 6.4 66.0 ± 3.4e 
 (n = 14) (n = 3) (n = 3) (n = 4) (n = 3) (n = 4) (n = 4) (n = 4) 
TNFRII−/− 12.0 ± 2.8d,e 4,291 ± 1,416d,e 24,660 ± 1,627d 27.1 ± 11.4d 15.5 ± 3.9e 87.1 ± 3.0 41.9 ± 4.0 62.9 ± 2.7 
 (n = 14) (n = 3) (n = 3) (n = 8) (n = 2) (n = 2) (n = 2) (n = 4) 
LTβR−/− 5.4 ± 2.3e 180 ± 81.3e 406 ± 212e 2.2 ± 1.4e c c 42.7 ± 7.5 72.2 ± 11.2e 
 (n = 7) (n = 3) (n = 3) (n = 4)   (n = 4) (n = 4) 
MouseFecal IgA (μg/ml)aLP Supernatant IgA (ng/ml)IgA SFC/106 LP Cellsb% of CD45+ LP Cells Which Are CD19+% of CD19+ LP Cells Which Are IgA+% of CD19+ LP Cells Which Are CD11b% of CD45+ LP Cells Which Are TCRβ+% of CD45+ Splenocytes Which Are CD19+
TNFRI−/− 49.4 ± 12.3d,e 7,766 ± 884d,e 51,610 ± 6,787d,e 21.9 ± 5.5d 20.7 ± 1.5e 90.5 ± 3.1 46.6 ± 6.4 66.0 ± 3.4e 
 (n = 14) (n = 3) (n = 3) (n = 4) (n = 3) (n = 4) (n = 4) (n = 4) 
TNFRII−/− 12.0 ± 2.8d,e 4,291 ± 1,416d,e 24,660 ± 1,627d 27.1 ± 11.4d 15.5 ± 3.9e 87.1 ± 3.0 41.9 ± 4.0 62.9 ± 2.7 
 (n = 14) (n = 3) (n = 3) (n = 8) (n = 2) (n = 2) (n = 2) (n = 4) 
LTβR−/− 5.4 ± 2.3e 180 ± 81.3e 406 ± 212e 2.2 ± 1.4e c c 42.7 ± 7.5 72.2 ± 11.2e 
 (n = 7) (n = 3) (n = 3) (n = 4)   (n = 4) (n = 4) 
a

Fecal IgA levels and supernatant IgA levels displayed as mean ± SEM.

b

ELISPOT and flow cytometric analysis displayed mean ± SD.

c

Too few cells to analyze.

d

Values of p < 0.05 when compared to LTα−/− mice.

e

Values of p < 0.05 when compared to C57BL/6 mice (Table I).

Table IV.

Analysis of LP lymphocytes after LTβR-Ig treatment

Treatment GroupaFecal IgA (μg/ml)b% of CD45+ LP Cells Which Are CD19+c% of CD19+ LP Cells Which Are CD11bc% of CD45+ LP Cells Which Are TCRβ+% of CD45+ Splenocytes Which Are CD19+
98.2 ± 17.8 43.1 ± 9.5 79.8 ± 14.7 37.6 ± 7.1 64.9 ± 6.0 
 (n = 24) (n = 3) (n = 3) (n = 3) (n = 3) 
0.15 ± 0.1e 5.8 ± 4.2e,f d 57.1 ± 5.5e,f 70.9 ± 2.0f 
 (n = 10) (n = 2)  (n = 2) (n = 2) 
0.24 ± 0.2e 4.3 ± 2.5e,f d 65.9 ± 1.6e,f 76.0 ± 13.9f 
 (n = 7) (n = 2)  (n = 2) (n = 2) 
13.3 ± 8.0 2.4 ± 0.2e,f d 53.6 ± 4.5 54.7 ± 13.4 
 (n = 3) (n = 2)  (n = 2) (n = 2) 
30.1 ± 14.4 22.1 ± 3.3e 76.3 ± 2.8 39.8 ± 5.2 55.5 ± 6.2 
 (n = 6) (n = 3) (n = 3) (n = 3) (n = 6) 
130.6 ± 58.8 58.3 ± 7.0 83.8 ± 5.2 29.6 ± 3.8 48.2 ± 3.8e 
 (n = 3) (n = 3) (n = 3) (n = 3) (n = 3) 
Treatment GroupaFecal IgA (μg/ml)b% of CD45+ LP Cells Which Are CD19+c% of CD19+ LP Cells Which Are CD11bc% of CD45+ LP Cells Which Are TCRβ+% of CD45+ Splenocytes Which Are CD19+
98.2 ± 17.8 43.1 ± 9.5 79.8 ± 14.7 37.6 ± 7.1 64.9 ± 6.0 
 (n = 24) (n = 3) (n = 3) (n = 3) (n = 3) 
0.15 ± 0.1e 5.8 ± 4.2e,f d 57.1 ± 5.5e,f 70.9 ± 2.0f 
 (n = 10) (n = 2)  (n = 2) (n = 2) 
0.24 ± 0.2e 4.3 ± 2.5e,f d 65.9 ± 1.6e,f 76.0 ± 13.9f 
 (n = 7) (n = 2)  (n = 2) (n = 2) 
13.3 ± 8.0 2.4 ± 0.2e,f d 53.6 ± 4.5 54.7 ± 13.4 
 (n = 3) (n = 2)  (n = 2) (n = 2) 
30.1 ± 14.4 22.1 ± 3.3e 76.3 ± 2.8 39.8 ± 5.2 55.5 ± 6.2 
 (n = 6) (n = 3) (n = 3) (n = 3) (n = 6) 
130.6 ± 58.8 58.3 ± 7.0 83.8 ± 5.2 29.6 ± 3.8 48.2 ± 3.8e 
 (n = 3) (n = 3) (n = 3) (n = 3) (n = 3) 
a

See Fig 1 and Materials and Methods for description of treatment regimens.

b

Fecal IgA levels measured time of sacrifice, displayed as mean ± SEM.

c

Flow cytometric analysis displayed mean ± SD.

d

Too few cells to analyze.

e

Values of p < 0.05 when compared with mice treated with LTβR-Ig day 16 pc (treatment group 1).

f

Values of p < 0.05 when compared with mice in which LTβR-Ig was discontinued (treatment group 5).

Table V.

Analysis of LP lymphocytes after bone marrow transfer

Bone Marrow DonorsBone Marrow RecipientsFecal IgA (μg/ml)a,b% of CD45+ LP Cells Which Are CD19+c% of CD19+ LP Cells Which Are CD11b% of CD45+ LP Cells Which Are TCRβ+% of CD45+ Splenocytes Which Are CD19+
LTα−/− LTα−/− 2.9 ± 1.4 3.8 ± 1.5 g 70.8 ± 3.5 62.4 ± 6.2 
  (n = 20) (n = 8)  (n = 8) (n = 9) 
C57BL/6 LTα−/− 73.6 ± 43.9d 19.1 ± 5.7d 84.2 ± 7.8 63.3 ± 4.6d 65.3 ± 9.1 
  (n = 7) (n = 5) (n = 5) (n = 5) (n = 5) 
C57BL/6 LTα−/− 35.4 ± 8.2e 19.4 ± 5.4d 91.8 ± 7.2 56.8 ± 7.5d — 
 (splenectomized) (n = 5) (n = 4) (n = 4) (n = 4)  
LTα−/− and RAG−/− LTα−/− 10.2 ± 3.5d 6.6 ± 1.9d,f 92.7 ± 4.5 60.5 ± 6.7d 60.3 ± 8.7 
  (n = 13) (n = 5) (n = 5) (n = 5) (n = 5) 
LTα−/− and JH−/− LTα−/− 21.1 ± 5.0d 5.9 ± 1.7d,f 92.1 ± 3.8 54.5 ± 7.5d,f 60.3 ± 1.9 
  (n = 8) (n = 5) (n = 6) (n = 5) (n = 5) 
C57BL/6 LTβR−/− 4.3 ± 1.6f 2.1 ± 1.1f g 70.1 ± 9.3 64.8 ± 8.9 
  (n = 8) (n = 4)  (n = 4) (n = 4) 
Bone Marrow DonorsBone Marrow RecipientsFecal IgA (μg/ml)a,b% of CD45+ LP Cells Which Are CD19+c% of CD19+ LP Cells Which Are CD11b% of CD45+ LP Cells Which Are TCRβ+% of CD45+ Splenocytes Which Are CD19+
LTα−/− LTα−/− 2.9 ± 1.4 3.8 ± 1.5 g 70.8 ± 3.5 62.4 ± 6.2 
  (n = 20) (n = 8)  (n = 8) (n = 9) 
C57BL/6 LTα−/− 73.6 ± 43.9d 19.1 ± 5.7d 84.2 ± 7.8 63.3 ± 4.6d 65.3 ± 9.1 
  (n = 7) (n = 5) (n = 5) (n = 5) (n = 5) 
C57BL/6 LTα−/− 35.4 ± 8.2e 19.4 ± 5.4d 91.8 ± 7.2 56.8 ± 7.5d — 
 (splenectomized) (n = 5) (n = 4) (n = 4) (n = 4)  
LTα−/− and RAG−/− LTα−/− 10.2 ± 3.5d 6.6 ± 1.9d,f 92.7 ± 4.5 60.5 ± 6.7d 60.3 ± 8.7 
  (n = 13) (n = 5) (n = 5) (n = 5) (n = 5) 
LTα−/− and JH−/− LTα−/− 21.1 ± 5.0d 5.9 ± 1.7d,f 92.1 ± 3.8 54.5 ± 7.5d,f 60.3 ± 1.9 
  (n = 8) (n = 5) (n = 6) (n = 5) (n = 5) 
C57BL/6 LTβR−/− 4.3 ± 1.6f 2.1 ± 1.1f g 70.1 ± 9.3 64.8 ± 8.9 
  (n = 8) (n = 4)  (n = 4) (n = 4) 
a

Fecal IgA levels measured 8 wk post bone marrow transfer, displayed as mean ± SEM.

b

Fecal IgA levels measured 10 wk post bone marrow transfer in splenectomized recipients, displayed as mean ± SEM.

c

Flow cytometric analysis displayed mean ± SD.

d

Values of p < 0.05 when compared with LTα−/− mice receiving LTα−/− bone marrow.

e

Values of p < 0.05 when compared with splenectomized LTα−/− mice receiving LTα−/− bone marrow.

f

Values of p < 0.05 when compared with LTα−/− mice receiving C57BL/6 bone marrow.

g

Too few cells to analyze.

Feces were collected from individual mice, diluted 1/10 wet weight to volume with PBS, vortexed into a uniform suspension, centrifuged at 12,000 rpm for 10 min in a table top microfuge, and supernatants removed. Fecal supernatants or IgA standards (Southern Biotechnology Associates, Birmingham, AL) diluted in PBS containing 0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO) were incubated in 96-well Immunlon 4 plates (Fisher Scientific, Pittsburgh, PA) previously coated with goat anti-mouse Ab (Southern Biotechnology Associates) and blocked with PBS containing 5% BSA and 0.05% Tween 20 at room temperature for 2 h. Plates were washed three times with PBS containing 0.05% Tween 20, then goat anti-mouse IgA alkaline phosphatase-conjugated Ab (Southern Biotechnology Associates) diluted in PBS containing 5% BSA and 0.05% Tween 20 was added to the plate and incubated for 2 h at room temperature. Plates were washed three times with PBS containing 0.05% Tween 20 and p-nitrophenyl phosphate alkaline phosphatase substrate (Sigma-Aldrich) was added. Plates were read at 405 nM using Bio-Tek Instruments Microplate Reader (Bio-Tek Instruments, Winooski, VT). Each sample was measured in duplicate in at least three dilutions. Data are reported as the concentration of IgA in the fecal supernatant prepared as above.

For the determination of LP culture supernatant IgA levels, LP cells, isolated as described below were cultured at a density of 6.25 × 105 cells/ml in RPMI 1640 media (BioWhittaker, Walkersville, MD) containing 10% FCS (HyClone, Logan, UT), 2 mM glutamax I (Life Technologies, Grand Island, NY), 10 mM HEPES (BioWhittaker), 1 mM sodium pyruvate (BioWhittaker), 50 U/ml penicillin-50 mg/ml streptomycin (Life Technologies), and 50 mM 2-ME (Fisher Scientific) for 72 h at 37°C and 5% CO2. After 72 h, the culture supernatants were removed and the concentration of IgA in the supernatant was determined as described above.

LP mononuclear cells and splenocytes were isolated as previously described (25). Briefly, PP were removed from small intestines, epithelial cells were removed by treatment with HBSS (BioWhittaker) containing 5 mM EDTA (Sigma-Aldrich), and single-cell suspensions were generated from the LP by dispase (Sigma-Aldrich) and collagenase (Sigma-Aldrich) digestion. Cells were counted for viability by trypan blue exclusion. Cellular populations with viability <75% were discarded. Typical yields for LP cell isolation was 1.5 × 107 viable cells/intestine. This population includes both stromal cells and bone marrow-derived cells. Resident peritoneal cells were obtained by flushing the peritoneal cavity with 10 ml of cold PBS. RBCs were lysed in all populations before analysis.

Single-cell suspensions obtained as above were resuspended in PBS with 1% BSA (Fisher Scientific) and 1 mg/ml human IgG (Sandoz Pharmaceuticals, East Hanover, NJ) at 2 × 107 cells/ml, or for the detection of LT expression using the LTβR-Ig fusion protein, cells were resuspended in PBS with 1% BSA, 5% normal mouse serum, 5% normal rabbit serum, and FC block (BD PharMingen, San Diego, CA) at 2 × 107 cells/ml. Cells were stained with the directly conjugated Abs, biotin-conjugated Abs, the LTβR-Ig fusion protein, or the appropriate isotype control Abs for 30 min on ice. Cellular suspensions were washed twice in PBS containing 1% BSA, and where appropriate, stained with directly conjugated secondary Abs, streptavidin-PE, or steptavidin-FITC for 30 min on ice. Cells were washed twice as above and fixed with 1% paraformaldehyde in PBS. Flow cytometric analysis was done on a triple-laser flow cytometer (FACScan; BD Biosciences, Mountain View, CA) and analysis was performed on a Macintosh G3 using the CellQuest program (BD Biosciences). Dead cells were excluded based on forward and side light scatter and 10,000 cells from the remaining population were analyzed for CD45 expression to determine the number of bone marrow-derived cells in the LP. For the analysis of lymphocyte subpopulations, 10,000 cells from the lymphocyte population (as determined by forward vs side scatter) were analyzed for the expression of lymphocyte markers. Gates for positive staining were defined such that 1% of the analyzed population stained positive with the appropriate isotype control Ab. Therefore, 1% positive staining is consistent with the absence of a positive population.

The following Abs and secondary staining reagents were used for flow cytometric studies: FITC-conjugated rat anti-mouse CD45, PE-conjugated rat anti-mouse CD19, biotin-conjugated rat anti-mouse CD11b, biotin-conjugated hamster anti-mouse TCRβ, PE-conjugated rat anti-mouse CD4, FITC-conjugated rat anti-mouse CD8β, PE-conjugated hamster anti-mouse TCRγδ, biotin-conjugated mouse anti-mouse NK1.1, appropriate isotype control Abs, streptavidin-FITC, streptavidin-PE (all from BD PharMingen), biotin-conjugated rat anti-mouse CD8α (Caltag Laboratories, Bulingame, CA), biotin-conjugated rat anti-mouse IgA (Southern Biotechnology Associates), and PE-conjugated donkey anti-human Ig (Jackson ImmunoResearch Laboratories, West-Grove, PA).

96-well multiscreen-HA plates (Millipore, Bedford, MA) were coated with goat anti-mouse Ig (Southern Biotechnology Associates) overnight at room temperature. Plates were washed three times in PBS, blocked with PBS containing 5% newborn calf serum (HyClone) for 1 h at 37°C, washed, and LP cellular suspensions in IMDM (BioWhittaker), 5% FCS (HyClone), 2 mM glutamax I (Life Technologies), 50 U/ml penicillin-50 mg/ml streptomycin (Life Technologies), and 50 mM 2-ME (Fisher Scientific) were added to the plates. Plates were incubated at 37°C 5% CO2 overnight, washed with PBS containing 0.05% Tween 20, and incubated with alkaline phosphatase-conjugated goat anti-mouse IgA Ab (Southern Biotech Associates) overnight at 4°C. Plates were washed with PBS and exposed to 5-bromo-4-chloro-3-indolyl phosphatase/nitro blue tetrazloium substrate (Sigma-Aldrich) and spot-forming cells were counted under a dissecting microscope.

Six to 10-wk-old LTα−/− and LTβR−/− mice (recipients) received 1000 Gy of γ irradiation in divided doses over two sequential days. Bone marrow was harvested from gender-matched adult C57BL/6, LTα−/−, JH−/−, and RAG−/− donors and treated with anti-Thy1.1 and Thy1.2 Ab (clone AT83; a gift from Dr. O. Kanagawa, Washington University School of Medicine) and low toxin rabbit complement (Cedarlane Laboratories Limited, Ontario, Canada) to deplete T lymphocytes. A total of 1 × 107 T lymphocyte-depleted bone marrow cells (5 × 106 cells from each donor in experiments involving multiple donors) from gender-matched donors were injected i.v. into recipients on the second day of irradiation. All recipient mice received 280 μg/ml sulfamethoxazole and 58 μg/ml trimethoprim in drinking water for 3 days before and 5 days following bone marrow transfer. Mice were allowed 12 wk for reconstitution with donor bone marrow before use for experiments involving flow cytometric analysis.

Laparotomy was performed on 8–10-wk-old anesthetized LTα−/− mice; the splenic artery was ligated and the spleen removed. Mice received 100 mg/kg ampicillin and 5 mg/kg gentamicin i.p. daily for 3 days following splenectomy. Two weeks following splenectomy, mice underwent bone marrow transfers as described above. The complete absence of the spleen was confirmed by examination at the time of sacrifice.

Soluble LTβR-Ig was purified from supernatants generated by a Chinese hampster ovary cell line producing the LTβR-Ig fusion protein (a gift from Dr. W. Yokoyama, Washington University School of Medicine). LTβR-Ig activity was confirmed by the ability to inhibit the formation of PP in offspring of pregnant females receiving 100 μg LTβR-Ig i.v. on day 16 postconception (pc).

Group 1: timed pregnant female C57BL/6 mice were injected with 100 μg LTβR-Ig via tail vein on day 16 pc. Group 2: offspring from timed pregnant female C57BL/6 mice injected with 100 μg LTβR-Ig on day 16 pc received 20 μg LTβR-Ig i.p. weekly for 5 wk beginning 7 days after birth. Group 3: offspring from untreated C57BL/6 female mice received 20 μg LTβR-Ig i.p. beginning 3 days after birth and weekly thereafter for 5 wk. Group 4: mice from group 2 were treated with 20 μg of LTβR-Ig i.p. weekly for an additional 10 wk. Group 5: mice from groups 2 and 3 were followed for 10 wk after the cessation of LTβR-Ig therapy. No differences were noted between groups 2 and 3 at any time point examined; and therefore, results from these two groups were combined and reported as group 5. Group 6: 10-wk-old mice were given 100 μg of LTβR-Ig i.p weekly for 3 wk.

Delta Soft 3 software (BioMetallics, Princeton, NJ) was used to determine the weighted mean ± the SEM of the fecal IgA concentration for each sample. Weighted mean = Σwi conci/Σwi; conci is the interpolated mean concentration for dilution i, wi = 1/(SEMi)2, SEMi is the SE of mean for conci. Data analysis using an unpaired Student’s t test was performed using GraphPad Prism (GraphPad, San Diego, CA).

The phenotype of mice deficient in the expression of LTα (lacking both LTα3 and LTα1β2) has been well described (4, 26). These mice lack organized lymphoid structures, including PP, have disrupted splenic architecture lacking GC and FDC, have diminished fecal IgA production, and have a diminished capacity to produce high affinity Ig upon immunization. Importantly, these mice have normal lymphatics, and studies of B and T lymphocyte function have not revealed deficits. To assess the role of LTα in directing the composition of the LP lymphocyte populations, we examined the LP cell populations from C57BL/6 and LTα−/− mice. As shown in Table I, LTα−/− mice have normal numbers of bone marrow-derived (CD45+) cells in their intestinal LP. Further analysis of the lymphocyte subpopulations revealed that LTα−/− mice have an increased proportion of αβ and γδ TCR+ T lymphocytes, and this increase is distributed between CD4+ and CD8+ T lymphocyte subpopulations. We did not observe CD4, CD8 double positive T lymphocytes in the LP of any mice examined. However, B lymphocytes are absent from the LP of the LTα−/− mice (Table I and Fig. 1). LTα−/− mice also lack LP lymphocytes expressing surface Ig (data not shown), confirming the absence of LP B lymphocytes as opposed to a failure of LTα−/− LP B lymphocytes to express an isolated cell surface marker. The absence of B lymphocytes in the LP of LTα−/− mice cannot be explained by a global deficiency of B lymphocytes, as an increased proportion of B lymphocytes were seen in the spleen of LTα−/− mice when compared with controls (Table I and Fig. 1). Notably, the LP B lymphocytes in C57BL/6 mice are overwhelmingly B-2 B lymphocytes (CD19+, CD11b) and, consistent with previous observations, contain a small population of CD19+IgA+ cells (19), suggesting that the deficiency of B lymphocytes in the LTα−/− mice is predominantly a deficiency in IgA B-2 B lymphocytes (Table I). The remainder of the CD45+ population in the LP of LTα−/− mice are also modestly increased, and include NK cells, granulocytes, and macrophages (our unpublished observations).

FIGURE 1.

Absence of LP B lymphocytes in LTα−/− mice. LP mononuclear cells were isolated from 7-wk-old C57BL/6 and LTα−/− mice and analyzed by flow cytometric analysis as described in Materials and Methods. CD19 and CD45 double positive cells are seen in the spleen of C57BL/6 and LTα−/− mice and intestinal LP of C57BL/6 mice, but are absent from the intestinal LP of LTα−/− mice. Identical results were obtained when analyzing for the presence of surface Ig, and CD45 double positive cells (data not shown), indicating that the lack of CD19+ expression is not due to the failure to express an isolated cell surface marker on LTα−/− B lymphocytes.

FIGURE 1.

Absence of LP B lymphocytes in LTα−/− mice. LP mononuclear cells were isolated from 7-wk-old C57BL/6 and LTα−/− mice and analyzed by flow cytometric analysis as described in Materials and Methods. CD19 and CD45 double positive cells are seen in the spleen of C57BL/6 and LTα−/− mice and intestinal LP of C57BL/6 mice, but are absent from the intestinal LP of LTα−/− mice. Identical results were obtained when analyzing for the presence of surface Ig, and CD45 double positive cells (data not shown), indicating that the lack of CD19+ expression is not due to the failure to express an isolated cell surface marker on LTα−/− B lymphocytes.

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Previous studies have demonstrated a peritoneal lymphocytosis in LTα−/− and aly/aly mice (5, 19). In aly/aly mice the peritoneal cavity contains an increased population of B lymphocytes with a disproportionate expansion of B-1 (CD11b+) B lymphocytes (19). Analysis of the resident peritoneal cell population from 14-wk-old LTα−/−, LTβR−/−, and C57BL/6 mice (Table II) reveals an increase in the number of resident peritoneal cells in both the LTα−/− and LTβR−/− mice with a preferential increase in B lymphocytes which are predominantly CD11b (B-2 B lymphocytes), the remaining CD19+ cells are CD11b+ and therefore, are B-1 B lymphocytes. No significant change is seen in the proportion of peritoneal B cells expressing IgA in either the LTα−/− and LTβR−/− mice. These findings are consistent with the inability of IgA B-2 B lymphocytes to migrate to appropriate effectors sites (intestinal LP) in the absence of LTβR signaling, resulting in their accumulation in the peritoneal cavity.

LTα−/− mice lack expression of both soluble LTα3 and membrane-bound LTα1β2; and therefore, the lack of B lymphocytes in the intestine of the LTα−/− mice could result from a lack of activation of TNFRI, TNFRII, or the LTβR. To determine the relevant events for the presence of B lymphocytes in the intestinal LP, LP lymphocyte populations were evaluated from mice deficient in TNFRI, TNFRII, and LTβR. As shown in Table III and Fig. 2,a, TNFRI- and TNFRII-deficient mice have a near normal population of LP B lymphocytes which is enriched for IgA+ B lymphocytes, while LTβR-deficient mice lack LP B lymphocytes. The deficiency of B lymphocytes in LTβR-deficient mice is not a global phenotype, as B lymphocytes are present in the spleen of LTβR-deficient mice in proportions equal to or exceeding wild-type mice. Consistent with their lack of LP B lymphocytes, LTβR−/− mice have diminished fecal IgA production equivalent to that of LTα−/− mice (compare Tables I and III). We also noted diminished fecal IgA production by TNFRI- and TNFRII-deficient mice when compared with that of C57BL/6 mice (compare Tables I and III). Notably, these mice have a population of LP B lymphocytes which is equivalent to that of C57BL/6 mice and a comparable or increased number of IgA producing plasma cells when compared with C57BL/6 mice (Table I and III). However, the ability of the LP plasma cells in the TNFRI- and TNFRII-deficient mice to produce IgA is diminished. These observations suggest that TNFRI- and TNFRII-dependent events play a role in augmenting Ab production by IgA plasma cells in the LP, and that defects in the epithelial transport of IgA in these mice strains are unlikely to account for their diminished fecal IgA production.

FIGURE 2.

LT-sufficient bone marrow-derived cells and LTβR-sufficient, non-bone marrow-derived, radio-resistant cells mediate postgestational LTβR-dependent events essential for the presence of LP B lymphocytes. a, LP mononuclear cells isolated from TNFRI−/−, TNFRII−/−, and LTβR−/− mice; b, Mice treated with LTβR-Ig during and/or after gestation as described in Materials and Methods; and c, Adult LTα−/− mice receiving LTα−/−, C57BL/6, or combinations of LTα−/− and RAG−/− or LTα−/− and JH−/− bone marrow or adult LTβR−/− mice receiving C57BL/6 bone marrow were analyzed by flow cytometric analysis for the presence of B lymphocytes. LP B lymphocytes are present in TNFRI−/− and TNFRII−/− mice, while LTβR−/− mice and mice treated with LTβR-Ig postgestation lack LP B lymphocytes, indicating that LTβR-dependent events occurring after birth are essential for the presence of LP B lymphocytes. Discontinuation of LTβR-Ig resulted in the presence of normal LP B lymphocyte populations, while treatment of adult mice with the LTβR-Ig for 3 wk was unable to modulate the LP B lymphocyte compartment. Splenectomized and unmanipulated adult LTα−/− mice receiving C57BL/6 bone marrow have LP B lymphocytes, indicating that postgestational LTβR-dependent events, independent of the presence of PP, LN, and the spleen, are sufficient for the presence of LP B lymphocytes. LTα−/− mice receiving LTα−/− and RAG−/− or LTα−/− and JH−/− bone marrow have diminished, but not absent, LP B lymphocyte populations, suggesting that the LT-dependent signal relevant for the presence of LP B lymphocytes is optimally transmitted by B lymphocytes. In contrast to LTα−/− mice receiving C57BL/6 bone marrow, LTβR−/− mice receiving C57BL/6 bone marrow lack LP B lymphocytes, indicating that a LTβR-sufficient, non-bone marrow-derived, radio-resistant cell is required for the presence of LP B lymphocytes.

FIGURE 2.

LT-sufficient bone marrow-derived cells and LTβR-sufficient, non-bone marrow-derived, radio-resistant cells mediate postgestational LTβR-dependent events essential for the presence of LP B lymphocytes. a, LP mononuclear cells isolated from TNFRI−/−, TNFRII−/−, and LTβR−/− mice; b, Mice treated with LTβR-Ig during and/or after gestation as described in Materials and Methods; and c, Adult LTα−/− mice receiving LTα−/−, C57BL/6, or combinations of LTα−/− and RAG−/− or LTα−/− and JH−/− bone marrow or adult LTβR−/− mice receiving C57BL/6 bone marrow were analyzed by flow cytometric analysis for the presence of B lymphocytes. LP B lymphocytes are present in TNFRI−/− and TNFRII−/− mice, while LTβR−/− mice and mice treated with LTβR-Ig postgestation lack LP B lymphocytes, indicating that LTβR-dependent events occurring after birth are essential for the presence of LP B lymphocytes. Discontinuation of LTβR-Ig resulted in the presence of normal LP B lymphocyte populations, while treatment of adult mice with the LTβR-Ig for 3 wk was unable to modulate the LP B lymphocyte compartment. Splenectomized and unmanipulated adult LTα−/− mice receiving C57BL/6 bone marrow have LP B lymphocytes, indicating that postgestational LTβR-dependent events, independent of the presence of PP, LN, and the spleen, are sufficient for the presence of LP B lymphocytes. LTα−/− mice receiving LTα−/− and RAG−/− or LTα−/− and JH−/− bone marrow have diminished, but not absent, LP B lymphocyte populations, suggesting that the LT-dependent signal relevant for the presence of LP B lymphocytes is optimally transmitted by B lymphocytes. In contrast to LTα−/− mice receiving C57BL/6 bone marrow, LTβR−/− mice receiving C57BL/6 bone marrow lack LP B lymphocytes, indicating that a LTβR-sufficient, non-bone marrow-derived, radio-resistant cell is required for the presence of LP B lymphocytes.

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The lack of B lymphocytes in the LP of the LTα−/− and LTβR−/− mice demonstrates that a LTα1β2/LTβR-dependent event is required for the presence of LP B lymphocytes. LTα1β2/LTβR-dependent events during gestation are essential for the formation of PP and LN, and LTα1β2/LTβR-signaling after birth cannot rescue the formation of PP and LN (9). To assess whether LTα1β2/LTβR-dependent events, distinct from the embryonic events required for the formation of PP and LN, are essential for the presence of LP B lymphocytes, we blocked gestational as well as postgestational LTβR signaling with a LTβR-Ig fusion protein (Fig. 3). As shown in Table IV and Fig. 2 b, treatment of pregnant mice on day 16 gestation, blocking PP formation, had no effect on the production of fecal IgA or the presence of LP B lymphocytes (group 1). However, LTα1β2/LTβR-dependent events after birth are essential for the presence of LP B lymphocytes as evidenced by the lack of LP B lymphocytes in mice receiving the LTβR-Ig fusion protein after birth (groups 2–4). There appeared to be no additive effect of blocking LTβR signaling both during and postgestation as compared with blocking LTβR signaling after birth alone (compare groups 2 and 3). Discontinuation of the LTβR-Ig fusion protein after 5 wk allowed for the population of the LP with B lymphocytes which are predominantly CD11b (B-2 B lymphocytes), while mice continually treated with the LTβR-Ig fusion protein over the same time period continued to lack LP B lymphocytes (compare groups 4 and 5). Notably, continuous postgestational treatment with the LTβR-Ig fusion protein did not completely suppress fecal IgA production. In addition, the LP B lymphocyte compartment could not be modulated by treatment of adult mice with 100 μg of LTβR-Ig weekly for 3 wk (group 6), a dosing schedule which is sufficient to cause disruption of splenic architecture and decreased BLC expression in the spleen (Ref. 13 and our unpublished observations). These observations suggest that the LTβR-dependent events required for the presence of LP B lymphocytes occur after birth and appear to be independent of the formation of PP and/or LN.

FIGURE 3.

LTβR-Ig treatment regimens. C57BL/6 mice were treated with LTβR-Ig during and/or after gestation as outlined in Materials and Methods. ↑, Time of conception, birth, and date of analysis. ↓, Time points of LTβR-Ig therapy at day 16 pc, or weekly intervals after birth as described in Materials and Methods. Vertical lines, Date of analysis for respective treatment groups. +, Mice in the respective treatment group received LTβR-Ig therapy at the indicated time point as described in Materials and Methods.

FIGURE 3.

LTβR-Ig treatment regimens. C57BL/6 mice were treated with LTβR-Ig during and/or after gestation as outlined in Materials and Methods. ↑, Time of conception, birth, and date of analysis. ↓, Time points of LTβR-Ig therapy at day 16 pc, or weekly intervals after birth as described in Materials and Methods. Vertical lines, Date of analysis for respective treatment groups. +, Mice in the respective treatment group received LTβR-Ig therapy at the indicated time point as described in Materials and Methods.

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As demonstrated above, postgestational LTβR events are essential for the presence of LP B lymphocytes. To determine whether these events are sufficient for the presence of LP B lymphocytes in the absence of LN and PP, we performed bone marrow transfers of wild-type (LTα-sufficient) bone marrow into adult LTα−/− recipients. These recipients lack LT-dependent events until the time of bone marrow transfer, and transfer of LT-sufficient bone marrow in adulthood cannot rescue LN and PP formation (Ref. 9 and our unpublished observations). As shown in Table V and Fig. 2,c, transfer of LTα−/− bone marrow to LTα−/− mice does not result in the production of fecal IgA or the presence of LP B lymphocytes, while the transfer of C57BL/6 bone marrow to LTα−/− recipients results in the production of normal levels of fecal IgA and the presence of LP B lymphocytes. LTα-sufficient B lymphocytes have been demonstrated to effect the splenic architecture of LTα−/− mice following bone marrow transfer, resulting in the formation of GC and the production of FDCs (11). To assess the contribution of LTα-dependent events in the spleen on the presence of LP B lymphocytes following bone marrow transfer, bone marrow transfers were performed in splenectomized recipients. As shown in Table V, splenectomy had no effect upon the presence of LP B lymphocytes or fecal IgA levels with the exception that fecal IgA levels in splenectomized recipients showed altered kinetics, with peak levels occurring 10 wk post bone-marrow transfer as opposed to 8 wk posttransfer in unmanipulated recipients. These findings demonstrate that bone marrow-derived cells can deliver the necessary and sufficient LT signal for the presence of LP B lymphocytes, independent of the presence of the spleen, PP, and LN.

To determine which bone marrow-derived cell lineage delivers the LT signal needed for the presence of LP B lymphocytes, mixed bone marrow transfers were performed using RAG−/− and JH−/− (LTα-sufficient) donors (Fig. 2,c and Table V). LTα−/− mice receiving a combination of LTα−/− and RAG−/− bone marrow produced fecal IgA which was significantly greater than LTα−/− mice receiving LTα−/− bone marrow alone. Thus, documenting that LT expression by lymphocytes is not required for the production of fecal IgA. However, LTα−/− mice receiving mixed LTα−/− and RAG−/− bone marrow had a diminished, but not absent, population of B lymphocytes when compared with LTα−/− mice receiving wild-type bone marrow, indicating that LT-expressing lymphocytes are necessary for the optimal population of the intestinal LP by B lymphocytes. LTα−/− mice receiving mixed LTα−/− and JH−/− bone marrow had normal production of fecal IgA, and a diminished, but not absent, population of LP B lymphocytes which was not different from that seen in LTα−/− mice receiving LTα−/− and RAG−/− bone marrow. These findings indicate that RAG-independent, LT-expressing, bone marrow-derived cells can transmit the relevant LT-dependent signal for the production of fecal IgA and can partially allow the population of the intestinal LP with B-2 B lymphocytes. However, an LT-expressing B lymphocyte is required for the optimal population of the intestinal LP with B lymphocytes.

The LTβR is expressed by stromal cells and a subpopulation of monocytes. To identify the LTβR-expressing cell population relevant for the entry of LP B lymphocytes, and to provide direct evidence for a role for LTβR in our bone marrow transfer experiments, we transferred C57BL/6 bone marrow into irradiated LTβR−/− mice. LTβR−/− mice receiving C57BL/6 bone marrow had diminished fecal IgA production and lacked LP B lymphocytes (Fig. 2,c and Table V). These observations demonstrate a requirement for LTβR-sufficient, radio-resistant, non-bone marrow-derived cell in directing the entry and residence of LP B lymphocytes.

The mucosa is a primary interface of higher organisms with the environment, and thus, the generation of appropriate immune responses at mucosal surfaces is essential for survival. Intrinsic to the development of these responses is the ability of lymphocytes to appropriately enter and reside in mucosal effector sites. Within the intestine the largest effector site is the LP, in which activated T lymphocytes, B lymphocytes, and plasma cells reside. How lymphocytes come to comprise the LP in the normal state is not well understood. Observations from multiple studies suggest the LP preferentially contains previously activated lymphocytes. It is unclear whether activation within organized lymphoid structures is required for lymphocytes to enter the LP, as well as whether this activation occurs exclusively in PP. In addition, it is unclear whether there are differential requirements for T lymphocytes and B lymphocytes to reside in the LP.

Using the LTα−/− mice, a well described induced mutant strain of mice lacking LN and PP, we examined the requirement of LT-dependent events for the normal composition of intestinal lymphocytes. We noted no differences in the number or composition of IEL in the LTα−/− mice (our unpublished observations). These findings are consistent with observations that the aly/aly mice, which have a spontaneous mutation in NIK, a signaling molecule necessary for the function of the LTβR (22), have normal composition of IELs (28). Examination of LTα−/− mice reveals that LTα-dependent events, including the development of PP and LN, are not required for T lymphocytes to enter and reside in the LP. Consistent with this observation, we noted a normal or increased population of TCRβ+ lymphocytes in the LP of TNFRI−/−, TNFRII−/−, and LTβR−/− mice, as well as LTα−/− mice reconstituted with LTα−/− bone marrow, and mice treated with LTβR-Ig. Flow cytometric analysis of CD69 and CD45RB expression revealed no differences between LP T lymphocytes from LTα−/− mice and C57BL/6 mice (our unpublished observations). These findings suggest that LT-dependent events including events occurring within organized lymphoid structures in general and PP specifically are not required for T lymphocytes to enter and reside in the LP. This contrasts with the current understanding that LP T lymphocytes are predominantly or exclusively comprised from T lymphocytes undergoing activation in PP. In a broader context, this observation suggests that events occurring in the PP directing Th cell development may not influence all T lymphocyte responses in the LP.

In contrast to the finding of a normal composition of LP T lymphocytes, we observed an absence of LP (CD19+) B lymphocytes in the LTα−/− mice. CD19, the earliest B lymphocyte lineage marker, is first expressed as early pro-B cells arise from stem cells in the bone marrow and is continually expressed by B lymphocytes until their differentiation into plasma cells. CD19 is a more specific marker for B lymphocytes when compared with B220 (29, 30, 31). We observed a small population of LP cells which are B220+, CD19 in LTα−/− and RAG−/− mice, suggesting that B220+ is not an accurate marker for the evaluation of intestinal B lymphocyte populations. Analysis of C57BL/6 mice revealed that the LP B lymphocyte population is overwhelmingly comprised of B-2 B lymphocytes, and thus, the LTα−/− mice are predominantly deficient in LP B-2 B lymphocytes.

Examination of the resident peritoneal cell population from the LTα−/− and LTβR−/− mice revealed an increase in cell number with a disproportionate expansion of B lymphocytes which were predominantly CD11b (B-2 B lymphocytes). Consistent with this finding, we also observed an increase in the splenic B lymphocyte population in LTα−/− and LTβR−/− mice. Together these observations suggest that the lack of LT/LTβR-dependent signals result in the inability of intestinal B lymphocytes to migrate appropriately to their effector site(s).

We were able to further demonstrate that the relevant event(s) for B lymphocytes to enter and reside in the LP are not dependent upon TNFRI and/or TNFRII binding soluble LTα3, but are dependent upon LTβR, which binds membrane-bound LTα1β2. Consistent with the absence of B lymphocytes in the LP in the LTα−/− and LTβR−/− mice, we saw an increase in the splenic B lymphocyte populations in these mice, suggesting an inability of B lymphocytes to migrate to their appropriate effector sites. In contrast to our observations in the knockout mice, we have not observed an increase in resident peritoneal cell number or a shift in the B-1 or B-2 B lymphocyte populations in mice treated with the LTβR-Ig fusion protein or in LTα−/− and LTβR−/− bone marrow recipients.

We also noted diminished production of fecal IgA by both TNFRI- and TNFRII-deficient mice; however, the levels were significantly higher than that seen in the LTα−/− and LTβR−/− mice. TNFRI-deficient mice have a normal population of B lymphocytes in their LP; however, they lack GC (10) and have hypoplastic PP which may contribute to their diminished fecal IgA production. Fecal IgA production by TNFRII-deficient mice was significantly lower than that seen for TNFRI-deficient mice. TNFRII-deficient mice are not known to have a defect in the formation of GC and FDC, are felt to have normal PP and LN formation, and by our observations have a normal population of LP B lymphocytes. In addition, we observed a normal or increased number of IgA producing cells in the LP of TNFRII−/− and TNFRI−/− mice. However, despite the normal or increased number of IgA producing plasma cells in these mice, the production of IgA by these plasma cells was significantly decreased in comparison with wild-type mice. These observations suggest that TNFRI- and TNFRII-dependent events may be necessary for the optimal production of IgA by LP plasma cells, and is consistent with the diminished Ig production in response to T lymphocyte-dependent Ags seen in TNF-deficient mice (32).

LTβR-dependent events occurring during embryogenesis are essential for the development of PP and LN. Using a LTβR-Ig fusion protein, we demonstrate that blocking LTβR-dependent events during gestation alone, which have been shown to be essential for the formation of PP, does not affect the production of fecal IgA or the population of the LP by B lymphocytes. However, blocking LTβR-dependent events after birth results in the absence of LP B lymphocytes and reduced, but not absent, fecal IgA production. The effects of LTβR blockade were reversible with the discontinuation of LTβR-Ig treatment in adulthood, demonstrating that the relevant LT-dependent signal for the entry of LP B lymphocytes can be delivered in adulthood. In contrast to the rapidly reversible effects of LTβR signaling on the normal segregation of lymphocytes in the spleen, we were unable to modulate the LP B lymphocyte compartment of adult mice with short-term LTβR-Ig treatment. Together these findings document a role for postgestational LT/LTβR interactions important for the presence of LP B lymphocytes.

To assess the requirement of PP and LN in the entry and residence of LP B lymphocytes, we performed adoptive transfers of wild-type bone marrow into adult LTα−/− mice. These mice lack LN and PP, and do not receive LT-dependent signals until after bone marrow transfer. LTα−/− mice reconstituted with wild-type bone marrow have normal fecal IgA and increased LP B lymphocytes when compared with LTα−/− mice reconstituted with LTα−/− bone marrow. Previous observations have suggested postgestational LTβR-dependent events may play a role in the formation of PP. Transgenic mice expressing concentrations of the LTβR-Ig fusion protein sufficient to block LTβR signal transduction on day 3 of life have decreased size and number of PP (33). However, we feel that the formation of PP is not the relevant LTβR-dependent event for the presence of LP B lymphocytes, as LP B lymphocytes are present in mice treated with the LTβR-Ig fusion protein at day 16 in utero, and in LTα−/− mice receiving C57BL/6 bone marrow, both of which lack PP. LT expressing B lymphocytes have been shown to direct normal compartmentalization of the spleen of LTα mice after adoptive bone marrow transfer (9, 34). To rule out a role for events in the spleen contributing to the presence of LP B lymphocytes, we performed bone marrow transfers on splenectomized LTα−/− recipients. Splenectomized recipients had normal production of fecal IgA and normal population of the LP with B lymphocytes. Together these findings suggest that postgestational LT/LTβR interactions, independent of the formation of LN and PP, and the independent of events occurring in the spleen, are both essential and sufficient for the entry and residence of LP B lymphocytes.

To determine the LT expressing bone marrow-derived cell lineage relevant for the presence of LP B lymphocytes, we performed mixed bone marrow transfers into LTα−/− recipients. Mice receiving combinations of LTα−/− with RAG−/− or JH−/− bone marrow had similar populations of LP B lymphocytes that were significantly increased over those seen in LTα−/− recipients receiving LTα−/− bone marrow and significantly less than those seen in LTα−/− recipients receiving wild-type bone marrow. These observations suggest that while LT expressing nonlymphocyte bone marrow cell lineages may provide the LT-dependent signal necessary for the production of fecal IgA and the entry of B lymphocytes into the intestinal LP, optimal population of the intestinal LP by B lymphocytes requires an LT expressing B lymphocyte. In vitro studies demonstrated up-regulation of LT expression upon lymphocyte activation (3, 35); however, the kinetics of LT expression in vivo is less well understood. Using flow cytometric analysis we observed 5–10% of CD19+ PP and mesenteric LN cells and <5% of CD19+ LP cells express LT in normal mice (our unpublished observations).

The LTβR is expressed by non-bone marrow-derived stromal cells and a subset of monocytes (3). To determine the LTβR expressing cell type relevant for the presence of LP B lymphocytes, and to provide direct evidence for the role of LTβR in our bone marrow transfer experiments, we transferred wild-type bone marrow to LTβR−/− recipients. In contrast to LTα−/− recipients receiving C57BL/6 bone marrow, LTβR−/− recipients of C57BL/6 bone marrow lacked LP B lymphocytes. These findings document a requirement for a LTβR-expressing, radio-resistant, non-bone marrow-derived cell for the presence of LP B lymphocytes.

The events leading to the production of fecal IgA are complex, and involve the maturation and migration of B-1 and B-2 B lymphocytes, both of which contribute to the pool of fecal IgA (19, 36, 37, 38). Multiple studies have investigated the role of LT in the development of IgA producing plasma cells and the generation of fecal IgA. It is currently not clear where the final development of B lymphocytes into IgA producing plasma cells occurs and it is also unclear as to what proportion of the LP B lymphocytes contribute to the pool of these plasma cells. Recent descriptions suggest that B lymphocyte differentiation into plasma cells may take place after B lymphocytes arrive in the LP (39). Given these observations, we feel it is important to make a distinction between our findings and the findings of previous investigations examining the roles LT plays in promoting the production of Ig. LT has been shown to play a role in the development of PP and LN which are essential to the production of high affinity Ab from B-2 B lymphocyte precursors (4, 5, 6, 7, 40, 41). In this study, we demonstrate a role of LT in directing the composition of B lymphocytes in the intestinal LP. Our observations suggest that the roles LT plays in the production of fecal IgA and in directing the composition of LP B lymphocytes may be independent as evidenced by the normal fecal IgA levels in mice with absent or diminished LP B lymphocytes (mice receiving continuous LTβR-Ig and mice receiving mixed bone marrow transfers, respectively).

Our observations parallel the role LT plays in directing the compartmentalization of the spleen. LT expressing B lymphocytes have been demonstrated to promote the development of splenic stromal cells which produce chemokines facilitating the normal segregation of splenic T and B lymphocytes (2, 11, 13, 34, 42). Similar to our findings, the LT-dependent signal important for the normal segregation of splenic lymphocytes can be transmitted by an LT-expressing B lymphocyte in adulthood. These observations suggest that an intestinal stromal cell with a function similar to the previously described splenic stromal cell may require LT for its development, and would expand the recently described roles of intestinal stromal cells in the intestinal immune response (39, 43).

Diminished BLC expression in the intestine is an attractive explanation for our observations, as BLC is a B lymphocyte selective chemokine, and its expression by splenic stromal cells is diminished in the absence of LTβR signal transduction (13). However, mice deficient in the Burkitt’s lymphoma receptor 1, the BLCR, have a normal number of IgA-producing cells in the LP (44), and BLC expression is not detected in non-PP bearing intestine (Ref. 19 and our unpublished observations), suggesting that the loss of LTβR-induced intestinal BLC expression is unlikely to explain our findings.

Recently, a primitive mechanism of intestinal IgA production independent of the presence of T lymphocytes and requiring lymphoid structures has been described (38). These observations were based upon the lack of IgA production in the LTα−/− and aly/aly mice (both lacking PP and LN). In addition, recent investigations have described a pathway of intestinal IgA production independent of the expression of the μ- or δ-chain (45). This pathway of IgA production is felt to require PP or LN based upon the inability of bone marrow from μ-chain-deficient mice to reconstitute IgA production in aly/aly mice. Based upon our observations, these pathways of IgA production may not be dependent upon the presence of organized lymphoid structures, but rather require LT/LTβR-mediated events, independent of the formation of PP and LN, necessary for the normal entry of B lymphocytes into the intestinal LP.

The mucosal immune system is a network of physically distinct lymphoid compartments with the capacity to transmit Ag-specific immune responses between distant surfaces. Essential to the generation of these coordinated responses is the appropriate entry and residence of lymphocytes in these lymphoid compartments. Recent investigations have defined roles for the β7 integrins and the CCR9/thymus-expressed chemokine receptor/chemokine pair in directing the compartmentalization of the intestinal immune system. We have demonstrated a role for the cytokine LTα1β2 and its receptor LTβR in directing the specific compartmentalization of B lymphocytes to the intestinal LP. Surprisingly, these LTα1β2/LTβR-dependent events are distinct and independent of the role LTα1β2/LTβR plays in the formation of PP, LN, and the segregation of lymphocytes in the spleen. Based on the severity of the deficit of LP B lymphocytes when LTβR signal transduction is blocked, we believe these events are of primary importance in directing the entry and residence of LP B lymphocytes.

We thank K. Sheehan and L. Peacock for technical assistance; J. Browning (Biogen, Cambridge, MA) for assistance with flow cytometric analysis using the LTβR-Ig; E. Newberry, S. Amadeus, and the members of the St. Louis Institute of Mucosal Immunology (St. Louis, MO) for assistance and advice with manuscript preparation.

1

This work was supported in part by a Glaxo Wellcome Institute for Digestive Health Basic Research Award (to R.D.N.), Research Project Grant no. 99-086-01 from the American Cancer Society (to R.G.L.), National Institutes of Health Grants DK-02608 (to R.D.N.), DK-57926 (to R.G.L.), and DK-57936 (to R.G.L.), and the Washington University School of Medicine Digestive Diseases Research Core Center Grant (P30-DK52574).

3

Abbreviations used in this paper: LT, lymphotoxin; PP, Peyer’s patch; FDC, follicular dendritic cell; GC, germinal center; SLC, secondary lymphoid tissue chemokine; BLC, B lymphocyte chemoattractant; LP, lamina propria; IEL, intraepithelial lymphocyte; NIK, NFκβ-inducing kinase; LN, lymph node; RAG, recombination-activating gene; pc, postconception.

1
Ware, C. F., T. L. VanArsdale, P. D. Crowe, J. L. Browning.
1995
. The ligands and receptors of the lymphotoxin system.
Curr. Top. Microbiol. Immunol.
198
:
175
2
Chaplin, D. D., Y. Fu.
1998
. Cytokine regulation of secondary lymphoid organ development.
Curr. Opin. Immunol.
10
:
289
3
Browning, J. L., I. D. Sizing, P. Lawton, P. R. Bourdon, P. D. Rennert, G. R. Majeau, C. M. Ambrose, C. Hession, K. Miatkowski, D. A. Griffiths, et al
1997
. Characterization of lymphotoxin-αβ complexes on the surface of mouse lymphocytes.
J. Immunol.
159
:
3288
4
De Togni, P., J. Goellner, N. H. Ruddle, P. R. Streeter, A. Fick, S. Mariathasan, S. C. Smith, R. Carlson, L. P. Shornick, J. Strauss-Schoenberger, et al
1994
. Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin.
Science
264
:
703
5
Alimzhanov, M. B., D. V. Kuprash, M. H. Kosco-Vilbois, A. Luz, R. L. Turetskaya, A. Tarakhovsky, K. Rajewsky, S. A. Nedospasov, K. Pfeffer.
1997
. Abnormal development of secondary lymphoid tissues in lymphotoxin β-deficient mice.
Proc. Natl. Acad. Sci. USA
94
:
9302
6
Futterer, A., K. Mink, A. Luz, M. H. Kosco-Vilbois, K. Pfeffer.
1998
. The lymphotoxin β receptor controls organogenesis and affinity maturation in peripheral lymphoid tissues.
Immunity
9
:
59
7
Rennert, P. D., J. L. Browning, R. Mebius, F. Mackay, P. S. Hochman.
1996
. Surface lymphotoxin α/β complex is required for the development of peripheral lymphoid organs.
J. Exp. Med.
184
:
1999
8
Banks, T. A., B. T. Rouse, M. K. Kerley, P. J. Blair, V. L. Godfrey, N. A. Kuklin, D. M. Bouley, J. Thomas, S. Kanangat, M. L. Mucenski.
1995
. Lymphotoxin-α-deficient mice: effects on secondary lymphoid organ development and humoral immune responsiveness.
J. Immunol.
155
:
1685
9
Mariathasan, S., M. Matsumoto, F. Baranyay, M. H. Nahm, O. Kanagawa, D. D. Chaplin.
1995
. Absence of lymph nodes in lymphotoxin-α (LTα)-deficient mice is due to abnormal organ development, not defective lymphocyte migration.
J. Inflamm.
45
:
72
10
Matsumoto, M., S. Mariathasan, M. H. Nahm, F. Baranyay, J. J. Peschon, D. D. Chaplin.
1996
. Role of lymphotoxin and the type I TNF receptor in the formation of germinal centers.
Science
271
:
1289
11
Matsumoto, M., Y. X. Fu, H. Molina, G. Huang, J. Kim, D. A. Thomas, M. H. Nahm, D. D. Chaplin.
1997
. Distinct roles of lymphotoxin α and the type I tumor necrosis factor (TNF) receptor in the establishment of follicular dendritic cells from non-bone marrow-derived cells.
J. Exp. Med.
186
:
1997
12
Mackay, F., G. R. Majeau, P. Lawton, P. S. Hochman, J. L. Browning.
1997
. Lymphotoxin but not tumor necrosis factor functions to maintain splenic architecture and humoral responsiveness in adult mice.
Eur. J. Immunol.
27
:
2033
13
Ngo, V. N., H. Korner, M. D. Gunn, K. N. Schmidt, D. S. Riminton, M. D. Cooper, J. L. Browning, J. D. Sedgwick, J. G. Cyster.
1999
. Lymphotoxin α/β and tumor necrosis factor are required for stromal cell expression of homing chemokines in B and T cell areas of the spleen.
J. Exp. Med.
189
:
403
14
Arroyo, A. G., J. T. Yang, H. Rayburn, R. O. Hynes.
1996
. Differential requirements for α4 integrins during fetal and adult hematopoiesis.
Cell
85
:
997
15
Wagner, N., J. Lohler, E. J. Kunkel, K. Ley, E. Leung, G. Krissansen, K. Rajewsky, W. Muller.
1996
. Critical role for β7 integrins in formation of the gut-associated lymphoid tissue.
Nature
382
:
366
16
Schon, M. P., A. Arya, E. A. Murphy, C. M. Adams, U. G. Strauch, W. W. Agace, J. Marsal, J. P. Donohue, H. Her, D. R. Beier, et al
1999
. Mucosal T lymphocyte numbers are selectively reduced in integrin αE (CD103)-deficient mice.
J. Immunol.
162
:
6641
17
Kunkel, E. J., J. J. Campbell, G. Haraldsen, J. Pan, J. Boisvert, A. I. Roberts, E. C. Ebert, M. A. Vierra, S. B. Goodman, M. C. Genovese, et al
2000
. Lymphocyte CC chemokine receptor 9 and epithelial thymus-expressed chemokine (TECK) expression distinguish the small intestinal immune compartment: epithelial expression of tissue-specific chemokines as an organizing principle in regional immunity.
J. Exp. Med.
192
:
761
18
Papadakis, K. A., J. Prehn, V. Nelson, L. Cheng, S. W. Binder, P. D. Ponath, D. P. Andrew, S. R. Targan.
2000
. The role of thymus-expressed chemokine and its receptor CCR9 on lymphocytes in the regional specialization of the mucosal immune system.
J. Immunol.
165
:
5069
19
Fagarasan, S., R. Shinkura, T. Kamata, F. Nogaki, K. Ikuta, K. Tashiro, T. Honjo.
2000
. Alymphoplasia (aly)-type nuclear factor κB-inducing kinase (NIK) causes defects in secondary lymphoid tissue chemokine receptor signaling and homing of peritoneal cells to the gut-associated lymphatic tissue system.
J. Exp. Med.
191
:
1477
20
Yamada, T., T. Mitani, K. Yorita, D. Uchida, A. Matsushima, K. Iwamasa, S. Fujita, M. Matsumoto.
2000
. Abnormal immune function of hemopoietic cells from alymphoplasia (aly) mice, a natural strain with mutant NF-κ B-inducing kinase.
J. Immunol.
165
:
804
21
Miyawaki, S., Y. Nakamura, H. Suzuka, M. Koba, R. Yasumizu, S. Ikehara, Y. Shibata.
1994
. A new mutation, aly, that induces a generalized lack of lymph nodes accompanied by immunodeficiency in mice.
Eur. J. Immunol.
24
:
429
22
Yin, L., L. Wu, H. Wesche, C. D. Arthur, J. M. White, D. V. Goeddel, R. D. Schreiber.
2001
. Defective lymphotoxin-β receptor-induced NF-κB transcriptional activity in NIK-deficient mice.
Science
291
:
2162
23
Gu, H., Y. R. Zou, K. Rajewsky.
1993
. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting.
Cell
73
:
1155
24
Peschon, J. J., D. S. Torrance, K. L. Stocking, M. B. Glaccum, C. Otten, C. R. Willis, K. Charrier, P. J. Morrissey, C. B. Ware, K. M. Mohler.
1998
. TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation.
J. Immunol.
160
:
943
25
Newberry, R. D., W. F. Stenson, R. G. Lorenz.
1999
. Cyclooxygenase-2-dependent arachidonic acid metabolites are essential modulators of the intestinal immune response to dietary antigen.
Nat. Med.
5
:
900
26
Matsumoto, M., Y. X. Fu, H. Molina, D. D. Chaplin.
1997
. Lymphotoxin-α-deficient and TNF receptor-I-deficient mice define developmental and functional characteristics of germinal centers.
Immunol. Rev.
156
:
137
27
Rocha, B., D. Guy-Grand, P. Vassalli.
1995
. Extrathymic T cell differentiation.
Curr. Opin. Immunol.
7
:
235
28
Nanno, M., S. Matsumoto, R. Koike, M. Miyasaka, M. Kawaguchi, T. Masuda, S. Miyawaki, Z. Cai, T. Shimamura, Y. Fujiura, et al
1994
. Development of intestinal intraepithelial T lymphocytes is independent of Peyer’s patches and lymph nodes in aly mutant mice.
J. Immunol.
153
:
2014
29
Renno, T., M. Hahne, J. Tschopp, H. R. MacDonald.
1996
. Peripheral T cells undergoing superantigen-induced apoptosis in vivo express B220 and upregulate Fas and Fas ligand.
J. Exp. Med.
183
:
431
30
Rolink, A., E. ten Boekel, F. Melchers, D. T. Fearon, I. Krop, J. Andersson.
1996
. A subpopulation of B220+ cells in murine bone marrow does not express CD19 and contains natural killer cell progenitors.
J. Exp. Med.
183
:
187
31
Krop, I., A. R. de Fougerolles, R. R. Hardy, M. Allison, M. S. Schlissel, D. T. Fearon.
1996
. Self-renewal of B-1 lymphocytes is dependent on CD19.
Eur. J. Immunol.
26
:
238
32
Marino, M. W., A. Dunn, D. Grail, M. Inglese, Y. Noguchi, E. Richards, A. Jungbluth, H. Wada, M. Moore, B. Williamson, et al
1997
. Characterization of tumor necrosis factor-deficient mice.
Proc. Natl. Acad. Sci. USA
94
:
8093
33
Ettinger, R., J. L. Browning, S. A. Michie, W. van Ewijk, H. O. McDevitt.
1996
. Disrupted splenic architecture, but normal lymph node development in mice expressing a soluble lymphotoxin-β receptor-IgG1 fusion protein.
Proc. Natl. Acad. Sci. USA
93
:
13102
34
Fu, Y. X., G. Huang, Y. Wang, D. D. Chaplin.
1998
. B lymphocytes induce the formation of follicular dendritic cell clusters in a lymphotoxin α-dependent fashion.
J. Exp. Med.
187
:
1009
35
Ware, C. F., P. D. Crowe, M. H. Grayson, M. J. Androlewicz, J. L. Browning.
1992
. Expression of surface lymphotoxin and tumor necrosis factor on activated T, B, and natural killer cells.
J. Immunol.
149
:
3881
36
Kroese, F. G., E. C. Butcher, A. M. Stall, P. A. Lalor, S. Adams, L. A. Herzenberg.
1989
. Many of the IgA producing plasma cells in murine gut are derived from self-replenishing precursors in the peritoneal cavity.
Int. Immunol.
1
:
75
37
Fagarasan, S., T. Honjo.
2000
. T-Independent immune response: new aspects of B cell biology.
Science
290
:
89
38
Macpherson, A. J., D. Gatto, E. Sainsbury, G. R. Harriman, H. Hengartner, R. M. Zinkernagel.
2000
. A primitive T cell-independent mechanism of intestinal mucosal IgA responses to commensal bacteria.
Science
288
:
2222
39
Fagarasan, S., K. Kinoshita, M. Muramatsu, K. Ikuta, T. Honjo.
2001
. In situ class switching and differentiation to IgA-producing cells in the gut lamina propria.
Nature
413
:
639
40
Matsumoto, M., S. F. Lo, C. J. Carruthers, J. Min, S. Mariathasan, G. Huang, D. R. Plas, S. M. Martin, R. S. Geha, M. H. Nahm, D. D. Chaplin.
1996
. Affinity maturation without germinal centres in lymphotoxin-α- deficient mice.
Nature
382
:
462
41
Tarlinton, D..
1998
. Germinal centers: form and function.
Curr. Opin. Immunol.
10
:
245
42
Ansel, K. M., V. N. Ngo, P. L. Hyman, S. A. Luther, R. Forster, J. D. Sedgwick, J. L. Browning, M. Lipp, J. G. Cyster.
2000
. A chemokine-driven positive feedback loop organizes lymphoid follicles.
Nature
406
:
309
43
Newberry, R. D., J. S. McDonough, W. F. Stenson, R. G. Lorenz.
2001
. Spontaneous and continuous cyclooxygenase-2-dependent prostaglandin e(2) production by stromal cells in the murine small intestine lamina propria: directing the tone of the intestinal immune response.
J. Immunol.
166
:
4465
44
Forster, R., A. E. Mattis, E. Kremmer, E. Wolf, G. Brem, M. Lipp.
1996
. A putative chemokine receptor, BLR1, directs B cell migration to defined lymphoid organs and specific anatomic compartments of the spleen.
Cell
87
:
1037
45
Macpherson, A. J., A. Lamarre, K. McCoy, G. R. Harriman, B. Odermatt, G. Dougan, H. Hengartner, R. M. Zinkernagel.
2001
. IgA production without μ or δ chain expression in developing B cells.
Nat. Immunol.
2
:
625