DOI: 10.1126/science.281.5373.96. Visualization of Specific B and T Lymphocyte Interactions in the Lymph Node

Visualization of Specific B and T Lymphocyte Interactions in the Lymph Node

10.1126/science.281.5373.96

Crossref journal-article

American Association for the Advancement of Science (AAAS)

Science (221)

Abstract

Early events in the humoral immune response were visualized in lymph nodes by simultaneous tracking of antigen-specific CD4 T and B cells after immunization. The T cells were initially activated in the T cell areas when the B cells were still randomly dispersed in the B cell–rich follicles. Both populations then migrated to the edges of the follicles and interacted there, resulting in CD154-dependent B cell proliferation and germinal center formation. These results provide visual documentation of cognate T-B cell interactions and localize them to the follicular border.

Bibliography

Garside, P., Ingulli, E., Merica, R. R., Johnson, J. G., Noelle, R. J., & Jenkins, M. K. (1998). Visualization of Specific B and T Lymphocyte Interactions in the Lymph Node. Science, 281(5373), 96â99.

Authors 6

Paul Garside (first)
Elizabeth Ingulli (additional)
Rebecca R. Merica (additional)
Julia G. Johnson (additional)
Randolph J. Noelle (additional)
Marc K. Jenkins (additional)

References 36 Referenced 634

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The DO11.10 TCR has been reported to possess a weak alloreactivity for the I-A b molecule expressed in C57BL/6 mice ( 25 ) raising the possibility that the DO11.10 T cells would be deleted during development in (BALB/c × C57BL/6)F 1 mice ( 26 ). Indeed DO11.10 (BALB/c × C57BL/6)F 1 mice consistently yielded a lower number of CD4 + KJ1-26 + lymph node T cells than their BALB/c counterparts probably as a consequence of a certain degree of thymic deletion. The peripheral KJ1-26 + cells in DO11.10 (BALB/c × C57BL/6)F 1 mice also contained more CD4 − CD8 − T cells than DO11.10 BALB/c mice. However most CD4 + KJ1-26 + cells in DO11.10 (BALB/c × C57BL/6)F 1 mice possessed the surface phenotype of naive T cells (CD45RB high ) and were clearly capable of undergoing extensive clonal expansion in vivo after adoptive transfer and immunization (Fig. 1).
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Before adoptive transfer spleen and lymph node cells from DO11.10 (BALB/c × C57BL/6)F 1 donor mice were depleted of CD8 T and B cells and a sample was stained with phycoerythrin (PE)-labeled anti-CD4 mAb (Pharmingen) and biotinylated KJ1-26 mAb followed by fluorescein isothiocyanate (FITC)-labeled streptavidin (Caltag) as described in ( 14 ). Similarly a sample of spleen and lymph node cells from MD4 (BALB/c × C57BL/6)F 1 mice was stained with PE-labeled anti-B220 mAb (Pharmingen) in combination with biotinylated HEL or biotinylated anti-IgM a mAb(Pharmingen) followed by FITC-labeled streptavidin. Stained cells (20 000 events per sample) were then analyzed on a flow cytometer and the percentages of DO11.10 CD4 T cells and MD4 B cells were determined. Cell suspensions containing 1 to 2.5 × 10 6 DO11.10 CD4 T cells and 2.5 to 5 × 10 6 MD4 B cells were then injected intravenously into (BALB/c-Igh b × C57BL/6)F 1 recipients.
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In a representative flow cytometry experiment CD4 + KJ1-26 + DO11.10 T cells accounted for 0.5% of viable lymph node cells before immunization of (BALB/c-Igh b × C57BL/6)F 1 recipients of DO11.10 T cells and MD4 B cells. This percentage increased to 4.1% 5 days after immunization with cOVA-HEL ( 11 ). We detected no CD4 + KJ1-26 + events in untransferred (BALB/c-Igh b × C57BL/6)F 1 before or after immunization which indicates that only transferred cells were detected. B220 + HEL-binding or IgM a+ MD4 B cells accounted for 0.5 or 1.5% of viable lymph node cells respectively before recipients of both transgenic populations were immunized. The disparity between these values is related to the fact that the anti-IgM a mAb detects slightly more background events in untransferred recipients (0.18%) than does HEL (0.06%). In addition a small percentage (about 10%) of B cells in the MD4 transgenic mice express IgM a but do not bind HEL probably because of expression of an endogenous light chain. The percentages of B220 + HEL-binding and IgM a+ MD4 B cells increased to 8.0 and 8.8% respectively 5 days after immunization with cOVA-HEL ( 11 ).
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Draining lymph nodes were harvested and frozen in liquid nitrogen in O.C.T. embedding medium (Miles). Tissue sections (10 μm) were cut on a Cryostat microtome fixed in acetone and blocked with goat serum. Sections were stained sequentially with biotinylated KJ1-26 anti-IgM a or peanut agglutinin; avidin-biotin complex–labeled alkaline phosphatase; and 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium substrate followed by biotinylated anti-B220 anti-Thy 1.2 or KJ1-26; avidin-biotin complex–labeled peroxidase; and 3 3′-diaminobenzidine substrate (Vector). Development of the first avidin-biotin complex–labeled enzyme destroyed residual avidin binding sites on the primary antibodies thereby preventing artifactual binding of the second avidin-biotin complex–labeled enzyme. The weak background anti-IgM a staining mentioned in ( 10 ) was not detected on tissue sections and thus has no bearing on the immunohistology experiments.
We thank J. Walter and M. Michels for technical assistance and T. Behrens D. Mueller M. Mescher J. Cyster and K. Hogquist for helpful discussions. Supported by grants from NIH (AI27998 AI35296 and AI39614 to M.K.J.; K08-AI01503 and HD33692 to E.I.; and AI07421 to R.R.M.) the University Children's Foundation (E.I.) the Vikings Children's Fund (E.I.) and the Wellcome Trust (P.G.).

Dates

Type	When
Created	23 years, 1 month ago (July 27, 2002, 5:35 a.m.)
Deposited	1 year, 7 months ago (Jan. 12, 2024, 10:20 p.m.)
Indexed	39 minutes ago (Sept. 2, 2025, 1:09 p.m.)
Issued	27 years, 1 month ago (July 3, 1998)
Published	27 years, 1 month ago (July 3, 1998)
Published Print	27 years, 1 month ago (July 3, 1998)

Funders 0

None

BibTeX

@article{Garside_1998, title={Visualization of Specific B and T Lymphocyte Interactions in the Lymph Node}, volume={281}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.281.5373.96}, DOI={10.1126/science.281.5373.96}, number={5373}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Garside, Paul and Ingulli, Elizabeth and Merica, Rebecca R. and Johnson, Julia G. and Noelle, Randolph J. and Jenkins, Marc K.}, year={1998}, month=jul, pages={96–99} }

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      "unstructured": "Draining lymph nodes were harvested and frozen in liquid nitrogen in O.C.T. embedding medium (Miles). Tissue sections (10 \u03bcm) were cut on a Cryostat microtome fixed in acetone and blocked with goat serum. Sections were stained sequentially with biotinylated KJ1-26 anti-IgM a or peanut agglutinin; avidin-biotin complex\u2013labeled alkaline phosphatase; and 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium substrate followed by biotinylated anti-B220 anti-Thy 1.2 or KJ1-26; avidin-biotin complex\u2013labeled peroxidase; and 3 3\u2032-diaminobenzidine substrate (Vector). Development of the first avidin-biotin complex\u2013labeled enzyme destroyed residual avidin binding sites on the primary antibodies thereby preventing artifactual binding of the second avidin-biotin complex\u2013labeled enzyme. The weak background anti-IgM a staining mentioned in ( 10 ) was not detected on tissue sections and thus has no bearing on the immunohistology experiments."
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