SCID Mice – Identifying the Main Source of Maternal IgA Production

This slide shows you the data that already I showed you. This is a number of IgA producing plasma cells in the mammary gland, during pregnancy, during lactation, after weaning or before pregnancy Again, as I told you, you can detect a very high number of IgA producing plasma cells in the mammary gland two weeks after parturition. But when we did this kind of experiment, we also noticed that the number of IgA producing plasma cells in the intestines is somehow reduced during lactation like this.

The data is like, the IgA producing plasma cells that should be in the intestines are somehow recruited from the mammary gland and then once recognitions of probably CCL28 they change directions, then they go to the mammary gland to produce IgA antibodies in milk, not in the intestine. So we have a hypothesis that the intestine is important tissues for producing IgA producing antibodies in the mammary gland, like this. But in contrast, we have known that there is one draining lymph node which are present in the mammary gland around the mammary gland alveoli, named “inguinal lymph node”. And there are still hypotheses that this inguinal lymph node is essential for the recruitment of IgA producing plasma cells in the mammary gland.

So we want to know which place, Peyer’s Patches, or gut immune system or inguinal lymph node is essential for the production of IgA antibodies in milk. Our experimental design is to distinguish or identify the important place to produce maternal IgA antibodies, Payer’s Patches, inguinal lymph node, or the others? So we did two experiments, Payer’s Patches, inguinal lymph nodes. To understand the importance of this kind of secondary lymph node tissues in inducing antibody productions We frequently use this special mice model named SCID. SCID is immunodeficient mice which lack of T-cells and B-cells. So we prepared pregnant SCID mice, and then we got SCID babies and simultaneously we also prepared wild-type mice after parturition.

And then we obtained the cells which are isolated from Peyer’s Patches well we also obtained cells which are isolated from inguinal lymph nodes. And then we individually transfer Peyer’s patches derived cells or inguinal lymph nodes derived cells into the SCID mice in order to see the migration process into the mammary gland.

So if you detect IgA producing plasma cells in the mammary gland of this SCID mice when you transfer, the Peyer’s patches derived wild-type cells that means Peyer’s patches are important places for IgA productions in the mammary gland So we did experiments, we prepared SCID mice, and then we isolated wild-type derived Payer’s patches cells or inguinal lymph node cells, then transfers into the SCID mice one week before harvest tissues. And then two weeks after parturition, we analyze the mammary gland. This graph shows you the number of IgA producing plasma cells in the mammary gland after adaptive transfer with either Peyer’s patches or inguinal lymph node cells.

Most left one is no adaptive transfer The SCID mice again do not develop any IgA antibodies. That’s why the number of IgA producing plasma cells is undetectable. And the right graph shows you the milk IgA, so no adaptive transfers are no IgA milk protein. But when you transfer with Peyer’s Patches derived cells, not inguinal lymph node cells, into the SCID mice you see a very high number of IgA-producing plasma cells in the mammary gland. You can detect a very large amount of IgA in milk. So meaning that Peyer’s patches in the intestinal tracts, were the inductive site of the gut immune system has a very important key role for the production of IgA producing plasma cells.

while secreting IgA, milk IgA antibodies. We also confirmed the importance of Peyer’s patches by generating Peyer’spatches null mice. The developmental process of Peyer’s Patches has been well known. So if you treated mice with anti-IL7Rα receptor antibodies, the baby through the mothers, pregnant mothers, the baby lacked the Peyer’s patches. Then these Peyer’s patches null mice are mated and then two weeks after parturition, we analyze the mammary gland tissues. And also we prepared surgically removed inguinal lymph nodes null mice. So before mating, we took out the inguinal lymph nodes surgically, and then we prepared the mice. Then analyzed the mammary gland tissues two weeks after parturition.

And this is the data, two weeks after parturition in the mammary gland The intact mice, that means they have Peyer’s patches, they have inguinal lymph nodes. Of course, they have a very high number of IgA-producing plasma cells in the mammary gland. However, in the Peyer’s patches null mice the number of IgA producing plasma cells in the mammary gland is reduced significantly. In contrast, in the inguinal lymph node null mice, the number of IgA producing plasma cells in the mammary gland was almost comparable to the intact mice.

And consistent with the number by IgA producing plasma cells the amount of milk IgA is reduced in the Peyer’s patches null mice suggesting that the Peyer’s patches has very important functions to produce maternal IgA antibodies.