The Hunger games- The quest for survival of B cells in the germinal center

The journey of constantly going back and forth, from the dark side to the light side, is not one that’s reserved just for the strongest Jedis of the galaxy but one that’s also shared by the maturing B cells in our immune system. But unlike the former, it’s not the galaxy testing their fate, but the dark and the light zones in the germinal centers situated in the secondary lymphoid organs. Initially, it was thought that endocytosis of the antigen by a cognate B cell and its presentation to a T helper follicular cell via the B cell receptor (BCR), was more than enough for it to get ‘positively selected’ i.e., getting the positive response for a B cell to become an antibody-producing plasma cell. However, that might be just a small chapter of a larger book.

Recently scientists from Rockefeller University came up with a unique tracker molecule to study this molecular handshake better and went on to prove that the former knowledge regarding positive selection might not be the complete depiction of BCR functioning.

For starters let’s talk about this novel tracker they made to study how B cells engage antigens in vivo. This tracker molecule, what they call NP-Eα is made up of several complex ingredients, but the 3 key ones are:

1.    NP or 4-hydroxy-3- nitrophenylacetyl, a hapten molecule that can’t be immunogenic on its own until it’s conjugated with a peptide 

2.    Eα, the peptide bound to NP

3.    AF647, a fluorescent molecule also conjugated with NP

How will it function as a tracker? Let me give you a simple explanation.

First, the researchers cultured B cells which were directed against the NP molecule, so when B cells come across it, they endocytose the whole package (NP-Eα). The moment they are inside the belly of the B cell, the fluorescent molecule starts making the B cell glow, which is one way of tracking these cells. Additionally, the B cell also presents the Eα peptide on its surface MHC, which is detected by an Eα specific antibody. 

Strangely, when they used this tracker on B cells present both in the dark zone and the light zone, they found that the majority of cells in the dark zone had a hard time gobbling up and presenting the NP-Eα tracker.

By using an affinity-determining technique called bio-layer interferometry, they concluded that BCRs of the dark zone B cells end up being non-functional and usually their surface expression goes down as well.

B cells that are positively selected by follicular helper T cells in the light zone usually display upregulated expression of a gene called Myc, which usually acts as a green signal for the cells to start multiplying. Talking about green signals, the scientists decided to tag this gene with a green fluorescent protein to study its expression during B cell signaling. They found that light zone B cells which tightly bound NP-Eα, showed a higher amount of green fluorescence, indicating that these cells had higher Myc expression. This meant that those specific B cells were more likely to get positively selected and undergo proliferation. 

Now as usual it was time to sniff out the pathways that were responsible for all these survival dramas. B cells with receptors that had a higher affinity for antigens had amped up the expression of genes of pathways that are usually expressed in active immune cells (like mTOR, NF-𝛋B). Adding onto the arsenal, these cells also pumped up the gene expression of growth-inducing factors (such as Il1r2, Sox2, and Sox3). These chosen ones were also most likely to enter the cell cycle and demonstrate enhanced metabolism, all of which are hallmarks of positive selection.

However, what about the not chosen ones? Well, the B cells with lower affinity BCRs which still expressed Myc got to join the memory squad and become memory B cells.

To explore the interaction between antigen binding and BCR signaling, they tried normalizing the delivery of antigens onto the receptors of the B cells. This made sure that all the cells have the same number of antigens on their surface, regardless of the affinity of the receptors. Even then, B cells having BCRs with higher affinity and better BCR signaling were getting positively selected at a higher rate.

Do the immune gods allow the B cells to live only if they get positively selected? To answer this, they took the help of activated caspase 3, a member of a family of proteins that facilitate apoptosis. Rolling with the hypothesis that the higher the love for antigens by BCRs, the lesser the chances of B cell suicide; light and dark zone B cells that bound antigens reeked less of activated caspase 3 than those with lesser or no affinity. Additionally, antigen-binding B cells, even with lower or nonexistent levels of Myc, still clung to life more than their lesser affinity-displaying counterparts. This hints that there is indeed some wiggle room for survival between antigen binding and being positively selected. 

But hold up! Don’t let this fool you into believing that our B cell is out of the woods yet.

Remember that scene in the movies, where some guy wakes up in this room and to his horror finds the walls closing in on him? We all know what happens if he does not think fast enough to get out. It’s the same with B cells here. Interactions on this level happen in a matter of microseconds and the B cells got to think fast. If they do not get the help of a T cell and join hands with it soon after antigen binding, and with the time window narrowing down (the walls), they’ll get turned into a disgusting paste (undergo apoptosis or programmed cell death). 

To bolster the fact that BCR signaling would enhance B cell survival, the scientists targeted the inhibition of a kinase of the B cell signaling pathway called Bruton’s tyrosine kinase. As speculated, even slight inhibition was enough to generate an increased self-destructive signal in light zone B cells. This further solidifies the hypothesis that BCR signaling gives that extra push for survival even when the B cells haven’t been positively selected yet.

The synergy between a B cell receptor and a T helper follicular cell is sort of like a Dutch and Dillon relationship and with the advent of modern tracking and detection techniques, deciphering this synergy is becoming more and more lucid. This knowledge will not only help us make better therapeutics but will also reveal secrets about how virgin B cells prey on and trap their antigens and feed on them, forging these cells into the ruthless archers of the immune system, who give most of the pathogens a run for their lives.

 Source:

Chen ST, Oliveira TY, Gazumyan A, Cipolla M, Nussenzweig MC. B cell receptor signaling in germinal centers prolongs survival and primes B cells for selection. Immunity. 2023 Mar 14;56(3):547-561.e7. doi: 10.1016/j.immuni.2023.02.003.

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