The unit ‘milliliter’ (mL) has little utility outside of laboratory. Perhaps it would be tremendously hard to quench thirst with one thousandth of a liter of water. White blood cells, the subject of my research, has a concentration of 4,000 – 11,000 of individual cells per mL of blood1.
With a vial of enriched peripheral blood cells2, the cell density hovered around 10,000,000 cells/mL. Perhaps one might think this would be enough to interrogate the diversity of immune cell populations, and one is not wrong. But that is not the case with certain cell populations and certain questions one would have in mind.
And this was the case with the work I carried for my doctoral dissertation.
Rarity Class S
In my public defense seminar, I made it clear that the odds were stacked against me. The primary subject of my research was antigen-specific memory B cells in human, a subset of white blood cells, which present in peripheral blood at a minute frequency. A manual counting with hemocytometer at 200 cells per minute allows for a person to count 50,000 cells in a little over 4 hours. If this person was lucky, s/he could have found 1 antigen-specific memory B cells between his/her breakfast and lunch. To obtain ~100 antigen-specific memory B cells would have taken more than 2 weeks with no breaks.
Without sophisticated technology, it was impossible to interrogate such rare population of immune cells. Indeed, it is very astounding that despite their extreme rarity, we are all not dead from infections.
Memory B cell is a subset of the B cell population. B cells respond to infection by producing antibodies that target the invading pathogens and clear them from our body. Total B cells themselves are not very high in frequency either, roughly present at 10 – 20% of the peripheral blood mononuclear cells. Antigen-specific memory B cells present in about 1 for every 25,000 – 50,000 peripheral blood mononuclear cells.
Technology allows for rapid progressions of discoveries in the field, and allowed me to interrogate probably close to 500 vials of enriched white blood cells. For a total of 5 billion cells, I probably had queried 100,000 – 500,000 of individual antigen-specific memory B cells within 36 months3 pursuing my dissertation projects.
In the game of chance, that is an awful bet. But immunology is much more complicated than that and this complexity allows us to… simply be alive.
Rarity and Utility
Combined rarity and utility fetch for a high price. Gold satisfies both attributes; its use extends from cosmetics to electronics. Sand, satisfies only the latter; but when carefully molded and manipulated, silicon also comes with an exorbitant price tag, made possible with technologies borderline wizardry.
B cells and its product, antibodies, exist between the two spectra of being gold and also being sand at the same time. Polyclonal preparation of antibodies is not a mythical art, and procuring them only takes several weeks. Inject several laboratory mice today with antigens and inject it again come two to three weeks afterwards, followed by another waiting period of several weeks, the mice would yield bountiful and presumably high quality antibodies4. Acquiring the polyclonal antibody mixture from mice requires minimal efforts nowadays.
However, this mixture is a finite resource. Use them frequently, you will run out of it. But worry not, because since 19755, the field has figured out approaches for immortalizing B cells that produce these antibodies. The first iteration of this approach is probably a little hacky, it involves coaxing antibody-producing B cells to physically fuse with myeloma cell partner6. The efficiency for generating these immortalized cells was not great, yet this technology revolutionized the field of biomedical research.
Antibodies are incredibly versatile. Hunting down invaders is their daytime job, but they are no slouch when moonlighting. Humanity has exploited antibodies for variety of applications. We use them as treatment for certain malignancies, for diagnosis of certain diseases (infectious or not), and a venerable tool in biomedical research. They are particularly useful as capturing and detection agents.
A single hybridoma (hybrid of antibody-producing B cell and myeloma fusion partner) produces antibody with a single specificity. Therefore, the antibody its produced is called ‘monoclonal antibody’, that is, of one clone. Hybridoma comes with two major advantages: (a) it is immortal, if you properly keep it in culture or freeze it away for later exploits, and (b) it continuously produce antibodies, so one could harvest the monoclonals over and over again.
It cannot be overstated how important this notion of ‘monoclonal antibody’ is. With polyclonal antibodies, you cannot be sure of the composition that makes up their constituents. It could be a mix of 70% high quality antibodies and the rest are of questionable characters. With monoclonal antibody, all doubts are removed. It does one job, recognizes its target in one way, and that’s it. Knowing precisely what they do enables for high fidelity dissections into many biological phenomena.
Close to half a century since the method was described, the field has many tricks to acquire for monoclonal antibodies. Searching for B cells with defined specificities became less of a gold mining but more of a refining sand to generate silicon. The efficiency soared higher from mere sub-10% with the 1975 method to close to 80% with contemporary strategies. Single B cell antibody cloning, which took off in the second half 2000s decade, is how the field prefers to acquire monoclonals.
Hybridoma allowed for mining monoclonals. Single B cell antibody cloning gives us ways to find the elusive ones.
To Many Billions More
A mouse spleen can yield 50,000,000 – 90,000,000 total cells. A spleen is similar to a big regional airport hub: everything routes through there. Every single blood cells transit through it, and some take residence within the spleen for a while. Spleens from roughly 20 mice already equals to 1 billion of cells.
I completed my doctoral dissertation without taking advantage of the advances in the single B cell antibody cloning methodologies. Though, antibody cloning requires a marriage between immunology, genetic engineering, and bioinformatics to be able to make sense of it all7. But for my postdoctoral work, I am eager to be at the bleeding edge of this technology. I am eager to hunt for the elusive monoclonals, and there are many of them out there awaiting characterization.
or PBMCs as we call them, short for peripheral blood mononuclear cells. PBMCs are collected from human by venipuncture of an arm vein. 4,000 – 11,000 cells/mL of cells is very dilute. To concentrate, it involves spinning the cells through a layer of density gradient carefully prepared, and the cells can be found later sitting at a nice interface and much more amenable for future interrogation. ↩
I enrolled in August 2017 and graduated in 2022 for a total 5 years, so this ‘36 months’ figure does not add up. Note that for most biomedical programs, the first year of PhD is for taking advanced classes and doing lab rotations. I joined a lab in April 2018. Unfortunately, the lab moved to another institution in 2019. I started in a new lab with a different set of projects mid 2019. ↩
Two-shot immunization schedule like this is called prime - boost, i.e., the first shot is for priming and the second shoot is for boosting. It is well established (and yet still being actively studied) concept where the first shot is crucial for generating B cells that produce antibodies and also B cells that become memory (which does not produce antibodies). The boosting phase would then activate the memory cells, which in turn generating antibodies with higher quality. ↩
This invention won the Nobel Prize in 1984. Georges J.F. Köhler and César Milstein both jointly received the prize for their work describing the methodology for generating hybridoma cell line. ↩
If you would like to, here’s a review on the generation of hybridoma cell line: “Hybridoma technology; advancements, clinical significance, and future aspects” by Sanchita Mitra & Pushpa Chaudhary Tomar (PubMed). ↩
(i) Immunology is for when you want to make sure you have the right samples and ways to tag your B cells with desired specificity, (ii) genetic engineering is when you start manipulating the genetic information that encodes for the antibody isolated from single B cells of interest, and (iii) bioinformatics is when you have to parse through mountains of genetic information post-cloning. The truth is, it is quite a lot to ask a person to be good in all three of them at the same time. ↩