It’s difficult enough to estimate the total number of B cells in the body, let alone the number of B cells specific for any given antigen, i.e., antigen-specific B cell frequency, aka precursor frequency. Additional obstacles include the fact that the pool of cells being analyzed include
- B cells at various stages of development, especially in the bone marrow.
- Not just conventional adaptive but also innate B cell subsets.
- Not just naive (antigen-inexperienced) but also memory B cells.
Though they all express antigen-specific receptors, the(BCR), which when secreted is called the antibody, B cells aren’t a monolithic entity. Rather, the classical B (as also T) cell subsets with somatically recombined antigen receptors ( ) belong to the adaptive immune system, which is characterized by remarkable diversity. Such classical or conventional B cells are B-2 or Follicular B cells. They constitute the bulk of B cells in the lymph nodes, spleen, bone marrow and in circulation.
However, other B cell subsets such asand (MZ B) also secrete antibodies, mostly IgM, some IgG3, usually also termed , but they tend to not somatically recombine their antigen receptors, i.e., they retain germline receptors, to not circulate, to not convert into memory cells, and to perform their effector functions without the help of T cells.
Thus, frequency of a given antigen-specific B cell is obviously very different between the ‘innate’ and ‘adaptive’ subsets of B cells. Greater the receptor diversity, lower the frequency of any specificity, simply for a practical reason, there just isn’t enough space in the body to house expanded numbers of each and every antigenic specificity, given that the total B cell pool in the body is estimated to be 1 to 2 X 10^11 (, ; also see below from ).
Frequency of individual B cell specificities in humans are also exceedingly difficult to estimate simply because of difficulty of access to source material. Blood, an obvious choice to sample, is estimated to harbor only ~2% of the total B cell pool (4, also see above from), with much higher numbers present in lymph nodes (~28%), spleen (~23%) and red bone marrow (~17%) ( , ).
Add the additional complexity that humans are estimated to have ~600 to 750 lymph nodes (, 5, , 7) and difficulty of enumerating antigen-specific B cell frequency becomes more than obvious. Further, even a mere ~2% of total body B cell numbers still amounts to ~ 2 to 4 X 10^9 B cells in blood. Reasonably accurate estimates crucially turn on how much needs to be minimally sampled and method(s) used to sequence and quantitate BCRs (see below from , emphasis mine).
The consequences of insufficient biological sampling have been investigated previously by Warren and colleagues : they showed that distinct 20 ml blood samples from the same individual captured only a portion of the TCR peripheral blood repertoire (biological undersampling). Furthermore, technological undersampling has been shown to compromise the detection of ‘public’ clones (clones shared across individuals), which are a common target in immune repertoire studies [27,28]. In fact, several studies indicated that there was a positive correlation between sequencing depth and the number of public clones detected [13,29,30]. Thus, the biological conclusiveness of a study benefits from the implementation of biological replicates (test for biological undersampling [26,31,32]) (Figure 1A) and technical replicates (test for technological undersampling [33–36]) (Figure 1A), which may be performed once for each cell population analyzed. It is important to note that biological undersampling can only be meaningfully addressed if sufficient technological sampling has been established . Furthermore, species accumulation and rarefaction analyses may be performed to quantify the extent of (under)sampling [29,33,35,37]
Thus, there is likely to be substantial margin of error in estimates based on blood B cells. As well, antigen-specific B cell frequency is unsurprisingly extremely dynamic, changing with age and unpredictably varying exposure to antigens over time.
For what it’s worth, a commonly bandied about estimate of antigen-specific B cell frequency in the circulating, naive repertoire is one in 10^5 to 10^6. Extrapolating from blood and totally disregarding the contribution of memory and innate B cells to the estimated total B cell number of 1 to 2 X 10^11, that means each B cell specificity could range from 1 to 2 X 10^5 to 10^6 as also a total of 1 to 2 X 10^5 to 10^6 different individual B cell specificities or capacity to bind that many different antigens. Obviously, since memory and innate B cells are indeed part of the total B cell number, actual numbers of naive B cells specific for a particular antigen are likely markedly lower.
Taking such estimates at face value, is that sufficient frequency and diversity, given that over the course of a lifetime an anticipatory defense system such as the B cell has to contend with a potential universe of antigens that is likely orders of magnitude higher? Important at this point to recall that in B cells, the naive or antigen-inexperienced repertoire diversity is bolstered, maybe even more than amply so, by three other cardinal features, namely, clonal proliferation,(which some refer to as polyreactivity) and (SHM), with that last, SHM, being a unique property of conventional B cells.
- Clonal proliferation is the capacity of an activated B or T cell to quickly proliferate (some estimates even suggest dividing every 16 to 20 hours). Progeny of each such single B or T cell are their clones having the exact same antigen receptor (BCR or TCR). Thus, at the height of an immune response, say during the acute phase of an infection, frequencies of individual B or T cells could increase to as many as 1 in 10^3 to 1 in 10^4, i.e., a 100 to 1000-fold increase, at least locally. Clonal proliferation helps bolster the sufficiency of antigen-specific B cell frequency.
- Cross-reactivity (aka polyreactivity) is the capacity for a given BCR (and antibody) to bind more than one antigen. Often but not always, this relates to structural similarity between different antigens. After all, though the antigenic universe is vast, biology still dictates its sequence and structural constraints. Cross-reactivity helps mitigate the potential insufficiency of antigen-specific B cell diversity.
- SHM is the process by which conventional B cells that bound their specific antigen and presented pieces of it in the MHC ( ) to cognate T cells receive T cell help that drives mutations within the V gene segment of the BCR. This creates BCR (and antibody) variants additional to those generated during primary B cell development by somatic recombination. Thus, though monozygotic (identical) twins have nearly identical primary antibody repertoire ( ), meaning it is largely the product of genetic factors, antigenic experience over time, which can be highly individual and variable, leads to a secondary repertoire that can vary substantially between individuals, even identical twins. SHM helps enhance antigen-specific B cell diversity.
1. Morbach, H., et al. “Reference values for B cell subpopulations from infancy to adulthood.” Clinical & Experimental Immunology 162.2 (2010): 271-279.
2. Greiff, Victor, et al. “Bioinformatic and statistical analysis of adaptive immune repertoires.” Trends in immunology 36.11 (2015): 738-749.
3. Apostoaei, A. Iulian, and John R. Trabalka. “Review, Synthesis, and Application of Information on the Human Lymphatic System to Radiation Dosimetry for Chronic Lymphocytic Leukemia.” Inc., Tennessee (2012).
4. Georgiou, George, et al. “The promise and challenge of high-throughput sequencing of the antibody repertoire.” Nature biotechnology 32.2 (2014): 158-168.
5. Trepel, F. “Number and distribution of lymphocytes in man. A critical analysis.” Klinische Wochenschrift 52.11 (1974): 511-515.
6. Valentin, Jack. “Basic anatomical and physiological data for use in radiological protection: reference values: ICRP Publication 89.” Annals of the ICRP 32.3 (2002): 1-277.
7. Agur, Anne MR, and Arthur F. Dalley. Grant’s atlas of anatomy. Lippincott Williams & Wilkins, 2009.
8. Greiff, Victor, et al. “Bioinformatic and statistical analysis of adaptive immune repertoires.” Trends in immunology 36.11 (2015): 738-749.
9. Glanville, Jacob, et al. “Naive antibody gene-segment frequencies are heritable and unaltered by chronic lymphocyte ablation.” Proceedings of the National Academy of Sciences 108.50 (2011): 20066-20071.