On plasma cell longevity or brevity

Abstract

The seemingly long half-life of antibody-producing plasma cells demonstrated by antigen-binding (ELISA type of) assays as compared with the short-livedness of neutralizing and protective antibody-producing plasma cells is explained here by the heterogeneity and multiple crossreactive antibodies detected by ELISA-type assays. While DNP-specific B cell frequencies are about 10^-2 that of virus, serotype-specific B cell frequencies are about 10^-5–10^-6. Therefore, the seemingly long-lived multiple low-affinity crossreactive antibody-producing plasma cells represent a collective of little if any biological or evolutionary relevance. The plasma cells producing high-affinity protective, neutralizing antibodies (>10⁹ M^-1) in mice are short-lived and therefore continued antibody production is dependent on antigen exposure from within (immune complexes and persistence of infections) or from without by epidemiologically circulating infectious agents, or by revaccinations.

Keywords::

• affinity
• antigen-driven
• antigenic site
• binding assay
• crossreactivity
• memory
• neutralization
• protection

The discussion about longevity of antibody-producing plasma cells is intimately linked to the questions of specificity and memory. Specificity is defined by the method used, mostly antigen-binding assays. The medically important read-out should, however, use protection by neutralizing antibodies. Memory has been defined in all textbooks as once exposed to an antigen the immune system reacts to a second exposure to the same antigen more quickly and more extensively [1–3]. Our laboratory has been attempting to evaluate the obvious alternative explanation of memory and of plasma cell half-life by assessing their antigen dependence [2,4,5]. These experiments indicate that classical studies and measurements illustrating long-lived plasma cells and memory have ignored their hidden antigen dependence by choosing read-outs that avoid the crucial question of the antigen kinetics involved [3,6,7]. The following paragraphs summarize some of the experimental findings with acutely lethal (ALV) (i.e., within 5–10 days) cytopathic virus infections (e.g., influenza, rabies or polio) [1,8–10] or with alternatively chronically persisting (CPV) noncytopathic and therefore often harmless virus infections such as HBV, HCV or HIV in man or lymphocytic choriomeningitis virus in mice [11–14]. In the latter infections, absence of an immune response avoids lethal immunopathology; such absence of a response results usually from transplacental and/or neonatal infections.

Serotypically defined protective sites on ALV usually represent only by neutralizing antibodies accessible site on an intact virus particle or the infected cell surface. The dense packaging of the relevant glycoproteins other than neutralizing antibodies prevents to bind to less peripheral or internal determinants of the intact virus. This was shown by mutually reciprocal competitive inhibition assays demonstrating complete cross competition of about 50 independent neutralizing antibodies but also of complex neutralizing sera from cows, humans or mice. Therefore, on an intact virus particle, serotype-specific neutralizing antibodies cover a small and unique site [10]. While this has not been formally shown for many serotypically defined related viruses, it is obvious for influenza viruses.

Early (days 2–4) protective, neutralizing IgM and IgG responses by days 4–7 are seen with ALV [4] but not with CPV viruses [13]. The latter finding may represent a hole in the host’s B cell repertoire for yet poorly understood genetic or other reasons.

In mice, the affinity of Fv, Fab or IgG antibodies is about 10⁹–10¹⁰ M⁻¹. Such antibodies are contained within the B cell repertoire and are found in the serum of uninfected hosts [15]. There is simply no time for affinity maturation [6,7]. These high-quality antibodies are, however, not found against CPV, and it takes about 70–300 days for neutralizing antibodies to affinity mature. By that time, the CPV has escaped by mutation and is no longer neutralized [13]. We have not found examples of an ALV–host relationship, where the neutralizing antibody specificity is not prominently represented in the B cell repertoire. Interestingly, in the example, we have tested more carefully, that is, vesicular stomatitis virus (VSV, Indiana or New Jersey) that there was no obvious repertoire difference between the usual hosts such as ruminants (including cows) versus unconventional hosts such as humans or mice [10]. This suggests that the B cell repertoire in mammals is about the same and includes anti-VSV neutralizing antibody titers in normal serum of 1/20–1/40 before infection. Interestingly, normal serum does not exhibit neutralizing activity against CPV (or less than half) [15].

For antibody-mediated protection by neutralization, there exists a threshold of affinity of about 10⁹ M⁻¹ and of concentration of 1 µg/ml blood in mice; both are available by day 7 of a primary infection [16]. Therefore, the textbook argument that by affinity maturation, increased antibody affinity contributing to memory cannot readily apply within 7 days. Our titrations in mice of the protective capacity of neutralizing monoclonal antibodies revealed that antibodies of 5 × 10⁸ or of 5 × 10¹⁰ M⁻¹ required the same concentration of about 1 µg/ml and not as could be expected much less for the higher affinity antibody [6,7]. The slow process of affinity maturation of specific but non-neutralizing antibodies against CPV eventually results in high-affinity neutralizing antibodies at efficient concentrations. This is however useless because the persisting virus has escaped by mutation of the protective antigenic site.

Plasma cells are usually measured by an antigen-binding assay (such as antibody-forming assay or by immunohistology). Antigen and antibody-binding assays have limitations due to usually low-affinity measurements as discussed above. We therefore used a nice trick to almost exclusively measure the neutralizing versus non-neutralizing antibody-producing plasma cells by exploiting some of the key parameters summarized above. We used intact VSV particles as specific probes to detect neutralizing antibody-producing plasma cells only in in vitro assays or by morphology. These experiments revealed short-lived neutralizing antibody-producing cells with a 2–4 day half-life versus seemingly much longer lived ELISA-type positive plasma cells [17]. Our adoptive transfer experiments showed that transfer of spleen or bone marrow cells together with antigen maintained plasma cells, while in the absence of antigen, plasma cells disappeared within 3 days. Thus, instead of multiple, undefined and uncontrollable low-affinity specificities of ELISA-type plasma cells, biologically protective specific antibody production is assessed; plasma cells are shortlived.

The so far summarized results impinge on our understanding of memory as follows: in the intact host maintenance of neutralizing antibody, titers usually reflects re-exposure to antigen. Plasma cells and protective antibody titers in fact are antigen driven [17] by re-exposure from the outside (mostly by epidemiologically circulating often subclinical and therefore unnoticed infections, sometimes by revaccination) by persisting antigen in the host, that is, immune complexes on follicular dendritic cells or persisting infections [2,13,17,18].

Broadly, cross-neutralizing antibodies are avoided by coevolution. The few examples published so far seem not to function by neutralization, but rather by some form of antibody-dependent plus complement or cell-dependent effector mechanisms. The great amount of antibodies (100 µg/ml) needed and the short effect on viremia is still unexplained [19].

Conclusion

Taken together, the summarized results indicate that as is often the case, our results and interpretations depend largely upon the tests and assays used. The only relevant read-out in medicine and evolution is the absence of disease and/or early death. Protection and species survival correlate with neutralizing antibody titers at the time of infection [8] but not necessarily with ELISA titers. Therefore, as reviewed here, maintenance of protective antibody titers is antigen dependent, and therefore is only guaranteed by frequent repetitive antigen re-exposure including revaccinations [20].

Financial & competing interests disclosure

R Zinkernagel was supported by the University of Zurich, Department of pathology. The author serves as a board member of Novartis which bears no relation to this manuscript. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.