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Antibody (Ab) affinity maturation (AAM) referred originally to the observed increase in average Ab affinity against a hapten (1). Later, it was found that AAM is associated with the formation of transient lymphoid structures in the B cell zones of lymphoid tissues, called germinal centers (GC), during T-cell dependent immune responses in higher vertebrates (2).  In another line of research, AAM was related to the occurrence of mutations in the variable (V) domain of Ab heavy (H) and light (L) chains, respectively, VH and VL. In those works, a mutational analysis of Ab V genes was performed, initially on bulk splenic plasma B cells and later on GC B cells vs. extrafollicular B cells, after successive immunizations. The results showed typically an increased number of mutated GC B cells (3–6), and an accumulation of mutations per Ab chain during the ongoing immune response, with many mutated B cells displaying higher affinity for the hapten used for immunization. This provided strong support to a previously suggested concept (7), according to which AAM is a B-cell receptor (BCR)-based Darwinian evolutionary process.  A few years later, two complementary hypotheses were proposed. The first one, based on a mathematical model, suggested that, for the fastest production of high affinity Abs, the mutation rate in GC B cells should be minimal before GCs reach a threshold size, and then switch abruptly to the maximal possible rate (8). The second hypothesis proposed, for the assumed Darwinian process, alternating cycles of B cell proliferation plus mutation plus selection (9). These ideas were soon extended in another modeling work, showing that Ab affinity can be maximized when the mutational mechanism switches on and off regularly (10). These results contributed considerably to strengthen the general belief in the recycling or multiple-step selection hypothesis. On the other hand, more recently, alternative B cell selection mechanisms were proposed that do not require multiple-step selection in order to be compatible with observed levels of Ab affinity increase during a primary immune response (11, 12).  There is still much to learn about AAM mechanisms (13–17), and there is a need to clarify some aspects of the GC physiology where overinterpretation and preconceptions prevail (18, 19). The multiple-step selection hypothesis is a prominent example of a concept that, having important basic and practical implications, has never been confirmed. Clearly, a direct way to establish it would be to observe multiple BCR-mediated selection events by tracking individual B cells via imaging of lymphatic tissue, observing SHM taking place between selection rounds. However, direct observation of even one selection event is not yet possible. At the same time, attempts to interpret indirect data must be faulty due to the need to use unverified assumptions on AAM mechanisms.  Therefore, we take here a radically different approach: we propose to consider the single-step selection concept to be a null-hypothesis which should be attempted to be falsified (Figure ​(Figure1).1). Because this ansatz puts the focus on a process of random non-directed acquisition of mutations, it minimizes the need for unverified assumptions. And because mutations carry the signature of the selection process, the data to be used should consist of Ab V gene sequences. In the following, we examine two possible falsifying strategies.    Figure 1  Sketch of proliferation plus SHM and selection history of a plasma or memory cell. (A) Single-step selection. Several cell division plus mutation cycles and a single final selection step before terminal differentiation into a plasma or memory cell. (B) ...     Falsifying the Null-Hypothesis with Phylogenetic Trees Let us consider all mutated VH or VL sequences belonging to a given B cell lineage. The corresponding phylogenetic tree is a result of the evolutionary process undergone by the initial sequence, and as such, is shaped by the various factors pertaining to the affinity maturation process.  Extensive work was performed on developing methods to build phylogenetic trees from V genes of a common lineage (20) and to analyze how shape measures depend on AAM mechanisms (21, 22). These simulations show that the tree shapes vary most on the initial clone affinity and the selection threshold, and much less in dependence on the rates of GC B cell recycling (22), not allowing for a unique mapping from tree shapes to selection mechanisms – likely because the investigated trees were small. In addition to global measures not always being helpful in pointing to mechanisms at the micro-evolutionary scale (15, 23, 24), simulation of global measures like peak total GC B cell numbers did not lead to results that contradict the single-step hypothesis (22).  Summing up, the null-hypothesis has never been falsified by examining the shapes of phylogenetic trees.

A Major Hindrance in Antibody Affinity Maturation Investigation: We Never Succeeded in Falsifying the Hypothesis of Single-Step Selection