PLASMA CELL DIFFERENTIATION

Once a GC B cell undergoes different cycles of mutations and selection for affinity maturation, it is ready to differentiate into PC and to produce Abs against a specific antigen. At this point a drastic change in expression of transcription factors is needed (Fig.10). Several transcription factors must be up-regulated such as BLIMP1 and XBP1 or down-regulated such as BCL6 and PAX5 [148, 149].

In this process, the BCR plays a key role, and the strength of the interaction with the antigen is critical in determining which B cells enter PC differentiation. In fact, the ligation of the BCR and the CD40 molecule leads to the expression of IRF4, downstream to NF-kB activation that inhibits BCL6 transcription and promotes the expression of PRDM1 but only after a strong signal from BCR with the help from cytokines or interleukins (IL-4, IL-5 or IL-2) [150]. B cells express distinct amounts of IRF4 during the different phases of development.

Naive resting B cells express a low basal amount of IRF4 that is rapidly increased during the maturation process [100]. However, it is not clear whether IRF4 alone is sufficient to induce expression of BLIMP1 and trigger PC differentiation. BCR activation stimulates also the MAPK cascade that leads to BCL6 inhibition by phosphorylation and subsequently proteasomal degradation, linking BCL6 turnover with BCR stimulation [151]. BCL6 as said before, is the master regulator of GC reactions and its repression is essential because allows the activation of BLIMP1 expression. BLIMP1, encoded by the PRDM1 gene, is the master regulator of PC differentiation and function as a transcriptional repressor. Within the B cell lineage BLIMP1 starts to be expressed in centrocytes [152] and is maintained in all antibody secreting cells (ASCs) [153], with cycling plasmablasts being distinguishable from long-lived PCs based on their expression of BLIMP1 in both mouse [153] and human [154]. Once BLIMP1 is activated it represses many important genes of GC B cell program in addition to BCL6 and PAX5, including genes encoding SPI-B, Id3, CIITA and MYC [111, 155, 156] thereby ensuring that, after PC development is induced, B cells cannot return to an earlier developmental stage. In particular, BLIMP1 expression is mutually exclusive with BCL6 and PAX5 expression ensuring that the phases of B cell maturation remained completely distinct.

37

Figure 10: interaction between centrocytes with FDC and TFH by BCR and CD40 respectively, allowed that PC differentiation take place. Once expressed, PC factors repress factors required for GC formation and B cell identity. NF-kB is activated after the stimulation by CD40 molecule on TFH.

NF-kB activates the expression of IRF4 that initiates plasmacytic differentiation by establishing a characteristic regulatory network, which extinguishes the mature B cell program while promoting terminal PC differentiation and antibody production through XBP1. IL-21secretion by TFH induces BLIMP1 in a STAT3-dependent manner while BCR stimulation by Ag on FDC inhibits BCL6 by inducing phosphorylation and degradation of it. Ca²⁺-loaded Calmodulin (CaM) is also triggered by BCR activation leading to GC genes down-regulation through E2A inhibition. Red lines with stop bars indicate inhibitory regulations blue arrows indicate activating regulations. Red wide arrow indicates down-regulation of all the alongside genes. Blue wide arrow indicates up-regulation of the alongside gene.

Hauser J. et al. [155] have tried to understand which genes trigger the switch into PC phenotype and the timeline of expression changes. From this study the primary regulatory events identified were inhibition of BCL6 as expected, together with PAX5, MITF, ETS1, FLI1 and SPI-B known as direct or indirect BLIMP1 repressors. These are very rapid events that occur within 30 minutes after BCR, CD40 or TLR stimulations [155]. Ca²⁺

signalling activated by BCR stimulation leads to inhibition of E2A by Ca²⁺-loaded Calmodulin (CaM) followed by BCL6, PAX5, ETS1 and FLI1 expression changes, with a weak increment of BLIMP1 [155]. BCR stimulation increases IRF4 and BLIMP1 mRNA levels relatively fast, but slightly slower than the rapid transcriptional down-regulations and this is

38

because IRF4 and BLIMP1 are more dependent on NF-kB activation than Ca²⁺-loaded Calmodulin (CaM) pathway [155]. The genes down-regulated through Ca²⁺-loaded Calmodulin (CaM) pathway could all be direct targets of E2A. This transcription factor functions in hematopoietic progenitor cells to activate expression of PAX5 and establishment of B cell specific gene expression program and when its expression is inhibited, BCR activation has no effect on the expression of any of previously described GC genes [155].

Besides repressing important GC transcription factors, BLIMP1 up-regulates genes involved in Ig secretion such as Ig genes themselves (IgH, IgL and the J chain) and X-box binding protein 1 (XBP1), a transcription factor essential for antibody secretion [149].

XBP1 is up regulated as part of the endoplasmic reticulum (ER) stress response and is expressed at a high level in PCs where is essential for inducing the secretory phenotype.

This gene is a mediator of unfolded protein response (UPR) activated by accumulation of misfolded protein in the ER [157]. XBP1 activity is further regulated by a splicing event that is also induced by the UPR [158], forming the splice active form XBP1s that present an excised 26-bp intron, causing a frameshift producing a new C-terminus that confers transcription activating activity [159]. XBP1 is the only transcription factor that is uniquely required for PC differentiation but it is not, apparently, required for any early stage of B cell development. In mature B cells, XBP1 is maintained at low levels thanks to PAX5 activity and the activation of BLIMP1 factor represses this regulation by repressing PAX5 [160]. The mutually exclusivity in their expression ensures that the phases of B cell maturation remained completely distinct.

2.4. REMARKS

The GCs are the histological structures where B cells undergo genetic remodeling of their Ig genes toward the production and selection of cells able to recognize the antigens with high affinity. As mentioned above, almost all B-NHL derived from GC B cell at different stages of differentiation maintaining several molecular and phenotypic characteristics of the normal B cell counterparts. Thus is very important to clarify molecular mechanisms governing normal GC and post-GC processes. A large number of studies have identified several transcription factors that have to be up-regulated or down-regulated during B cell differentiation. However, a lot of questions about the role of several genes whose expression is regulated during B cell differentiation are still opened.

In particular, ETS1 and FLI1 are described as GC players that must be silenced to allow

39

BLIMP1 up-regulation and PC differentiation. But, what is their specific role in GC? It is also known that ETS1 and FLI1 interact with other important GC transcription factors such as PAX5 and that ETS1 lack lead to a propensity in PC differentiation under TLR9 stimuli, while the lack of FLI1 leads to the onset of autoimmune disease. But, how ETS1 and FLI1 contribute in the regulation of PC differentiation? What are their direct targets? These are some questions to which I will try to answer.

40

3. Genetics of Diffuse Large