II. ZUSAMMENFASSUNG
1.4 The histone methyltransferases SUV420H
2.1.6 Suv420h2 ko; VavBcl2 mice have increased proportion of follicular helper T cells
of a specific B cell promoter does not result in spontaneous activation of the germinal centers.
These data proved that excessive germinal center formation in the VavP-Bcl2 mice could not be simply a function of the amount of transgenic Bcl2 in B cells, but rather other splenic populations are capable to induce uncontrolled B cell proliferation (Egle et al., 2004). In the same work, VavP-Bcl2 mice were crossed with GK5 mice, which lack CD4 cells. Like GK5 mice, bitransgenic VavP-Bcl2-GK5 mice had no CD4+ T cells. When age-matched littermates were analysed at 18 weeks of age, it became clear that germinal centers were not detectable by histology. This finding suggested that expansion of the germinal centers in VavP-Bcl2 mice and the substantial increase in the frequency of activated B cells is dependent on CD4+ T-cell help.
Because germinal center reaction is strictly connected to the presence of TFHs in the follicles we first investigated TFHs in the VavBcl2 spleens. Surprisingly, VavBcl2 TFHs were comparable, in percentage, to the wild type population (Fig. 2.7 a). This observation suggests that the signal which primed germinal center hyperplasia in VavBcl2 mice did not come from TFHs; therefore another population of T helper cells must induce B cell proliferation. Because Suv420h2 ko; VavBcl2 mice showed an increase in germinal center formation when compared to VavBcl2, we asked if these additional PNA+ FAS+ B cells were induced by TFHs.
Remarkably, Suv420h2 ko; VavBcl2 TFHs were consistently increased by 3 folds (Fig. 2.7 a and b).
Suv420h2 ko; VavBcl2 bitransgenic mice represent another context were we could confirm, with better resolution, what we already observed in Suv420h2 ko mice. Moreover, these mice represent a very useful tool to perform in vitro analysis and additional molecular studies, as increased spleen cellularity will allow us to harvest enough material for further investigation.
In fact, although Suv420h2 ko mice have increased TFHs and germinal center B cells, the total number of cells might be still limiting.
Results (I)
Figure 2.7 TFH cells are significantly expanded in Suv420h2 ko; VavBcl2 spleen
a) TFH cells (CD4+, CXCR5+, PD1+) population comparison between VavBcl2 and Suv420h2 ko;
VavBcl2 ko spleens. b) Frequencies of TFH cells shown as percentage of CD4+ cells (n=6). ***P <
0.001 (unpaired two-tailed Student‟s t-test). c and d) FACS plot and statistics (n=4) of resting naive T cells (CD4+, CD62L high, CD44- /low, CD25-) cells, T effector-like (CD4+, CD44+, CD62L low, CD25-) and memory T cells ( CD4+, CD44+, CD62L+, CD25-) frequencies in VavBcl2 and Suv420h2 ko; VavBcl2 spleens. NS, not significant and *P < 0.05 (unpaired two-tailed Student‟s t-test).
Discussion (I)
2.2 Discussion (I)
Antigen presentation to naive T cells by APCs triggers a number of differentiation events which initiate the immune response to pathogens. Germinal center formation is one of the most elaborated immune answers which results from antigen presentation and mediates production of highly specific antibodies. Failure to induce proper immune responses results in immunodeficiency; while, at the opposite, aberrant activation of the immune system is the primary source for inflammatory diseases, autoimmune disorders and lymphoma.
Although the signalling pathways which trigger and modulate the immune response are abundantly explained, we still lack some knowledge regarding the functional role that histone modifications exert in this context. An effort in this direction has been recently made by Beguelin and colleagues demonstrated that EZH2, the enzyme responsible for H3K27me3 establishment, is required for germinal center formation. In fact, it seems that the repressive H3K27me3 is necessary to keep silent genes involved in plasma cell differentiation, as their derepression antagonizes and bypasses the germinal center reaction (Beguelin et al., 2013).
Additional evidences which underlined the importance of repressive histone marks during mature T cell differentiation have been collected in the Almouzni laboratory. This group demonstrated that maintenance of the SUV39H1–H3K9me3–HP1α pathway is critical to ensure TH2 cell stability by inhibiting TH1 cell-associated genes, indicating that H3K9me3 repressive mark is essential to silence inappropriate transcriptional programs (Allan et al., 2012).
The role of the repressive mark H4K20me3 during hematopoietic development has been already demonstrated by Schotta et al. In this paper, loss of Suv420h1 and Suv420h2 during haematopoiesis impaired lymphoid differentiation and resulted in ineffective B cell class switch recombination. Up to the present, this publication remains the only descriptive study which established an in vivo link between SUV420H-mediated H4K20me3 and hematopoietic development. Interestingly, in this thesis we described that the sole loss of Suv420h2 did not impair lymphoid development, but instead resulted in spontaneous germinal center B cell activation. In homeostatic condition germinal center B cells are not present; therefore loss of Suv420h2 and its associated H4K20me3 must generate positive signals to initiate the germinal center reaction. In this regard the work published by Stender and colleagues, encouraged us to believe that H4K20me3 represent a suppressive signal for several immune-related genes and its removal is required to initiate their expression. Since these results were obtained from in vitro derived macrophages and the histone methyltransferase which mediates H4K20me3 silencing of pro inflammatory genes is SMYD5 and not SUV420H2, we cannot decipher our
Discussion (I)
phenotype in the light of these findings; however it is exciting to observe the presence of H4K20me3 outside its conventional heterochromatic location (Stender et al., 2012).
In the first place, we connected the production of Suv420h2 ko germinal center B cells mice to the B cell defects observed in Suv420h conditional knockout animals, implying a major role of SUV420h enzymes during B cell differentiation. For this reason we assumed that the observed defects were B cell intrinsic. However; in vitro experiments demonstrated that Suv420h2 ko B cells activated by different stimuli behaved comparably to wild type cells, suggesting that aberrant Suv420h2 ko B cell activation is not cell autonomous.
Making a step backwards in the germinal center formation timeline, we encounter a very specialized T helper cell population, known as TFH. TFHs differentiate from resting CD4+ T cells and gradually spread into follicular areas where they start to interact with B cells, initiating germinal center reaction. Notably, increased germinal centers nicely correlated with presence of TFHs in the absence of Suv420h2. Furthermore, effector T cells were also increased in the absence of Suv420h2 (Fig. 2.5 d and e), confirming signs of activation within CD4+ T cell population. Recent work demonstrated that TFHs survive in the blood as memory T cells (Schmitt et al., 2014). Nicely, we found that also this population was increased compared to wild type mice, reinforcing the idea that Suv420h2 ko mice are able to differentiate more TFHs in the absence of immune stimuli.
Because germinal center B cells undergo massive apoptosis due to removal of potential self-reactive-antibody-producing cells, we verified whether releasing apoptotic check point had any impact on the observed phenotype. To do this we generated Suv420h2 ko; VavBcl2 transgenic mice and checked how germinal center B and TFH cells behaved in young mice.
Importantly, young VavBcl2 animal‟s germinal centers were not significantly increased and TFHs were comparable to wild type mice. Differently, Suv420h2 loss in the VavBcl2 background showed a significant increase in the germinal center B cell population accompanied by a 3 fold increase of TFHs. These observations consistently confirmed what we already observed in Suv420h2 ko mice.
Because Suv420h2 ko mice are full knockout animals, we could not attribute the origin of the phenotype to any specific cell type, however we planned to perform bone marrow transplantation with Suv420h2 ko; VavBcl2 B cells or naive T cells to reveal which of these populations is the actual inducer of spontaneous germinal center formation. Also, Suv420h2 ko; VavBcl2 mice represent a promising tool to better investigate the molecular details of the described phenotype. In fact, we are going to test whether in vitro stimulation of Suv420h2 ko;
VavBcl2 B or/and T cells will show different behaviour compared to control cells.
Discussion (I)
VavBcl2 mice are prone to get follicular lymphoma after 1 year of age. Interestingly, preliminary data showed that Suv420h2 ko; VavBcl2 mice die earlier compared to VavBcl2 (data not shown). This phenotype needs to be further investigated, since we do not really know at the moment why loss of Suv420h2 would further compromise VavBcl2 mice viability. One hypothesis could be related to the onset of a more aggressive form of follicular lymphoma or a combination of tumour onset and persistent inflammation. To better address this question we set up an aging experiment were a cohort of mice is monitored for the appearance of any obvious disease.
In the near future we aim to identify genome wide H4K20me3 and SUV420H2 binding sites, in B and T cells. So far, ChIP-Seq analysis has been performed in our laboratory in MEF cells and revealed that SUV420H2-mediated H4K20me3 mainly covers retrotransposable elements and constitutive heterochromatic regions (telomeres, centromeres). However, the genomic distribution of this mark might change according to the cellular context we take in account. Once these target regions will be identified, we are going to analyse whether loss of H4K20me3 also results in gene activation which might promote terminal differentiation of mature immune cells.
Noteworthy, stacks of confocal images showed that only a very small percentage of CD4+ T cells carried a clear H4K20me3 methylation pattern, while the others seemed to be totally refractory to the establishment of this repressive mark (Fig. 2.8). To better elucidate this point, we will sort CD4+ T cell subpopulations to verify whether there is a preferential accumulation of H4K20me3 in a certain cell type. Moreover, we also noticed that the H4K20me3 signal not always overlapped with DAPI dense regions, suggesting that the H4K20me3 methylation pattern might be slightly different from the one observed in MEFs.
Although the limited amounts of data which have been collected so far leave little room for speculations and conclusions, we are pretty confident that H4K20me3 plays a role during germinal center formation. As yet, how this happens is unclear; however additional experiments will hopefully reveal more about the molecular basis of this phenotype.
Discussion (I)
Figure 2.8 Heterogeneous H4K20me3 mark distribution within CD4+ T cell population
a) Confocal picture showing CD4+ T cells stained with H4K20me3 primary antibody detected with Alexa488 secondary staining.