The aim of our study was to investigate key mechanisms and molecules involved in the formation of B cell aggregates in the CNS of MP4-immunized mice. A particular focus was set on the role of LTi cells. Some studies have already provided evidence for the involvement of LTis in the formation of TLOs. Meier and colleagues have shown that an overexpression of interleukin-7 (IL-7) in transgenic mice induced the accumulation of LTi cells and the development of new Peyer’s patches, ectopic lymph nodes, and cecal patches [
22]. Further experiments have demonstrated the formation of intestinal lymphoid tissue after transfer of LTi cells into CXCR5
−/− newborn mice [
23]. Besides LTi cells, T
H17 cells also seem to play an important role in TLO development. The transfer of MOG-specific T
H17 cells led to the formation of ectopic lymphoid tissue in the CNS of mice. IL-17 and the T
H17 cell surface molecule podoplanin have initially been suggested to be involved in this process [
25], while further investigation has demonstrated that podoplanin also has an inhibitory effect on T cells [
29]. Hence, podoplanin might play a dual role in TLO development, which needs to be further investigated. Here, we set out to identify the roles of LTi cells in the CNS of mice suffering from EAE, in particular in the MP4 model, which is characterized by ectopic lymphoid tissue formation in the chronic stage of the disease [
12]. B cell aggregates were also described in patients suffering from secondary progressive MS and were associated with more rapid disease progression and cortical histopathology [
3,
4]. Although MP4-immunized mice do not display disease progression so that B cell aggregate formation cannot be correlated to clinical disease parameters, MP4-induced EAE represents a convenient model to study key molecules and mechanisms involved in the development of ectopic lymphoid organs [
12]. Our data demonstrate the absence of LTi cells in the cerebella of MP4- and MOG:35–55-immunized mice throughout the disease course. While LTi cells have been shown to induce the development of ectopic lymphoid tissue in the periphery of mice [
22,
23], our results provide no evidence of a key role of this cell population in the CNS of EAE mice. As positive control, we analyzed the presence of LTi cells in the embryonic spleen. LTi cells migrate from the liver to the target tissue, where they induce the development of SLOs [
21]. Therefore, LTi cells can be detected in the spleen during embryogenesis [
30]. For adult mice, we used the intestine as a positive control. Studies have shown that LTi cells persisted in the intestine and were important to support the innate immune system [
31,
32]. Along these lines, we detected a low number of LTi cells both in the embryonic spleen and in the intestine of adult mice. In humans, the occurrence of CD3
−RORγt
+ ILCs has been described in the CNS of a subgroup of MS patients in association with B cell follicle-like structures [
24]. According to our definition of LTi cells, being described as CD3
-CD5
−CD4
+RORγt
+, the results of Serafini and colleagues are not in conflict with our data because of no further characterization of CD3
−RORγt
+ cells in their paper [
24]. Nevertheless, we cannot exclude that few LTi cells still migrate into the CNS of MP4-immunized mice or are present at an earlier time point to induce lymphoid tissue formation. Detection of small cell populations always implicates difficulties. LTi cells represented a minority of the total leukocyte population in our control tissues and, therefore, it might be hard to detect a possibly even smaller amount in the cerebellum. Similar to the paper of Serafini, we also detected CD3
−RORγt
+ cells in the cerebellum of EAE mice, which we defined as CD3
−CD5
−CD4
−RORγt
+ ILCs. Overall, the numbers detected in our study were quite low. Only a slight increase could be observed during the acute stage of MP4-induced EAE and in the MOG:35–55 model. While B cell aggregates are a hallmark of the chronic stage of MP4-induced EAE, these structures do not occur in MOG:35–55-immunized mice [
11]. In particular, chronic MP4-immunized mice showed almost no ILCs. Moreover, the amount of ILCs, which were detected in the acute stage of MP4-induced EAE, was comparable to the numbers of MOG:35–55-immunized mice. Therefore, it remains unclear if ILCs play any important role in ectopic lymphoid tissue formation in the CNS. Next to LTi cells and ILCs, we focused on the presence of CD3
+CD5
+CD4
+RORγt
+ T
H17 cells in the cerebellum of EAE mice. According to the human study, which showed a much higher frequency of CD3
+RORγt
+ than of CD3
−RORγt
+ cells [
24], we also detected CD3
+CD5
+CD4
+RORγt
+ T
H17 cells in the CNS of both mouse models. Sharing a lot of common markers with LTi cells [
27], it becomes obvious that T
H17 cells could possibly undertake the tasks of LTi cells in the process of lymphoid tissue formation. Yet, they may not be the only decisive factor. On the one hand, one would expect the highest number of T
H17 cells in the acute MP4 cerebellum. On the other hand, although the MOG:35–55 model is characterized by lack of B cell aggregates in the CNS [
11], the frequencies of T
H17 cells were comparable between the MP4 and MOG:35–55 model during the acute stage of the disease. Only a slight increase in the number of T
H17 cells was observed in chronic MP4-immunized mice, which may also be attributed to the gating strategy. Overall, the formation of B cell aggregates and their evolution into ectopic lymphoid structures is certainly a complex process that involves more than one cell type and molecule. Indeed, when performing RNA sequencing on dissected B cell aggregates from chronic MP4-EAE mice, we detected a range of upregulated molecules compared to SLOs and to the MOG:35–55 model. In particular, we detected a significantly higher expression of
Il17f, which is characteristic of T
H17 cells [
33]. Moreover, B cell aggregates expressed two members of the
Mmp gene family, i.e.,
Mmp3 and
Mmp10. On the one hand, MMP-3 has been shown to assert neuroprotective function, e.g., by reducing Fas/FasL-mediated apoptosis [
34]. On the other hand, MMP-3 could be involved in the pathophysiology of MS by degrading the blood brain barrier [
35]. Unlike MMP-3, MMP-10 has not been mentioned in association with MS before. Yet, MMP-10 has been described to play a role in autoimmune processes in synovial pathology [
36]. Furthermore, HSPs were highly expressed in MP4-related B cell aggregates. It is a moot question, whether heat shock proteins of the HSP70 family play a negative or beneficial role in MS pathogenesis, because of their contradictory function. Besides a neuroprotective role, HSP70 acts as an immunomodulator, for example by triggering the expression of pro-inflammatory cytokines [
37,
38]. Moreover, it has been suggested that FAM19A2 has an immunomodulatory function, acts as a neurokine, and might be involved in axonal sprouting [
39]. In addition, RNA sequencing showed further genes to be upregulated in B cell aggregates of the MP4 model. Although all of these genes have not been described in association with neurological diseases and autoimmunity before and there are currently no reports that they are involved in the process of ectopic tissue formation, they should not be excluded from further investigation.