3. La teoría del gen 15
3.1. Especializaciones de la teoría del gen
In order to investigate the expression of CD248 in activated lymphoid tissues, a well-described immunisation schedule was followed. The immunisation protocol employed in which NP-CGG is administered with alum adjuvant is fully discussed in the introduction to this thesis and in the materials and methods section. In these experiments, 100µg NP-CGG with alum adjuvant was administered as an intra-peritoneal injection (i.p.) on day 0 in order to induce an immune response in the spleen. In the rechallenge experiments, the mice were reimmunised on days 21 and 41, where appropriate. In order to investigate the immune response in the draining lymph nodes, 10µg NP-CGG in alum was administered as a sub-cutaneous injection (s.c.) into the foot pad of the mice at the same time points as mentioned for the i.p. immunisations. Using this immunisation schedule in order to investigate the expression of CD248 in activated lymphoid tissue, I was able to identify a population of cells with very high levels of CD248 expression within the inner part of the follicle and separated from the CD248+ capsule. These cells were located within the B cell zones of repeatedly challenged lymph nodes and further co-staining with follicular dendritic cell markers such as FDC-M1 revealed that these cells were in fact FDCs (fig. 4.1a).
As shown in figure 4.1b, the FDC network formed in response to immunisation in CD248-/- lymph nodes
appear histologically normal. The FDCs in WT lymph nodes have been shown to express CD248, as shown in figure 4.1a. However, in the CD248-/- nodes, as shown in figure 4.1b, although there is no
CD248 expression in the CD248-/- lymph nodes, as expected, the CD248-/- FDC networks have formed
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In the spleen following primary immune response, the follicular dendritic cell network form normally within the germinal centres (fig. 4.2a & b). However, as shown in figure 4.2d, following secondary immunisation, the FDC networks that form in the CD248-/- spleens display an abnormal histology, with
aberrant interconnected network. As shown in figure 4.2d, the FDC networks formed in the CD248-/-
spleens have a punctate histology, lacking the interconnected networks displayed in their WT counterparts shown in figure 4.2c. Interestingly, there is no abnormal structure in the CD248-/-
following primary immunisation as shown in figure 4.2a. This is different to the expression seen in the CD248-/- lymph nodes in figure 4.1, demonstrating a differential role in different tissues.
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CD3, FDC M1, CD19
X40 CD248, FDC M1, CD45
WT
WT
WT
CD248-/-
A
B
Figure 4.1 Analysis of FDC networks in immunised WT and CD248-/- draining lymph nodes. A) Identification of a population of CD248+ FDCs in WT LN as described by staining
with CD248 (green), FDC-M1 (red) and CD245 (blue) at x25 magnification. B) Analysis of the effect of losing CD248 on the development of FDCs in the LN stained with CD3 (green), FDC-M1 (red) and CD19 (blue) at x10 magnification.
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B
Figure 4.2 Analysis of FDC networks in immunised WT and CD248-/- spleens. FDCs in A) WT and B)
CD248-/- primary immunised spleens were stained using DAPI (grey), CD3 (green), FDC-M1 (red). FDCs in C) WT and D) CD248-/- secondary immunised spleens were stained using CD248 (green), FDC-M1 (red) and CD45 (blue). Images all taken at x40 magnification.
DAPI CD3FDC-M1
CD248
FDC-M1CD45
C
D
WT
CD2
48
-/
-
WT
CD2
48
-/
-
A
B
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This histological defect was further investigated throughout the whole spleens, using tile scans at multiple cutting levels and different staining protocols. Representative images are shown in figure 4.3. FDC morphology was analysed, staining the tissue with both FDC-M1 and CD35 using a number of different methods (fig. 4.3).
Spleens isolated from CD248-/- mice and stained with FDC-M1 showed abnormal FDC development
with a complete absence of the interconnected networks demonstrated in the WT spleen in figure 4.3a, in agreement with the previous data demonstrated in figure 4.2. There also appears to be a reduction in the number of germinal centres across the whole spleen when compared to the WT. When the histology of the FDCs is analysed by staining with CD35 (fig. 4.3d), which is a different FDC marker, known to stain a functional molecule on the surface of the FDCs, the structures of the networks did not seem to be affected. The CD248-/- FDCs demonstrate the same structure as their
WT counterparts, but the total size and number of FDC networks appears to be was reduced in comparison to the WT spleens shown in figure 4.3c.
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A B
C D
CD3, FDC M1, CD19
CD3, CD35, CD19
Figure 4.3 Identification of a defect in FDC networks of CD248-/- spleens. Representative
images of WT and CD248-/- spleens stained with different markers as follows: A) WT and B) CD248-/- spleens stained with CD3 (green), FDC-M1 (red) and CD19 (blue) and C) WT D) CD248- /- spleens stained with CD3 (green), CD35 (red) and CD19 (blue). Overview images at 10x magnification with inserts at 40x magnification.
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These defects were quantified using a number of methods. Initially, the size of the follicular dendritic cell networks was measured in µm2, this revealed that the area covered by FDC-M1 positive staining
within the germinal centre was reduced in the CD248-/- spleens compared to the WT (fig. 4.4a). This
was also true of the area of CD35 positive staining, with a significant reduction in the size of the of the follicular dendritic cell areas in the CD248-/- when compared to the WT (fig. 4.4b). The defect in
the FDCs when investigated using FDC-M1, as previously observed, was not a significant reduction in the size of the areas, rather a reduction in the development of processes and the interconnectivity of the cells. In order to further quantify this defective development, the pixel density of the FDC region was analysed. The FDC-positive stained area was selected using Zen software; this software then calculated the number of positive pixels within this region. Using this data, I was able to calculate the mean pixel density per unit area, therefore quantifying the distribution of the FDC-M1 positive staining and giving an indication of the interconnectivity of the dendritic processes and network formation. No significant difference in the mean number of FDC-M1 positive pixels between the CD248-/- and the WT was observed in mice immunised once (figure 4.4c). Following secondary
rechallenge with the same antigen, however, the CD248-/- spleens revealed a significant reduction in
the mean FDC positive pixel density per unit of FDC area compared to the WT (figure 4.4d). The defect in the immunised CD248-/- lymph nodes was also analysed in this manner, and as can be seen
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E
A B
C D
Figure 4.4 Analysis and quantification of the observed defect in FDC networks of CD248-/- spleens and LN.
A) The area of the single largest FDC-M1 positive area was calculated using Zen software in µm2. B) The area of
the single largest CD35 positive area was calculated using Zen software in µm2. Data shown in A) & B) is
representative of 4 spleens, with 3 independent cutting levels per spleen. Error bars demonstrate the mean ± SD with significance calculated using a Student’s t test. Mean pixel density per unit area of the germinal centre was calculated from 3 representative images per spleen, with 4 independent spleens used per group. This was calculated following primary immunisation (C) and secondary immunisation (D). Error bars demonstrate the mean ± SEM, and significance was calculated using a Student’s t test with p < 0.0005 = ***. Mean pixel density was also calculated for the germinal centres found within immunised bLN (E) (n = 24) and errors bars describe mean ± SEM
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This analysis gave some interesting insights into the defect in the CD248-/- germinal centres, although
it did not completely address the abnormal structure of the FDC networks observed in the CD248-/-
spleens. Therefore, an imagej macro was designed in order to more fully understand the reduction in connections between the individual follicular dendritic cells. The steps in the macro analysis are described in figure 2.2, and also shown in fig. 4.5a. However, in brief, the percentage of GC covered by FDC-M1 positive staining was calculated. The aim of this analysis was to give a better representation of the defect in network formation by analysing the connections between the FDC-M1 positive regions. As shown in figure 4.5b, the CD248-/- spleens display a significantly reduced coverage over the
germinal centre with follicular dendritic cells, again indicating that there is significant reduction in the interconnectivity of the networks.
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A
B
Figure 4.5 Design and use of an imageJ macro to quantify the defect in FDC networks of CD248-
/- spleens. A) Description of the macro designed to analyse this FDC defect, described in detail in
chapter 2. The analysis of the FDC networks in three times challenged spleens is demonstrated in
(B) with error bars describing the mean ± SD. Significance was calculated using a Student’s t test
with p < 0.0005 = ***.
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