For assessing the kinetics of GC macrophages (Mφ) serial spleen sections of phOx-CSA immunized mice, derived at different time points after immunization, were triple stained for proliferating cells (mAb Ki-67), FDC-networks (mAb FDC-M2) and Mφ (mAb CD68). In the following, stained sections were analyzed by confocal microscopy for the presence of GC that were identified as cell clusters of Ki-67+ proliferating cells in the context of FDC-networks. The collectivity of all GC within individual sections was subsequently recorded by image acquisition (x400 magnifi-cation). Image evaluation was performed as exemplary depicted for a GC image obtained at day 10 after immunization in Figure 5.8. In total, 1290 GC out of 52 sections derived from 26 mice were evaluated. A detailed survey of all analyzed sections and evaluated GC is given in Table 5.3, the recorded parameters (direct and derived quantities) are listed in Table 5.4.
days p.i. Σ(GC)/
Table 5.3: Survey of spleen sections analyzed for tracing the kinetics of GC Mφ. Eachrepresents one analyzed spleen section at the given time point after immunization. Identical patterns ( ⊗ ) reflect sections (interspace>500 µm) derived from the same animal. The number of evaluated GC for each section is given in parentheses.
Parameter Parameter description Calculation formula
(D) areat total area of GC
(D) areaF DC+ area of FDC-rich light zone (D) nKit total number of Ki-67+ cells
(D) nKiF DC+ number of Ki-67+ cells within FDC-rich light zone
(D) nMF DC+ number of Mφwithin FDC-rich light zone (D) nMF DC− number of MφFDC-poor dark zone
(d) areaF DC− area of FDC-poor dark zone =areat- areaF DC+
(d) nKiF DC− number of Ki-67+ cells within FDC-poor dark zone
=nKit- nKiF DC+
(d) nMt total number Mφ =nMF DC+ + nMF DC−
Table 5.4: Survey of recorded parameters for assessing the kinetics of GC Mφ. Listed are the recorded direct (D) and derived quantities (d).
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Figure 5.8: Data acquisition for assessing the kinetics of GC Mφexemplified by an image of a GC obtained at day 10 after immunization. GC were identified as clusters of proliferating cells (mAb Ki-67) within the context of FDC networks (mAb FDC-M2). GC Mφ(M) were specified as CD68+ cells within the manually assigned boundaries of GC (ROIs), by further discriminating between the FDC-rich light zone (FDC+) and the FDC-poor dark zone (FDC-) of GC. Scale bar 50µm.
days p.i. Σ(B cell zones)/
naive (0) (69)(60) ⊗(53)⊗(46) (228)/4
4 (39)(62) (101)/2
6 (52)(57) (109)/2
10 (36)(54) (90)/2
21 (43)(53) (96)/2
x (68)(79) (147)/2
(771)/14
Table 5.5: Survey of spleen sections analyzed for assessing the frequency of Mφwithin B cell zones of naive and immunized BALB/c. Each represents one analyzed spleen section at the given time point after immunization. Identical patterns (⊗) reflect sections (interspace>500 µm) derived from the same animal. The number of evaluated B cell zones for each section is given in parentheses.
5.2.1 The number of GC Mφ reflects cross-sectional GC size
Likewise to GC T cells, the recorded numbers of Mφ per cross-sectional GC feature prominent inner-section variation based on the underlying broad size distribution of cross-sectional GC. The total numbers as well as the numbers of Mφwithin dark and light zone of GC appear to be dependent on the size of the appendant compartments (Figure 5.9). The strict correlation of numbers of Mφand cross-sectional GC size is observed throughout all sampled time points, irrespective of the delineation of cross-sectional GC size in terms of either areas or numbers of Ki-67+ cells (Figure 5.9, A and B). Notably, the regulation of numbers of macrophages appears to be very tight, as the vast majority of evaluated spleen sections feature squared correlation coefficients (R2) substantially above 0.9.
Figure 5.9: Inner-section correlation of numbers of GC Mφand cross-sectional GC size exemplarily depicted for spleen sections obtained at day 10 (A) and day 16 (B) after immunization. The cross-sectional size of GC is given as both cross-cross-sectional GC area (left panel) and number of Ki-67+ cells per cross-sectional GC (right panel). Correlations are rated applying simple linear regression.
Regression line (black) for total numbers of macrophages and squared correlation coefficient (R2) are specified for each scatter plot.
5.2.2 Mean kinetics of GC Mφ
The mean kinetics of GC Mφ follows the overall GC growth kinetics, marked by an increment of mean numbers of GC Mφ in the early phase of GC formation up to
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the peak (day 4 to 10 after immunization), followed by a gradually but moderate decline until the last sampled time point (day 21 after immunization). The herein described kinetics holds true for both, the total numbers of GC Mφand the numbers of GC Mφ in the two differentiated GC compartments (Figure 5.10, A). The mean numbers of GC Mφ in the FDC-rich light zone and FDC-poor dark zone do not differ significantly, at the utmost a trend towards slightly elevated numbers GC of Mφ in the FDC devoid compartment can be anticipated.
The frequency of GC Mφ in relation to Ki-67+ cells ranges between 8-11% (Fig-ure 5.10, B). Hence GC Mφ constitute a rather small GC cell population, if not the smallest, given the recorded frequency of GC T cells (10-16%). As a hallmark, the frequency of GC Mφ in relation to Ki-67+ cells additionally turns out to be sus-tained in an almost fabulous fashion throughout the immune response, displaying pronounced robustness (Figure 5.10, B). Taken together, the recorded, rather in-variant mean frequency of numbers of GC Mφper Ki-67+ cells and virtually absent standard deviations with regard to the total GC as well as the two GC compart-ments, indicate a very tight regulation.
Figure 5.10: Mean kinetics of GC Mφ. Illustrated are the mean numbers and standard deviations of GC Mφ per cross-sectional GC. Each mean is representative of the entirety of analyzed spleen sections at the given time point (see Table 5.3). (A) Mean kinetics of the overall number of GC Mφand numbers of GC Mφin the FDC- and FDC+ GC compartment (dark and light zone) (B) Mean frequencies of GC Mφ in relation to Ki-67+ cells per cross-sectional GC. Depicted are the frequencies of total GC Mφand GC Mφwithin the FDC+ and FDC- GC compartment (dark and light zone).
5.2.3 GC Mφ do not invade B cell zones due to GC forma-tion but are already detectable in B cell zones of naive mice
The course of the mean kinetics of numbers and frequencies GC Mφhighly suggests an influx of GC Mφ into B cell zones due to the onset of GC formation, as do T cells (Section 5.1.3). Indeed, influx of Mφ only as a consequence of GC formation (simplified: first comes the GC, then the Mφ follow) is a current doctrine that is taken up to allege that GC Mφ cannot play a role for initiation and the early phase
of GC development at all [45]. Astonishingly, juxtaposing images of follicular niches of naive to follicular niches of immunized mice explicitly refutes the "Mφ influx doctrine". In contrary to T cells, Mφ are already frequently detected in B cell zones of naive mice (Figure 5.11, A). The presence of Mφ in B cell zones was repeatedly verified in naive mice that were checked for the absence of GC by PNA staining.
Based on this finding, Mφ have to be understood as a component of the primary follicular niche that is a prerequisite for GC formation (simplified: first comes the Mφ, then GC follow).
Figure 5.11: Mφ do not invade B cell zones due to GC formation but are already detectable in B cell zones of naive mice. Images are representative of triple immunofluorescent stainings of B cell zones of naive (A) and immunized mice at day 10 after immunization (B). B cell zones of naive mice were visualized applying mAb B220, GC of immunized mice were identified as clusters of Ki-67+cells within the context of FDC networks (mAb FDC-M2). Scale bar 50µm. Note: Images are representative of both the same B cell zone and GC as shown for GC T cells (figure 5.6).
5.2.4 The frequency of Mφ per cross-sectional GC is regu-lated in a superordinate fashion by the overall B cell zone size
The comparison of images of follicular niches of naive to immunized mice gives the impression that the frequencies of Mφ in the corresponding B cell zones are quite equivalent (Figure 5.11). To further elucidate the regulation of numbers of Mφ, the frequencies of Mφ per cross-sectional B cell zone size was assessed in naive and im-munized mice. For this purpose spleen sections were triple stained for B cells (mAb B220), T cells (mAb CD4) and macrophages (mAb CD68) and both the size of cross-sectional B cell zones and associated numbers of Mφwere recorded (Figure 5.12, A).
Within individual sections the number of Mφstrictly correlates with cross-sectional B cell zone size (R2 ≥0.9) (Figure 5.12, B). The quantitative analysis verifies the prementioned qualitative impression. As illustrated in Figure 5.12, C, the frequen-cies of Mφ within B cell zones of naive (35±6 Mφ/25,000µm2) and immunized mice (30±3 Mφ/25,000 µm2) are rather steady. The overall average frequency of Mφ within follicles of BALB/c mice, ultimately adds up to 31±5 Mφ/25,000 µm2.
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Besides affirming the involvement of Mφ in formation of primary follicular niches (Section 5.2.3) this finding further implies that the frequency of Mφ is governed by the overall B cell zone size. Therefore, the executing regulation mechanism does not take charge of GC B cells in particular, but of B cells in general. Figuratively, GC formation and growth lead to an expansion of the overall B cell zone that is accompanied by an influx of Mφ because the number of B cells increases, not by reason that there are "specialized" GC B cells. As an aside, the superordinate regu-lation of the frequency of Mφis not confined to naive and immunized BALB/c mice but is also valid for other mouse strains, as well as its perturbation is potentially associated with autoimmunity (Section 5.4.2).
Figure 5.12: Steady frequencies of Mφ within B cell zones of naive and immunized mice. (A) Evaluation of the frequency of Mφ within B cell zones exemplarily shown for a spleen section derived from a naive mouse. B cell zones were identified as B220+ areas (ROI, highlighted in yellow) adjacent to T cell zones (mAb CD4). Mφwithin B cell zones were identified and counted by CD68 reactivity. Scale bar 50µm. (B) The number of Mφstrictly correlates with cross-sectional size of the B cell zone. Data is representative of a spleen section derived from a naive BALB/c mouse. Correlation is rated applying simple linear regression. Regression line (black) and squared correlation coefficient (R2) are specified for the scatter plot. (C) The frequency of Mφwithin B cell zones remains sustained upon immunization. Illustrated are the mean frequencies of Mφ within B cell zones of two naive and five immunized mice at different time points after immunization. Data is each representative of two evaluated sections per mouse. In order to improve interpretability, frequencies are quoted as numbers of Mφper 25,000µm2, corresponding to the area comprised by the white selection in (A).