Acute SIV infection in sooty mangabey monkeys is characterized by rapid virus clearance from lymph nodes and absence of productive infection in germinal centers

Abstract

Lymphoid tissue immunopathology is a characteristic feature of chronic HIV/SIV infection in AIDS-susceptible species, but is absent in SIV-infected natural hosts. To investigate factors contributing to this difference, we compared germinal center development and SIV RNA distribution in peripheral lymph nodes during primary SIV infection of the natural host sooty mangabey and the non-natural host pig-tailed macaque. Although SIV-infected cells were detected in the lymph node of both species at two weeks post infection, they were confined to the lymph node paracortex in immune-competent mangabeys but were seen in both the paracortex and the germinal center of SIV-infected macaques. By six weeks post infection, SIV-infected cells were no longer detected in the lymph node of sooty mangabeys. The difference in localization and rate of disappearance of SIV-infected cells between the two species was associated with trapping of cell-free virus on follicular dendritic cells and higher numbers of germinal center CD4(+) T lymphocytes in macaques post SIV infection. Our data suggests that fundamental differences in the germinal center microenvironment prevent productive SIV infection within the lymph node germinal centers of natural hosts contributing to sustained immune competency.

Figure 1. Follicular development and apoptosis within GC.

A, (Top) IHC for Ki67 expression in activated lymphocytes within GC of SM (wk 6) and PM (wk 5). Positive cells indicated by brown 3,3′ diaminobenzadine (DAB) chromogenic substrate; Mayer’s hematoxylin counterstain. (Bottom), Dual IHC for CD20⁺ B lymphocytes to delineate follicles (labeled with Vector Blue chromogenic substrate), and cleaved-caspase 3 to identify cells undergoing programmed cell death (labeled with brown DAB chromogenic substrate). Counterstain was omitted. All photos taken at original magnification 200x. B, Follicular development is reported as total CD20⁺ area in mm² per biopsy. C, Percent germinal center area was quantified as the Ki67⁺ region that included the zones of centroblasts (i.e., dark zones) and activated centrocytes (i.e., light zones) within follicles, divided by the total LN section area. D, Apoptosis was measured by number of cells positive for cleaved-caspase 3 (caspase 3⁺) by IHC in B cell areas (CD20⁺). Both SM and PM had progressive germinal center reactions shown by the trend of increasing germinal center size with increasing apoptosis within germinal centers. SM have more apoptotic cells in GC at 6 wpi compared to PM at 5 wpi (p = 0.035). The number of caspase 3⁺ cells decrease in SM by 24 wpi, while the number of apoptotic cells in PM continues to increase (trend). CD8 depleted animals were included in the statistical analysis and are indicated by boxes. Pairs of time points were analyzed (MWU) for difference between medians (bars).

Figure 2. Plasma and tissue viremia in SM and PM during acute SIV infection.

A, SIV viral copies per ml of plasma longitudinally in CD8-depleted and non-depleted SM versus PM. B, SIV infected cells per mm² LN area. Infected cells indicated by RNA in situ hybridization in short-fixed paraffin embedded tissue sections of peripheral lymph node at 2, 6 and 24 wpi in SM and 2, 5 and 21 wpi in PM. Number of infected cells was quantified as NBT/BCIP positive cells per mm² lymph node section area. PM have higher median number of infected cells per mm² at 5 wpi (p = 0.03), and 21 wpi (p = 0.03). Differences in medians were analyzed by MWU. Median virus burden was not significantly different at 2 wpi between SM and PM. SIV+ cell counts for CD8-depleted SM are indicated in boxes. C, Correlation between plasma SIV viral copies at 2 wpi and LN virus burden as measured by number of ISH⁺ cells per mm² LN. No correlation is seen, Spearman’s correlation coefficient (r) = 0.033, p = 0.91.

Figure 3. ISH for SIV RNA in peripheral LN from SM and PM.

ISH was performed using short-fixed paraffin embedded tissue sections of peripheral lymph node collected at 2, 6 and 24 wpi in SM and 2, 5 and 21 wpi in PM. A, SIV-infected cells were not observed in GC of immune competent SM but were readily apparent in GC of PM at all time points. Diffuse hybridization signal characteristic for the presence of immune-complexed virions trapped by FDC was not observed in SM, but was identified in PM at all time points, with the most significant trapping seen at 5 and 21 wpi. B, Despite higher tissue viral burden in CD8 depleted SM compared to PM, germinal centers typically lack infected cells and virus trapping despite close proximity to numerous infected cells in the lymph node paracortex. C, GC in CD8-depleted SM at 2, 6 and 24 wpi. Only one infected cell was localized within a GC from a SM at any given time point in CD8-depleted SM. Virus trapping was also extremely rare in CD8 depleted SM (bottom).

Figure 4. Immunophenotype of resident GC cells in SM and PM by IHC. A, (Top)

CD4 IHC at 2 wpi in SM and PM. (Bottom), Iba-1 IHC at 2 wpi in SM and PM, All images are original magnification 200x. B, (Top) CCR5 IHC at 6 wpi in SM and PM. Original magnification 200x (Middle), CCR5 positive cells within germinal center, (Bottom) isotype negative control. Original magnification 600x. Immunoreactive cells in all images are labeled with brown DAB chromogen; blue hematoxylin counterstain reveals tissue morphology. All photos are representative examples.

Figure 5. Immunophenotype of resident GC cells by confocal microscopy.

A, Localization of CD3⁺ and CD4⁺ resident cells in germinal centers. Multiparameter confocal microscopy was used to identify CD3⁺ and CD4⁺ cells within germinal centers (PCNA⁺) of lymph nodes from SM and PM at baseline and 2 wpi by simultaneous localization of CD3 with Alexafluor 633 (blue), CD4 with Alexafluor 568 (red) and PCNA with Alexafluor 488 (green). CD4⁺ T lymphocytes (CD3⁺/CD4⁺) were identified by purple/pink co-localization of Alexafluors 633 (blue) and 568 (red). Macrophages (CD3^−/CD4⁺) were identified by localization of red Alexafluor 568. By default, CD8⁺ T lymphocytes (CD3⁺/CD4) were identified by localization of Alexafluor 633 (blue). Five randomly selected GC were imaged for most animals; however, all available GC were imaged when fewer than 5 GC were present per section. Magnitude of fluorescence (panels B through E) is reported as ratio of area fraction (AF) of fluorescent pixels within GC to area fraction of PCNA⁺ (total germinal center) fluorescent pixels, as calculated using an algorithm in the imaging software (Fovea Pro). Each point represents a germinal center, with horizontal bars indicating the mean. B, PM had greater numbers of CD4⁺ cells in GC at 2 wpi than SM (p = 0.02). C, No difference was noted in the mean number of CD3⁺ cells in GC between SM and PM at baseline and 2 wpi. However, numbers of CD3⁺ cells were higher at baseline than at 2 wpi in SM (p = 0.02). Values for CD8-depleted SM were excluded from the 2 wpi data set. D, CD3^−/CD4⁺ fluorescence (macrophages), calculated by subtracting CD3⁺ AF from the total CD4⁺ AF. Negative ratios reported as zero. PM had higher numbers of GC macrophages at 2 wpi compared to SM (p = 0.05). Mean number of GC macrophages increased in PM from baseline to 2 wpi (p = 0.003). E, CD3⁺/CD4⁻ fluorescence calculated by subtracting total CD4⁺ AF from CD3⁺ AF. Negative ratios reported as zero. Low numbers of CD3⁺/CD4⁻ cells (likely cytotoxic CD8⁺ T lymphocytes) were observed in GC for both species at all time points. SM have higher baseline numbers of (CD8⁺ cells) compared to 2 wpi (p = 0.02). Differences in mean area fractions between time points were analyzed using unpaired Student’s t-tests. Welsh’s correction was used if variances were unequal. Values for CD8-depleted SM were excluded from the2 wpi data set.