Measles immune suppression: lessons from the macaque model

Abstract

Measles remains a significant childhood disease, and is associated with a transient immune suppression. Paradoxically, measles virus (MV) infection also induces robust MV-specific immune responses. Current hypotheses for the mechanism underlying measles immune suppression focus on functional impairment of lymphocytes or antigen-presenting cells, caused by infection with or exposure to MV. We have generated stable recombinant MVs that express enhanced green fluorescent protein, and remain virulent in non-human primates. By performing a comprehensive study of virological, immunological, hematological and histopathological observations made in animals euthanized at different time points after MV infection, we developed a model explaining measles immune suppression which fits with the "measles paradox". Here we show that MV preferentially infects CD45RA(-) memory T-lymphocytes and follicular B-lymphocytes, resulting in high infection levels in these populations. After the peak of viremia MV-infected lymphocytes were cleared within days, followed by immune activation and lymph node enlargement. During this period tuberculin-specific T-lymphocyte responses disappeared, whilst strong MV-specific T-lymphocyte responses emerged. Histopathological analysis of lymphoid tissues showed lymphocyte depletion in the B- and T-cell areas in the absence of apoptotic cells, paralleled by infiltration of T-lymphocytes into B-cell follicles and reappearance of proliferating cells. Our findings indicate an immune-mediated clearance of MV-infected CD45RA(-) memory T-lymphocytes and follicular B-lymphocytes, which causes temporary immunological amnesia. The rapid oligoclonal expansion of MV-specific lymphocytes and bystander cells masks this depletion, explaining the short duration of measles lymphopenia yet long duration of immune suppression.

Figure 1. MV infects high percentages of B-lymphocytes and CD45RA⁻ memory T-lymphocytes.

(A–D) Macroscopic detection of EGFP in lymphoid tissues of the gastro-intestinal tract in three different macaques: mesenteric lymph nodes (A), gut-associated lymphoid tissue (GALT) (B and C), including the Peyer's patches (C and D). Panel D is an enlargement of panel C (indicated by asterisk); (E–H) MV infection percentages in lymphocyte subsets during the approximate peak viremia. T-lymphocyte subpopulations were identified as Tⁿ (CD45RA⁺), T^CM (CD45RA⁻CCR7⁺) or T^EM (CD45RA⁻CCR7⁻), B-lymphocytes were identified as Bⁿ (CD27⁻IgD⁺) or B^M (CD27⁺IgD⁻). Box plots were chosen since the data were not normally distributed, and show the median infection percentages with the 25^th–75^th percentiles, error bars indicate the 10^th–90^th percentiles, dots the 5^th–95^th percentiles. The 10^th–90^th percentiles and 5^th–95^th percentiles are only shown if the number of observations is at least ten; **, P<0.01. *, P<0.05. In panel E, F and G 14 animals were included; in panel H 3 animals were included.

Figure 2. Susceptibility of human or macaque T-lymphocyte subsets to in vitro MV infection.

(A) Human or macaque PBMC were sorted into naive (CD45RA⁺, Tⁿ) or memory (CD45RA⁻, T^M) CD4⁺ or CD8⁺ T-lymphocytes, and infected with MV in vitro. Percentages MV-infected T-lymphocytes were determined 2 d.p.i. by measuring EGFP fluorescence by flow cytometry. CD4⁺ (human and macaque) and CD8⁺ (human only) T^M were significantly more susceptible to MV infection than the corresponding Tⁿ. For macaque CD8⁺ T-lymphocytes the difference was significant in two out of three experiments; (B) Unsorted human and macaque PBMC were infected, MV infection percentages in the different T-lymphocyte subsets were determined 2 d.p.i. by flow cytometry. Both in human and macaque PBMC the CD4⁺ and CD8⁺ T^CM and T^EM were significantly more susceptible to MV infection than the corresponding Tⁿ subpopulations. In addition, CD4⁺ T^EM and, to a lesser extent, CD8⁺ T^EM proved more susceptible to MV infection than T^CM. (C–F) Levels of CD150 expression on the different T-lymphocyte subsets in human and macaque PBMC. (C) PBMC collected from human or macaque donors were stained for memory markers as described in Figure S1, in combination with an IgG1 isotype control or CD150^FITC staining. CD150 expression on the different subsets is shown as geometric mean fluorescence intensity (Gmean FI) ± SD. Both for humans and macaques CD150 expression on CD4⁺ and CD8⁺ T^CM and T^EM was significantly higher than on Tⁿ. Interestingly, in CD4⁺ T-lymphocytes CD150 expression was significantly higher on T^EM than on T^CM, whereas in CD8⁺ T-lymphocytes an inverse pattern was observed. (D–F) An IgG1 isotype control was used to determine the level of background staining, and is shown in combination with the CD150 staining for each subset. **, P<0.01. *, P<0.05. Experiments were performed with sorted cells from 3 macaque and 2 human donors, and unsorted cells from 4 macaque and 5 human donors. Data are shown as means ± standard deviation (SD) of representative donors.

Figure 3. Histology and immunohistochemistry of lymphoid tissues obtained from macaques euthanized between 5 and 15 d.p.i.

Serial sections were stained for histological changes (H&E), MV infection (EGFP), B-lymphocytes (CD20), T-lymphocytes (CD3) or proliferating cells (Ki67). Multiple lymphoid tissues from multiple animals collected at each time-point were analyzed, panels shown are representative for the tissues that have been examined. The color intensity of the blue bars below the photomicrographs indicates the relative levels of viremia, lymphocyte depletion or proliferation. The green and red bars at the bottom indicate the appearance and disappearance of viremia and rash and correspond to the bars in Figure 5B.

Figure 4. MV infection causes temporary immunological amnesia.

(A) T-lymphocyte responses to PPD and MV were measured by IFN-γ production after in vitro stimulation of PBMC collected from 4 BCG-vaccinated macaques. Measurements were performed in triplicate, graphs shows means ± SD; (B) Mantoux tests were performed 7 days before (n = 4) and 8 (n = 2) or 10 (n = 2) days after MV infection. Images were collected 3 days after intra-dermal injection with tuberculin. Before MV infection classical delayed-type hypersensitivity responses were observed, associated with diffuse swelling and redness (indicated by arrows), after MV only a small localized papule was observed (indicated by arrow). H&E and CD3 staining of the corresponding skin tissues showed infiltration of T-lymphocytes in the dermis of the pre-infection Mantoux response, which was absent after MV infection. Representative images from 4 animals are shown.

Figure 5. A model for measles immune suppression.

(A) Relative population sizes of CD4⁺ or CD8⁺ Tⁿ, T^CM and T^EM in PBMC at different d.p.i., expressed as fold changes relative to 0 d.p.i. Means ± SEM of 9 animals are shown. (B). A model describing the changes in the relative size of pre-existing naive lymphocytes (blue), pre-existing memory lymphocytes (red, sum of T^M and follicular B-lymphocytes) and newly induced MV-specific (and bystander) lymphocytes (green) before, during and after measles. The relative WBC counts obtained from the macaques included in this study have been overlaid (black circles, means ± SEM). Thirty-four animals were included to obtain the WBC count graph. The red line indicates the time-point of MV-infection, bars above the graph indicate the approximate period of MV viremia (green), rash (red) and immune suppression (black).