CD8+ T cells target cerebrovasculature in children with cerebral malaria

Abstract

BACKGROUNDCerebral malaria (CM) accounts for nearly 400,000 deaths annually in African children. Current dogma suggests that CM results from infected RBC (iRBC) sequestration in the brain microvasculature and resulting sequelae. Therapies targeting these events have been unsuccessful; findings in experimental models suggest that CD8+ T cells drive disease pathogenesis. However, these data have largely been ignored because corroborating evidence in humans is lacking. This work fills a critical gap in our understanding of CM pathogenesis that is impeding development of therapeutics.METHODSUsing multiplex immunohistochemistry, we characterized cerebrovascular immune cells in brain sections from 34 children who died from CM or other causes. Children were grouped by clinical diagnosis (CM+ or CM-), iRBC sequestration (Seqhi, Seqlo, Seq0) and HIV status (HIV+ or HIV-).RESULTSWe identified effector CD3+CD8+ T cells engaged on the cerebrovasculature in 69% of CM+ HIV- children. The number of intravascular CD3+CD8+ T cells was influenced by CM status (CM+ > CM-, P = 0.004) and sequestration level (Seqhi > Seqlo, P = 0.010). HIV coinfection significantly increased T cell numbers (P = 0.017) and shifted cells from an intravascular (P = 0.004) to perivascular (P < 0.0001) distribution.CONCLUSIONWithin the studied cohort, CM is associated with cerebrovascular engagement of CD3+CD8+ T cells, which is exacerbated by HIV coinfection. Thus, CD3+CD8+ T cells are highly promising targets for CM adjunctive therapy, opening new avenues for the treatment of this deadly disease.FUNDINGThis research was supported by the Intramural Research Program of the National Institutes of Health.

Keywords: Infectious disease; Malaria; Neuroimaging; Neuroscience; T cells.

Figure 1. Flow chart of patient classification.

Patients were first grouped by antimortem CM status as diagnosed on admission to the Paediatric Research Ward. Patients were further subcategorized based on analysis of postmortem H&E-stained sections from the same cerebral biopsy sample analyzed by MP-IHC. An iRBC sequestration cut-off of 23.1% between “hi” and “lo” was prospectively determined based on previous studies. These 3 groups were then further subcategorized based on HIV status.

Figure 2. CD3⁺CD8⁺ T cells within venous cerebrovasculature of CM children.

Shown are representative images of brain sections from CM⁺ Seq^hi HIV^– (A), CM⁺ Seq^lo HIV^– (B), and CM^– Seq⁰ HIV^– (C) patients. Images depict the distribution of CD3⁺ (red) CD8⁺ (green) T cells in relation to CD31⁺ (white) cerebrovasculature. DAPI-stained cell nuclei are shown in blue. Scale bars: 20 μm. Yellow asterisks denote the vascular lumen. Normalized counts of luminal, abluminal, and total CD3⁺CD8⁺ T cells per vessel area (log₂((number of cells/μm²/10⁴) + 2)) are described in D–F. Each symbol within a plot represents the normalized count of CD3⁺CD8⁺ T cells per vessel area for 1 of the 20 vessels examined for each child. Luminally, a significant increase in CD3⁺CD8⁺ T cells/vessel area was observed in CM⁺ Seq^hi (n = 8) patient brain sections relative to CM⁺ Seq^lo (n = 5; FDR P = 0.010) and CM^– Seq⁰ (n = 7; FDR P = 0.004) sections (D). P values were obtained via post hoc analysis using the difflsmeans function under FDR correction conditions following mixed-effects modeling with the lmer function. Significant differences remained so under bootstrap conditions 100% of the time when any normalized cell count for a single vessel was removed or when all vessels for any 1 child were removed. Error bars represent mean ± SD. Asterisks denote statistical significance: FDR **P ≤ 0.01.

Figure 3. Quantification of CD8⁺ T cells in cerebral arteries versus veins.

Normalized counts of CD3⁺CD8⁺ T cells per vessel area (log₂((number of cells/μm²/10⁴) + 2)) are described in A–E. Each symbol within a plot represents the normalized count of CD3⁺CD8⁺ T cells per vessel area for 1 of the 20 vessels examined for each child with the color denoting the vessel type (artery: blue; vein: red). A significant increase in venous CD3⁺CD8⁺ T cell counts was observed for CM⁺ Seq^hi HIV^– (A, n = 8; FDR P < 0.001) and CM⁺ Seq^hi HIV⁺ (B, n = 8; FDR P = 0.035). No significant difference in normalized CD3⁺CD8⁺ T cell counts was observed between arteries and veins for CM⁺ Seq^lo HIV^– (C, n = 5), CM^– Seq⁰ HIV^– (D, n = 7), or CM^– Seq⁰ HIV⁺ (E, n = 4). P values were obtained via post hoc analysis using the difflsmeans function under FDR correction conditions following mixed-effects modeling with the lmer function. Significant differences remained so under bootstrap condition 100% of time when any normalized cell count for a single vessel was removed or when all vessels for any 1 child were removed. Error bars represent mean ± SD. Asterisks denote statistical significance: FDR *P ≤ 0.05; ***P ≤ 0.001.

Figure 4. Granzyme B–loaded CD8⁺ T cells target cerebrovasculature during CM.

Shown are representative confocal images captured from a CM⁺ Seq^hi HIV^– patient brain section. Images depict the distribution of granzyme B (green) and CD8⁺ T cells (red) in relation to CD31⁺ (white) cerebrovasculature and autofluorescent RBCs (orange). Representative RBCs in A and B are denoted with small white asterisks. The vascular lumen is denoted with large yellow asterisks or the word “lumen.” The dotted pink lines in C and D delineate the border of the blood vessel walls. Cyan arrowheads denote granzyme B⁺ CD8⁺ T cells engaged with (A–C) or depositing granzyme B⁺ onto (D) CD31⁺ vasculature. The pink arrowheads in A denote a CD8^– granzyme B⁺ cell. Scale bars: 10 μm (A and B), 4 μm (C), and 2 μm (D).

Figure 5. Impact of HIV infection on the accumulation of CD8⁺ T cells in venous cerebrovasculature.

Representative images of brain sections from CM⁺ Seq^hi HIV⁺ (A), CM⁺ Seq^hi HIV^– (B), and CM^– Seq⁰ HIV⁺ (C) children. Images show the distribution of CD3⁺ (red), CD8⁺ (green) T cells in relation to CD31⁺ (white) cerebrovasculature. DAPI-stained cell nuclei are shown in blue. Yellow asterisks denote the vascular lumen. Scale bars: 20 μm. Normalized counts of luminal, abluminal, and total CD3⁺CD8⁺ T cells/vessel area (log₂((number of cells/μm²/10⁴) + 2) are provided in D–F. Each symbol represents the number of CD3⁺CD8⁺ T cells/vessel area for 1 of the 20 vessels examined per child. In CM⁺ Seq^hi children, HIV coinfection was associated with a decrease in CD3⁺ CD8⁺ T cells/vessel area, luminally (D, n = 8 for both; FDR P = 0.004). Conversely, in CM⁺ Seq^hi children, HIV coinfection was associated with an increase in CD3⁺CD8⁺ T cells/vessel area abluminally. HIV⁺ cases with (n = 8) or without CM (n = 4) also showed significantly more CD3⁺CD8⁺ T cells/vessel area abluminally than cases without HIV (n = 8) coinfection (E, FDR P < 0.0001 and FDR P < 0.005, respectively). Furthermore, CM⁺ Seq^hi HIV⁺ children had a greater total number of CD3⁺ CD8⁺ T cells/vessel area compared with CM⁺ Seq^hi HIV^– (F, FDR P < 0.017). P values were obtained via post hoc analysis using the difflsmeans function under FDR correction conditions following mixed-effects modeling with the lmer function. Significant differences remained so under bootstrap conditions 100% of time when any normalized cell count for a single vessel was removed or when all vessels for any 1 child were removed. Error bars represent mean ± SD. Asterisks denote statistical significance: FDR *P ≤ 0.05; **P ≤ 0.01; ****P < 0.0001.

Figure 6. Activated CD68⁺IBA1⁺ monocytes/macrophages in the venous cerebrovasculature.

Representative images of brain sections from CM⁺ Seq^hi HIV^–, CM⁺ Seq^lo HIV^–, and CM^– Seq⁰ HIV^– (A) patients and CM⁺ Seq^hi HIV⁺, CM⁺ Seq^hi HIV^–, and CM^– Seq⁰ HIV⁺ (E) patients. Images show the distribution of IBA1⁺ (green), CD68⁺ (red) monocytes/macrophages in relation to CD31⁺ (white) cerebrovasculature and DAPI-stained cell nuclei (blue). Yellow asterisks denote the vascular lumen. Scale bars: 20 μm. Normalized counts of luminal, abluminal, and total CD68⁺IBA1⁺ inflammatory monocytes/macrophages/vessel area (log₂((number of cells/μm²/10⁴) + 2) in the same ROIs used previously are given in B–D and F–H. Each symbol represents the number of CD68⁺IBA1⁺ cells/vessel area for each of the 20 vessels examined per child. There were significantly more activated monocytes/macrophages luminally in CM⁺ (n = 8) versus CM^– (n = 7) children (B, FDR P < 0.0001). CM⁺ Seq^lo HIV^– in B–D, n = 5. This is also reflected when comparing total cell numbers between both groups (D, FDR P < 0.030). Comparison of CM patients with (n = 8) and without (n = 8) HIV shows that CM alone promotes recruitment of activated monocytes/macrophages on the luminal aspect of cerebrovasculature (F, FDR P < 0.006). This observation is also evident when comparing CM⁺ Seq^hi HIV^– and CM^– Seq⁰ HIV⁺ (n = 4) patients (F, FDR P < 0.002). P values were obtained via post hoc analysis using the difflsmeans function under FDR correction conditions following mixed-effects modeling with the lmer function. Significant differences remained so under bootstrap conditions 100% of the time when any single vessel normalized cell count was removed or when all vessels for any 1 child were removed. Error bars: mean ± SD. Asterisks denote statistical significance: FDR *P ≤ 0.05; **P ≤ 0.01; ****P < 0.0001.