The central nervous system is a target of acute graft versus host disease in mice

Abstract

Despite significant advances in prevention and management, graft versus host disease (GVHD) is still a leading complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although skin, gut, liver, thymus, and lung are GVHD targets, neurological complications (NC) have also been reported following allo-HSCT. We demonstrate that the central nervous system (CNS) can be a direct target of alloreactive T cells following allo-HSCT in mice. We found significant infiltration of the CNS with donor T lymphocytes and cell death of neurons and neuroglia in allo-HSCT recipients with GVHD. We also found that allo-HSCT recipients with GVHD had deficits in spatial learning/memory and demonstrated increased anxious behavior. These findings highlight CNS sensitivity to damage caused by alloreactive donor T cells and represent the first characterization of target cell subsets and NC during GVHD. Therefore, these clinically relevant studies offer a novel and rational explanation for the well-described neurological symptoms observed after allo-HSCT.

Figure 1

Donor T cells with a distinct phenotype infiltrate the CNS during acute GVHD. Flow cytometric analysis and histopathological CNS findings in syngeneic and allogeneic transplanted animals. Lethal GVHD (0.5 × 10⁶ T cells) assessed at days 7, 14, and 21. (A-G) Mononuclear cells were isolated from brains of mice given syngeneic (5 × 10⁶ Thy1.1 BALB/c T cell–depleted bone marrow (TCD BM) cells + 5 × 10⁵ CD5+ Thy1.1 T cells transplanted into Thy1.2 BALB/c recipients) or allogeneic (5 × 10⁶ Ly5.1 B6 TCD BM cells + 5 × 10⁵ CD5+ B6 T cells transplanted into Thy1.2 BALB/c recipients) HSCT after complete perfusion on days 7 (n = 10-13), 14 (n = 13-14), and 21 (n = 48). (A) Representative flow cytometry plots detailing expression of CD3. (B) Absolute number of CNS infiltrating donor CD3+ T cells. (C) Origin of allogeneic CD3+ T cells (donor = CD45.2+H-2b+). (D) Absolute number of CD4+ or CD8+ allogeneic CD3+ T cells. (E) Absolute number of CD4+ and CD8+ T cells in syngeneic and allogeneic HSCT recipients in spleen (n = 8-10). (F) Representative plots and absolute numbers of naive (N, CD62L+CD44-), effector memory (EM, CD62L+CD44+), and central memory (CM, CD62L-CD44+) T cells in allo-HSCT recipients at day 21 (n = 31-37). (G) Expression of PSGL-1, α4-Integrin, LFA-1, LPAM, CXCR3, and CCR4 on CD4+ and CD8+ T cells in CNS and spleen at day 21 (n = 10-15). (H-M) Coronal sections were taken from paraffin-embedded brains 21 days after syn- (n = 10) or allo-HSCT (n = 10). (H) CD3 or CD4 staining. (I) Hematoxylin and eosin–stained section from an untreated mice demonstrating observed areas of the brain examined; cortex (Co), hippocampus (Hc), midbrain (Mb: thalamus, basal ganglia, hypothalamus), cerebellum (Ce), medulla oblongata (Mo), and choroid plexus (CP). Quantification of total CD3+ T cells (J) and CD4+ T cell (K) numbers in indicated areas. (L) Relative and (M) absolute distribution of CD3+ T cells in meninges/ependyma, parenchyma, and parenchymal vessels. Bar graphs represent mean ± standard error of the mean. All data are representative of at least 2 independent experiments. *P < .05; **P < .01; ***P < .001.

Figure 2

Cerebral alloreactive T-cell infiltration leads to cell damage and cognitive deficits. (A) Histopathological CNS findings in syngeneic and allogeneic transplanted animals. Lethal GVHD (0.5 × 10⁶ T cells) assessed at day 21 (Allo) or nonlethal GVHD (0.25 × 10⁶ T cells) assessed at days 42 (Allo) and 63 (Allo). Control groups received equal number of syngeneic T cells. GVHD score (0 within normal limits, ≥ 1 pathology) and estimated infiltration with lymphocytes (0 none/rare, 1 mild, 2 moderate, 3 marked, 4 severe) using immunohistochemistry for CD3. (B) Terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling staining in representative coronal brain sections 21 days after allo-HSCT in cerebellum gray matter (1, 2, 4), cortex (3), corpus callosum (5), hypothalamus (6), and hippocampus (7). (C) Wet brain weights after allo- and syn-HSCT (n = 13-14). (D) Accelerated Rotorod test measuring motor coordination by latency to fall. (E) Grip test measuring strength and neuromuscular function where latency to fall was measured. (F) Exploratory activity and anxiety was measured in an open field test where distance moved in center squares was measured. (G-H) Spatial memory and learning were assessed in the gold standard Morris water maze. (G) Time spent finding the platform over 5 consecutive days or (H) time spent in each quadrant were measured. (I) Motor function and mobility were assessed by measuring the total distance moved in the open field test. (J) Mobility was measured by examining the velocity in the Morris water maze. *Represents a comparison between syn-HSCT and allo=HSCT; ^Represents a comparison between untreated and allo-HSCT. For all behavioral tests, n = 20/treatment group. Bar graphs represent mean ± standard error of the mean of at least 2 independent experiments. *P < .05; **P < .01, ***P < .001.