A case report of clonal EBV-like memory CD4+ T cell activation in fatal checkpoint inhibitor-induced encephalitis

Abstract

Checkpoint inhibitors produce durable responses in numerous metastatic cancers, but immune-related adverse events (irAEs) complicate and limit their benefit. IrAEs can affect organ systems idiosyncratically; presentations range from mild and self-limited to fulminant and fatal. The molecular mechanisms underlying irAEs are poorly understood. Here, we report a fatal case of encephalitis arising during anti-programmed cell death receptor 1 therapy in a patient with metastatic melanoma. Histologic analyses revealed robust T cell infiltration and prominent programmed death ligand 1 expression. We identified 209 reported cases in global pharmacovigilance databases (across multiple cancer types) of encephalitis associated with checkpoint inhibitor regimens, with a 19% fatality rate. We performed further analyses from the index case and two additional cases to shed light on this recurrent and fulminant irAE. Spatial and multi-omic analyses pinpointed activated memory CD4⁺ T cells as highly enriched in the inflamed, affected region. We identified a highly oligoclonal T cell receptor repertoire, which we localized to activated memory cytotoxic (CD45RO⁺GZMB⁺Ki67⁺) CD4 cells. We also identified Epstein-Barr virus-specific T cell receptors and EBV⁺ lymphocytes in the affected region, which we speculate contributed to neural inflammation in the index case. Collectively, the three cases studied here identify CD4⁺ and CD8⁺ T cells as culprits of checkpoint inhibitor-associated immune encephalitis.

Extended Data 1.. Inflammatory and myeloid/microglial infiltrate in infarct regions of brain.

A) H&E stain shows a dense chronic inflammatory infiltrate in the meninges (upper left; black arrow), focal hemorrhage in the underlining brain parenchyma (lower left; red arrow), and gliotic gray matter; 20x. B) CD68 positive cells at the interface of the necrotic area (black arrow) and the adjacent brain parenchyma (red arrow) 20x. C) Limited expression of CD20 staining in perivascular and parenchymal regions of brain; 20x.

Extended Data 2.. Lymphocytic and myeloid infiltrate in meninges, perivascular, and parenchymal regions.

A) The lymphocytic infiltrate involving the meninges (upper image; upper left of image, black arrow), parenchyma and brain perivascular regions (upper image; lower right of image, red arrow and lower image) includes CD4 and (B) CD8 positive T cells; 20x. C) CD68 positive cells accompanying lymphocytic infiltrate in meninges (upper left of image, black arrow) and brain parenchyma (lower right, red arrow); 20x.

Extended Data 3.. Absence of substantial inflammatory infiltrate in radiologically and macroscopically non-affected area.

A) H&E corresponding to radiologically and macroscopically non-affected area with preserved architecture and no apparent inflammatory infiltrate; 20x. B) CD4 and (C) CD8 stains demonstrating sparse presence or absence of T cell infiltrates in radiologically and macroscopically non-affected area; 20x.

Extended Data 4.. PD-1 expression in inflamed region of brain.

PD-1 positive perivascular lymphocytes and pericytes; 20x.

Extended Data 5.. Expression of T cell and NK cell markers of immune cell exhaustion.

Inflamed and non-inflamed adjacent regions of neural tissue were immunostained for CD244, CD160, and LAG-3; 20x.

Extended Data 6.. RNAseq analysis of encephalitic and unaffected tissue.

A) Absolute and relative quantification of immune subsets by CIBERSORT. B) Interferon-γ-inducible genes (HALLMARK_INTERFERON_GAMMA_RESPONSE; M5913; 177 overlapping genes) quantified in inflamed and unaffected regions by RNAseq, as well as additional cases identified in Supplementary Table 3. *adjusted P value <0.0001 versus all other samples via ANOVA with Tukey’s post-hoc test.

Extended Data 7.. Analysis of repertoire overlap between inflamed brain and other resected and biopsied tissues.

In all panels, we display the frequency of TCRs detected in both the inflamed brain and the listed tissue using the ArcherDX Immunoverse platform, as well as the linear regression and its 95% confidence intervals on a log₁₀ scale. A) Lymph node biopsy. B) Prior mesentery resection. C) Brain recurrence scar; the TCR repertoire of the the brain recurrence scar and the D) uninflamed brain overlapped significantly with that of the inflamed brain (r² of 0.46 and 0.17, p < 0.0001 and p = 0.02 respectively). EBV-specific clones in both the inflamed brain and the spleen (E and F) were observed at high frequency, though the shared TCR repertoires detected in the brain and spleen were poorly correlated. G) Hierarchical clustering on the F2 distance metric was used to evaluate the similarity of the TCR repertoires from each of the tissue sites sampled from the patient. As also observed above, we the brain and spleen samples from 2016 and the time of death were most similar to each other, and that the brain-resident TCR repertoire appeared to be somewhat stable over time. The TCR repertoire of the lymph node nearest to the original tumor was highly distinct in comparison to the other samples.

Extended Data 8.. Overlap of HLA-A*02:01-restricted known EBV-specific TCR with CD8+ Ki67+ and GZMB+ phenotypes.

Representative images of the TCRβ RNAish probe overlaid with IHC markers.

Extended Data 9.. Evidence of latent EBV infection at the site of encephalitic inflammation.

A) EBER(1/2) staining of lymphocytes by RNA in situ hybridization in the cortex and meninges (B) of the encephalitic infarct. C) EBNA1 staining of lymphocytes by RNA in situ hybridization in the cortex and meninges (D) of the encephalitic infarct. E) Positive EBER staining of rare lymphocytes in lymph node resection pre-dating anti-PD-1 therapy, suggesting historic EBV infection. F) Positive EBER staining of rare tumor cells in brain metastasis resection pre-dating anti-PD-1 therapy, also suggesting historic EBV infection.

Extended Data 10.. Lack of detection of EBER+ cells in 2 additional cases of checkpoint-inhibitor encephalitis.

A) EBER stain of neural tissue from additional case 1 and additional case 2 (details of case in methods).

Figure 1:. Clinical course of anti-PD-1-induced encephalitis and histologic findings at autopsy.

A) Timeline of index patient diagnosis, prior therapies and anti-PD-1 therapy. B) (left) Magnetic resonance image (MRI) demonstrating restricted diffusion and hyper-intensity in the right temporal lobe and left basal ganglia from the day of hospital admission. (right) MRI showing progressive bilateral medial temporal encephalitis with bilateral putamen involvement. C) H&E stain shows intense perivascular lymphocytic infiltrate extending to adjacent brain parenchyma, next to a region of infarction (lower left); 20x. D) CD4 and CD8 IHC showing the lymphocytic infiltrate is composed of approximately equal ratios of CD4+ and CD8+ T cells; 20x. E) Diffuse PD-L1 positivity staining in cells with macrophage morphology surrounding the region of infarction; 20x. *Of particular note, tissues collected and assayed in this study anteceded treatment with steroids and anti-TNFα, which could impact the immunologic status of the patient.

Figure 2:. Digital spatial profiling of immune-related protein markers across inflamed and non-inflamed neural tissue.

A) Schematic describing DSP workflow. B). Representative region-of-interest selection under immunofluorescence for analysis. Tissue was stained with anti-GFAP (pink) and anti-CD3 (green) in addition to SYTO 83 nuclear staining for ROI visualization and selection. Circular regions 200 μm in diameter were excited with UV light to release barcodes for collection and analysis. C) Heatmap of area-normalized barcode counts from inflamed and unaffected tissue sections across 12 ROIs. Key antigens representing activation, T cell, and B cell markers are identified by arrows. D) Spatio-regional protein expression patterns across 8–10 ROIs from brain tissues demonstrating enrichment for T cell markers in encephalitis cases, but only memory activated markers in (Ki67^HI CD45RO^HI GZMB^HI) in ICI-encephalitis. E) Representative images of CD3+ T cells gated for barcode collection across 2 spatial ROIs in the inflamed region. F) Area-normalized counts for key antigens demonstrating enrichment of memory activated phenotypes (Ki67^HI CD45RO^HI GZMB^HI) in CD3+ population ROIs from ICI-induced encephalitis versus non-ICI-encephalitis.

Figure 3:. TCR sequencing identification of oligoclonal CD4+ cytotoxic T cells in inflamed encephalitic tissue.

A) ImmunoSEQ statistics of total cellular content, composition (fraction of T cells) and total T cells sequenced in inflamed and unaffected neural tissue. B) Enrichment of productive clonality in inflamed region of encephalitic tissue. C) Circos plot depicting the V(D)J rearrangements in TCRβ in the repertoire. D) Biplot demonstrating enrichment of shared TCRs among the unaffected (likely regional presence) and inflamed tissues. The amino acid sequence predicted from the most highly represented DNA sequence is identified (CASSFPSGSYEQYF). E) Representation of top 25 clones according to known EBV-specificity based on patient HLA haplotype. F) Sequence similarity in TCRβ amino acid sequence to a known EBV clone with specificity for the immediate-early HLA-A2-restricted epitopes GLC (BMLF1). G) Clonotype tracking over time and across tissue samples of EBV-specific and non-EBV-specific dominant clones found in the inflamed brain tissue. H) Dual IHC/RNA-ISH analysis for CD4/CD8 and EBV-like CASSFPSGSYEQYF RNA sequence. Upper left: only the TCR probe (pink); upper right: exclusion of TCR probe with CD8+ T cells, lower: Co-expression of CD4 with TCR probe. I) Co-expression of granzyme B with TCR probe J) Co-expression of CD45RO with TCR probe. K) Quantification for Ki67, CD45RO, and granzyme B (GZMB) expressed as percent of all CASSFPSGSYEQYF TCRβ+ cells, or the comparator CASSRGQGSADTQYF TCRβ sequence, a known HLA-A*02:01 restricted EBV clone.