Immune function in X-linked retinoschisis subjects in an AAV8-RS1 phase I/IIa gene therapy trial

Abstract

This study explored systemic immune changes in 11 subjects with X-linked retinoschisis (XLRS) in a phase I/IIa adeno-associated virus 8 (AAV8)-RS1 gene therapy trial (ClinicalTrials.gov: NCT02317887). Immune cell proportions and serum analytes were compared to 12 healthy male controls. At pre-dosing baseline the mean CD4/CD8 ratio of XLRS subjects was elevated. CD11c+ myeloid dendritic cells (DCs) and the serum epidermal growth factor (EGF) level were decreased, while CD123+ plasmacytoid DCs and serum interferon (IFN)-γ and tumor necrosis factor (TNF)-α were increased, indicating that the XLRS baseline immune status differs from that of controls. XLRS samples 14 days after AAV8-RS1 administration were compared with the XLRS baseline. Frequency of CD11b+CD11c+ DCc was decreased in 8 of 11 XLRS subjects across all vector doses (1e9-3e11 vector genomes [vg]/eye). CD8+human leukocyte antigen-DR isotype (HLA-DR)+ cytotoxic T cells and CD68+CD80+ macrophages were upregulated in 10 of 11 XLRS subjects, along with increased serum granzyme B in 8 of 11 XLRS subjects and elevated IFN-γ in 9 of 11 XLRS subjects. The six XLRS subjects with ocular inflammation after vector application gave a modestly positive correlation of inflammation score to their respective baseline CD4/CD8 ratios. This exploratory study indicates that XLRS subjects may exhibit a proinflammatory, baseline immune phenotype, and that intravitreal dosing with AAV8-RS1 leads to systemic immune activation with an increase of activated lymphocytes, macrophages, and proinflammatory cytokines.

Keywords: AAV8 vector; T cells; X-linked retinoschisis; cytokines; gene therapy clinical trial; immune function; ocular inflammation; retinoschisin.

Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Copyright © 2021. Published by Elsevier Inc.

Figures

Graphical abstract

Figure 1

CD4/CD8 ratio in XLRS at baseline and correlation to ocular inflammation (A) Representative flow cytometric plots for CD4 and CD8 populations gated on CD45+CD3+ cells for all groups. (B) The baseline CD4/CD8 ratio was calculated for XLRS subjects (n = 11) and compared to healthy controls (n = 12). Bar shows standard deviation. (C) Linear regression analysis for the baseline CD4/CD8 ratio in subjects manifesting ocular inflammation after vector dosing showed positive association to ocular inflammation scores.

Figure 2

Cell-mediated immune and serum cytokine changes in XLRS at baseline Cell-mediated immune cell populations were analyzed by flow cytometry, and serum analytes were analyzed by cytometric bead array in 11 XLRS subjects and 12 healthy controls. Bars represent standard deviation. Significant frequency changes are identified in DCs. (A) Representative flow cytrometric plots for CD11c+ mDCs and CD123+ pDCs gated on CD56−HLA-DR+ cells. (B and C) CD123+ pDCs had an elevated frequency (B), while CD11c+ mDCs showed decreased proportions compared to healthy controls (C). Baseline values for serum cytokines/chemokines of XLRS subjects were evaluated compared with controls. (D–F) Serum levels of both IFN-γ and TNF-α were elevated (D and E), and EGF was downregulated (F). p < 0.05 was considered significant.

Figure 3

Systemic immune cell alteration after AAV8-RS1 vector dosing Relative immune cell proportions in peripheral blood were evaluated for each XLRS subject before and after vector dosage. Representative flow cytometric plots and heatmaps were constructed for evaluating changes in each subject. (A and B) CD68+CD80+ macrophages (A) and CD8+HLA-DR+ activated cytotoxic T cells (B) were upregulated in 10 XLRS subjects 14 days after vector injection. (C) CD11b+CD11c+ regulatory DCs were downregulated in 8 of 11 XLRS subjects after vector injection.

Figure 4

Serum cytokine changes after AAV8-RS1 vector dosing (A and B) Pro-inflammatory cytokine granzyme B (A) and IFN-γ (B) levels were elevated on day 14 after vector injection. Heatmap analysis of each subject showed that granzyme B was upregulated in five of six subjects that had ocular inflammation at day 14 after vector dosing. IFN-γ did not show any particular trend with vector dose and was generally upregulated.

Figure 5

Association of pro-inflammatory systemic immune analytes to ocular inflammation after vector dosing Linear regression analysis identified two immune analytes associated with the ocular inflammation scores in XLRS subjects after vector dosing. (A and B) CD68+CD80+ macrophages (R2 = 0.69; p = 0.04) (A), and granzyme B (R2 = 0.50; p = 0.11) (B) are both pro-inflammatory immune factors that play a major role in cytotoxic immune reaction.