Impact of HLA alloimmunization on clinical outcomes of severe aplastic anemia treated with immunosuppressive therapy

Abstract

Immune aplastic anemia (iAA) frequently results in transfusion dependence on platelets and packed red blood cells, increasing the risk for complications. The most common immune-mediated cause for platelet-transfusion refractoriness is alloimmunization with HLA antibody (Ab) to nonself class I antigens. The clinical impact of the HLA alloimmunization has not been well studied in patients with iAA. We investigated the clinical relevance of HLA alloimmunization in our large cohort of patients with iAA from 5 prospective trials and correlated with disease outcomes. Of 444 patients with severe AA treated with immunosuppressive therapy (IST), 99 (22%) had HLA alloimmunization. The presence of HLA Ab was associated with shorter overall survival, reduced responses to IST and higher risk of clonal evolution. Our data suggest that HLA alloimmunization is a marker of disease outcome. Furthermore, using single-cell RNA sequencing, we show enhanced activation of both complement-mediated pathways and the adaptive immune system in alloimmunized patients, indicating an interconnection between immune compartments.

Graphical abstract

Figure 1.

Overall survival. (A) OS curve for the combined cohort of 442 patients censored for HSCT, 88.4% at a median time to follow-up of 3.6 years (1320 days). (B) Kaplan-Meier survival curve depicting worse OS in HLA alloimmunized vs nonimmunized patients, P = .007. (C) Kaplan-Meier survival curves of patients segregated by sex and HLA alloimmunization status. Alloimmunized male patients had the worst reported OS, P = .004. (D) Kaplan-Meier survival curves at median survival time (1320 days): HLA class I Ab-negative nonresponders = 71.3%; HLA class I Ab-positive nonresponders = 51.4%; HLA class I Ab-negative responders = 96.3%; HLA class I Ab-positive responders = 93.5%. Statistically significant difference in OS between nonresponders to IST with HLA class 1 Ab-positive (pink curve) compared to responders with HLA class I Ab (blue curve), 51.4% vs 93.5%; P= .002. No difference in the OS between nonresponders regardless of HLA alloimmunization; P = .1. Pos, positive; Neg, negative.

Figure 2.

Response and clonal evolution. (A) Response rates to IST regimens in HLA alloimmunized patients compared to nonalloimmunized patients separated out by the degree of response. Total number of patients n = 430. Response to IST in HLA alloimmunized (positive) patients (48/85; 57%) and HLA nonalloimmunized (negative) patients (252/345; 73%); P = .004. (B) Alluvial plots of the improvement in response between 3-month and 6-month time points in HLA alloimmunized vs nonalloimmunized patients. (C) Kaplan-Meier curves depicting statistically significant difference in the risk of clonal evolution based on HLA alloimmunization status; P= .03. NE, not evaluable; NR, no response.

Figure 3.

Single cell RNA sequencing. (A) UMAP plot of single-cell gene expression in BMMNCs of HLA alloimmunized (n = 8) and nonalloimmunized (n = 12) patients with SAA. Cells are colored by type (HSPC; ProB; erythroblast, neutrophil at different differentiation stages, monocyte at different differentiation stages, CD8⁺ T cell, CD4⁺ T cell, NK cell, B/plasma, DC, stromal cell, and platelet). Frequency (% in BMMNCs) of cell populations in HLA alloimmunized SAA (n = 8) and nonalloimmunized (n = 12) patients with SAA, by 2-sided unpaired Mann-Whitney test. No statistical significance is observed. (B) A bar chart showing enriched gene sets by Gene Set Enrichment Analysis (GSEA). Enriched plots of differentially expressed genes for Hallmark complement, Hallmark interferon gamma response, Hallmark TNF-α signaling via NFkB, and Hallmark complement in BMMNCs of HLA alloimmunized compared with nonalloimmunized patients with SAA. GSEA based on the Kolmogorov-Smirnov test. (C) A network of upregulated genes involved in interferon gamma response, TNF-α signaling via NFkB, and complement pathway in those with postive HLA class I Ab (n = 8) compared to nonalloimmunized (n = 12) patients with SAA. B/Plasma, B cell and plasma cell; DC, dendritic cell; HSPC, hematopoietic stem and progenitor cells; IFN-γ, interferon gamma; NES, normalized enrichment score; NK, natural killer; ProB, B-cell progenitors.