Tracking Immune Cell Subsets and Activation Profiles in Adults with Haemophagocytic Lymphohistiocytosis Treated with Anakinra

Introduction

HLH is a hyper-inflammatory syndrome characterised by fevers, cytopenias and hemophagocytosis. The IL1-antagonist anakinra is approved by NHS-England, although efficacy across different triggers is unclear. We used full spectral flow cytometry and cytokine analysis pre and post anakinra for adult HLH to describe immune cell subsets and identify potentially targetable immune cells or cytokines.

Methods

Patients were diagnosed using the H-score. Blood was taken pre- and 72 hours post-anakinra. Leukocytes were analysed using full spectrum flow cytometry, using both manual and unsupervised analysis. Cytokine, chemokine and growth factor levels were measured using 48-Plex Bio-Plex Pro-Human Cytokine Screening Panel.

Results

Patient 1: 44 year old with lupus and EBV reactivation - complete response to anakinra and rituximab. At baseline high IL1ra, IL2, IL3, IL5, IL6, IL13, CXCL10 and HGF were seen; post treatment, IL-1 family members IL1α , IL1β and IL-18 decreased with >20 fold drop in IL2, IL6, IL13, CCL2, CXCL9, CXCL10 and HGF. IFNγ, elevated at baseline further increased. High % of monocytes and low % of CD4+ T-cells were present. T-cells were activated (CD38+/HLADR+) with high PD1 expression. The balance of CD8 memory phenotype was perturbed with high (early) effector and TEMRA T-cells. Only 7.43% B-cells were naïve and only 7.84% CD56^DIMCD16+ conventional NK-cells but very high (87.64%) unconventional CD56^DIMCD16- NK-cells. Post-treatment there were reduced monocytes, increased % CD8+ cells and marked increases in CD4+ and CD8+ effector memory cells and activated PD1+ CD4+ and CD8+ cells.

Patient 2: 71 year old with EBV+ve plasmablastic lymphoma - progression on anakinra. Baseline IL10 levels were very high with elevated IL1Rα, IL2, IL6, IL9, IL18, IFNγ, TNFα IL18, IL2ra and CXCL1. Post-treatment an 11-fold increase in IFN-γ was seen, and >2 fold increase in IL1α, IL2, IL4, IL7, IL12(p40). IL10 rose further. At baseline high monocytes and low total T-cells, particularly CD4+. Within CD8+ T-cells, low % of naive and central memory cells with high TEMRA cells, and high PD-1 expression on CD4+ and CD8+ cells were seen, increasing with treatment. High levels of CD56+ monocytes and unconventional NK-cells were seen. Post treatment increased T-regs and central memory CD4+ T-cells occurred.

Patient 3: 74 year old with T-cell lymphoma - no response to anakinra. Baseline elevated IL1Rα, IL2, IL9, IL12(p40), IL18, IFNγ, TNFα, CXCL1 and CXCL9 was seen, and post-treatment a 7-fold increase in IFN-γ, and >2 fold increase in IL1α, IL4, IL7, IL12(p40), TNFα and b-NGF. Neutropenia was present but relatively normal % monocytes and T-cells. In CD8+ but not CD4+ there were high levels of TEMRA. CD4+ and CD8+ cells showed high activation levels and PD-1 expression. 8.65% monocytes expressed CD56 rising post-treatment with concomitant increase in intermediate monocytes and reduced classical monocytes. NK-cells were predominately conventional.

Patient 4: 75 year old with EBV+ve Hodgkin lymphoma - no response to anakinra. Elevated IL1Rα, IL2, IL6, IL9, IL12(p40), IL18, MIF, IFNγ, TNFα, CXCL1 and CXCL9 was seen. Post treatment 8-fold increase in IFN-γ, and <2-fold increase in IL2 and b-NGF. As per patients 1 and 2, there were low monocytes and CD4+ T-cells and reduced % of naïve and central memory CD8+ T-cells and very high TEMRA. High activation and PD-1 expression were present in CD4+ and CD8+ cells. Very low baseline naïve B-cells were seen, partly reversing post treatment. High unconventional NK-cells were seen. There were more CD11c+ dendritic cells (DCs) and less plasmacytoid DCs than other patients which reversed post anakinra.

Discussion

Patient 1 had high levels of nearly all cytokines which, other than IFNγ, markedly reduced post-treatment. Patients 2-4 all had modestly raised IL-1 family and T-cell related cytokines and post-treatment increases in IL1α, ILβ, TNFα, IL4, IL17A, CCL2, LIF, bFGF and NGF. All patients had increased IFNγ post treatment. All patients displayed T-cell hyper-activation and exhaustion. EBV positive patients 1, 2 and 4 had a pattern of high % of monocytes, reduced CD4 T-cells, and CD8 compartment dominated by effector memory and TEMRA phenotype combined with a high % of unconventional NK-cells. We conclude there is marked variation in immune status in HLH. All patients had increased IFN-γ post anakinra, suggesting IFNγ or JAK1-2 inhibitors might allow combination strategies.

No relevant conflicts of interest to declare.

Anakinra is a recombinant, non-glycosylated form of the human Interleukin-1 receptor antagonist (IL-1Ra) that differs from the naturally occurring human interleukin-1 (IL-1) receptor antagonist by the addition of one N-terminal methionine. Anakinra is commissioned in England for the treatment of Haemophagocytic Lymphohistiocytosis (HLH) for adults and children of all ages, but does not have a license for this indication. Anakinra does have a license for other inflammatory diseases.