Background Hemophagocytic lymphohistiocytosis (HLH) is an often fatal disorder of infancy resulting from homozygous mutations in proteins involved in cytolysis (e.g. MUNC13-4, RAB27a, Perforin 1, Syntaxin11, STXBP2) [1]. HLH treatment is an etoposide based aggressive chemotherapy, and associated mortality remains problematic. Secondary forms of HLH, or macrophage activation syndrome (MAS), frequently result beyond infancy from rheumatologic, infectious, and oncologic conditions. MAS is typically treated with immunosuppression, including high dose corticosteroids, cyclosporine, and, recently, the IL-1 receptor antagonist, anakinra [2].

Objectives Of late, mutations in HLH associated cytolytic pathway genes have been identified in children with MAS [3-5]. The importance of these mutations in the pathophysiology of MAS, and the appropriate treatment for MAS in these settings remains unclear. Our goal was to assess the effect on NK cell lytic activity following over-expression of single copy mutations in HLH genes identified from MAS patients.

Methods We screened 21 children with MAS and identified 13 with mutations in 1 of the HLH genes. Three mutations [RAB27A (A87P), PRF1 (A91V), STXBP2 (A433V)] were studied for their effect on NK cell lytic activity, CD107a expression (degranulation), intracellular perforin and granzyme B levels, and granzyme B polarization (confocal microscopy). Lentiviral expression vectors were generated with wild-type (WT) or specific gene mutations, sequenced, and introduced into NK-92 cells to assess their effect on NK cell function. Instiutional Review Board approval was obtained from the University of Alabama at Birmingham for these studies.

Results Transduction of WT RAB27A into NK-92 had no effect on cytolysis of K562 cells, whereas the mutation decreased cytolytic activity by ∼50%. Both the PRF1 and STXBP2 mutants inhibited cytolysis to lesser degrees. The RAB27A mutant, but not the PRF1 or STXBP2 mutants, inhibited degranulation (diminished CD107a upregulation). All 3 mutants had no effect on intracellular perforin or granzyme B levels, or on conjugate formation with target cells. Confocal microscopy revealed the RAB27A mutant disrupted granzyme B polarization to the immunologic synapse.

Conclusions Single copy mutations in HLH genes are relatively common in MAS patients. These single copy HLH gene mutations can partially disrupt cytolytic activity via a dominant negative effect, thus, blurring the genetic distinction between HLH and MAS.

References

1. Janka GE. Annu Rev Med. 2012;63:233.

2. Ravelli A, Grom A, Behrens EM, Cron RQ. Genes Immun. 2012;13:289.

3. Zhang K, et al. Arthritis Rheum. 2008;58;2892.

4. Vastert SJ. Rheumatology (Oxford). 2010;49:441.

5. Zhang K, et al. Blood. 2011;118:5794.

Acknowledgements We acknowledge the patients and their families for participating.

Disclosure of Interest R. Cron Consultant for: Genentech and Novartis for Actemra and Canakinumab use in sJIA, respectively, M. Zhang: None Declared