Functional and genetic testing in adults with HLH reveals an inflammatory profile rather than a cytotoxicity defect

Abstract

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory condition. Primary HLH occurs early in life as a result of monogenic biallelic mutations affecting lymphocyte cytotoxicity. Secondary HLH occurs mostly in adults secondary to infection, lymphoma, or rheumatic disease. In this latter setting, lymphocyte cytotoxicity status is not known. We conducted a systematic evaluation of natural killer (NK) cell cytotoxicity in adult patients with secondary HLH. Adult patients with secondary HLH were prospectively studied ex vivo for total lymphocyte count and subtype, NK cell phenotype, perforin expression and degranulation, and natural or antibody-dependent cell cytotoxicity, in comparison with patients affected by the same underlying disease without HLH (disease controls [DCs]) and with healthy controls (HCs). Screening for variants of cytotoxity genes was systematically performed. 68 patients were included in the HLH group and 34 each in the DC and HC groups. In HLH patients, severe and transient lymphopenia, activated NK cell phenotype (eg, increased CD69, ICAM-1, HLADR, and CCR5 expression), and decreased capacity of interferon γ production were observed; mean perforin expression was normal; and degranulation tests and NK cell cytotoxicity were not different from those in DCs. A monoallelic variant of uncertain significance affecting a lymphocyte cytotoxicity gene or the perforin variant A91V was observed in almost 50% of the patients. We detected no major intrinsic cytotoxicity dysfunction in secondary HLH patients compared with DCs and no predicted pathogenic gene variant. The activated NK phenotype profile associated with decreased interferon γ production seems similar to those of other hyperinflammatory diseases such as sepsis or systemic juvenile idiopathic arthritis.

Graphical abstract

Figure 1.

Lymphocyte counts in the 3 groups. Total lymphocytes (A), CD19⁺ B cells (B), NK cells (C), CD4⁺ T cells (D), and CD8⁺ T cells (E) in HC group (white plots), DC group (light gray plots), and HLH group (dark gray plots). Each point represents an outlier (error bars, 10th and 90th percentiles). *P < .05, ***P < .001, ****P < .0001 by Mann-Whitney U test.

Figure 2.

Evolution of lymphocyte counts during (HLH⁺) and after HLH recovery in 15 patients. Each line represents 1 patient. Lymphopenia (A) and NK (B) and T-cell (C) deficiency were corrected, but not B cells (D). **P < .01, ***P < .001 by Mann-Whitney U test compared with respective controls. ns, not significant.

Figure 3.

NK cell phenotyping in the 3 groups. Each point represents an outlier (error bars, 10th and 90th percentiles). ***P < .001, ****P < .0001 by Mann-Whitney U test (2 groups) and Kruskal-Wallis test (3 groups). ns, not significant.

Figure 4.

NK cell perforin expression in the 3 groups. (A) Percentage of NK cells expressing perforin (left) and perforin MFI (right). Each point represents 1 patient; bars indicate median values. (B) Evolution of the percentage of NK cells expressing perforin (left) and perforin MFI (right) during (HLH⁺) and after HLH recovery in 15 and 14 patients, respectively. Each line represents 1 patient. *P < .05 by Mann-Whitney U test (2 groups) and Kruskal-Wallis test (3 groups). ns, not significant.

Figure 5.

NK cell functional tests. (A) Degranulation evaluated by CD107/LAMP expression on NK cell surface after activation by K562 and Ig-coated P815 target cells. (B) IFN-γ–producing NK cells after activation by K562 and Ig-coated P815 target cells. Each point represents an outlier (error bars, 10th and 90th percentiles; box plots, median values and 25th and 75th percentiles). ***P < .001, ****P < .0001 by Mann-Whitney U test (2 groups) and Kruskal-Wallis test (3 groups). ns, not significant.

Figure 6.

Direct NK cell cytotoxicity depending on cell percentage (<5%, 5%-10%, >10%) of total lymphocytes. (A) Natural cytotoxicity is represented for the different E:T ratios. Each line represents 1 HLH patient. The gray waves represent normal values derived from HCs. (B) Evaluated in an E:T ratio of 25:1 (K25 for K562 targets and A25 for ADCC); no differences between the 3 groups. Each symbol represents an outlier (error bars, 10th and 90th percentiles). ns, not significant (P > .05) by Wilcoxon nonparametric test.