Hemophagocytic lymphohistiocytosis (HLH) is a rare but severe and life-threatening immune disorder; the prompt initiation of immunosuppressive treatment is therefore crucial for survival. HLH is characterized by the uncontrolled activation of lymphocytes and macrophages and the subsequent secretion of large amounts of pro-inflammatory cytokines (i.e. a “cytokine storm”) (1, 2).

Many different diseases can lead to HLH. Most forms of primary (or familial) HLH are caused by pathogenic genetic variants in the lymphocyte’s cytotoxic machinery (3, 4). Other genetic causes for HLH and macrophage activation syndrome (which are distinct from deficiencies in cytotoxicity) have been discovered recently (5). The so-called “acquired” forms of HLH can appear in a setting of severe infection, auto-inflammatory conditions, autoimmune disease, (lymphoid) malignancies or drug treatment (6).

The initial diagnosis of HLH or macrophage activation syndrome is based on a set of criteria (7, 8). The subsequent work-up seeks to identify a potential trigger and detect an underlying genetic predisposition to HLH. Prompt determination of the patient’s genetic predisposition to HLH significantly influences the subsequent care and the prognosis. While the clinician is awaiting a molecular diagnostic confirmation, rapid assays of protein expression and cell function have proven immensely beneficial in accelerating the diagnosis of primary HLH.

One of the best-established functional assays of degranulation deficiencies involves the detection of extracellular lysosomal associated membrane protein 1 (Lamp-1, CD107a); this protein is transiently exposed on the surface of cytotoxic cells after degranulation, which enables its measurement using flow cytometry (9). This assay is typically used to evaluate natural killer (NK) cell degranulation in response to the incubation of patient-derived peripheral blood mononuclear cells (PBMCs) with the erythroleukemia K562 cell line. The absence of the major histocompatibility complex class I antigen on K562 cells initiates cytotoxic granule delivery, which can be indirectly quantified by the measurement of CD107a expression on the NK cells. At present, this is the “gold standard” degranulation assay in diagnostic laboratories worldwide. It has outstanding sensitivity (nearly 100%) and slightly lower specificity (88%) (10, 11).

Although the K562-cell-line-based assay is well established, it has several limitations. Firstly, it is time-consuming and labor-intensive and requires a relatively large blood sample. Secondly, many external confounding factors (such as temperature changes during sample storage, and the K562 cells’ growth status, culture conditions, variations in K562 cell sourcing and passage) can introduce variability and reduce sensitivity. These variables can only be standardized in specialized laboratories with experience of performing NK cell degranulation assays (10, 11). Nevertheless, the introduction of the K562-based flow cytometry assay was a significant improvement over the previous cytotoxicity assays, which relied on radioactive chromium and thus required cumbersome radioactive safety monitoring procedures (12).

In order to overcome variability issues and make degranulation assays available to nonspecialized centers, other means of diagnosing deficiencies in cytotoxicity are needed. We therefore looked at whether the K562 cell line could be replaced by small antigenic molecules. When considering cells capable of cytotoxicity, the responsiveness of Vγ9Vδ2 T cells to phosphoantigens (pAgs) offers unique advantages (13). These include the homogeneity of the cell population’s response to stimulation and their robustness vis-à-vis long storage periods and cryopreservation. In some respects, yδ T cells expressing the Vγ9Vδ2 T-cell receptor (TCR) are similar to conventional cytotoxic αβ T lymphocytes because they form immune synapses and share the resulting signaling pathways (14). Vγ9Vδ2 T cells are not involved in major histocompatibility complex antigen presentation but rely on an alternative antigen presentation mechanism featuring butyrophillin 3 (BTN3) and BTN2A1 (15, 16). The Vγ9Vδ2 TCR combination is the most abundant among yδ T cells in the peripheral blood of healthy adults. Furthermore, the number of functional, perforin-producing cytotoxic Vγ9Vδ2 T cell is already high in the fetus and at birth relative to the less responsive and still immature NK cells (17).

HMBPP ((E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate, a pyro-phosphorylated metabolite of isoprenoid synthesis) is a pAg produced by eubacteria, cyanobacteria, plants, and apicomplexan protozoa. Its subsequent recognition by Vγ9Vδ2 T cells triggers a potent immune response (18).

The primary objective of the present study was to develop and evaluate a novel, rapid, easy-to-use functional assay based on Vγ9Vδ2 T cell degranulation, for the diagnosis of patients with suspected HLH. The novel assay was compared with the conventional K562 cell/NK cell co-incubation assay. We evaluated cell samples from patients with subsequently confirmed familial HLH and degranulation deficiency (hereafter referred to as the “fHLH group”) and from blood donors with no known degranulation deficiencies (hereafter referred to as the “control group”). The study’s secondary objective was to determine a threshold that could serve as a prospective cut-off for validation of the novel assay.

This was an empirically designed prospective study. To assess degranulation capacity, the percentage of CD107a expression on the cell surface was analyzed after stimulation of either NK cells with K562 cells or Vγ9Vδ2 T cells with HMBPP. The effect of cryopreservation on isolated PBMCs and the effect of interleukin (IL)-2 prestimulation were also explored.

The blood sample analysis was approved by the institutional-review-board, Zürich, Switzerland (BASEC; reference: PB_2016_02280) and registered at Clinicaltrials.gov (NCT02735824).

A total of 213 samples ( Table 1 ) from 182 individuals (n=12 with confirmed primary HLH and degranulation deficiency; n=170 in whom these conditions were excluded or unknown) were assessed with at least one of the assays (K562- or HMBPP-induced expression of CD107a on NK cells or Vγ9Vδ2 T cells, respectively). Repeated samplings and sample analyses were included. Of those individuals presenting with primary HLH, we analyzed 23 samples in total (20 samples of 10 individuals with UNC13D deficiency; 1 sample of 1 individual with STXBP2 deficiency; 2 samples of 1 individual with RAB27A deficiency, Table 2 ).

A novel functional assay of CD107 expression on Vγ9Vδ2 T cells in response to in vitro stimulation with HMBPP proved to be a user-friendly, efficient means of diagnosing degranulation deficiencies. Relative to the established NK cell degranulation assay, the new assay showed a higher specificity (78.9% vs. 86.2%, respectively) and the same, high sensitivity (100%). Furthermore, the novel assay was more reliable, less labor-intensive, more rapid, and less susceptible to errors than the NK cell degranulation assay. Given its decreased vulnerability to errors, we expect that the Vγ9Vδ2 T cell degranulation will necessitate fewer repetitions of analysis, making it especially suitable for testing infants.

The new Vγ9Vδ2 T cell degranulation assay showed a lower cut-off than the conventional NK cell degranulation assay and also distinguished more clearly between positive results (for healthy individuals) and negative results (individuals with a degranulation deficiency). These advantages reduced the need to repeat the assay, which is a crucial advantage in an urgent medical situation. We set the %CD107a cut-off to 7.5%, with a grey area (in which the test should be repeated) between 7.5% and 13.4% (the 25^th percentile of the CD107a expression for samples from control individuals without a degranulation deficiency). We also showed that the assay could be performed on cryopreserved PMBCs and that it revealed an increase in degranulation capacity after incubation of the PBMCs with IL-2 (19). The latter feature could be used to check results close to the cut-off and thus further characterize the PBMCs’ degranulation capacity.

As reported by Bryceson et al. (2012) and by our group, the NK cell degranulation assay shows high intra-sample variability in the %CD107a; this is mainly due to factors that influence the quality of the K562 cells and the PBMC sample. The novel Vγ9Vδ2 T cell degranulation assay is less susceptible to this variation. Moreover, there is no need to culture a cell line. Nevertheless, the interpretation of the novel assay’s results might not be reliable if too few Vγ9Vδ2 T cells are counted by flow cytometry. Detection of CD107a upregulation in both Vγ9Vδ2 T cells and NK cells poses inherent challenges due to their low frequency, particularly in scenarios where blood sample volumes are limited, as is often the case with infants. Furthermore, we found that the event count in the Vγ9Vδ2 T cell degranulation assay was sometimes low for cryopreserved PBMC samples. It remains to be established whether the cell membrane and/or other components were damaged by freezing. Furthermore, the literature data on the mechanism of HMBPP’s activation of the Vγ9Vδ2 T cells are contradictory: some reports mention an intracellular mechanism, whereas others mention an extracellular mechanism involving a transmembrane protein from the butyrophilin family (20).

We tested both assays with freshly isolated and cryopreserved PBMC samples, which is consistent with routine clinical practice. Although the use of cryopreserved samples in the Vγ9Vδ2 T cell degranulation assay expanded the sample size, the number of samples from patients with fHLH remained small. Other study limitations included the lack of double-blind testing, the single-center study design, and the relative short sampling period.

In the future, larger multicenter cohort studies will be required to define a consistent %CD107a cut-off for the Vγ9Vδ2 T cell degranulation assay. Furthermore, the degranulation of cord blood samples and PBMCs from adults, adolescents and infants should be analyzed to investigate potential age-related differences in control individuals. Moreover, sources of error in the Vγ9Vδ2 T cell degranulation assay and reasons for false negative results should be determined.

It is thought that a significant proportion of patients with primary HLH are not diagnosed because of a lack of awareness of the condition or its resemblance to sepsis. The lack of an accurate diagnosis or timely treatment ultimately lead to severe organ failure or death. Hematopoietic stem cell transplantation (HSCT) has potential as a curative treatment for primary HLH (21, 22). Post-HSCT outcomes are notably better when patients are free from active disease at the time of transplantation. In families with primary HLH, the overall survival rate is better for a second affected child than for the index sibling (23–26). Newborn screening therefore has significant potential for improving the prognosis in cases of primary HLH. Most rare disease experts agree that DNA sequencing could be used to screen apparently healthy newborns for treatable genetic disorders (27). Before sequencing-based newborn screening programs can be implemented, we must be able to distinguish between disease-causing variants and benign variants of all the screened genes (28). The Vγ9Vδ2 T cell degranulation assay might be a valuable addition to a newborn screening program as a means of functionally validating a positive genetic test results. It might also constitute a rapid screening method for prompt treatment initiation in an emergency context. Lastly, the low volume of blood required for the assay increases its feasibility.

In conclusion, a novel Vγ9Vδ2 T cell degranulation assay with HMBPP stimulation is more specific and faster than the conventional NK cell degranulation assay. The novel assay is less sensitive to error, is less labor-intensive, and enables the rapid diagnosis of a degranulation deficiency. In vitro IL-2 prestimulation prior to the assay helps to classify samples close to the cut-off. This Vγ9Vδ2 T cell degranulation assay is a feasible, very sensitive method for detecting degranulation deficiencies in freshly isolated and cryopreserved PBMCs.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by institutional-review-board, Zürich, Switzerland. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin.

OJ-M: Writing – original draft, Writing – review & editing, Investigation, Formal analysis, Methodology, Validation, Visualization. DP: Resources, Software, Investigation, Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Formal analysis. RT: Writing – review & editing, Formal analysis, Writing – original draft. HYT: Writing – original draft, Writing – review & editing, Investigation, Formal analysis. CR: Writing – original draft, Investigation, Writing – review & editing, Formal analysis. SP: Writing – review & editing, Resources, Writing – original draft. SV: Writing – original draft, Writing – review & editing, Conceptualization, Methodology. JPS: Supervision, Writing – review & editing, Writing – original draft, Resources, Project administration, Funding acquisition.