Background/Purpose: VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is a severe auto-inflammatory disorder associated with acquired mutations in the UBA1 gene that occur in hematopoietic stem cells and persist in peripheral mature myeloid cells, but also, as recently described, in natural killer (NK) cells. VEXAS syndrome is associated with a high incidence of severe infections, especially with opportunistic pathogens. We hypothesized that the NK cell immune response may be impaired in VEXAS syndrome.

Methods: This was a non-interventional study in the setting of a local biological samples collected in routine care. A written informed consent was collected for all participants. Patients included were adults with severe autoinflammatory disease with (VEXAS) or without (VEXAS-like) UBA1 somatic mutations. We compared with gender-matched healthy controls (HCs). We performed an integrated immune analysis of peripheral NK cells including multiparameter phenotyping using CyTOF, single-cell gene expression analyses, cytokine profiling and whole blood cytokine response to Toll-like receptor (TLR) agonists challenge using the TrueCulture system.

Results: Forty-two VEXAS patients, 20 VEXAS-like and 36 HCs were included in the CyTOF analysis. Peripheral NK cells from UBA1-mutated individuals were quantitatively impaired with a median (IQR) count of 49/mm3 (19.5-122) vs 217 (138-346) in HCs (p< 0.0001) and 192 (135-276) in VEXAS-like, (p=0.0004). NK cells from VEXAS patients exhibited features of impaired cytotoxic capacity with decreased CD8a expression compared to HCs (58.3% vs 26.9% p< 0.0001), exhaustion with increased PD-1 (59.6% vs 29% in HCs p< 0.0001 and 44.2% in VEXAS-like p=0.03) and Tim3 expression (57% vs 27% in HCs p< 0.0001), and had aberrant CCR4 and CCR6 expression. VEXAS NK cells also had decreased CXCR3 expression (7.1% in VEXAS vs. 14.6% in HCs p=0.0002 and 14.2% in VEXAS-like p=0.001), which play a role in NK cell recruitment to tissues and promotes NK cell-mediated cytotoxicity.

Single-cell gene expression analysis revealed increased inflammatory signatures (including NFkB, TNFa, IL-6 and IL-1 signaling), unfold protein response, apoptosis and P53 signatures in NK cells from VEXAS patients. They also showed decreased cytotoxicity and Granzyme A-mediated apoptosis signatures, as well as IL-2 and IFN-y production signatures.

Peripheral blood cytokine levels showed similar results between the 3 groups for IL-2, IL-12, IL-15, IFN-y and granzyme B. In contrast, circulating levels of PDL1 were significantly increased in VEXAS patients with a median (IQR) of 135 pg/mL (115-166) vs 81 (67-91), p=0.0006 in HCs and 110 (87-140) in VEXAS-Like, p=0.02).

TruCulture assays shows impaired cytokine (IL-2, IL-15 and IFN-y) and Granzyme B production after whole blood stimulation with TLR4 (LPS), TLR7/TLR8 (R8478) and TLR3 (polyl:C) agonists.

Conclusion: Our results suggest that peripheral NK cells from VEXAS patients exhibit an exhausted phenotype with impaired cytotoxicity. This may play a role in the increased risk of severe infections associated with VEXAS syndrome.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/deep-phenotyping-characterization-of-peripheral-natural-killer-cells-reveals-impaired-cytotoxicity-and-exhaustion-during-vexas-syndrome/