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Activating Killer-cell Immunoglobulin-like Receptors are associated with the severity of COVID-19

Abstract Background Etiopathogenesis of the clinical variability of the coronavirus disease 2019 (COVID-19) remains mostly unknown. Here we investigate the role of Killer-cell Immunoglobulin-like receptor (KIR)/Human Leukocyte Antigen Class-I (HLA-I) interactions in the susceptibility and severity of COVID-19. Methods KIR and HLA-I genotyping and NK cell (NKc) receptors immunophenotyping in 201 symptomatic patients and 210 non-infected controls. Results NKcs with a distinctive immunophenotype, suggestive of recent activation (KIR2DS4 low CD16 low CD226 low CD56 high TIGIT high NKG2A high), expanded in patients with severe COVID-19. This was associated with a higher frequency of the functional A-telomeric activating KIR2DS4 in severe than mild/moderate patients and controls (83.7%, 55.7% and 36.2%, p<7.7x10 -9). In mild/moderate patients HLA-B*15:01 was associated with higher frequencies of activating B-telomeric KIR3DS1 compared to patients with other HLA-B*15 subtypes and non-infected controls (90.9%, 42.9% and 47.3%, p<0.002, Pc=0.022). This strongly suggests that HLA-B*15:01 specifically presenting SARS-CoV-2 peptides could form a neo-ligand interacting with KIR3DS1. Similarly, a putative neo-ligand for KIR2DS4 could arise from other HLA-I molecules presenting SARS-CoV-2 peptides expressed on infected/activated lung antigen presenting cells. Conclusions Our results support a crucial role of NKcs in the clinical variability of COVID-19 with specific KIR/Ligand interactions associated to disease severity.


INTRODUCTION
The new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) associated with the coronavirus disease-2019 (COVID-19) emerged in Wuhan (China) and spread worldwide, creating a global pandemic and a historic health and economic crisis [1]. Clinical spectrum of COVID-19 ranges from asymptomatic to pneumonia and the appearance of multiple complications such as acute respiratory distress syndrome, multiple-organ failure and death [2].
HLA-F, which is upregulated in local macrophages during viral infections, is a ligand for KIR3DS1 and KIR2DS4 [15,16].
To regulate activating signals, a repertoire of inhibitory receptors that survey HLA-I repress NKcs to protect healthy cells from inappropriate killing [17]. Although it may seem contradictory, NKcs become fully competent during a process known as "licensing", where inhibitory receptors interact with A c c e p t e d M a n u s c r i p t 6 (Sequential Organ Failure Assessment) scale was used to assess disease severity [26]. The disease was considered severe when patients required invasive mechanical ventilation (IMV). Serum and EDTA blood samples were obtained to determine biochemical, microbiological, hematological and immunological variables.

Modeling B-telomeric aKIR affinity for HLA-B*15 allotypes presenting SARS-CoV-2 peptides
Since KIR3DS1 residues interacting with HLA-B*15 presenting peptides have not been described, potential interacting residues (C-alpha8Å) were obtained by using the structure of its homologous KIR3DL1 (with 97% amino acid similarity of extracellular domains [31]) in complex with HLA-B*57:01 presenting peptide (PDB-ID: 5T70), and aligning the protein sequence of KIR3DL1 with that of KIR3DS1 (NCBI accession number: NP_001077008.1). Interaction score between the receptor interacting residues and the SARS-CoV-2 peptide was obtained as the mean affinity alignment score of total interacting residues. Interaction score of KIR3DL1/KIR3DS1 with its Bw4 ligand [32] was calculated to ascertain the affinity of natural occurring KIR/ligand interactions. For this analysis, potential KIR3DS1 residues interacting with the Bw4 epitope (HLA-B positions 77, 80, 81, 82, and 83 [33]) were identified based on the structure of KIR3DL1 in complex with HLA-B*57:01 (PDB ID: 3VH8).
Similar procedure was used to model KIR2DS1/HLA-B*15-peptide interactions. KIR2DS1 interacting residues were obtained using KIR2DL1 structure in complex with HLA-Cw4 presenting a peptide (PDB-ID: 1IM9) [34] and aligning the protein sequence of KIR2DL1 with that of KIR2DS1 (NCBI accession number: XP_011546300.1).

Statistical analysis
Data were analyzed using SPSS-24.0 (SPSS-Inc., Chicago, IL). Categorical variables were analyzed using frequency tables and Pearson's- 2 or two-tailed Fisher's exact tests. Bonferroni correction (Pc) was applied when needed. Mean and standard deviation (SD) were used for continuous variables and differences were estimated with ANOVA, Student's-t or Mann-Whitney-U tests based on the presence or absence of normal distribution. Association between variables was assessed with A c c e p t e d M a n u s c r i p t 8 binary logistic regression analysis, adjusted for confounding variables. Variables with p<0.05 in the univariate regression analysis were included in the multivariate analysis. The impact of fKIR2DS4 on the mean time for admission to the intensive care unit (ICU) was assessed using Kaplan-Meier and the Mantel-Haenszel log-rank test. The time was censored 30 days after the onset of symptoms. P<0.05 was considered significant. Table 1 summarizes biological, clinical and evolutionary characteristics of patients according to IMV requirements. Although no significant differences in age or sex were observed between patients requiring or not IMV, the former showed worst comorbidity and severity scores, biochemical and hematological parameters, more intense treatments and worst clinical evolution. Higher frequency of Caucasians than Latin-Americans required IMV probably due to their older age (59.0% vs. 53.5%, p=0.019).

fKIR2DS4 is associated with severe COVID-19
First we explored the role of HLA-I allotypes in the susceptibility and severity of SARS-CoV-2 infection.
A c c e p t e d M a n u s c r i p t No differences were found either for inhibitory or activating KIRs, AA and Bx centromeric and telomeric genotypes, or HLA-ligands (C1, C2, Bw4, or KIR2DS4-ligands), except for fKIR2DS4, which showed higher frequency in COVID-19 patients than in controls (61.7% vs. 36.2%, Pc<9.5x10 -6 ).
Consequently fKIR2DS4 could be considered as a risk factor for symptomatic COVID-19. Besides, fKIR2DS4 was associated to severe diseases with frequencies of 83.7% vs. 56.3% for patients who did or did not require IMV (p<0.001) ( Table 2).

KIR2DS4 + NKcs with a distinctive immunophenotype were expanded in COVID-19
To understand the relationship that KIR2DS4 + NKcs may have with disease severity, leukocyte subtypes and KIR + NKc repertoire were analyzed in healthy controls and patients without or with IMV.
Although the numbers of CD56 bright and CD56 dim NKcs as well as the repertoire of KIR + NKcs did not show significant differences between controls and patients, NKcs expressing KIR2DS4 as the only KIR receptor (single-KIR2DS4 + ) showed higher frequency in patients requiring IMV than in patients without IMV and controls (9.1%, 5.1%, and 4.3%, p<0.05) ( Figure 3B). When the expression as MFI of each KIR was studied in single-KIR + NKcs, no differences were found between the three study groups for KIR2DL1 + , KIR2DL2/S2 + , KIR2DL3 + , KIR2DS1 + , or KIR3DS1 + NKcs, whereas a slight increase of KIR3DL1 was found in patients requiring IMV compared to those that did not or to controls (7869.5, 6076.1 and 6083.9, MFI, p<0.01). However, the expression of KIR2DS4 showed a prominent downmodulation in both groups of patients compared to controls (5392.5, 6500 and 8721.6 MFI, p<0.001) ( Figure 3C). Since the expression of KIR receptors is down-modulated when interacting with their specific HLA-I ligand [35], we analyzed the expression of KIRs in presence of their HLA-ligands in COVID-19 patients. As already described, the presence of C2-ligand very significantly induced the down-modulation of KIR2DL1 (1847.5 vs. 1296.8, p<0.001). However, C1-ligands, Bw4-ligands and HLA-A*11/C*2/C*4/C*5/C*16 induced slight non-significant down-modulations of their respective KIR2DL2/S2-L3, KIR3DL1 and KIR2DS4 receptors ( Figure 3D). Therefore, the strong down-modulation observed for KIR2DS4 in COVID-19 patients did not appear to be associated with the presence of its HLA-ligands.
Additional immunophenotypic characteristics of KIR2DS4 + NKcs are shown in Figure 3E. CD16 expression showed no differences between patients and controls, but, in general, NKcs expressing aKIRs (KIR2DS1, KIR3DS1 or KIR2DS4) showed lower levels of CD16 than NKcs expressing iKIRs (p<0.05). CD56 expression was higher in patients than in controls on NKcs expressing activating A c c e p t e d M a n u s c r i p t 11 KIR2DS1 (p<0.01), KIR3DS1 (p<0.001) and KIR2DS4 (p<0.05), but not on NKcs expressing iKIRs.
CD226 expression was higher on all NKc subsets in patients that did not require IVM (p<0.01, for all KIR + subsets) than in controls and patients with IMV. CD226 showed lower expression on NKcs bearing aKIRs than in those bearing iKIRs (p<0.001). NKG2D expression was lower in patients than in controls for all NKc subsets, with reductions statistically significant on KIR2DL1 + (p<0.05), KIR2DL3 + (p<0.01) or KIR2DS1 + (p<0.001) NKc subsets. In contrast, TIGIT expression was higher in patients (particularly in patients with IMV) than in controls (p<0.05, for all KIR + subsets). Finally, although no significant differences were found between controls and patients in NKG2A expression , in general, KIR2DS4 + NKcs showed higher NKG2A expression than KIR3DS1 + (p<0.01) and the other KIR + NKc subsets (p<0.001). Altogether, KIR2DS4 + NKcs in severe COVID-19 showed a distinctive phenotype different from other NKc subsets consisting in KIR low CD16 low CD226 low CD56 high TIGIT high NKG2A high .

B-telomeric aKIRs are associated to milder COVID-19 in HLA-B*15:01 patients
Since the frequency of KIR2DS4-ligands HLA-A*11, C*02, C*04, C*05, and C*16 [36], all together or individually, did not show differences between controls and patients, we studied other putative HLAligands. Frequency of fKIR2DS4 remained within the ranges described for the global COVID-19 group   Bx-telomeric genotype was more frequent in HLA-B*15:01 patients that did not require IMV (84.6% vs. 15.4%, p=0.09), and therefore it was associated with milder COVID-19, which is reasonable since the frequency of the A-telomeric fKIR2DS4 (associated to severe disease) was decreased in these patients. Functionality and number of NKcs are significantly decreased in COVID-19 [3,4,37], and yet the underlying causes have not been explored. During the acute phase of the infection peripheral blood NKcs increase the express of inhibitory (NKG2A), regulatory (TIM-3) and exhaustion (PD-1) molecules and reduce the expression of activating receptors (DNAM-1 and NKG2D) and the secretion of IFNγ [38]. Two months after infection, some of these effects were still observed in patients from our study, particularly in those requiring IMV: reduced number of total lymphocytes, non-classical monocytes, neutrophils and CD4 + T-cell and increased number of CD8 + T-cells and single-KIR2DS4 + NKcs. These single-KIR2DS4 + NKcs showed a distinctive phenotype with a clear down-modulation of its KIR compared to healthy controls, most probably due to active interaction with specific ligands. KIRs are generally down-modulated when interacting with their HLA-ligand [35]. Although in our study KIR/HLAligand-induced down-modulation was observed for most single-KIR + NKc subsets, down-modulation induced by normal-constitutive HLA-I expression was much lower than that observed in single-KIR2DS4 + NKcs, suggesting that KIR2DS4 might interact with unconventional ligands. Like other single-aKIR + NKc subsets (KIR2DS1 + and KIR3DS1 + ), single-KIR2DS4 + NKcs showed reduced expression of CD16, CD226 and NKG2D, but highly expressed CD56 and TIGIT, suggesting that these cells had been recently involved in an activation event [39,40]. Supporting this idea is the NKG2A overexpression specifically observed in single-KIR2DS4 + NKcs, which is consistent with the strong NKG2A A c c e p t e d M a n u s c r i p t 14 over-expression described during the acute phase [41] and supports a primary role of KIR2DS4 + NKcs in the SARS-CoV-2 response.
In fact, the frequency of fKIR2DS4 gene was significantly increased in all COVID-19 patients, and particularly in those requiring IMV, 83.7% vs. 36.2% in controls. It has been recently suggested that frequency of HLA-C*05 (a KIR2DS4 ligand) was related to mortality variability in different COVID-19 populations [42]. However, in our series no differences between patients and controls were observed in the frequency of HLA-C*05 or KIR2DS4/HLA-C*05 interaction. In fact, fKIR2DS4 frequency did not change in patients bearing its putative ligands (HLA-A*11/C*02/C*04/C*05/C*16 [36]), which suggests that new ligands could be interacting with KIR2DS4 in COVID-19 patients. In 2013 Goodridge et al. suggested that open-conformer HLA-F could be a ligand for KIR2DS4 [43]; however, more recent results demonstrate that HLA-F open-conformer is a ligand for KIR3DS1 and KIR3DL2 but not for KIR2DS4 [44]. In contrast, peptide-bound HLA-F is not a ligand for KIR3DS1 because peptides directly hinder KIR3DS1/HLA-F interaction [44]. However, it is possible that a SARS-CoV-2 peptide presented in HLA-F could form a neo-ligand for KIR2DS4, in the same way that HLA-C*05:01 does present bacterial 9-mer peptides carrying a tryptophan at position-8 to become a ligand for KIR2DS4 [45].
Indeed, HLA-F can present peptides of unconventional length dictated by the R62W mutation that produce an open-ended groove accommodating long peptides [44]. In contrast, this putative neo-ligand formed by HLA-F presenting SARS-CoV-2 peptides will not be recognized by KIR3DS1, as previously indicated [44] and supported by our data, since the frequency of KIR3DS1 in COVID-19 patients did not differ from that in controls.
HLA-F is mainly expressed in activated lymphocytes and monocytes [46]. Alveolar macrophages, the predominant leukocytes in the lung, are strongly activated in COVID-19 patients and include viral particles within their cytoplasm [47], and therefore, they would be able to present SARS-CoV-2 peptides in HLA-F. Besides, SARS-CoV-2 induces the expression of CXCR3 ligands (CXCL9-11) in lung tissue [48] and macrophages [5] facilitating NKc recruitment from the peripheral blood [37].          A c c e p t e d M a n u s c r i p t 28