Dose-Related Aberrant Inhibition of Intracellular Perforin Expression by S1 Subunit of Spike Glycoprotein That Contains Receptor-Binding Domain from SARS-CoV-2

Studies had shown that severe cases of COVID-19 tend to have high viral loads and correlate with functional impairment of cytotoxic lymphocytes, and the features of cytokine storm syndrome are similar to manifestations of severe influenza that have been partially explained by suppressed perforin expression. To test the hypothesis that the spike glycoprotein from SARS-CoV-2 may inhibit the perforin expression, we determined the kinetics of immune responses of CD8+ T cells to low dose (LD) or high dose (HD) of S1 stimulation through an in vitro dendritic cell (DC)-T cell model over seven days of incubation. The cytotoxic activity and intracellular perforin expression of CD8+ T cells induced by HD-S1-presenting DCs were aberrantly lower than those induced by LD-S1-presenting DCs from day three of incubation. Discrepantly, the levels of lymphoproliferation and cytokine (interferon-γ and tumor necrosis factor-α) production induced by HD-S1-presenting DCs were significantly higher than those induced by LD-S1-presenting DCs from day four. The dose-related responses between doses of S1 and intracellular perforin expression showed a significant linear correlation with a negative slope. In conclusion, the S1 subunit may suppress the perforin expression in CD8+ T cells to decrease the cytotoxic capacity to kill spike-presenting cells in a dose-dependent manner; the persistence of antigen presentation may result in an overproduction of interferon-γ and subsequent proinflammatory cytokines. That may help explain the insufficient cytotoxicity against high quantities of viruses or highly replicated strains of SARS-CoV-2 in severe cases of COVID-19.

Cytotoxic efficiency of human CD8+ T cell memory subtypes

Immunological memory is an important concept to protect humans against recurring diseases. Memory CD8+ T cells are required for quick expansion into effector cells but also provide immediate cytotoxicity against their targets. Whereas many functions of the two main cytotoxic subtypes, effector memory CD8+ T cells (TEM) and central memory CD8+ T cells (TCM), are relatively well defined, single TEM and TCM cell cytotoxicity has not been quantified. Here, we analyze human CD8+ subtype distribution following SEA stimulation and quantify the expression of death mediators, including perforin, granzyme B, FasL and TRAIL. We find higher perforin, granzyme B and FasL expression in TEM and compared to TCM. To quantify single TEM and TCM cytotoxicity, we develop a FRET-based fluorescent assay with NALM6 target cells stably transfected with a GFP-RFP FRET construct based on a caspase-cleavage sequence (apoptosis sensor Casper-GR). Applying this assay, TEM or TCM induced target cell apoptosis or necrosis can be quantified. We find that single TEM are much more effective than TCM in killing their targets mainly by apoptosis and secondary necrosis. The reason for this is the higher perforin expression and on a more efficient lytic immunological synapse during TEM-target contact compared to TCM-target contact. Defining and quantifying single TEM and TCM cytotoxicity and the respective mechanism should be helpful to optimize future subset-based immune therapies.

Hemophagocytic Lymphohistiocytosis Gene Mutations in Adult Patients Presenting With CLIPPERS-Like Syndrome

ObjectiveTo determine whether adult cases of Chronic Lymphocytic Inflammation with Pontine Perivascular Enhancement Responsive to Steroids (CLIPPERS) may be related to familial hemophagocytic lymphohistiocytosis (HLH) causes, we have screened patients with adult-onset CLIPPERS for mutations in primary HLH-associated genes.MethodsIn our cohort of 36 patients fulfilling the criteria for probable or definite CLIPPERS according to the CLIPPERS-2017 criteria, we conducted a first study on 12 patients who consented to genetic testing. In these 12 patients, systemic HLH criteria were searched, and genetic analysis of 8 genes involved in primary HLH was performed.ResultsFour definite and 8 probable CLIPPERS were enrolled (n = 12). Mutations involved in HLH were identified in 2 definite and 2 probable CLIPPERS (4/12). Three of them had biallelic PRF1 mutations with reduced perforin expression in natural killer cells. The remaining patient had biallelic UNC13D mutations with cytotoxic lymphocyte impaired degranulation. None of the mutated patients reached the criteria for systemic HLH. During follow-up, 3 of them displayed atypical findings for CLIPPERS, including emergence of systemic non-Hodgkin lymphoma (1/3) and confluent gadolinium-enhancing lesions on brain MRI (3/3).ConclusionsIn our patients presenting with adult-onset CLIPPERS, one-third have HLH gene mutations. This genetic treatable condition should be searched in patients with CLIPPERS, especially in those presenting with atypical findings.

Natural Killer (NK) Cell Expression of CD2 as a Predictor of Serial Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

NK cell ADCC supports monoclonal antibody anti-tumor therapies. We investigated serial ADCC and whether it could be predicted by NK phenotypes, including expression of CD16A, CD2 and perforin. CD16A, the NK receptor for antibodies, has AA158 valine or phenylalanine variants with different affinities for IgG. CD2, a costimulatory protein, associates with CD16A and can augment CD16A-signaling. Pore-forming perforin is essential for rapid NK-mediated killing. NK cells were monitored for their ADCC serial killing frequency (KF). KF is the average number of target cells killed per cell by a cytotoxic cell population. KF comparisons were made at 1:4 CD16pos NK effector:target ratios. ADCC was toward Daudi cells labeled with 51Cr and obinutuzumab anti-CD20 antibody. CD16A genotypes were determined by DNA sequencing. CD2, CD16A, and perforin expression was monitored by flow cytometry. Serial killing KFs varied two-fold among 24 donors and were independent of CD16A genotypes and perforin levels. However, high percentages of CD2pos of the CD16Apos NK cells and high levels of CD16A were associated with high KFs. ROC analysis indicated that the %CD2pos of CD16Apos NK cells can predict KFs. In conclusion, the extent of serial ADCC varies significantly among donors and appears predictable by the CD2posCD16Apos NK phenotype.

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

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.

Decrease of Perforin Expressing Lymphocytes after On-Pump Coronary Artery Bypass Grafting Surgery Irrespective of Carbohydrate Preoperative Oral Feeding

Background: Coronary artery bypass grafting (CABG) surgery continues to be the gold standard for treating the patients with coronary artery disease. CABG surgery can be performed on or off cardiopulmonary bypass, termed as on-pump or off-pump CABG, respectively. It has been shown that CABG surgery, preferably on-pump CABG surgery, leads to the changes of cell immunity during perioperative and early postoperative period. The mechanisms of regulation of the immune response in patients during and early after surgical revascularization are not fully understood. The aim of this study was to investigate the influence of carbohydrate preoperative oral feeding on frequency and perforin expression in peripheral blood lymphocytes in patients after on- or off-pump CABG surgery in early postoperative period. Patients and methods: In this prospective clinical study, 80 patients scheduled for CABG surgery were included in the study. The patients were randomly allocated into four groups (20 in each group): patients in Group 1 underwent on-pump CABG and did not receive carbohydrate preoperative oral feeding; patients in Group 2 underwent on-pump CABG and were preoperatively fed; patients in Group 3 underwent off-pump CABG and did not receive carbohydrate preoperative oral feeding; while patients in Group 4 underwent off-pump CABG and received carbohydrate preoperative oral feeding. Blood samples were collected immediately before (T1), 24 (T2) and 72 (T3) hours after the surgery. Peripheral blood mononuclear cells were isolated by gradient centrifugation and simultaneously labelled by antigens using fluorochrome-conjugated monoclonal antibodies. Frequency of T lymphocytes, NK and NKT cells, their subsets as well as their perforin expression were detected, and analyzed by flow cytometry. Results: There was significant decrease in frequency of CD3+ and CD3+CD4+ cells, as well as perforin expressing CD3+CD8+ cells in patients who underwent on-pump CABG in comparison to patients who underwent off-pump CABG 24 hours after the surgery. Carbohydrate preoperative oral feeding did not effect changes in lymphocytes subpopulations and perforin expression at any time point. Conclusion: Decreases of CD3+ cells on account of CD3+CD4+ subsets, and perforin expressing cells on account of CD3+CD8+ perforin+ cells were found in patients who had undergone on-pump CABG, but not in patients who had undergone off-pump CABG surgery, irrespectively of carbohydrate preoperative oral feeding.

Clinical and Genetic Features of Epstein-Barr Virus-Triggered Late-Onset Primary Hemophagocytic Lymphohistiocytosis: 10 Pedigrees Study

Introduction: It is widely accepted that primary Hemophagocytic Lymphohistiocytosis (PHLH) in infants and children is a genetic disease inherited in an autosomal recessive manner. However, it is proposed recently that the late-onset PHLH could result from the synergetic effects of the atypical heterozygous HLH-associated mutations and trigger of environmental factors such as viral infection. EBV is the most common viral pathogen linked to the development of HLH. EBV is known as a trigger to induce secondary HLH in some patients; it also can be a trigger of PHLH. However, the subpopulation of EBV infection in PHLH is unknown. Identification of EBV-infected lymphocyte subsets will help to clarify the role of EBV in the development of late-onset PHLH. This study identified the EBV infected cells subpopulation and summarized the clinical and genetic characteristics of 10 late-onset PHLH patients. Methods: Between June 2013 and June 2018, 10 patients with Epstein-Barr virus-triggered Late-onset PHLH and their family members were investigated, with a median age of 22.3 years (range, 12-45 years). We quantified EBV load by PCR, examined Natural killer (NK) cell activity by Lactate dehydrogenase (LDH) assay, and performed flow cytometry to analyze perforin expression and degranulation in addition to collecting clinical characteristics of 10 EBV-triggered PHLH patients. Next-generation sequencing was used to detect HLH-associated genes mutations. Immunobead sorting followed by quantitative PCR and fluorescence in situ hybridization were conducted to identify EBV-infected cells. We also performed pedigrees investigation and detected EBV loads and HLH-associated genes mutations in these patients' family members. Results: All patients had the acute onset and rapid progression of a disease and met the Diagnostic Guidelines for HLH (2004) proposed by the Histiocyte Society. Reduced NK-cell activity was observed in 6 late-onset PHLH patients (normal range: 30%-50%), while NK-cell activity defect (<5%) was found in 4 patients. Substantially reduced perforin expression (normal range: > 50%) on CD3-CD56+ NK cells were detected in 7 of 10 patients (70%). One of 10 cases (10%) had abnormal resting NK-cell degranulation, and 4 of 10 (40%) had defective resting NK-cell degranulation. Abnormal activated NK-cell degranulation (normal range: > 20%) was recorded in 3 of 10 cases (30%). Nine of 10 cases (90%) had either reduced perforin expression or decreased activated NK-cell degranulation. The EBV-DNA load and genetic characteristics of the late-onset PHLH cases and their families were summarized in Figure A. All the details of 10 pedigrees are shown in Figure B. The family members had the same type of mutations as the patients but did not develop the disease. High load of EBV was detected in the peripheral blood mononuclear cells of the probands, whereas a much lower load of EBV were found in the probands' family members by quantitative PCR. The results of sorting-PCR demonstrated that three group of cells (T cells, B cells, and NK cells) in all the patients were infected with EBV, in 9 of them EBV significantly infected NK cells, and only in 1 patient EBV infected both NK cells and T lymphocyte cells primarily. More than half of the patients (7 of 10) died of disease progression or complications, and median survival time is only 95 days. Only three patients who received allogeneic hematopoietic stem cell transplantation from unrelated donor acquired long-term survival, currently alive. Conclusions: The late-onset PHLH is a devastating disorder, and the prognosis is poor. Unlike sporadic HLH, EBV-associated late-onset PHLH patients usually have 1 or 2 pathologic mutations of PHLH-associated genes, reduced perforin expression or degranulation. Some of them have abnormal T or NK cells in bone marrow or lymph node; most of them have high load EBV infection, and the target cells of EBV infectious are usually NK cells, sometimes combined with T cells. HLH gene sequencing and pedigree investigation can help clinicians classify patients as having late-onset PHLH or sporadic HLH. Based on genetic mutations, EBV-Infected NK/T lymphocyte subtypes may be involved in the development of Late-onset PHLH. Figure. Figure. Disclosures No relevant conflicts of interest to declare.