key: cord-0728358-lougobpy
authors: deKay, Joanne T.; May, Teresa L.; Riker, Richard R.; Rud, Jonathan; Gagnon, David J.; Sawyer, Douglas B.; Seder, David B.; Ryzhov, Sergey
title: The number of circulating CD26 expressing cells is decreased in critical COVID‐19 illness
date: 2022-03-16
journal: Cytometry A
DOI: 10.1002/cyto.a.24547
sha: 421c34f0834e1c4f89c40f9f4cf0726dc3761b76
doc_id: 728358
cord_uid: lougobpy

We evaluated the number of CD26 expressing cells in peripheral blood of patients with COVID‐19 within 72 h of admission and on day 4 and day 7 after enrollment. The majority of CD26 expressing cells were presented by CD3(+)CD4(+) lymphocytes. A low number of CD26 expressing cells were found to be associated with critical‐severity COVID‐19 disease. Conversely, increasing numbers of CD26 expressing T cells over the first week of standard treatment was associated with good outcomes. Clinically, the number of circulating CD26 cells might be a marker of recovery or the therapeutic efficacy of anti‐COVID‐19 treatment. New therapies aimed at preserving and increasing the level of CD26 expressing T cells may prove useful in the treatment of COVID‐19 disease.

resolution of inflammation via purinergic signaling modulation [8] . In this study, we characterized T cell subpopulations, and specifically CD26 expressing cells, in patients with COVID-19 and healthy donors to identify associations between the number of T cells and the severity, clinical features, and outcomes of COVID-19.

The study and informed consent process were approved by the Maine Medical Center Institutional Review Board. Informed consent was obtained from the patient or the legally authorized representative (LAR) using secure electronic consent to prevent disease transmission.

Patients were enrolled between July and December 2020 within 72 h of being hospitalized with PCR-confirmed SARS-CoV-2 infection (real-time RT-PCR test, NorDx Laboratories, Portland, ME). We excluded patients who were under 18 years of age, represent a vulnerable population, had a hemoglobin <8 g/dl, or from whom consent was not obtained.

COVID-19 disease severity was defined using clinical criteria from the US Centers for Disease Control and Prevention(CDC) [9] :

(1) mild illness: any signs and symptoms of COVID-19 (e.g., fever, cough, sore throat, malaise, headache, muscle pain) without shortness of breath, dyspnea, or abnormal chest imaging, (2) severe illness: respiratory rate > 30 breaths per minute, SpO 2 < 94% on room air at sea level (or, for patients with chronic hypoxemia, a decrease from baseline of >3%), a ratio of arterial partial pressure of oxygen to fraction of inspired oxygen (PaO 2 /FiO 2 ) <300 mmHg, or lung infiltrates >50%, and (3) critical illness: respiratory failure, septic shock, and/or multiple organ dysfunction.

Peripheral blood samples were obtained by the Maine Medical Center BioBank from asymptomatic and SARS-CoV-2 negative subjects as a control population. Pertinent demographic and clinical data were collected from the electronic medical record for all study participants.

The standard of care during hospitalization of patients with COVID-19 evolved during the study period as new information became available. It often included the antiviral drug remdesivir and synthetic adrenocortical glucocorticoid dexamethasone; many received systemic anticoagulation, and antibiotics for communityacquired pneumonia until it was determined they did not have concurrent bacterial infection. IL-1 or IL-6 inhibitors were not administered to these patients. Our COVID-19 cohort included a few asymptomatic patients admitted for other reasons who were determined to have SARS-CoV-2 infection on administrative testing, although most had severe or critical illness as described above.

Mechanically ventilated patients received lung-protective ventilation, proning for refractory hypoxemia, and standard critical care therapies (e.g., neuromuscular blocking agents, sedation, and analgesia). Patients may also have received renal replacement therapy (i.e., intermittent hemodialysis or continuous veno-venous hemofiltration) or arteriovenous or venovenous extracorporeal membrane oxygenation, as indicated.

Subjects underwent phlebotomy on the day of enrollment and, when feasible, at day 4 and day 7 post-enrollment. Blood (8.5 ml) was collected from COVID-19 and control subjects using BD Vacutainer ACD tubes.

Platelet-free plasma was prepared at room temperature using 2-step centrifugation, each at 2000 x g for 20 min. After processing, plasma was stored at À80 C until further analysis. No more than one freeze/thaw cycle was allowed for PFP samples to prevent protein degradation. 

Data are expressed as mean value and standard error for normal distribution or as median and interquartile range if the distribution is skewed. Comparisons between two groups were performed using Mann-Whitney tests. Comparisons between more than two groups were performed using Kruskal-Wallis test with Dunn's multiple-comparisons test, or two-way ANOVA with Tukey multiple comparisons test. Correlation analysis was performed using a Spearman (skewed distribution) correlation. Statistical analyses were performed with GraphPad Prism 7.05. p <0.05 was considered statistically significant.

A convenience sample of 29 patients with COVID-19 and 12 control subjects were analyzed. There were no differences between the study subjects and control subjects in terms of age, sex, and body mass index (Table 1) .

We examined the immune cell phenotype by flow cytometry. ( Figure 1D -F) and control subjects ( Figure 1G -I). CD73 is expressed on immunosuppressive T cells [10, 11] as opposed to CD26, which is highly expressed on Th1 and Th17 T effector cells [12] . The analysis revealed that the majority of CD26 pos cells are represented by CD3 + CD4 + T lymphocytes in both groups. The majority of CD73 expressing cells were B lymphocytes (data not shown). 

Low number of circulating lymphocytes is associated with severe and critical COVID-19 illness [13] [14] [15] . While cytotoxic T cells are crucial for the prevention of virus replication [16] , it has been shown that the activation of helper T cells contributes to the efficient immune Although the expression of CD26 can be found at low levels on natural killer cells, B cells, and monocytes [18] [19] [20] , only T cells are characterized by high levels of CD26 cell surface expression [4] . T cells also contribute to the pool of circulating soluble CD26 [21] .

and CD4 T cells [5, 7, 12] . Our data demonstrate that CD4 T cells represent a major subset of CD26 expressing cells in the circulation of patients with COVID-19. We also found that the number of CD4 but not CD8 T cells expressing CD26 is inversely associated with poorer outcomes. While we did not assess T cell functional status, it is plausible to suggest that this relationship reflects the role of CD26 + CD4 + T cells in the modulation of T cell cytotoxic activity, and that a higher number of these cells promote a more efficient immune response against SARS-Cov2.

The important role of CD26 expressing cells in the immune response to viral infections has been highlighted by previous studies.

and resolution [22] . A recent study demonstrated a decreased number of CD26 expressing T cells in HIV, specifically in progressors, symptomatic individuals with high-viral loads [23] . However, in a different study, no differences were found between the control group and progressors [24] . Both studies performed characterization of CD26 cells in relatively small cohorts of subjects, which may partially explain the controversy. Our study adds to the increasing evidence that CD26 expressing cells play a role in the pathogenesis of viral infections, including SARS-Cov2, and requires further investigation.

Sex is a contributing factor in infectious disease pathology [25] . A higher number of CD26/CD4 T lymphocytes have been found in healthy women compared to men. However, this difference has been abrogated in progressive HIV-1 [26, 27] . In this context, it is interesting that several studies demonstrated lower HIV viral load in women early during infection and no differences in disease progression between the sexes [28] . Female sex is associated with lower risks of death and intensive care unit treatments in patients with COVID-19 [29] . In our study, we also found an association between sex and lymphocytes, with a higher number of CD26/CD4 T cells in women. Age represents another risk factor in COVID-19 [30] . A complex CD26 expression pattern was found on CD8 T lymphocytes associated with a significant decrease in aging [31] . No differences, however, were found for CD26/CD4 cells. We also did not find any association between age and the number of CD26/CD4 T cells or the severity of COVID-19.

CD26 is also a binding protein for adenosine deaminase, an enzyme that catalyzes the conversion of adenosine to inosine [32] . Adenosine is an endogenous purine nucleoside characterized by potent immunosuppressive properties [33] [34] [35] [36] and may mediate inhibition of effective cytotoxic T cells response against the SARS-Cov2. T cells are highly mobile;

after egress from lymphoid organs, they circulate for a very short time before entering nonlymphoid tissue [37] . In agreement with the potential role of CD4 + CD26 + T cells in the regulation of adenosine availability, we found that only a small fraction of these cells co-expressed CD73, an adenosine-generating enzyme. Our data also demonstrated that the total number of CD73 expressing cells is significantly lower compared to indicating the potential role of these cells in recovery. It should be noted that the coordinated action of CD73 and adenosine deaminase results in the generation of inosine, a nucleoside that contributes to T cell cytotoxic activity [38] and down-regulation of inflammatory cytokine production [39] .

CD26 has been described as a candidate binding target for the SARS-Cov2 spike protein [3, 40] . This has led to the hypothesis that 

The co-authors have no disclosures.

The peer review history for this article is available at https://publons. com/publon/10.1002/cyto.a.24547.

https://orcid.org/0000-0002-7930-9814

Dipeptidyl peptidase-4 (DPP4) inhibition in COVID-19

Use of DPP-4 inhibitors in patients with COVID-19

Emerging WuHan (COVID-19) coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human CD26

Expression of CD26 (dipeptidyl peptidase IV) on resting and activated human T-lymphocytes

CD26: a multifunctional integral membrane and secreted protein of

The crystal structure of dipeptidyl peptidase IV (CD26) reveals its functional regulation and enzymatic mechanism

CD26-mediated co-stimulation in human CD8(+) T cells provokes effector function via pro-inflammatory cytokine production

CD26, adenosine deaminase, and adenosine receptors mediate costimulatory signals in the immunological synapse

COVID-19 Treatment Guidelines Panel. Coronavirus disease 2019 (COVID-19) treatment guidelines. National Institutes of Health

Therapeutically expanded human regulatory T-cells are super-suppressive due to HIF1A induced expression of CD73

CD73 is expressed by human regulatory T helper cells and suppresses proinflammatory cytokine production and helicobacter felisinduced gastritis in mice

Human CD26high T cells elicit tumor immunity against multiple malignancies via enhanced migration and persistence

Functional exhaustion of antiviral lymphocytes in COVID-19 patients

Lymphopenia predicts disease severity of COVID-19: a descriptive and predictive study

Lymphopenia as a biological predictor of outcomes in COVID-19 patients: a Nationwide cohort study

Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19

Antigen-specific adaptive immunity to SARS-CoV-2 in acute COVID-19 and associations with age and disease severity

Soluble CD26/dipeptidyl peptidase IV enhances the transcription of IL-6 and TNF-α in THP-1 cells and monocytes

Expression and functional role of dipeptidyl peptidase IV (CD26) on human natural killer cells

Functional role of CD26 on human B lymphocytes

Lymphocytes are a major source of circulating soluble dipeptidyl peptidase 4

Differential expression of CD26 on virus-specific CD8(+) T cells during active, latent and resolved infection

Systemic DPP4/CD26 is associated with natural HIV-1 control: implications for COVID-19 susceptibility

Low relative frequencies of CD26(+) CD4(+) cells in long-term nonprogressing human immunodeficiency virus type 1-infected subjects

SeXX matters in infectious disease pathogenesis

CD26/dipeptidyl peptidase IV (CD26/DPPIV) is highly expressed in peripheral blood of HIV-1 exposed uninfected female sex workers

Integration of microarray data and literature mining identifies a sex bias in DPP4+CD4+ T cells in HIV-1 infection

Sex differences in HIV infection

Male sex identified by global COVID-19 metaanalysis as a risk factor for death and ITU admission

Is older age associated with COVID-19 mortality in the absence of other risk factors? General population cohort study of 470,034 participants

T cell subset-specific susceptibility to aging

Characterization of adenosine deaminase binding to human CD26 on T cells and its biologic role in immune response

Adenosine receptors in regulation of dendritic cell differentiation and function

Adenosinergic regulation of the expansion and immunosuppressive activity of CD11b+Gr1+ cells

Role of A2a extracellular adenosine receptor-mediated signaling in adenosine-mediated inhibition of T-cell activation and expansion

The development and immunosuppressive functions of CD4(+) CD25(+) FoxP3(+) regulatory T cells are under influence of the adenosine-A2A adenosine receptor pathway

Lymphocyte subsets in the blood: a diagnostic window on the lymphoid system?

Inosine is an alternative carbon source for CD8+-T-cell function under glucose restriction

Inosine inhibits inflammatory cytokine production by a posttranscriptional mechanism and protects against endotoxin-induced shock

Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC

Sitagliptin treatment at the time of hospitalization was associated with reduced mortality in patients with type 2 diabetes and COVID-19: a multicenter, case-control, retrospective, observational study

Impact of comorbidities and glycemia at admission and dipeptidyl peptidase 4 inhibitors in patients with type 2 diabetes with COVID-19: a case series from an academic Hospital in Lombardy

Association between DPP-4 inhibitors and COVID-19-related outcomes among patients with type 2

An inflammatory cytokine signature predicts COVID-19 severity and survival

Interleukin-6 receptor inhibition in Covid-19-cooling the inflammatory soup

The potential for repurposing anti-TNF as a therapy for the treatment of COVID-19

Dexamethasone in hospitalized patients with Covid-19

The number of circulating CD26 expressing cells is decreased in critical COVID-19 illness