Thymosin Alpha 1 Reduces the Mortality of Severe Coronavirus Disease 2019 by Restoration of Lymphocytopenia and Reversion of Exhausted T Cells

Abstract

Background

Thymosin alpha 1 (Tα1) had been used in the treatment of viral infections as an immune response modifier for many years. However, clinical benefits and the mechanism of Tα1 treatment for COVID-19 patients are still unclear.

Methods

We retrospectively reviewed the clinical outcomes of 76 severe COVID-19 cases admitted to 2 hospitals in Wuhan, China, from December 2019 to March 2020. The thymus output in peripheral blood mononuclear cells from COVID-19 patients was measured by T-cell receptor excision circles (TRECs). The levels of T-cell exhaustion markers programmed death-1 (PD-1) and T-cell immunoglobulin and mucin domain protein 3 (Tim-3) on CD8⁺ T cells were detected by flow cytometry.

Results

Compared with the untreated group, Tα1 treatment significantly reduced the mortality of severe COVID-19 patients (11.11% vs 30.00%, P = .044). Tα1 enhanced blood T-cell numbers in COVID-19 patients with severe lymphocytopenia. Under such conditions, Tα1 also successfully restored CD8⁺ and CD4⁺ T-cell numbers in elderly patients. Meanwhile, Tα1 reduced PD-1 and Tim-3 expression on CD8⁺ T cells from severe COVID-19 patients compared with untreated cases. It is of note that restoration of lymphocytopenia and acute exhaustion of T cells were roughly parallel to the rise of TRECs.

Conclusions

Tα1 treatment significantly reduced mortality of severe COVID-19 patients. COVID-19 patients with counts of CD8⁺ T cells or CD4⁺ T cells in circulation less than 400/μL or 650/μL, respectively, gained more benefits from Tα1. Tα1 reversed T-cell exhaustion and recovered immune reconstitution through promoting thymus output during severe acute respiratory syndrome–coronavirus 2 infection.

thymosin alpha 1, COVID-19, SARS-CoV-2, T-cell exhaustion, immune reconstitution

The 2019 novel coronavirus disease 2019 (COVID-19) epidemic, which was first reported in December 2019 in Wuhan, China, has been recognized as a pandemic by the World Health Organization [1–3]. As of 13 May 2020, China had reported 84 451 cases of confirmed COVID-19 and 4644 fatalities. Meanwhile, the number of confirmed COVID-19 cases and fatalities worldwide were 4 098 018 and 283 271, respectively [4].

Lymphocytes play an essential role in fighting against viral infections; therefore, boosting the number and enhancing the antiviral function of T cells in COVID-19 patients is of paramount value for successful recovery. However, most COVID-19 cases displayed severe lymphocytopenia, especially in the elderly and severe cases [2, 5–7]. Thymosin-α-1 (Tα1), a type of polypeptide hormone produced by thymic epithelial cells, can effectively increase T-cell numbers; support T-cell differentiation and maturation; and reduce cell apoptosis [8–10]. Therefore, Tα1 has been successfully used in clinical practice to treat patients infected with hepatitis B, hepatitis C (HCV), and human immunodeficiency viruses (HIV), and its efficacy has been proven by pathological observation [11–13]. To enhance immunity, all medical support team members from China received a Tα1 injection before being deployed to Hubei Province, and no infectious cases have been reported, suggesting Tα1 might have the potential to prevent severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2) infection. Nevertheless, no data are available on whether Tα1 treatment has any benefits for critically ill COVID-19 patients.

Here, we retrospectively analyzed the clinical data from 76 critically ill COVID-19 patients who were admitted to General Hospital of the Central Theatre Command and Wuhan Pulmonary Hospital in Wuhan, China, from December 2019 to March 2020. We also compared the expression of exhaustion markers programmed death 1 (PD-1) and T-cell immunoglobulin and mucin domain protein 3 (Tim-3) on CD8⁺ T cells and analyzed the thymus output ability of COVID-19 patients after Tα1 treatment. Our results provide a preliminary demonstration that Tα1 has benefits for COVID-19 patients, especially those with severe lymphocytopenia.

METHODS

Patients

Medical records of severe/critical COVID-19 patients (aged 21–92 years) admitted to General Hospital of the Central Theatre Command or Wuhan Pulmonary Hospital were collected and retrospectively analyzed. Diagnosis and classification of clinical types of COVID-19 were based on the New Coronavirus Pneumonia Prevention and Control Program (5th edition) published by the National Health Commission of China. In brief, a patient who meets any of the following conditions is diagnosed as severe: respiratory distress with respiratory rate ≥30 breath/min, oxygen saturation ≤93% on room air, and arterial blood oxygen partial pressure/fraction of inspired oxygen ≤300 mm Hg (1 mm Hg  =  0.133 kPa). A patient who meets any of the following conditions is diagnosed as critical: respiratory failure and requiring mechanical ventilation support, shock, and multiple organ dysfunction syndrome and requires intensive care unit (ICU) admission. Only severe and critical COVID-19 patients with hospitalization duration of at least 10 days were included in the study. Based on the presence or absence of 7 days of continuous Tα1 treatment, patients were placed into treatment group or the nontreatment group. The study’s endpoint was recovery or death.

Tα1 Management

In the treatment group, patients received subcutaneous injections of 1.6 mg Tα1 once a day for at least 7 consecutive days; continued use was recommended until the end of the study. Prior to administration, the lyophilized powder was reconstituted with 1 mL of the provided diluent (sterile water for injection). After reconstitution, the final concentration of Ta1 was 1.6 mg/mL. The workflow chart is shown in Figure 1.

Figure 1.

Workflow chart. Receipt of Tα1 treatment in combination with standard or conventional medical therapies is defined as the treatment group and treatment with standard or conventional medical therapies only is defined as the control group. Abbreviations: COVID-19, coronavirus disease 2019; PD-1, programmed death 1; Tα1, thymosin alpha 1; Tim-3, T-cell immunoglobulin and mucin domain protein 3; TREC, T-cell receptor excision circle.

Data Collection

We reviewed clinical records, nursing records, laboratory findings, and chest X rays or computed tomography (CT) scans for all patients. All information was obtained and curated with a customized data collection form. Two investigators (YP Liu and Y Chen) independently reviewed the data collection forms to verify data accuracy.

PD-1 and Tim-3 Assay

Peripheral blood samples from patients were harvested with anticoagulants (EDTA-K₂). Lymphocytes were gated from CD45⁺ leukocytes using anti-CD45-APC antibodies (SK1, BD Biosciences). The expression of exhaustion markers on CD8⁺ T cells was further detected using CD8-PE (SK1, Biolegend), PD-1-PE-CY5 (EH12.2H7, Biolegend), and TIM-3-FITC (F38-2E2, Biolegend) antibodies. After being stained, the cells were measured using flow cytometry on an LSRFortessa cell analyzer (BD Biosciences), and data were analyzed using FlowJo software (TreeStar). All experimental procedures were completed under biosafety level II plus condition.

T-cell Receptor Excision Circle Assay

Peripheral blood mononuclear cells (PBMCs) were harvested by density gradient centrifugation in the central laboratory of General Hospital of the Central Theatre Command. The T-cell receptor excision circle (TREC) assay was performed using DNA extracted from the patients’ PBMCs. Amplification reactions (20 μL) contained approximately 20 ng of genomic DNA, 10 μL of 2× Premix Ex Taq master mix (Takara Bio Inc, Japan), and the appropriate primers and probes. Polymerase chain reaction (PCR) conditions, including primers and probe sequences, have been described previously [14]. Reactions were carried out in a Roche LightCycler96 detection system (Roche Applied Science, Germany). TREC copies in a given sample were estimated by comparing the cycle threshold (Ct) value with a standard curve obtained from PCRs performed with 10-fold serial dilutions of an internal standard. Amplification of RNAseP (Applied Biosystems) was used to verify the quantity and presence of genomic DNA. TREC values were adjusted for total DNA content.

RESULTS

Tα1 Reduces COVID-19 Patient Mortality

To fight against virus infection, it is necessary to boost T-cell numbers and their antiviral functions in COVID-19 patients because there is no specific drug for SARS-CoV-2 infected patients. Tα1, a type of polypeptide hormone that can regulate T-cell production, differentiation, and activity, was selected to treat COVID-19 patients. A total of 76 severe or critical COVID-19 patients from 2 designated hospitals were enrolled in this retrospective cohort study based on the preset inclusion criteria. Based on the presence or absence of 7 days of continuous Tα1 treatment, 36 patients were placed into the treatment group and 40 patients were placed into the nontreatment group. There were no significant differences between the 2 groups in terms of demographic characteristics and clinical findings at the time of admission, such as age, sex, coexisting conditions, total lymphocyte count, T cells, CD8⁺ T cells, CD4⁺ T cells, partial pressure of carbon dioxide (PCO₂) on admission, and PCO₂ on discharge. Moreover, all patients received antiviral and antibacterial treatment during hospitalization. Glucocorticoid and oxygen inhalation were also very common. The Tα1-treated group had a slightly higher level of interleukin (IL)-6 than the untreated group (P = .047), whereas partial pressure of oxygen levels on admission and at discharge were slightly higher in the Tα1-treated group than in the nontreatment group (Table 1). Ta1 has been approved by the US Food and Drug Administration (FDA) and the China FDA and is a very well tolerated drug. No side effects associated with Ta1 were found after a thorough review of all medical records of the 36 participants in the treatment group. Two patients in the treatment group underwent noninvasive mechanical ventilation, while 11 patients underwent noninvasive mechanical ventilation in nontreatment group. Nine of the 11 cases in the control arm underwent subsequent invasive mechanical ventilation owing to hypoxia that could not be adjusted by noninvasive mechanical ventilation. None in the treatment arm were intubated. More interestingly, 11.11% (4/36) of patients who received Tα1 treatment died, whereas the mortality of severe COVID-19 patients without Tα1 treatment reached 30% (12/40) (Figure 2, Table 1), suggesting that Tα1 treatment can reduce mortality.

Table 1.

Clinical Characteristics of Severe/Critically Ill Coronavirus 2019 Patients With or Without Thymosin Alpha 1 Treatment

Characteristic	Treatment Group (N = 36)	Nontreatment Group (N = 40)	P Value
Thymosin treatment time (days)	17.5 (14–27.75)	Not applicable
Age (y)	56 (41.3–69.8)	57.5 (53.50–73.75)	.3620
Male (%)	69.44 (25/36)	50 (20/40)	.0850
Coexisting disorders (%)	38.89 (14/36)	42.5 (17/40)	.7494
Mortality (%)	11.11 (4/36)	30 (12/40)	.0437
Intensive care unit length of stay of survivors (days) (N = 32 vs 27)	6.5 (4.25–10.75)	4 (1–14.0)	.2300
Hospital stay of survivors (days) (N = 32 vs 28)	22.5 (16.85–34.25)	20.4 (18.76–26.31)	.3140
Total T cells on admission (*10^6/L) (N = 34 vs 20)	632.5 (365–812)	612 (293–461)	.7880
CD8+ T cells on admission (*10^6/L) (N = 34 vs 20)	208 (95–298)	190.5 (118–280)	.9860
CD4+ T cells on admission (*10^6/L) (N = 34 vs 20)	330 (175–411)	320 (132–597)	.6800
Total T cells of discharge-total T cells on admission Ratio (N = 21 vs 20) (%)	1.65 (1.23–2.32)	3.78 (3.65–5.27)	.1410
CD8+ T cells of discharge-CD8+ T cells on admission ratio (N = 21 vs 20) (%)	1.68 (1.23–2.18)	3.05 (2.50–4.44)	.1030
CD4+ T cells of discharge-CD4+ T cells on admission ratio (N = 21 vs 20) (%)	1.5 (1.28–1.50)	4.13 (3.30–9.70)	.1200
Neutrophils on admission (*10^9/L)	3.95 (2.54–5.31)	3.95 (2.55–6.36)	.818
Lymphocytes on admission (*10^9/L)	0.97 (0.64–1.35)	0.73 (0.46–0.70)	.0770
Neutrophil-Lymphocyte ratio on admission	3.88 (2.54–7.29)	4.37 (2.74–9.83)	.657
Interleukin-6 on admission (pg/mL) (N = 36 vs 33)	24 (17.13–48.48)	10.4 (7.30–37.25)	.0470
PCT on admission (ng/mL) (N = 36 vs 33)	0.07 (0.05–0.115)	0.06 (0.04–0.125)	.838
C-reactive protein on admission (mg/L) (N = 36 vs 39)	15.95 (8.95–48.37)	19.55 (13.96–65.45)	.560
Ferritin on admission (ng/mL) (N = 11 vs 12)	890.8 (437.4–1707.0)	472.4 (254.4–1016.1)	.684
PO2 on admission (N = 25 vs 17) (kPa)	10.7 (9.75–15.3)	9.33 (7.74–13.32)	.0320
PO2 of discharge (N = 20 vs 18) (kPa)	12.6 (9.77–16.68)	9.7 (6.57–12.35)	.0420
PO2 of discharge-PO2 on admission ratio (N = 20 vs 17) (%)	1.1 (0.96–1.28)	0.95 (0.76–0.95)	.1700
Time from admission to measurement of PO2 (days) (N = 20 vs 17)	8 (5.00–18.50)	8 (5.50–9.00)	.3740
Antiviral treatment (%)	100 (36/36)	100 (40/40)	1.0000
Oseltamivir (%)	88.9 (32/36)	87.5 (35/40)	.852
Lopinavir/Ritonavir (%)	50 (18/36)	40 (16/40)	.381
Ribavirin (%)	50 (18/36)	40 (16/40)	.381
Interferon-α (%)	58.3 (21/36)	47.5 (19/40)	.345
Arbidol (%)	5.6 (2/36)	7.5 (3/40)	.733
Traditional Chinese medicinea (%)	91.7 (33/36)	80 (32/40)	.149
Antibacterial treatment (%)	100 (36/36)	100 (40/40)	1.0000
Moxifloxacin (%)	75.0 (27/36)	62.5 (25/40)	.242
Ceftriaxone (%)	58.3 (21/36)	22.5 (9/40)	.001
Cefoperazone sulbactam (%)	52.8 (19/36)	65.05 (26/40)	.279
Meropenem (%)	52.8 (19/36)	25.0 (10/40)	.013
Levofloxacin (%)	8.3 (3/36)	27.5 (11/40)	.031
Azithromycin (%)	5.6 (2/36)	7.5 (3/40)	.733
Oxygen inhalation (%)	83.33 (30/36)	95 (38/40)	.0980
Glucocorticoid (%)	66.67 (24/36)	95 (38/40)	.0015
Intravenous (%)	100 (24/24)	100% (38/38)	1.000
Duration of corticosteroid treatment (days)	7 (4.25–16.75)	5 (4.00–8.00)	.121
Total dose of methylprednisolone (mg)	885 (205–1390)	400 (260–585)	.076
Dose of methylprednisolone per day (mg)	81.18 (57.28–116.69)	68.57 (50.29–91.66)	.326
Low-to-moderate doses of corticosteroids (%) (25–150 mg/d methylprednisolone or equivalent)	91.7 (22/24)	92.1 (35/38)	.951
High-dose corticosteroid therapy (%) (>150 mg/d methylprednisolone or equivalent)	8.3 (2/24)	7.9 (3/38)	.951
Noninvasive mechanical ventilation (%)	5.56 (2/36)	27.5 (11/40)	.0198
Invasive mechanical ventilation (%)	0 (0/36)	22.5 (9/40)	.0027
Blood purification (%)	2.78 (1/36)	0 (0/40)	.2886

Based on the presence or absence of 7 days of continuous thymosin alpha 1 treatment, the 36 patients were in the treatment group and 40 patients were in the nontreatment group. Data are median (interquartile range) or % (n/N), where N is the total number of patients with available data. P values are from the χ ² test or Mann-Whitney U test.

Abbreviations: PO₂, partial pressure of oxygen; PCT, procalcitonin.

^aTraditional Chinese medicine primarily included lianhuaqingwen capsules and ganmao qingre granules.

Figure 2.

(A) Survival rate, (B) NMV (free-%), and (C) IMV (free-%) of severe or critical coronavirus 2019 patients with or without Tα1 treatment. Two patients underwent NMV in the treatment group, and 11 patients underwent NMV in the nontreatment group. Nine of the 11 patients underwent IMV because hypoxia could not be adjusted by NMV. *P < .05. Abbreviations: IMV, invasive mechanical ventilation; NMV, noninvasive mechanical ventilation; Tα1, thymosin alpha 1.

Tα1 Enhances T-cell Counts in COVID-19 Patients With Severe Lymphocytopenia

Next, we investigated whether Tα1 can restore T-cell numbers in COVID-19 patients with severe lymphocytopenia. A total of 34 cases with a record of T-cell numbers on admission and after 7 continuous days of Tα1 treatment were included in this part of the study. Results showed that Tα1 treatment effectively restored T-cell numbers in cases with counts of CD8⁺ T cells or CD4⁺ T cells less than 400/μL or 650/μL, respectively. Patients whose T-cell numbers were higher than the above-mentioned levels gained no benefits from Tα1 treatment (Figure 3A). Tα1 also dramatically increased the CD8⁺ and CD4⁺ T-cell counts in elderly patients (>60 years old; Figure 3B) and in patients with comorbidities of hypertension or cardiovascular disease (Figure 3C). Collectively, these data demonstrate that Tα1 promotes timely T-cell recovery in COVID-19 patients with severe lymphocytopenia.

Figure 3.

Tα1 significantly enhanced T-cell counts in COVID-19 patients with severe lymphocytopenia. A, Tα1 treatment effectively restored T-cell numbers in cases with counts of CD8⁺ T cells or CD4⁺ T cells less than 400/μL or 650/μL, respectively. B, Tα1 dramatically increased the counts of CD8⁺ and CD4⁺ T cells in the elderly (>60 years). Enhancement of T-cell numbers by Tα1 treatment was also observed in cases with comorbidities of hypertension (C) or cardiovascular diseases (D). *P < .05; **P < .01; ***P < .001. Abbreviation: ns, not significantly different; Tα1, thymosin alpha 1.

Tα1 Reveres CD8⁺ T-cell Exhaustion by Enhancing Thymus Output

In our previous studies, we demonstrated that SARS-CoV-2 infection induces acute T-cell exhaustion, which might lead to ineffective elimination of viruses in vivo [7]. In the current study, we determined whether Tα1 affects T-cell exhaustion in severe COVID-19 patients. Hence, the expression of PD-1 and Tim-3 on CD8⁺ T cells was detected using flow cytometry. Because of the retrospective nature of this study, peripheral blood samples from 22 enrolled patients, including 10 patients from the nontreatment group and 12 patients from the Tα1-treatment group, were available at the time of PD-1/Tim-3 analysis. Results showed that Tα1 effectively downregulates both PD-1 and Tim-3 on CD8⁺ T cells in those who received Tα1 treatment compared with those in the nontreatment group (Figure 4A), suggesting that Tα1 can partially reverse T-cell exhaustion during SARS-CoV-2 infection.

Figure 4.

Tα1 treatment enhanced thymus output and reversed T-cell exhaustion in COVID-19 patients. A, The percentage of PD-1 and Tim-3 on CD8⁺ T cells in severe COVID-19 patients without or with 7 continuous days of Tα1 treatment. B, T-cell receptor excision circle levels in peripheral blood mononuclear cells of severe COVID-19 patients before and after 7 days of Tα1 treatment. **P < .01; ****P < .0001. Abbreviations: PD-1, programmed death 1; Tα1, thymosin alpha 1; Tim-3, T-cell immunoglobulin and mucin domain protein 3; TREC, T-cell receptor excision circle.

Next, we determined whether the reversion of CD8⁺ T-cell exhaustion in COVID-19 patients by Tα1 is due to enhancement of thymus-derived naive T cells in circulation. TRECs are specific circular DNA by-products that are formed during the random rearrangement of T-cell receptors (TCRs), and they are only present in cells exported from the thymus but not found in replicating cells within PBMCs [15]. To determine whether Tα1 administration affects thymus output in COVID-19 patients, we measured TRECs in PBMCs of patients before and after 7 days of Tα1 treatment. Interestingly, the Tα1-treated group manifested significantly higher TREC levels than untreated controls (Figure 4B), suggesting that Tα1 has the capacity to enhance thymus output and thus enhance TCR diversity and finally reverse CD8⁺ T cell exhaustion.

DISCUSSION

Here, we retrospectively reviewed and found that Tα1 significantly reduces mortality of severe COVID-19 patients compared with those in the untreated group (Figure 2). Further study showed that Tα1 effectively enhances T-cell counts in COVID-19 patients with severe lymphocytopenia, especially in cases with counts of CD8⁺ or CD4⁺ T cells less than 400/μL or 650/μL, respectively (Figure 3A). Based on these results, we strongly recommend that COVID-19 patients especially whose CD8⁺ T-cell or CD4⁺ T-cell counts are less than 400/μL or 650/μL, respectively, receive Tα1 treatment to improve their immune function. Additionally, the duration of Tα1 treatment should be at least 7 days. Importantly, enhancement of T cells was also observed in elderly patients and in patients with coexisting conditions, including hypertension and cardiovascular disease, after 7 days of continuous Tα1 treatment (Figure 3B–D). We therefore suggest that healthy people aged >60 years receive Tα1 treatment to prevent potential SARS-CoV-2 infection because the elderly’s immune systems also respond to Tα1, even if the thymus is extremely atrophied.

Immune system damage is very common in COVID-19 patients, and most critically ill cases manifest severe lymphocytopenia [6, 16]. Tα1 has been found to regulate T-cell development and enhance T-cell numbers. It has also been used in many clinical settings in which T-cell immunity is involved, including aging, viral infectious diseases, autoimmune disorders, and immune reconstitution after bone marrow transplantation [17–19]. Tα1 also showed promise in larger clinical studies of acute infections. Several clinical studies demonstrated that Tα1 has benefit in the treatment of severe sepsis, which begins with a bacterial or fungal infection [20]. Interestingly, Tα1 treatment also significantly increased the number of CD4⁺ and CD8⁺ cells in patients with cytomegalovirus infection accompanied by acute respiratory distress syndrome after renal transplantation [21]. We extended this field of study and found that Tα1 treatment effectively boosts both CD4⁺ and CD8⁺ T-cell numbers in COVID-19 cases with severe lymphocytopenia.

The naive CD8⁺ T cells undergo robust proliferation and clonal expansion to differentiate into effector CD8⁺ T cells that directly kill target cells during acute infection. CD4⁺ T cells, on the other hand, primarily assist B cells in producing antibodies and clearing pathogens. However, some CD8⁺ T cells lose their effector functions and become exhausted during chronic infections and cancer where antigen stimulation persists. Nevertheless, revitalization of exhausted T cells can reinvigorate immunity. The exhausted T cells express elevated inhibitory receptors such as PD-1 or TIM-3 [22]. Interestingly, recent work has confirmed that exhausted T cells exist in acute viral infections, and T cells from patients in the acute phase of Ebola infection manifest with enhancing PD-1 expression but impaired interferon (IFN)-γ production [23]. Moreover, blocking the Tim-3 signal efficiently enhanced IFN-γ secretion from T cells following H1N1 infection [24]. In our previous work, we were the first to report that SARS-CoV-2 triggers the expression of PD-1 and Tim-3 on T cells, suggesting that exhausted T cells were persistent in COVID-19 patients during SARS-CoV-2 acute infection [7]. Here, we found that Tα1 effectively downregulates both PD-1 and Tim-3 on CD8⁺ T cells in COVID-19 patients (Figure 4A), suggesting Tα1 might also boost immune response in hosts by reversing T-cell exhaustion during acute viral infection.

Inflammation promotes exhausted T-cell differentiation. For example, IL-6, a pleiotropic cytokine that plays an essential role in regulating immune responses, can rescue lymphocytes from exhaustion following HCV infection [25]. IL-10 is an inhibitory cytokine that has the capacity to induce T-cell exhaustion, and blocking IL-10 function has been shown to successfully prevent T-cell exhaustion following chronic lymphocytic choriomeningitis virus infection [26, 27]. Severe and critically ill COVID-19 patients have very high levels of serum IL-6 and IL-10, and these patients also display high levels of PD-1 and Tim-3 on T cells, suggesting that both IL-6 and IL-10 might be mechanistically responsible for mediating exhausted T-cell differentiation in COVID-19 patients [7]. Moreover, serum levels of other inflammation factors, including procalcitonin (PCT), C-reaction protein (CRP), and ferritin, on admission were also very high in COVID-19 patients (Table 1). However, the outbreak of SARS-CoV-2 infection was an extreme emergency in February 2020, and we regret not having the capacity to measure serum IL-10, IL-6, and inflammation markers, including PCT, CRP, and ferritin, in Tα1-treated patients. Additional work is needed to clarify the exact mechanism that underlies Tα1 downregulation of PD-1 and Tim-3 expression on CD8⁺ T cells.

The number of lymphocytes from the thymus decreases in elderly persons due to thymus dysregulation and atrophy. Consequently, the TCR repertoire diversity of lymphocytes in the peripheral blood of the elderly is decreased, which likely makes the elderly more susceptible to virus infection [28–30]. This phenomenon was also seen in severe COVID-19 patients as the overwhelming number of patients in ICUs are aged >60 years [1, 2, 31]. An altered T-cell repertoire diversity occurs in exhausted T cells [32], and reduced thymic output might be a major mechanism of immune reconstitution failure in patients living with HIV after long-term antiretroviral therapy [33]. In this study, we found that Tα1 can promote thymus output in COVID-19 patients based on the levels of TREC (Figure 4B), which is an accurate method for detecting thymus output in circulation T cells [34], demonstrating that Tα1 enhances immune reconstitution by promoting thymus output, leading to enhancement of TCR repertoire diversity and lymphocyte numbers in circulation.

We acknowledge that our study has several limitations. One issue is the normalization of TREC levels among individuals. There are no specific guidelines on how to normalize circulating TREC copies, and we chose to adjust measured TREC levels to the total DNA content in the sample. Second, we only investigated PD-1 and Tim-3 on CD8⁺ T cells. Additional studies using peptides from SARS-CoV-2 virus to activate T cells in vitro and clarify whether Tα1 treatment also can enhance IL-2 and IFN-γ secretion from peptide- activated T cells are needed. Third, because detection kits to quantify the virus titer after Tα1 treatment are not available, we did not know whether Tα1 treatment could control virus titers until now. Fourth, although our results show Tα1 treatment has some benefits for COVID-19 patients, they should be interpreted with caution because of the inherent nature of retrospective studies and the small sample size. Additional longitudinal studies on a larger cohort are urgently needed. Fifth, it is very difficult to achieve a clinical improvement–related index, which is why we chose mortality as our primary clinical outcome. This is very unlike recent randomized, controlled trial research [35] in that study a 7-category ordinal scale was designed prior to the start of research. Additionally, mortality from this study was crude mortality, which included both COVID-19–attributable mortality and mortality related to underlying disease. Four of the 36 patients who received Tα1 treatment died, accounting for 11.11%. Serious complications occurred in these 4 cases during the clinical courses of COVID-19, including hemorrhagic shock that resulted from gastrointestinal hemorrhage in case 1, a heart attack in case 2, and septic shock in cases 3 and 4. It is very difficult to know what exactly contributed to their deaths. Last, the characteristics of enrolled patients at baseline were generally matched between the 2 groups. However, some confounders during the clinical courses, including but not limited to glucocorticoids dosage and exposure levels, cannot be avoided and are difficult to evaluate.

In conclusion, we demonstrated that Tα1 treatment has the capacity to improve and restore T-cell counts in COVID-19 patients with severe lymphocytopenia. Importantly, Tα1 treatment can reverse T-cell exhaustion and induce immune reconstitution by inducing thymus output in COVID-19 patients with SARS-CoV-2 infection. All of these factors have collectively contributed to the reduction in mortality in this study cohort. Though our results should be interpreted with caution, they do provide additional information regarding treatment of COVID-19 patients and the need for future similar studies.

Notes

This study was approved by the Ethics Commission of General Hospital of the Central Theatre Command and Wuhan Pulmonary Hospital. Written informed consent was waived by the Ethics Commission of the designated hospital for emerging infectious diseases and anonymous analysis of data.

Author contributions. B. D., Y. W., and Y. C. were involved in final development of the project and manuscript preparation; Z. H., Z. Y., Y. P., C. W., and Z. F. analyzed the data; B. D., Ying Liu, and Yueping Liu performed T-cell receptor excision circle assays; C. M., Y. T., and L. C. conducted serum enzyme-linked immunosorbent assays; and M. L. and G. W. performed flow cytometry analysis.

Disclaimer. The funding agencies did not participate in study design, data collection, data analysis, or manuscript writing.

Financial support. This work was supported by the National Natural Science Foundation of China (81971478 and 8177169).

Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.

Author notes

Y. L., Y. P., and Z. H. contributed equally to this work.

Y. C., Y. W. and B. D. are cocorresponding authors and equally contributed to this work.