key: cord-0987353-vfe3wmk9
authors: Liu, Yueping; Pang, Yue; Hu, Zhenhong; Wu, Ming; Wang, Chenhui; Feng, Zeqing; Mao, Congzheng; Tan, Yingjun; Liu, Ying; Chen, Li; Li, Min; Wang, Gang; Yuan, Zilin; Diao, Bo; Wu, Yuzhang; Chen, Yongwen
title: Thymosin alpha 1 (Tα1) reduces the mortality of severe COVID-19 by restoration of lymphocytopenia and reversion of exhausted T cells
date: 2020-05-22
journal: Clin Infect Dis
DOI: 10.1093/cid/ciaa630
sha: 0e3295fdd2a1c1756bcefb41c4f644adc34f0f6e
doc_id: 987353
cord_uid: vfe3wmk9

BACKGROUND: We previously reported that lymphocytopenia and T cell exhaustion is notable in acute COVID19 patients, especially in aged and severe cases. 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 mechanism of Tα1 supplement to COVID-19 are still unclear. METHODS: We retrospectively reviewed the clinical outcomes of 76 severe cases with COVID-19 admitted into two hospitals in Wuhan from December 2019 to March 2020. The thymus output in peripheral blood mononuclear cells (PBMCs) from COVID-19 patients was measured by T cell receptor excision circles (TREC). The levels of T cell exhaustion markers PD-1 and Tim-3 on CD8(+) T cells were detected by flow cytometry. RESULTS: Compared with untreated group, Tα1 treatment significantly reduces mortality of severe COVID-19 patients (11.11% vs. 30.00%, p=0.044). Tα1 timely enhances blood T cell numbers in COVID-19 patients with severe lymphocytopenia (the counts of CD8(+) T cells or CD4(+ )T cells in circulation lower than 400/μL or 650/μL, respectively). Under such conditions, Tα1 also successfully restores CD8(+) and CD4(+) T cell numbers in aged patients. Meanwhile, Tα1 reduces PD-1 and Tim-3 expression on CD8(+) T cells from severe COVID-19 patients in comparison with untreated cases. It is of note that restoration of lymphocytopenia and acute exhaustion of T cells are roughly parallel to the rise of TRECs. CONCLUSIONS: Tα1 supplement significantly reduce mortality of severe COVID-19 patients. COVID-19 patients with the counts of CD8(+) T cells or CD4(+) T cells in circulation lower than 400/μL or 650/μL, respectively, gain more benefits from Tα1. Tα1 reverses T cell exhaustion and recovers immune reconstitution through promoting thymus output during SARS-CoV-2 infection.

The epidemic of 2019 novel coronavirus (SARS-CoV-2), which was first reported in December 2019 in Wuhan, China, has been recognized as a pandemic by the World Health Organization (WHO) [1] [2] [3] . As of May 13, 2020, China has reported 84451 cases of confirmed COVID-19 and 4644 fatalities. Meanwhile, the number of confirmed COVID-19 and fatalities in the whole world were 4098018 and 283271, 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 aged and severe cases [2, [5] [6] [7] . Thymosin-α1 (Tα1), a kind of polypeptide hormone produced by thymic epithelial cells, can effectively increase T cell numbers, support T cell differentiation, maturation, and reduce cell apoptosis [8] [9] [10] . Therefore, Tα1 has been successfully used in clinical practice to treat patients infected with hepatitis B (HBV), hepatitis C (HCV) and human immunodeficiency viruses (HIV), and its efficacy was proved by pathological observation [11] [12] [13] . To enhance immunity, actually, the medical support team members from all over the country got Tα1 injection before being deployed to Hubei Province, and no infectious cases were reported till now, suggesting Tα1 might have the potential to prevent SARS-CoV-2 infection.

Nevertheless, no data are available whether Tα1 treatment has any benefits to critically ill COVID-19 patients yet.

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

Patients Medical records of severe/critical COVID-19 patients (aged from 21 years to 92 years) admitted into General Hospital of the Central Theatre Command or Wuhan Pulmonary Hospital in Wuhan 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. To be brief, patient who meets any of the following conditions is diagnosed as severe type: patients present respiratory distress with M a n u s c r i p t 4 / 19 respiratory rate ≥ 30 breath/min, SpO2 (oxygen saturation) ≤ 93% on room air, and PaO2 (arterial blood oxygen partial pressure)/FiO2 (fraction of inspired oxygen) ≤ 300 mmHg (1 mmHg = 0.133 kPa); patient meets any of the following conditions is diagnosed as critical type: patient presents respiratory failure and requires mechanical ventilation support, patient presents shock, and patient presents multiple organ dysfunction syndrome and requires ICU admission. Only severe or critical COVID-19 patients with at least 10 days' hospitalization duration were finally included in this study. According to the presence or absence of 7 days continuous Tα1 injection, the enrolled patients under study were categorized as treatment group and non-treatment group. The endpoint of this study was recovery or death.

Tα1 management In the treatment group, patients received subcutaneous injections of 10 mg Tα1 once per day for at least seven consecutive days, then still recommended to continue to use till the endpoints of the present study. Prior to administration, the lyophilized powder is to be reconstituted with 1 ml of the provided diluent (sterile water for injection). After reconstitution, the final concentration of Ta1 is 10 mg/ml. The workflow chart was showed in Figure 1 .

We reviewed clinical records, nursing records, laboratory findings, and chest x-rays or CT scans for all the patients. All information was obtained and curated with a customized data collection form. Two investigators (Y Liu and Y Chen) independently reviewed the data collection forms to verify data accuracy. (Takara Bio Inc. Japna), and the appropriate primers and probes. PCR conditions including primers and probe sequences have been described previously [14] . Reactions were carried out in an Roche

LightCycler®96 detection system (Roche Applied Science, Germany). The copies of TRECs in a M a n u s c r i p t

given sample was estimated by comparing the CT value with a standard curve obtained from PCRs performed with tenfold 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.

To fight against virus infection, it is necessary to boost T cell numbers and their antiviral functions in COVID-19 patients due to no specific drugs for SARS-CoV-2 coronavirus are available till now. Tα1, a kind 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 from two designated hospitals were enrolled in this retrospective cohort study based on the pre-set inclusion criteria. According to the presence or absence of 7 days continuous Tα1 injection, the patients under study were categorized as treatment group in 36 patients and nontreatment group in 40 patients. There were no significant differences between the two groups in demographic characteristics and clinical findings at the time of admission, such as age, sex, coexisting disorders, the counts of total lymphocytes, T cells, CD8 + T cells, CD4 + T cells, PCO2 on admission and PCO2 of discharge. Moreover, all of these patients got antiviral and antibacterial treatment during hospitalization. Glucocorticoid and oxygen inhalation are also very common. Tα1treated group has slightly higher level of IL-6 than untreated group (p=0.047), whereas, the levels of PO2 on admission and PO2 of discharge are a little higher in Tα1-treated group than those in non-treatment group ( Table 1) . Ta1 has been approved by FDA and China FDA, and it is a very well tolerated drug. No side effects associated with Ta1 were found after thorough reading of all the medical records of 36 participants in the treatment group. There were 2 patients in treatment group underwent noninvasive mechanical ventilation while 11 patients underwent in non-treatment group. 9 out of the 11 cases in the control arm underwent subsequent invasive mechanical ventilation owing to hypoxia cannot by adjusted by noninvasive mechanical ventilation whereas none in the treatment arm were intubated. More interestingly, 11.11% (4/36) cases with Tα1 treatment succumbed, whereas, the mortality of severe COVID-19 patients without Tα1 treatment reaches 30% (12/40) (Figure 2 , Table 1 ), suggesting Tα1 supplement can reduce mortality.

We next investigated whether Tα1 can restore T cell numbers in COVID-19 patients with severe M a n u s c r i p t 6 / 19 lymphocytopenia. 34 cases with recording of T cell numbers on admission and after continuous 7 days of Tα1 treatment were involved in this section. Results showed that Tα1 supplement effectively restores T cell numbers in cases with the counts of CD8 + T cells or CD4 + T cells lower than 400/μL or 650/μL, respectively, whereas, patients whose T cell numbers are higher than the above-mentioned levels gain no benefits from Tα1 treatment ( Figure 3A ). More interestingly, Tα1 also dramatically increases the counts of CD8 + and CD4 + T cells in aged patients (> 60 years old) ( Figure 3B ), and cases with comorbidities of hypertension or cardiovascular diseases ( Figure 3C) .

Collectively, these data demonstrate that Tα1 promotes timely T cell recovery in COVID-19 patients with severe lymphocytopenia.

Our previous studies have demonstrated that SARS-CoV-2 infection induces acute T cell exhaustion, which might lead to ineffectively eliminate viruses in vivo [7] . We then analyzed whether Tα1 affects T cell exhaustion in severe COVID-19 patients, hence the expression of We next investigated whether the reversion of CD8 + T cell exhaustion in COVID-19 patients by Tα1 is due to enhancement of thymus-derived naïve T cells in circulation. TRECs are specific circular DNA byproducts formed during the random rearrangements of T cell receptors, and they are only present in cells exported from thymus but not found in replicating cells within PBMCs [15] . To explore whether Tα1 administration affects thymus output in COVID-19 patients, we measured TRECs in PBMCs of patients before and after 7 days' Tα1 injection. Interestingly, the Tα1 treated groups manifested significantly higher TREC levels than untreated control (Figure 4B) , suggesting Tα1 has the capacity to enhance thymus output, by thus enhance TCR diversity and finally reverse CD8 + T cell exhaustion.

We here retrospectively reviewed and found that Tα1 significantly reduces mortality of severe COVID-19 patients compared with those in untreated group (Figure 2) . Further study showed that M a n u s c r i p t

Tα1 effectively and timely enhances T cell counts in COVID-19 patients with severe lymphocytopenia, especially in cases with the counts of CD8 + or CD4 + T cells lower than 400/μL or 650/μL, respectively ( Figure 3A) . Based on these results, we strongly recommend COVID-19 patients whose CD8 + T or CD4 + T cell counts lower than 400/μL or 650/μL, respectively, apply Tα1 injection to improve their immune function. Additionally, the duration of Tα1 injection should be at least 7 days. Importantly, enhancement of T cells was also observed in aged patients and cases with coexisting disorders including hypertension and cardiovascular disease after 7 days continuous Tα1 supplement ( Figure 3B～D) . We therefore suggest healthy people aged above 60 receive Tα1 supplement to prevent potential SARS-CoV-2 infection due to the immune system of the elderly also have responses to Tα1, even the thymus might be extremely atrophy.

Immune damage is very common in COVID-19 patients, and most critically ill cases manifested severe lymphocytopenia [6, 16] . Tα1 has been described 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] [18] [19] . infection [7] . We here found that Tα1 effectively down-regulates 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 through revering T cell exhaustion during acute viral infection.

Inflammation would promote exhausted T cell differentiation. For example, IL-6, a pleiotropic cytokine that plays an essential role in regulating immune responses, can rescue lymphocyte 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 LCMV infection [26, 27] . The severe/critically ill COVID-19 patients have very high levels of serum IL-6 and IL-10, and these patients also displayed high levels of the 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 PCP, CRP and ferritin on admission were also very high in COVID-19 patients ( Table 1) . However, due to the outbreak of SARS-CoV-2 infection is extremely emergency in February, we regret having not the capacity to measure serum IL-10, IL-6 and inflammation markers including PCP, CRP and ferritin in Tα1-treated patients. Further work is needed to clarify the exact mechanism underlying Tα1 down-regulates PD-1 and Tim-3 expression on CD8 + T cells.

The number of lymphocytes from thymus will decrease in aged person due to thymus dysregulation and atrophy, so TCR repertoire diversity of lymphocytes in the peripheral blood of the elderly are decreased, which might make the elderly more susceptible to virus infection [28] [29] [30] .

This phenomenon was also seen in severe COVID-19 patients due to overwhelming cases in ICU are aged above 60 [1, 2, 31] . Actually, altered T cell repertoire diversity occurs in exhausted T cells [32] , and reduces thymic output might be a major mechanism of immune reconstitution failure in HIV-infected patients 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 to detect thymus output in circulation T cells [34], demonstrating Tα1 enhances immune reconstitutions through promoting thymus output leading to enhancement of TCR repertoire diversity and lymphocyte numbers in circulation.

However, we acknowledge that our study has several limitations. One issue is the normalization of TREC levels among individuals, there are no specific guidelines as to how to normalize circulating TREC copies, and we chose to adjust to total DNA content in the sample. The second issue is we only investigated PD-1 and Tim-3 on CD8 + T cells, further studies using peptides from SARS-CoV-2 virus to activate T cell in vitro and clarify whether Tα1 treatment also can enhance IL-2 and IFN-γ secretion from peptides-activated T cells is needed. The third issue is no detective A c c e p t e d M a n u s c r i p t 9 / 19 kits are available to quantify virus titer after Tα1 treatment, so we did not know whether Tα1 treatment can control virus titers till now. Fourthly, although our results show Tα1 treatment has some benefits to COVID-19 patients, it should be interpreted with caution because of inherent nature of retrospective study and small sample size, future longitudinal studies on a larger cohort are eagerly needed. Fifthly, it's very difficult to retrieve clinical improvement related index, which is why we just chose mortality as our primary clinical outcome and is very unlike a recent RCT research [35] , in that study a seven-category ordinal scale was designed prior to the commence of research. Additionally, mortality of this study was crude mortality, which included both COVID-19attributable mortality and mortality related to underlying disease. 4 out of the 36 cases receiving Tα1 treatment succumbed, accounting for 11.11%. Some serious complications occurred in these 4 cases during clinical courses of COVID-19, such as hemorrhagic shock resulting from gastrointestinal hemorrhage in case 1, heart attack in case 2, septic shock in both case 3 and case 4.

It is very difficult to tell which exactly contributes to their corresponding deaths. Last but not the least, the characteristics of enrolled patients at baseline were generally matched between the two groups, however, some confounders during clinical courses, including but not limited to glucocorticoids dosage or exposure levels, are inevitably to avoid and difficult to evaluate.

In conclusion, we here demonstrated that Tα1 supplement has the capacity to improve and restore 

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CRP on admission

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.

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

This work was supported by the National Natural Science Foundation of China (NSFC; No. 81971478, and 8177169).

The authors declare no financial or commercial conflict of interest.A c c e p t e d M a n u s c r i p t

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