Immunological and inflammatory profiles in mild and severe cases of COVID-19

COVID-19 is associated with 5.1% mortality. Although the virological, epidemiological, clinical, and management outcome features of COVID-19 patients have been defined rapidly, the inflammatory and immune profiles require definition as they influence pathogenesis and clinical expression of COVID-19. Here we show lymphopenia, selective loss of CD4+ T cells, CD8+ T cells and NK cells, excessive T-cell activation and high expression of T-cell inhibitory molecules are more prominent in severe cases than in those with mild disease. CD8+ T cells in patients with severe disease express high levels of cytotoxic molecules. Histochemical studies of lung tissue from one fatality show sub-anatomical distributions of SARS-CoV-2 RNA and massive infiltration of T cells and macrophages. Thus, aberrant activation and dysregulation of CD8+ T cells occur in patients with severe COVID-19 disease, an effect that might be for pathogenesis of SARS-CoV-2 infection and indicate that immune-based targets for therapeutic interventions constitute a promising treatment for severe COVID-19 patients. Immunophenotyping of patients with COVID-19 is ongoing, but much remains to be learned. Here the authors analyze 41 hospitalized patients with COVID-19 and show a higher degree of lymphopenia in various immune cell subsets as well as cytotoxicity and T cell inhibitory marker expression in severe cases compared with mild.

A wide spectrum of clinical manifestations occurs from mild to severe disease with extensive lung involvement leading to acute respiratory distress syndrome (ARDS) and death [2][3][4][5][6][7]10,13 . Severe lung disease with extensive alveolar damage and progressive respiratory failure leads to fatal outcomes [3][4][5]7,11 . Higher fatality rates occur in the elderly, and in individuals who have diabetes, co-morbidities and other causes of immunocompromise 4 . We recently reported a case where significant interstitial lymphocytic infiltrates in the lung tissue, lymphopenia, and overactivation of T cells in peripheral blood 10 . Inflammatory and immune responses are important for elimination of the infection, but may have a significant impact on SARS-CoV-2 pathogenesis, and may play a role in the expression of the clinical spectrum of COVID-19 disease.
In this study, we perform a cross-sectional observational study of immunological and inflammatory markers and cells in 41 COVID-19 patients (29 with mild disease and 12 with severe disease including 2 fatal cases). We find COVID-19 patients in severe group are characterized by profound lymphopenia, strong T-cell activation, and increased expression of T-cell inhibitory molecules compared to mild group of patients.
Laboratory findings. Routine laboratory test results are shown in Table 2. There were no significant differences in the absolute number of leukocytes, neutrophils, and platelets counts between mild and severe group. The severe group exhibited a significantly lower absolute lymphocyte count than the mild group (p < 0.0001, two-tailed Mann-Whitney U-test). The severe group showed increased D-dimer (p = 0.0065, two-tailed Mann-Whitney Utest) and serum ferritin (p = 0.0024), and had a lower albumin levels (p = 0.0005, two-tailed Mann-Whitney U-test). Fig. 1a. The severe group had decreased numbers of CD3+ T cells, CD4+ T cells, CD8+ T cells, and NK cells  (Fig. 1b).

CD8+ T cells in severe patients
. T-cell activation plays a critical role in defense of pathogen infection. Thus, we evaluated the degree of T-cell activation. The levels of T-cell activation were statistically higher in SARS-CoV-2 infected patients than cells from healthy controls (HC), as reflected by higher proportions of  CD38+CD8+T cells, HLA-DR+CD8+T cells, and CD38+HLA-DR+CD8+T cells (Fig. 2a, b). Notably, the frequencies of CD38 +HLA-DR+CD8+T cells in patients with severe symptoms were significantly higher compared with patients in the mild symptom group (p = 0.0372, two-tailed Mann-Whitney U-test) (Fig. 2b). of CD38+CD4+T cells and HLA-DR+CD4+T cells among HC, mild, and severe patients. However, compared with HC, the proportion of CD38+HLA-DR+CD4+T cells was higher in both mild and severe patients, but without difference between the two groups of SACR-CoV-2 infected patients (Fig. 2c). There is no difference in the absolute number of CD38+HLA-DR+CD8+T and CD38+HLA-DR+CD4+T cells between mild and severe group ( Supplementary Fig. 2b).
PD-1 and TIM-3 expression on CD8+ T cells in severe patients.
The activation/inhibitory costimulatory molecules expression on circulating T cells is shown in Fig. 3. The proportion of PD-1+ CD8+T cells was higher in severe patients compared to HC and mild patients, but there was no statistical difference in the proportion of PD-1+CD4+T cells among HC, mild and severe patients (Fig. 3a, b). In addition, the frequencies of PD-1+CD8+ T cells correlated positively (R = 0.4271, p = 0.0053, Pearson correlation test) with time after disease onset ( Supplementary  Fig. 2c). Severe patients also exhibited higher frequencies of TIM-3+CD8+T cells compared to HC and mild patients. Increased frequencies of TIM-3+CD4+T cells were also observed in mild and severe patients compared with HC. However, no statistically significant difference was observed in the frequencies of TIM-3+ CD4+T cells between mild and severe patients (Fig. 3c, d).
CD4+ T cells differentiation. The naïve/memory subsets of CD4+ T cells were analyzed according to CD45RA, CD27, and CCR7 expression ( Supplementary Fig. 3a). Compared with HC, SARS-COV-2 infection leads to increased proportion of T N cells in severe patients and increased T CM cells in mild patients. Decreased proportion of T TM cells was observed in mild patients compared with HC. No statistical differences were observed between mild and severe group in the proportions of memory subsets.
The functional polarization of CD4+ T cells was defined according to the chemokine receptors CCR4, CCR6, and CXCR3 expression ( Supplementary Fig. 3b). Compared with HC, the percentages of Th1 and Th1Th17 cells were higher in mild patients, while the percentages of Th2 and Th17 cells were lower. No statistical differences were observed in the percentages of Th1, Th2, and Th17 cells between mild and severe patients. The frequencies of Treg and pTfh cells were also assessed, but there was no statistical difference among HC, mild and severe groups ( Supplementary Fig. 3c, d).
Cytokines and chemokines. Cytokines and chemokines in plasma of COVID-19 patients and HC were measured using flow cytometry based Aimplex kit (Supplementary Fig. 4). Inflammatory cytokines and chemokines in SARS-CoV-2 infected patients, such as IL-6, TNF-α, IL-17A, IFN-γ, and MCP-1, were significantly elevated than HC, although there was no significance between mild and severe patients. Cytokines related tissue repair, such as IL-10, IL-4, and IL-5, were also upregulated in COVID-19 patients, but without statistical significance between mild and severe patients. In summary, similar to SARS-CoV 14 and MERS-CoV 15 infections, COVID-19 patients showed higher plasma concentrations of cytokines/chemokines than HC, albeit there was no difference between mild and severe cases. Fig. 5a). Among the CD8+ T cells, the frequencies of T TM subset significantly decreased in SARS-CoV-2 infected patients, but without difference between mild and severe individuals (Supplementary Fig. 5b). Moreover, in severe patients, there was an increase of T E subset compared to HC and a decrease of T CM subset compared to mild patients ( Supplementary Fig. 5b). No significant changes were seen in T N or T EM subsets. The expressional levels of cytotoxic molecules (granzyme B, GZMB; perforin, PRF and granulysin, GNLY) in CD8+ T cells were analysed. Based on cytolytic ability, CD8+ T cells were divided into four subpopulations: monocytotoxic T lymphocytes (M-CTL) expressing only one of GZMB, PRF, and GNLY; dicytotoxic T lymphocytes (D-CTL) expressing two kinds of cytotoxic molecules; tricytotoxic T lymphocytes (T-CTL) expressing all three cytotoxic molecules; and noncytotoxic T lymphocytes (N-CTL) which express none of the molecules. The data showed that the frequencies of T-CTL are the highest in CD8+ T cells from severe patients and the lowest in HCs (Fig. 4a, b). There were also higher expressional levels of GZMB and PRF double positive D-CTL in SARS-CoV-2 infected patients compared to HCs (Fig. 4b).

Multifunctional cytotoxic CD8+ T cells. Phenotypes of circulating CD8+ T cells were analyzed (Supplementary
The high cytotoxicity of CD8+ T cells coincides with their overactivated status in severe patients, which might contribute to the ongoing immune injury in lung tissue. Immunobiological findings. Pulmonary infiltration of lymphocytes and viral distribution in tissues are shown in Fig. 5a-d. There was diffuse alveolar damage with cellular fibromyxoid exudates and desquamation of pneumocytes in both lungs. Massive CD4+ T cell, CD8+ T cell, macrophages and GZMB+ cells infiltration were observed in the interstitial area of lung tissues (Fig. 5a). We used an in situ hybridization assay to detect viral RNA in fixed tissue sections. Intense dot signals were observed both in the left and right lungs (Fig. 5b), suggesting active replication in these cells. Indeed, these cells with intense signals showed atypical enlarged pneumocytes nuclei and prominent nucleoli, which are typical characteristics for multinucleated syncytial cells. Punctate dots were mainly distributed in the intraalveolar spaces, which might be dissociative viral particles. Notably, viral signal was not observed in liver (Fig. 5c) or heart tissues (Fig. 5d).

Discussion
To our knowledge this is the first detailed study of the immunological and inflammatory profiles of COVID-19 patients with severe disease. Cellular immune responses, consisting of both CD4+ T and CD8+ T cells, are essential for the control of viral infection. Much work has been aimed to elucidate the virological features 3,6,9,[11][12][13][16][17][18][19] as well as clinical characteristics [3][4][5]7,16,17 of COVID-19 patients. However, little is known about the immunological and inflammatory profiles of patients and their association with COVID-19 severity.
The clinical, imaging, and routine laboratory findings of enrolled patients are similar to recently published data 2-7,10,11,13 . Lymphocytopenia has been observed in COVID-19 patients showing different spectrum of clinical disease 20 , although the changes in different lymphoid subsets were not yet defined. In a previous study, mainly in noncritical patients infected with SARS-CoV-2, 35% of patients had only mild lymphocytopenia 4 . In contrast, in other two reports with patients with severe disease, the incidences of lymphopenia were 63% 3 and 70.3% 7 , respectively. Moreover, lymphocytopenia occurred in more than 80% of severe COVID-19 patients 11 . Our results show that the degree of lymphocytopenia may reflect the severity of COIVD-19 disease since the lymphocyte counts were slightly decreased in COVID-19 patients with mild disease, and significantly decreased in patients with severe disease. In addition, our data showed that several immune subsets, such as CD4+ T, CD8+ T, and NK cells, are lower in patients with severity disease.  The reason for patients developing lymphopenia remains unknown. Given that lymphocytes express low level of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for both SARS-CoV-2 and SARS-CoV 12,16 , and the viral genome is rarely detectable in peripheral blood of SARS-CoV-2 infected patients 5,18 , it is reasonable to speculate that the decrease of peripheral lymphocytes is not directly attributed to SARS-CoV-2 infection. An alternative explanation is that the decease of peripheral lymphocytes is a result of activation-induced apoptosis or aggressive migration from peripheral blood to the lungs, where robust viral replication occurs. Further studies should elucidate the mechanisms responsible for the lymphopenia. It is noteworthy that CD4+ T-cells were profoundly decreased in peripheral blood of severe SARS-COV-2 infected patients. Lessons from HIV infection indicated that a low CD4+ T-cell counts increases the risk of opportunistic infections and lower antiviral immune surveillance, suggesting more attention should to be given to patients in critical condition. Given the limitations of our cross-sectional study, and the clinical significance of lymphopenia and of CD4+ T-cell count, further longitudinal studies with serial sampling are needed to define this further.
The immune response is essential for eradication of the pathogen and resolution of active disease. A recent study demonstrated robust multi-factorial immune responses can be induced by SARS-CoV-2 in a mild case, which is similar to the avian H7N9 disease 21 and Flavivirus infection 22 . However, the immune and inflammatory response can also be detrimental and cause immune mediated tissue injury, especially in severe cases. It was observed in the 2003 SARS epidemic as well as in the COVID-19 pandemic that pulmonary disease often worsens at 1-2 weeks after onset of respiratory symptoms, coinciding with the onset of virus clearance 17,18 . We recently reported the histology of COVID-19 by obtaining biopsy specimens at autopsy 10,17,23 . Histologic examination revealed diffuse alveolar damage, prominent desquamation of pneumocytes and hyaline membrane formation within left and right lungs with significant interstitial lymphocytic infiltrates. We showed in the histochemical examination of lung tissue obtained from one of our patients with severe disease who died, intense CD4+ and CD8+ T cells infiltrates, with strong granzyme B expression (Fig. 5). Our current study further suggests that the involvement of immunemediated injury is plausible in the pathogenesis of COVID-19.
In severe patients, despite the absolute counts of CD8+ T cells decreased, these cells showed over-activation, increased T-cell inhibitory molecules expression and increased multiple cytotoxic granules expression, resembling other acute inflammatory processes such as malaria infection, Q-fever, and sepsis 24,25 . Whether these activated cells are antigen-specific or bystander ones warrants further study. However, PD-1 positive T-cells in the peripheral circulation may not only indicate T-cell exhaustion, but could also suggest the presence of antigen-specific T-cells, which needs to be further evaluated 26 . We found that T-cell activation appears to correlate with disease severity in COVID-19 patients as a higher frequency of activated CD8+ T cells (defined by CD38+ and HLA-DR+) occurred in severe patients with longer time after disease onset. Taken together, these findings highlight the involvement of CD8+ T cells for the immunopathogenesis of severe COVID-19. Considering the possibility of clinical application, lymphocyte subset cell counts and activated phenotypes of CD8+ T cells could be used to predict disease outcomes and evaluate new interventions for COVID-19 patients. For example, lymphocyte count is suggested to be closely monitored in COVID-19 patients receiving ECMO 27 .
In conclusion, we provided immunological and inflammatory markers linking disease severity in mild and severe cases of COVID-19 and suggested aberrant activation and dysregulation of CD8+ T cells occur in patients with severe COVID-19 disease which may play an important role in pathogenesis of SARS-CoV-2 infection. Immune-based therapeutic interventions 28 , such as IL-6 receptor antagonist (ChiCTR2000029765) and mesenchymal stem cells (NCT04252118), are undergoing clinical trials. Further studies are required to define immune-mediated tissue injury and identify specific host-directed therapy targets for treatment of the disease.

Methods
Ethical approval. This study was approved by the ethics committee of the Fifth Medical Center of PLA General Hospital and written informed consent was obtained from all patients enrolled in this study.
Patients. Between January 19th and 20th February, a total of 41 patients with laboratory confirmed COVID-19 disease hospitalized at the Fifth Medical Center of PLA General Hospital in Beijing, China, were studied. All patients were diagnosed according to World Health Organization (WHO) interim guidance and were positive for new coronavirus nucleic acid by using SARS-CoV-2 specific RT-PCR test. The patients were classified on basis of disease severity: The mild disease group were defined as with or without pneumonia who were admitted to general wards not requiring intensive care. The severe disease group were those who required critical care and met one or more of these criteria: dyspnea and respiratory rate ≥30/min, blood oxygen saturation ≤93%, PaO 2 /FiO 2 ratio <300 mmHg, and lung infiltrates on CT scan >50% within 24-48 h, or those who exhibited respiratory failure, septic shock, and/or multiple organ dysfunction/failure. Patient demographics, clinical history, presentations, and findings were recorded. All patients had routine laboratory investigations (full blood count, liver and renal function tests, and coagulation tests).
Clinical samples. Peripheral venous blood samples were obtained on admission of the COVID-19 patients within 24 h and placed into the vacutainer tube, then centrifuged at 400×g for 5 min at 4°C. The time of sampling relative to the onset of symptoms was recorded. Plasma samples were collected and stored at −80°C until used. In one patient with severe disease who died, lung tissue samples were obtained post-mortem for immuno-histological analysis.
Lymphocyte counts and subsets. Absolute lymphocyte counts and subsets were determined using lymphocyte detection kit (Beijing Tongshengshidai Biotechnology Co., LTD, Beijing, China) following the manufacturer's instructions. Immunohistochemical staining. Formalin-fixed paraffin-embedded 4-μm sections of lung tissue were subject to immunohistochemistry. Following deparaffinization and rehydration, sections were incubated in 3% H 2 O 2 in methanol for 30 min at room temperature to block endogenous peroxidase. The sections were then boiled in citrate buffer or EDTA buffer in a microwave oven. The staining was performed using primary antibodies against CD4 (ZSGB-BIO, Beijing, China), CD8 (Abcam, Cambridge, MA), CD68 (ZSGB-BIO), GZMB (Abcam), and incubated at 4°C overnight. The sections were visualized using the diaminobenzidine solution (DAKO, Carpinteria, CA) and then lightly counterstained with hematoxylin. Images were captured with an inverted fluorescence microscope (PerkinElmer, Norwalk, CT).
RNAscope. SARS-CoV-2 RNA in formalin-fixed paraffin-embedded (FFPE) tissue were probed by RNAscope reagents, following the manufacture's protocol. The probes (v-nCoV2019-S, cat. 848561)) targeting SARS-CoV-2 Spike gene and control probes (cat. 320751) are designed by ACD. Briefly, after H 2 O 2 treatment and protease digestion, FFPE slides were incubated 2 h at 40°C with probes. The amplifiers and detection solution in the RNAscope 2.5 HD Duplex Reagent kit were added sequentially for hybridization signal amplification for the indicated time. The slides were counterstained with 50% Hematoxylin staining solution for 30 s, and washed with water immediately. Tissue slides were cover slipped with Vectamount Permanent Mounting Medium (Vector Labs, 321584, Burlingame, California). Images were obtained with Perkin Elmer Vectra 3.0 (PerkinElmer).
Statistical analysis. GraphPad Prism statistical software version 8.0 (GraphPad Software, San Diego, CA) was used. Continuous measurements were displayed as median (IQR) and two group comparisons performed using Mann-Whitney Utest. Categorical variables were expressed as count (%) and compared by χ² test or Fisher's exact test between mild and severe group. Pearson correlation test was conducted to assess association between two quantitative variables. p-value < 0.05 were considered statistically significant.
Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.