key: cord-321960-p4twpm2z
authors: Thibaudin, Marion; Fumet, Jean-David; Bon, Marjorie; Hampe, Léa; Limagne, Emeric; Ghiringhelli, Francois
title: Immunological features of coronavirus disease 2019 in patients with cancer
date: 2020-09-07
journal: Eur J Cancer
DOI: 10.1016/j.ejca.2020.08.013
sha: 
doc_id: 321960
cord_uid: p4twpm2z

BACKGROUND: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has caused a major pandemic. Patients with cancer are at higher risk of severe COVID-19. We aimed to describe and compare the immunological features of cancer patients hospitalized for COVID-19 or other concomitant, cancer-related illness. METHODS: In this prospective study, the clinical and immunological characteristics of 11 cancer patients with COVID-19 and 11 non-COVID-19 cancer patients hospitalized in the same unit at the same period for other medical issues were analyzed. We also used 10 healthy volunteers as controls. Peripheral immune parameters were analyzed using multiparametric flow cytometry. RESULTS: The median age of COVID-19-positive cancer patients was 71.1 years, and 66.4 years for controls. Compared with non COVID-19 cancer patients, COVID-19 positive cancer patients had more extensive lymphopenia and hypoalbuminemia, with higher levels of C-reactive protein. In COVID-19 patients, elevated procalcitonin was associated with a higher risk of death. By phenotypic analysis, COVID-19 positive patients presented CD3 lymphopenia, with inversion of the CD4/CD8 ratio and modification of monocyte activation, with accumulation of mMDSC-like cells and a decrease in activated monocytes. Analysis of the T-cell compartment revealed a T-dependent inflammatory response with accumulation of Th17 cells and cytotoxic CD8 T cells producing TNFα, a decrease in HLA-DR positive CD8 T cells and Treg/CD8 ratio. CONCLUSION: SARS-CoV-2 infection in cancer patients is associated with CD4 T cell lymphopenia with induction of an inflammatory T-cell response, accumulation of IFNγ(+) TNFα(+) CD8 T and Th17 cells, and a concomitant modification of monocyte activation status.

Since early 2020, the coronavirus disease 2019 (COVID- 19) pandemic has affected millions of people worldwide. Although harmless for 85% of the population, COVID-19 can be lifethreatening in vulnerable (e.g. older, immunosuppressed or comorbid) subjects. Estimated mortality from COVID-19 is 1 to 3%, i.e. 10 to 30 times higher than the death rate from seasonal influenza. The causative agent of COVID-19 is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1] .The virus spreads via small droplets projected from the mouth and nose. Analysis of data on the propagation of SARS-CoV-2 in China suggest that close contact with an infected person is necessary. In fact, propagation is mainly limited to family members, health professionals and other persons in close contact with the infected individual [2] . This suggests that quarantine is the best means to contain the epidemic. To this end, lockdown was implemented in many countries around the world starting in March 2020.

Many comorbidities, such as older age, obesity, or diabetes, pathologies that affect the immune system, and cancer can all incur an increased risk of severe forms of COVID-19 infection [3] . Accordingly, emerging data from China, Italy and North America indicate that severe disease is more frequent, and the death rate higher in patients with cancer who contract COVID-19 [4] [5] [6] [7] . Recent data from the British National Health System (NHS) show that organ transplant, immunosuppression status, presence of cancer or haematological malignancy are associated with a high risk of death [8] .

Lymphopenia, increased proinflammatory markers and cytokines, and potential blood hypercoagulability characterize severe COVID-19 cases, with features reminiscent of cytokine release syndromes. While immunosuppression and cancer could enhance the risk of severe COVID-19, one might also suspect that such patients would develop an unusual form of immune response after exposure to COVID-19. Therefore, we decided to explore this question by performing peripheral blood immunomonitoring on patients with cancer J o u r n a l P r e -p r o o f hospitalized in our Cancer center for mild to severe COVID-19 infection over a one-month period. We further compared them to cancer patients hospitalised for intercurrent non-COVID-19 medical problems during the same period, and also to healthy volunteers.

From March 30 th to May 9 th , 2020, 11 consecutive patients with positive diagnosis of SARS-CoV-2 during the care of their cancer disease were hospitalized in Georges Francois Leclerc center. Concomitantly 11 matched cancer patients without SARS-CoV-2 infection were hospitalized in Georges Francois Leclerc center during the same period for another intercurrent complication during the care of their cancer disease. We used as control group of healthy blood donor from Etablissement Francais du Sang. The study was declared as an ancillary study of the NCT02281214 and NCT02840604. The study was approved by the CNIL (national commission for data privacy) and the local ethics committee, and was performed according to the Helsinki declaration and in compliance with the European reglementation.

We reprospectively obtained the medical history, physical examination, and hematological, biochemical, radiological, and microbiological. The data collection forms were reviewed independently by 2 researchers.

Real-time reverse transcription PCR assay for SARS-CoV-2. Respiratory specimens were collected by the local CDC and then shipped to designated authoritative laboratories to detect SARS-CoV-2. The presence of SARS-CoV-2 in respiratory specimens was detected by realtime reverse transcription (RT-PCR) methods. The SARS-CoV-2 VIASURE Real Time PCR Detection kit (supplied by CerTest BIOTEC) containing the primers and probe targeting the CoV envelope gene were used. The use of this kit for the targeting of SARS-CoV-2 gene has been validated by the CNR virology respiratory laboratory in Lyon. Conditions for the J o u r n a l P r e -p r o o f amplifications were 45°C for 15 minutes, 95°C for 2 minutes, followed by 45 cycles of 95°C for 10 seconds and 60°C for 50 seconds.

Initial clinical laboratory investigations included a complete blood count, serum biochemical test (including liver and renal function, creatine kinase, LDH, and electrolytes), and coagulation profile.

Leucocyte population identification and numeration Liquid reagents: Antibody clones CD127-BrilliantViolet605 (clone A019D5), CCR7-BrilliantViolet650 (clone G043H7) and CD45RA-BrilliantViolet785 (cloneHI100) were purchased from BioLegend. Coulter) was added on vortex and incubated for 15 min at room temperature. Then, 2 mL of PBS 1X was added and after centrifugation, the pellet was resuspended in 25 µL of FBS (Foetal Bovine Serum, Dutscher) and 300 µL of Perfix-NC R2 buffer was added. A 325 µL aliquot was transferred to a Duraclone tube containing the liquid antibody, vortexed immediately for 15s and incubated for 1 hour at room temperature in the dark. PBS 1× (3 mL) was added to the tubes, incubated for 5 min at room temperature in the dark before centrifugation for 5 min at 500g . After supernatant removal, the cells was resuspended in 3 mL of 1X Perfix-NC R3 buffer prior to another 5-min centrifugation at 500g. The pellet was dried and resuspended in 300 µL of 1X R3 buffer. Acquisition was done on Cytoflex cytometer. The proportion of IFNγ + , TNFα + , IL-2 + , IL-17A + or IL-4 + expression by CD4 + T J o u r n a l P r e -p r o o f or CD8 + T cells and the proportion of CD25 + Foxp3 + CD4 + or CD8 + cells were studied with this labelling procedure. The gating strategy is described in Supplementary Figure 7 .

All analyses were done with Kaluza 1.3 software (Beckman Coulter).

Continuous variables are expressed as median (IQR) and compared with the Mann Whitney U test. Categorical variables are expressed as number (%) and compared by χ 2 test or Fisher's exact test between COVID and none COVID patients. A 2-sided α of less than 0.05 was considered statistically significant. Statistical analyses were done using PRISM software.

Results:

Between March 30 th and May 9 th , 2020, a total of 22 cancer patients were admitted to our cancer center for suspicion of SARS-CoV-2 infection. The diagnosis was confirmed for 11 patients, including 5 with positive PCR, and 6 with negative PCR but compatible CT scan images and no other plausible etiology. Among the 11 COVID-19 positive patients, median age was 71.1 years, and few comorbidities were reported. Nine of the 11 had metastatic disease, and 4 had received more than 2 lines of therapy. All 11 patients received antibiotic therapy, 9 required oxygen therapy and 8 had fever. At baseline, mean oxygen saturation (SpO 2 ) was 90% (58 -95)). Table 1 summarizes the characteristics of the patients, and

supplementary Table 1 represents individual data for each patient.

Among the 11 non-COVID cancer patients, median age was 66.4 years. This group was comparable to the COVID-19-positive patients, with similar metastatic status, cancer type and comorbidity status ( Table 1) . As a second control dataset, we also used data from 10 healthy volunteers (HV) with a mean age of 60. 

We first examined the number and proportion of immune cells in peripheral blood [9] . Total leukocyte number did not vary between healthy donors, COVID-19 positive and non-COVID cancer patients ( Figure 3A ). When looking at the proportion of myeloid cells, we observed no difference in the proportion of monocytes, but a marked increase in the proportion of neutrophils in cancer patients, with a higher proportion in COVID-19 positive cancer patients ( Figure 3A ). No particular variation was observed for the frequency of CD15 + CD16 low eosinophils ( Figure SD1 ). For monocyte subsets, we observed a decrease in absolute number of CD14 low CD16 + monocytes in COVID-19-positive patients compared to both healthy volunteers and non-COVID-19 cancer patients. CD14 high CD16 + monocytes accumulated in all cancer patients, but more marked extent in COVID-19 negative cancer patients ( Figure   SD2 ). Regarding maturation of monocytes, we observed that HLA-DR low monocytes, which harbor a monocytic MDSC phenotype, were increased in COVID-19-positive cancer patients, while HLA-DR high -activated monocytes were decreased ( Figure 3B ).

When looking at lymphoid cells, we did not observe any difference in the proportion of NK subsets, γδT cells or NKT cells between COVID-19-positive and non-COVID cancer patients ( Figure SD3 ). However, the frequency of these 3 cell types was lower in cancer patients than J o u r n a l P r e -p r o o f in healthy volunteers ( Figure SD3 ). We observed a significant decrease in the proportion of CD3 T cells and a respective increase in the B cell proportion in cancer patients compared to healthy volunteers ( Figure 3C and not shown). We noted a modification of the CD4/CD8 ratio between COVID-19 positive and negative patients, with an increase in the CD8 proportion and a decrease in the CD4 proportion in COVID-19 positive patients ( Figure 3D and E).

Together these data underline the induction of CD3 lymphopenia, with an inversion of the CD4/CD8 ratio, a change in monocyte activation, accumulation of mMDSC-like cells and a decrease in activated monocytes in COVID-19 positive cancer patients.

Looking at CD4 and CD8 subsets, we observed a decrease in effector memory cells in CD8 T cells and an increase in effector memory RA + cells (EMRA), suggesting an accumulation of terminally differentiated cells in COVID-19-positive patients ( Figure 4A ). For the CD4 subset, we did no observe any difference for each subtype in COVID-19 positive patients with exception of a decrease of naïve CD4 T cells ( Figure 4B ). The Treg proportion in CD4 T cells did not change in COVID-19-positive patients, but as for the overall increase in CD8 T cells, we observed a decrease in the Treg/CD8 ratio ( Figure 4C ). Interestingly, we observed in the basal peripheral blood a marked decrease in HLA-DR expression in CD8 T cells in COVID-19-positive patients ( Figure 4D) , which is marker of T cell activation [10, 11] . Concerning secretory function of T cells, we observed an increase in IL-17A and Th17 cells in COVID-19-positive patients ( Figure 4E ) and an accumulation of cytotoxic (IFNγ + ) CD8 T cells producing TNFα ( Figure 4F ). The study of CD8 T cells secretion capacities showed no other differences between COVID-19 positive and negative patients ( Figure SD4 ). To the best of our knowledge, this is the first preliminary study to describe the immunological characteristics of cancer patients with SARS-CoV-2 infection. A growing body of data is emerging regarding the clinical and epidemiological features of patients with COVID-19 [9] .

In addition, there have been some reports on immune response during SARS-CoV-2 infection [12] [13] [14] . The results obtained in this study could be strengthened by a larger number of patients and by the addition of analyses on COVID-19 positive non-cancer patients.

With regard to the myeloid compartment, we observed an accumulation of HLA-DR low CD14 + cells, similar to mMDSC cells, as well as concomitant decrease in HLA-DR high CD14 + cells.

Previous studies in non-cancer, symptomatic COVID-19 patients have shown a similar accumulation of HLA-DR low CD14 + inflammatory monocytes (IM) [15] [16] [17] . The expansion and activation of these cells frequently depends on the cytokines IL-1 and IL-6 [18, 19] . This accumulation could be linked to an inflammatory signature found during COVID-19 [20] .

Significantly elevated systemic levels of the pro-inflammatory cytokine IL-6 have been reported in several COVID-19 patient cohorts, and shown to correlate with disease severity [21] and the presence of inflammatory cytokines in serum. As in non-cancer patients, SARS-CoV-2 infection appears to affect CD14 myeloid cell differentiation.

this disease is lymphopenia, with drastically reduced numbers of both CD4 and CD8 T cells in moderate and severe COVID-19 cases [9, [22] [23] [24] [25] . Previous reports have shown predominant CD8 T cell depletion, which seems to correlate with COVID-19-associated disease severity and mortality [9, 22, 24, [26] [27] [28] [29] . In cancer patients, we also observed major CD3 T cell depletion. COVID-19 accentuates cancer-related CD3 lymphopenia, but CD4 lymphopenia is more pronounced than CD8 depletion.

Regarding the T cell phenotype, many studies have underlined the increased presence of activated T cells during COVID-19 [19, [30] [31] [32] [33] [34] [35] . Functional analysis showed impaired function J o u r n a l P r e -p r o o f of both CD4 and CD8 T cells, with reduced frequencies of polyfunctional T cells [9, 25, 35] .

Our data showed marked CD4 lymphopenia, with a trend towards a decreased naïve CD4 T cell fraction, which may suggest an overall reduction in CD4 T cell help, with absence of precursors. From a functional point of view, we observed a strong accumulation of IL-17Aproducing Th17 cells. These are inflammatory cells that are involved in neoangiogenesis and accumulation of myeloid cells at infection or tumor sites [36] , as frequently observed during SARS-Cov2 infection [37] . While IL-6 production is observed during SARS-Cov2 infection [21] , and because this cytokine is involved in Th17 differentiation, the accumulation of Th17 is logical and suggests IL-6-dependent inflammation in these patients.

Surprisingly, we observed a substantial reduction in HLA-DR expression on CD8 T cells.

HLA-DR is recognized as a marker of T cell activation [10, 11] and has been shown to be increased in cytotoxic T lymphocytes in autoimmune diseases [38] and in patients with HIV infection [39] . Thus, CD8 immune response is ambivalent, with on the one hand, a decrease in T cell count and activated HLA-DR CD8 T cells, and on the other hand, a concomitant decrease in the Treg/CD8 ratio and an accumulation of polyfunction CD8 T cells, suggesting a marked CD8 dependent inflammatory response that might be deleterious. J o u r n a l P r e -p r o o f 

A pneumonia outbreak associated with a new coronavirus of probable bat origin

A new coronavirus associated with human respiratory disease in China | Nature n

Drinking no-links to the severity of COVID-19: a systematic review and meta-analysis

Clinical Characteristics of Coronavirus Disease 2019 in China

Clinical characteristics of COVID-19-infected cancer patients: a retrospective case study in three hospitals within Wuhan, China

Cancer patients in SARS-CoV-2 infection: a nationwide analysis in China

Do Patients with Cancer Have a Poorer Prognosis of COVID-19? An Experience

OpenSAFELY: factors associated with COVID-19-related hospital death in the linked electronic health records of 17 million adult NHS patients. Epidemiology

Clinical and immunological features of severe and moderate coronavirus disease 2019

MHC Class II Expression Identifies Functionally Distinct Human Regulatory T Cells

CD8+HLA-DR+ T lymphocytes are increased in common variable immunodeficiency patients with impaired memory B-cell differentiation

A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study

Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China

Complex Immune Dysregulation in COVID-19 Patients with Severe Respiratory Failure

COVID-19 infection induces readily detectable morphological and inflammation-related phenotypic changes in peripheral blood monocytes, the severity of which correlate with patient outcome

Aberrant pathogenic GM-CSF + T cells and inflammatory CD14 + CD16 + monocytes in severe pulmonary syndrome patients of a new coronavirus

Immune cell profiling of COVID-19 patients in the recovery stage by single-cell sequencing

Tocilizumab treatment in severe COVID-19 patients attenuates the inflammatory storm incited by monocyte centric immune interactions revealed by single-cell analysis

A Dynamic Immune Response Shapes COVID-19 Progression

COVID-19: consider cytokine storm syndromes and immunosuppression

Characteristics of Peripheral Lymphocyte Subset Alteration in COVID-19 Pneumonia

Metabolic disturbances and inflammatory dysfunction predict severity of coronavirus disease 2019 (COVID-19): a retrospective study

Mortality of COVID-19 is Associated with Cellular Immune Function Compared to Immune Function in Chinese Han Population

Functional exhaustion of antiviral lymphocytes in COVID-19 patients

Reduction and Functional Exhaustion of T Cells in Patients With Coronavirus Disease 2019 (COVID-19)

Longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of SARS-CoV-2 infected patients

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

Pathogenic T cells and inflammatory monocytes incite inflammatory storm in severe COVID-19 patients

Presence of SARS-CoV-2 reactive T cells in COVID-19 patients and healthy donors

Characterization of antiviral immunity in recovered individuals infected by SARS-CoV-2

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

Breadth of concomitant immune responses prior to patient recovery: a case report of nonsevere COVID-19

Analysis of adaptive immune cell populations and phenotypes in the patients infected by SARS-CoV-2

Elevated exhaustion levels and reduced functional diversity of T cells in peripheral blood may predict severe progression in COVID-19 patients

Controversies on the role of Th17 in cancer: a TGF-β-dependent immunosuppressive activity?

Pulmonary Vascular Endothelialitis, Thrombosis, and Angiogenesis in Covid-19

HLA-DR expression on lymphocyte subsets as a marker of disease activity in patients with systemic lupus erythematosus

HIV controllers exhibit potent CD8 T cell capacity to suppress HIV infection ex vivo and peculiar cytotoxic T lymphocyte activation phenotype