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Letters to the Editor
Ibrutinib interferes with innate immunity in chronic lymphocytic leukemia patients during COVID-19 infection
Severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) is a novel coronavirus that from December
2019 is spreading throughout the world causing a pan-
demic of Coronavirus Disease 2019 (COVID-19).1
COVID-19 is characterized by severe activation of
inflammatory response that is thought to be a major
cause of disease severity and death. Immune response to
COVID-19 infection is constituted by two steps: during
the phase of incubation, an adaptive immune response is
able to control virus proliferation preventing disease pro-
gression. In the later phase, an uncontrolled inflammato-
ry response could determine major clinical complications.
This response, known as cytokine storm, is induced by
the activation of T cells, natural killer (NK) cells, mono-
cytes/macrophage, neutrophils and any cells that run into
Blood samples from patients that matched standard diagnostic criteria for CLL were obtained from the Hematology Unit of Modena Hospital, Italy with a pro- tocol approved by the local Institutional Review Board and adhered to the tenets of the Declaration of Helsinki. Peripheral blood mononuclear cells (PBMC) were isolated and used fresh or cryopreserved until use. This study was performed on samples isolated from CLL patients who have not experienced SARS-CoV-2 infection. PBMC iso- lated from CLL patients were treated with ibrutinib or vehicle then stimulated with SARS-CoV-2 Peptide Pools Protein S, S1, S+, N and M (Miltenyi Biotech) and ana- lyzed using cytokine secretion assay (CSA) for TNF-α and IFN-γ (Miltenyi Biotech). In order to identify the mono- cytic and T-cell population, PBMC were stained with CD14 and CD3 antibody respectively. For the ex vivo analysis, PBMC from CLL patients before and after 3 months of ibrutinib therapy were collected and stored in liquid nitrogen. PBMC were stimulated with SARS-CoV-2 Peptide Pools Protein and monocytes or T cells were identified by staining with CD14 or CD3. Secretion of TNF-α and IFN-γ was analyzed by CSA. Conditioned media were collected by centrifugation at 1,600 rpm for 10 minutes and stored at -20°C before being assayed. The levels of IL-6 were determined by human enzyme linked immunosorbent assay kit high sensitivity (Abcam). All data are presented as mean ± standard error of the mean. The t-test or Wilcoxon matched-pair signed rank test were used to determine statistical significance (GraphPad v6, GraphPad Software Inc).
CLL is typically characterized by perturbations of the immune system, involving both innate and adaptive immune responses. Firstly, we examined the impact of SARS-CoV-2 infection in vitro on the adaptive and innate immunity in CLL collected from treatment-naϊve patients. As shown in Figure 1, following stimulation with SARS-CoV-2 peptides, we measured a strong and significant release of pro-inflammatory cytokines both by CD3+ T cells and CD14+ monocytes characterized by sig- nificant increase of TNF-α and IFN-γ secretion (*P<0.05; **P<0.01). Then, we analyzed if treatment with ibrutinib can modify the immunological response during infection. Ibrutinib targets T cells by irreversibly binding Tec family kinase, ITK, skewing toward Th1 phenotype and affect- ing Th2 immunity.9 ITK is critically involved in T-cell- mediated immune response and depletion of its expres- sion impairs influenza virus replication.11 As shown in Figure 1A and B and in the Online Supplementary Figure S1 ibrutinib did not determine a significant alteration in TNF-α secretion either in presence or absence of stimula- tion with SARS-CoV-2 peptides (P=not significant [ns]), in addition secretion of IFN-γ was not altered by ibrutinib and after stimulation we detected a slight increase (*P<0.05). Ibrutinib targets BTK in CLL-derived macrophages (nurse-like cells [NLC]) potentiating its immunosuppressive M2-skewed features.10 BTK inhibi- tion hampered the inflammatory response of NLC during A. fumigatus infection.12 According to this evidence, we aimed to characterize the response of CLL monocytes to SARS-CoV-2 in the presence of ibrutinib. As reported in Figure 1C and D and in the Online Supplementary Figure S1, ibrutinib strongly impaired the release of TNF-α and IFN-γ by CLL monocytes induced by SARS-CoV-2 (*P<0.05). IL-6 is one of the key mediators of inflamma- tion and cytokine storm in COVID-19 patients. Therefore, we measured the amount of secreted IL-6 in conditioned media collected after stimulation with sSARS-CoV-2 peptides either in the presence or absence
the virus itself or degrade viral products. During the
cytokine storm TNF-α, IFN-γ, IL-1b, IL-6, IL-12 and IL-17
are of crucial importance.2 Given the aberrant response of
immune cells to viral infection, acute respiratory distress
syndrome appears to be the most acute and fatal event of
this disease. It’s evident that COVID-19 poses several
challenges to the management of patients with hemato-
logic malignancies since patients appear to be vulnerable
to COVID-19.3 Chronic lymphocytic leukemia (CLL) is
the most common leukemia in Western countries that
mainly affects older people. CLL is characterized by sev-
eral clinical complications related to alterations in the
immune system. Predisposition to infections in CLL
patients includes both immunodeficiency related to the
leukemia itself and the results of cumulative immunosup-
pression caused by treatments. Given these evidences
CLL patients might represent a high-risk group for SARS-
CoV-2 infection and the outcome of patients needed
admission is poor with high mortality rate.4 As supposed
by different studies, BTK inhibitors (BTKi), in particular
ibrutinib, may have a possible protective effect against
lung injury in patients with COVID-19, tempering the
hyper-inflammatory state.5,6 Some different retrospective
studies, in particular an observational study conducted in
Europe identified treatment with ibrutinib or BTKi as a
factor associated with better outcome during COVID-19,
with a shorter hospitalization of CLL patients. On the
contrary, a similar study conducted in US did not confirm
this observation. Of importance, BTKi treatment was
interrupted at the onset of COVID-19 in the majority of
4,7
cases. On this line, even though off-label administra-
tion of acalabrutinib in patients with severe COVID-19 had demonstrated reduction of the host inflammatory immune response and improved clinical outcomes, the CALAVI phase II trial for acalabrutinib in patients hospi- talized with respiratory symptoms did not meet the pri- mary efficacy endpoint of increasing the proportion of patients who remained alive and free of respiratory fail- ure (https://www.astrazeneca.com/media-centre/press-releas- es/2020/update-on-calavi-phase-ii-trials-for-calquence-in- patients-hospitalised-with respiratory-symptoms-of-covid- 19.html).8 Ibrutinib exerts an immunomodulatory action, both on innate and adaptive immunity, sustaining a Th1 polarization and, at the same time, an anti-inflammatory polarization of macrophages.9,10
In the present study, we examined the modulation of ibrutinib treatment on the cytokine release by T cells and monocytes in CLL patients during infection with SARS- CoV-2.
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