Rituximab efficiently depletes B cells in lung tumors and normal lung tissue

Rituximab is a monoclonal antibody that targets the CD20 B-cell-specific antigen and is widely used as therapy for B-cell lymphoma. Since rituximab depletes both malignant and normal B cells, it is increasingly being used to treat various conditions in which normal B cells have a pathogenic role, such as rheumatoid arthritis and multiple sclerosis. It is well-established that rituximab efficiently eliminates B cells in blood, lymph nodes, and spleen. In contrast, the effect of rituximab in non-lymphoid tissues remains poorly documented and is debated. Here, we report a rheumatoid arthritis patient who was treated with rituximab before receiving thoracic surgery for non-small cell lung cancer. Using flow cytometry and immunohistochemistry, we show that rituximab efficiently depleted CD20-positive B cells in a primary lung tumor, in lung-associated lymph nodes, and in normal lung tissue. We conclude that rituximab may be very efficient at depleting normal B cells in the lungs. This property of rituximab may potentially be exploited for the treatment of conditions in which pathogenic B cells reside in the lungs. On the other hand, the clearance of lung B cells may provide an explanation for the rare cases of severe non-infectious pulmonary toxicity of rituximab.

Rituximab was the first monoclonal antibody to be approved for the treatment of cancer and it is estimated that >4 million people have been treated with rituximab worldwide 1 . Rituximab is a depleting chimeric anti-CD20 monoclonal antibody routinely used for the treatment of B-cell lymphoma 2-4 . The B cell-specific antigen CD20 is expressed on all normal B cells, except for early B cell precursors and antibody-secreting plasma cells, and by nearly all B-cell lymphomas. Since rituximab depletes both malignant and normal B cells, its use has been extended to non-cancerous conditions in which normal B cells are believed to play a central role in pathogenesis. Significant clinical benefits have been reported for the treatment of autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, vasculitis, Sjögren's syndrome, and scleroderma 5-9 . The mechanism whereby rituximab depletes B cells is not fully understood but there is evidence for complement-dependent cell lysis and for antibody-dependent cellular cytotoxicity 2,10,11 . It has been shown that rituximab efficiently eliminates normal and malignant B cells in blood and in lymphoid organs such as lymph nodes, spleen, and bone marrow 12-14 . In contrast, the effect in non-lymphoid tissues remains poorly documented. Here, we report the effect of rituximab in the lungs of a patient who was treated with rituximab because of rheumatoid arthritis before receiving thoracic surgery for non-small cell lung cancer.

Ethics approval
The Regional Committee for Medical and Health Research Ethics (Oslo, Norway) has approved the study (permit number: REK S-05307). Written informed consent for publication of the clinical details was obtained from all patients included in the study. .2g/L), whereas low IgG levels (4.2-5.6g/L) were observed several times over the past three years. The last rituximab cycle (2×1000mg) was given 6 months pre-operatively.
Upon informed consent, the patient was included in a research project. A pre-operative blood sample and biopsy samples from the tumor, a lung-associated lymph node, and normal lung were collected for flow cytometric analysis. For comparison, samples from a control patient with lung adenocarcinoma (not treated with rituximab) were analyzed. The control patient had CD19 + B cells in blood and tumor ( Figure 1A,B). In the rituximab-treated patient, CD19 + B cells were virtually absent from the blood ( Figure 1C) and strongly reduced in the tumor (0.2% of all CD45-positive leukocytes, Figure 1D). The remaining CD19 + B cells in the tumor were mostly HLA-DR-negative ( Figure 1H) indicating that they were plasma cells which typically lack the CD20 antigen. Rituximab did not deplete other types of immune cells, such as monocytes/ macrophages, CD4 + T cells, or CD8 + T cells ( Figure 1I-P). Flow cytometric analysis of a lung-associated lymph node and normal lung tissue revealed virtual absence of CD19 + B cells in the rituximab-treated patient (Figure 2).
Immunohistochemistry of formalin-fixed paraffin-embedded routine specimen was performed by staining for CD3 and CD20. In the control patient, the inflammatory infiltrate in and around the tumor contained both CD20 + B cells ( Figure 3A,C) and CD3 + T cells ( Figure 3E,G). In contrast, the inflammatory infiltrate in the rituximab-treated patient contained T cells ( Figure 3F,H) but virtually no B cells ( Figure 3B,D), in accordance with the flow cytometry data.
The same pattern was observed in normal lung and in lungassociated lymph nodes. In the control patient, normal lung tissue contained peribronchial lymphoid foci with both CD20 + B cells ( Figure 4A) and CD3 + T cells ( Figure 4C). In contrast, the peribronchial lymphoid foci from the rituximab-treated patient contained T cells ( Figure 4D) but virtually no B cells ( Figure 4B). In control lung-associated lymph nodes, a normal lymphocyte distribution was observed with typical germinal centers with a high density of CD20 + B cells ( Figure 5A), whereas CD3 + T cells were mostly present outside the germinal centers ( Figure 5C). In sharp contrast, lung-associated lymph nodes from the rituximab-treated patient contained virtually no B cells ( Figure 5B) and were homogenously and densely populated by T cells ( Figure 5D). The black dots in Figure 5B represent anthracotic pigment in macrophages. Thus, rituximab therapy resulted in efficient depletion of CD20positive B cells throughout the lungs, including in a lung tumor, in normal lung tissue, and in lung-associated lymph nodes.

Discussion
Rituximab was initially developed with the goal of eradicating B-lymphoma cells which typically reside in blood and lymphoid organs [1][2][3][4] . It is now well established that rituximab efficiently eliminates normal and malignant B cells in those anatomical locations 12-14 .
In contrast, current knowledge on the effect of rituximab therapy in non-lymphoid tissues remains fragmentary. This is problematic because rituximab is being considered as a therapeutic option for a number of non-malignant conditions such as autoimmune diseases 5-9 and myalgic encephalopathy/chronic fatigue syndrome 15 . In autoimmune diseases, depletion of pathogenic B cells in inflamed tissues is likely to be required to obtain clinical benefits. In the cerebrospinal fluid of patients with multiple sclerosis, rituximab therapy was shown to result in 90-95% depletion of B cells 16,17 . In the salivary glands of patients with Sjögren's syndrome, the efficiency of rituximab remains controversial because both complete and partial depletion of B cells have been reported 8,18 . Similarly, the effect of rituximab on synovial B cells is debated since various levels of depletion have been reported in patients with rheumatoid arthritis 12,19,20 .
Our case report illustrates that rituximab may efficiently deplete B cells in the lungs, including lung tumor, normal lung tissue, and lung-associated lymph nodes. This property of rituximab is of particular interest for the treatment of conditions in which pathogenic B cells reside in the lungs, such as antisynthetase syndrome, granulomatosis with polyangiitis, and scleroderma-associated interstitial lung disease 9, 21,22 . On the other hand, the strong B cell-depleting effect in the lungs may provide an explanation for the rare cases of severe non-infectious pulmonary toxicity of rituximab 23,24 . Rituximabassociated lung disease is a rare but potentially fatal complication of rituximab therapy, whose pathogenic mechanism remains to be elucidated 23 .
Rituximab therapy was associated with virtual absence of tumorinfiltrating B cells in a patient with lung adenocarcinoma. Nonsmall cell lung cancer (NSCLC) tumors typically contain tertiary lymphoid structures with a high frequency of CD20 + follicular B cells 25 . Tumor-infiltrating immune cells, including B cells, may represent an ongoing protective immune response against the malignant cells 26,27 . In fact, it has recently been reported that a high density of follicular B cells correlated with longer patient survival in NSCLC 25 . Therefore, rituximab-mediated depletion of tumor-infiltrating normal B cells may potentially have a detrimental impact on the antitumor immune response, particularly in NSCLC.
Author contributions HA, OTB, IØ, and AC conceived the study. AC supervised the study. JS and ÅH managed the patient. AJB, CH, BS, JS, and ÅH acquired the data. AJB, CH, BS, JS, and AC analyzed the data and prepared the figures. AC prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.

Competing interests
The authors declare that they have no competing interests. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.