A 48-year-old Caucasian man was referred to our department for progressive asthenia, anorexia, and cachexia (-12 kg in 9 months).
His main medical history was a hematological malignancy (OMS 2008 grade 1-2 follicular lymphoma) diagnosed 3 years before and involving the lacrimal gland and the bowel. Biopsies (right part of the colon) revealed a monotypic lymphoid infiltrate made of CD20+, CD5-, CD10+, and Bcl6+ lymphocytes with a Ki67 index at 50%. Lungs were not involved at this time (Supplementary Figure S1). He was treated with combined chemotherapy (R-CHOP) and a maintenance therapy with Rituximab every 2 months, for 18 months. He had experienced neither opportunistic and severe infections nor autoimmune diseases. Timeline of his medical history is resumed in Figure 1.
Seven months after the last Rituximab therapy, the patient presented fever, splenomegaly (17 cm), and fine dry crackles at pulmonary auscultation with dyspnea (New York Heart Association Classification class II-IV). An interstitial lung disease with nodular lesions was diagnosed on CT-scan with a decline of alveolar exchanges on breath tests (Figure 2A). Broncho-alveolar lavage (BAL) revealed an alveolar lymphocytosis (230,000 cells/ml, 67% lymphocytes, 28% macrophages) with inverted CD4/CD8 ratio and without any virus (screened by PCR), bacteria, or parasite in culture. Biology showed inflammatory syndrome, acute renal failure, pancytopenia with severe lymphopenia, mild neutropenia, thrombopenia and anemia, as well as hypogammaglobulinemia (Supplementary Table S1). Bone marrow aspiration revealed a poor cellularity and fat involution (reflecting likely the nutritional deficiency state) without malignant cells. Considering kidney failure, a biopsy was made and revealed an acute interstitial nephritis without glomerular lesions. 18F-FDG PET/CT-scan showed several hypermetabolic lesions (tonsil, lung nodules, and parenchyma) but no hypermetabolism of the spleen. Within a couple of days, the condition of the patient worsened with hypoxemia requiring increasing level of oxygen despite probabilistic antibiotherapy (sulfamethoxazole/trimethoprim for Pneumocysis jirovecii PCR+ in BAL and large-spectrum antibacterial therapy with piperacillin/tazobactam). Corticosteroids were added and transiently improved the respiratory state. A lung biopsy was performed that finally revealed invasive infection by M. bovis BCG, suspected by Ziehl-Neelsen coloration and confirmed by PCR and culture (Figure 2B). The M. bovis BCG strain was also found in the bone marrow and blood cultures. The strain was phenotypically susceptible to rifampicin, isoniazid, and ethambutol. Cryptococcus neoformans was also identified in lung biopsy but without central nervous system involvement. HIV PCR and serology were negative. Phagocytosis assays were normal, discarding the hypothesis of chronic granulomatous disease.
Despite a treatment regimen of isoniazid, rifampin, pyrazinamide, and ethambutol for BCGosis, initiated before the strain identification, and fluconazole for Cryptococcus, the patient state worsened with need for intensive care unit support. Corticosteroids were re-introduced, and G-CSF therapy was added two times per week. We hypothesized a primary or secondary defect in IFNgamma pathway, such as an autoimmunization against IFNgamma, and we decided to introduce Imukin  as an add-on therapy (50 microg S.C. three times/week and after 2 weeks at 100 microg S.C. three times/week) (Supplementary Table S1). This strategy finally allowed an improvement of the patient's condition with CT-scan and TEP-scan complete normalization after 3 and 6 months, respectively (Figure 2A). Interferon therapy was maintained during 12 months with bi-antibiotherapy (rifampcin and isoniazid) and fluconazole after the complete remission in order to avoid any relapse, and 12 months after treatment's discontinuation, the patient remained in complete remission.
The patient harbored a severe post-chemotherapy lymphopenia with delayed immune reconstitution of B-cell and T-cell compartment, i.e., very low level of CD19+ cells with a selective lack of IgM production, and low levels of naive CD45RA+ T cells. CD4/CD8 ratio was conserved in proportion (Supplementary Table S1). The absolute number of monocytes was normal with 55% of classical, 18% of intermediate, and 27% non-classical monocytes.
Potential IFN pathway defects that may lead to ineffective response towards BCG were explored. IFNgamma stimulation of peripheral mononuclear cells (PBMCs) from a healthy donor (HD) in the presence of 25% (v/v) allogenic HD or patient's serum (from different time points, i.e., before, during, and after IFNgamma therapy) did not impair STAT1 phosphorylation. Similarly, no significant difference in STAT1 phosphorylation was observed when we mixed the IFNgamma and the patient's serum before using it to stimulate PBMCs from HD. These results suggested for the absence of anti-IFNgamma autoantibodies or any other autoantibodies that may directly hinder/block IFNgamma signaling (Figures 3A-C). Absence of anti-GM-CSF autoantibodies was also confirmed (data not shown). STAT1 phosphorylation, after IFNgamma stimulation of monocytes from the patient, was preserved (Figure 3D). CD4+ T cells from the patient responded to BCG (Figure 3E) and showed no apparent defect of IFNgamma production after BCG +- IL12 stimulation (Figure 3F). Altogether, these results did not support the hypothesis of an IEI or acquired immunodeficiency related to defective host defense mechanisms against BCG.
Whole exome sequencing (WES) was performed but did not reveal/detect any known mutations or unknown variants with high in silico predicted consequence (CADD > 10, MAF < 0.005%) involved in MSMD (i.e., IL12RB1, IL12B, IL12RB2, IL23R, IFNGR1, IFNGR2, STAT1, CYBB, IRF8, SPPL2A, TYK2, ISG15, RORC, JAK1, and NEMO). Similarly, no candidate genes of IEI were found (Supplementary Table S2).
Whole genome sequencing (WGS) of M. bovis BCG strain and comparative phylogenomic analysis with the genomes of reference M. bovis BCG strains were performed at the National Reference Center for Mycobacteria and Resistance of Mycobacteria to Antituberculosis (CNR-MyRMA). The results showed that the patient's strain (M. bovis BCG_ 1811074784, Figure 4) was most closely related to the M. bovis BCG Tice strain.