Case Report: Sarcoid-Like Reactions and Tertiary Lymphoid Structures Following Dual Checkpoint Inhibition in a Patient with Early-Stage Lung Adenocarcinoma.

Immune checkpoint inhibitor-induced sarcoid-like reactions and tertiary lymphoid structures (TLSs) are increasingly recognized but rarely reported in the same patient. We report a patient with lung adenocarcinoma who displayed sarcoid-like reactions in intrathoracic lymph nodes and tertiary lymphoid structures in surgical tumor after neoadjuvant therapy with nivolumab plus ipilimumab. Pathological examination revealed 50% residual tumor cells after treatment, and the CT evaluation of the primary tumor showed a stable disease. The patient experienced a recurrence eight months after surgery. To identify immune correlates of the limited response to immunotherapy, we conducted genomic and transcriptional assays, multiplex immunoassay, and multiplex immunohistochemistry on the pre- and post-immunotherapy tumor, lymph node, and plasma samples. TP53 R181C, KRAS G12C and SMAD4 R361H were identified as driver mutations of the tumor. In addition to abundant infiltrated lymphocytes, immunotherapy induced high levels of inhibitory components in post-treatment tissue samples, especially the FOXP3+ regulatory T cells in tumor and PD-L1 expression in the lymph node. Despite abundant TLSs in the post-treatment tumor, most TLSs were immature. Moreover, increasing levels of circulating checkpoint proteins BTLA, TIM-3, LAG-3, PD-1, PD-L1, and CTLA4 were observed during immunotherapy. Collectively, our observations revealed that high levels of immunosuppressive molecules in tumor, lymph nodes and/or in peripheral blood might indicate poor outcomes after immunotherapy, even in the setting of a patient with concurrent sarcoid-like reactions and tertiary lymphoid structures.

Suppression of plasmacytoid dendritic cell migration to colonic isolated lymphoid follicles abrogates the development of colitis.

BACKGROUND: Dendritic cells (DCs) play a pivotal role in maintaining immunological homeostasis by orchestrating innate and adaptive immune responses via migration to inflamed sites and the lymph nodes (LNs). Plasmacytoid DCs (pDCs) have been reported to accumulate in the colon of inflammatory bowel disease (IBD) patients and dextran sulfate sodium (DSS)-induced colitis mice. However, the role of pDCs in the progression of colonic inflammation remains unclear. METHODS: 80 compounds in natural medicines were searched for inhibitors of pDC migration using bone marrow-derived pDCs (BMpDCs) and conventional DCs (BMcDCs). BALB/c mice were given 3% DSS in the drinking water to induce acute colitis. Compounds, which specifically inhibited pDC migration, were administrated into DSS-induced colitis mice. FINDINGS: Astragaloside IV (As-IV) and oxymatrine (Oxy) suppressed BMpDC migration but not BMcDC migration. In DSS-induced colitis mice, the number of pDCs was markedly increased in the colonic lamina propria (LP), and the expression of CCL21 was obviously observed in colonic isolated lymphoid follicles (ILFs). As-IV and Oxy reduced symptoms of colitis and the accumulation of pDCs in colonic ILFs but not in the colonic LP. Moreover, in a BMpDC adoptive transfer model, BMpDC migration to colonic ILFs was significantly decreased by treatment with As-IV or Oxy. INTERPRETATION: pDCs accumulated in the colon of colitis mice, and As-IV and Oxy ameliorated colitis by suppressing pDC migration to colonic ILFs. Accordingly, the selective inhibition of pDC migration may be a potential therapeutic approach for treating colonic inflammatory diseases.

Tertiary lymphoid structures with overlapping histopathologic features of cutaneous marginal zone lymphoma during neoadjuvant cemiplimab therapy are associated with antitumor response.

The development of immune checkpoint inhibitor (ICI) therapy with anti-CTLA-4 and anti-PD-1/L1 monoclonal antibodies has led to a paradigm shift in cancer therapy. ICI neoadjuvant therapy followed by surgery has become the standard of care for several advanced-stage cancers. The pathology associated with ICI therapy is vast and includes neoadjuvant-associated tissue reactions and activation of tertiary lymphoid structures (TLSs) at the site of the tumor bed and off-target immune-related adverse events. TLSs are thought to recapitulate lymph node function and may act as localized immune machinery to mount an antitumor response. B-cell activation in TLSs during neoadjuvant ICI therapy has been correlated with antitumor response. We report a patient with a history of sarcomatoid squamous cell carcinoma treated with neoadjuvant ICI cemiplimab who developed clonal expansion of B-cells in the TLSs of the tumor bed. The TLSs morphologically mimicked a cutaneous marginal zone lymphoma with plasmacytic differentiation. Awareness of clonal expansion of B-cells in TLSs during neoadjuvant ICI therapy is critical to recognize a response to ICI therapy and to avoiding an incorrect diagnosis of low-grade B-cell lymphoma.

Tertiary lymphoid organs in the inflammatory myopathy associated with PD-1 inhibitors.

BACKGROUND: Programmed cell death 1 inhibitors have revolutionized therapy for cancer by their outstanding effectiveness. However, they may cause adverse effects, among which inflammatory myopathy is one of the most disabling. To elucidate its mechanism, we analysed muscle biopsies and compared them with other inflammatory myopathies. METHODS: Muscle biopsies from three patients with inflammatory myopathy after treatment with PD-1 inhibitors for cancer were subjected to immunohistochemical and ultrastructural analyses to localize CD8+ cytotoxic cells and markers of lymphoid follicles. For comparison, two cases of polymyositis and one of juvenile dermatomyositis were examined. RESULTS: Nearly identical pathological features were observed in the three cases. In the island-like foci of inflammation, muscle fibers were undergoing degeneration. CD8+ cytotoxic T cells, macrophages, CD4+ cells, and B cells were observed in the foci. CD8+ cells were seen outside and inside the basal lamina of non-necrotic muscle fibers. Lymphoid follicle-like structures with CD21+ follicular dendritic cells were present. The blood vessels in the foci showed features consistent with the high endothelial venules, on which their markers, PNAd and CCL21, were expressed. In polymyositis, blood vessels stained only faintly for PNAd and CCL21, while in juvenile dermatomyositis, in which tertiary lymphoid follicle-like structure was reported in the past, they stained positively. CONCLUSIONS: In inflammatory myopathy associated with PD-1 inhibitors, CD8+ cells appear to predominantly destruct muscle fibers. The presence of lymphoid follicle-like structures and expression of PNAd and CCL21 on the endothelial cells suggest the tertiary lymphoid organs are formed, and involved in the leakage of lymphocytes. Thus, in the three cases examined, formation of the tertiary lymphoid organs is likely to play an important role in genesis of the PD-1 myopathy.

Role of p53 in the chronic pulmonary immune response to tangled or rod-like multi-walled carbon nanotubes.

The fiber-like shape of multi-walled carbon nanotubes (MWCNTs) is reminiscent of asbestos, suggesting they pose similar health hazards when inhaled, including pulmonary fibrosis and mesothelioma. Mice deficient in the tumor suppressor p53 are susceptible to carcinogenesis. However, the chronic pathologic effect of MWCNTs delivered to the lungs of p53 heterozygous (p53+/-) mice has not been investigated. We hypothesized that p53+/- mice would be susceptible to lung tumor development after exposure to either tangled (t-) or rod-like (r-) MWCNTs. Wild-type (p53+/+) or p53+/- mice were exposed to MWCNTs (1 mg/kg) via oropharyngeal aspiration weekly over four consecutive weeks and evaluated for cellular and pathologic outcomes 11-months post-initial exposure. No lung or pleural tumors were observed in p53+/+ or p53+/- mice exposed to either t- or rMWCNTs. In comparison to tMWCNTs, the rMWCNTs induced the formation of larger granulomas, a greater number of lymphoid aggregates and greater epithelial cell hyperplasia in terminal bronchioles in both p53+/- and p53+/+ mice. A constitutively larger area of CD45R+/CD3+ lymphoid tissue was observed in p53+/- mice compared to p53+/+ mice. Importantly, p53+/- mice had larger granulomas induced by rMWCNTs as compared to p53+/+ mice. These findings indicate that a combination of p53 deficiency and physicochemical characteristics including nanotube geometry are factors in susceptibility to MWCNT-induced lymphoid infiltration and granuloma formation.

Germinal Centers Determine the Prognostic Relevance of Tertiary Lymphoid Structures and Are Impaired by Corticosteroids in Lung Squamous Cell Carcinoma.

In solid tumors, the presence of lymph node-like structures called tertiary lymphoid structures (TLS) is associated with improved patient survival. However, little is known about how TLS develop in cancer, how their function affects survival, and whether they are affected by cancer therapy. In this study, we used multispectral microscopy, quantitative pathology, and gene expression profiling to analyze TLS formation in human lung squamous cell carcinoma (LSCC) and in an experimental model of lung TLS induction. We identified a niche of CXCL13+ perivascular and CXCL12+LTB+ and PD-L1+ epithelial cells supporting TLS formation. We also characterized sequential stages of TLS maturation in LSCC culminating in the formation of germinal centers (GC). In untreated patients, TLS density was the strongest independent prognostic marker. Furthermore, TLS density correlated with GC formation and expression of adaptive immune response-related genes. In patients treated with neoadjuvant chemotherapy, TLS density was similar, but GC formation was impaired and the prognostic value of TLS density was lost. Corticosteroids are coadministered with chemotherapy to manage side effects in LSCC patients, so we evaluated whether they impaired TLS development independently of chemotherapy. TLS density and GC formation were each reduced in chemotherapy-naïve LSCC patients treated with corticosteroids before surgery, compared with untreated patients, a finding that we confirmed in the experimental model of lung TLS induction. Overall, our results highlight the importance of GC formation in TLS during tumor development and treatment.Significance: Corticosteroid treatment during chemotherapy negatively affects the development of tertiary lymphoid structures and abrogates their prognostic value in patients with lung cancer. Cancer Res; 78(5); 1308-20. ©2018 AACR.

Role of BAFF in pulmonary autoantibody responses induced by chronic cigarette smoke exposure in mice.

Emerging evidence suggests that autoimmune processes are implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). In this study, we assessed the expression of B-cell activating factor (BAFF) in smokers, and investigated the functional importance of BAFF in the induction and maintenance of cigarette smoke-induced pulmonary antinuclear antibodies (ANA) and tertiary lymphoid tissues (TLTs) using a preclinical mouse model. We observed that BAFF levels were elevated in smokers and mice exposed to cigarette smoke. In mice, BAFF expression was rapidly induced in the lungs following 4 days of cigarette smoke exposure and remained elevated following 8 and 24 weeks of exposure. Alveolar macrophages were the major source of BAFF Blockade of BAFF using a BAFF receptor-Fc (BAFFR-Fc) construct prevented pulmonary ANA and TLT formation when delivered concurrent with cigarette smoke exposure. Under these conditions, no impact on lung inflammation was observed. However, administration of BAFFR-Fc following smoking cessation markedly reduced the number of TLTs and ANA levels and, of note, reduced pulmonary neutrophilia. Altogether, this study shows for the first time a central role of BAFF in the induction and maintenance of cigarette smoke-induced pulmonary ANA and suggests that BAFF blockade following smoking cessation could have beneficial effects on persistent inflammatory processes.In this study, we assessed the expression of B-cell activating factor (BAFF) in smokers, and investigated the functional importance of BAFF in the induction and maintenance of cigarette smoke-induced pulmonary antinuclear antibodies (ANA) and tertiary lymphoid tissues (TLTs) using a preclinical mouse model. Data presented show that BAFF plays a central role in the induction and maintenance of cigarette smoke-induced pulmonary ANA and suggest a therapeutic potential for BAFF blockade in limiting autoimmune processes associated with smoking.