Tertiary Lymphoid Structures Validated as Biomarker.

New trial data confirm the potential of tertiary lymphoid structures to serve as a predictive biomarker of responsiveness to an immune checkpoint inhibitor-based drug regimen. According to study results presented at the Society for Immunotherapy of Cancer Annual Meeting, treatment with a PD-L1-targeted agent plus a multikinase inhibitor with anti-angiogenic activity yielded clinical responses in patients with TLS-positive tumors, even in cancer types typically considered resistant to checkpoint inhibitors.

Association of NRAS mutations and tertiary lymphoid structure formation with clinical outcomes of adjuvant PD-1 inhibitors for acral melanoma.

OBJECTIVES: This study evaluates the efficacy of programmed death-1 (PD-1) inhibitors as adjuvant therapy for acral melanoma (AM) and the predictive value of genetic mutations and tertiary lymphoid structures (TLSs). METHODS AND RESULTS: A single-center retrospective longitudinal cohort study was conducted between October 1, 2018, and September 31, 2022. Patients with stages II-III completely resected AM were treated with at least two doses of adjuvant PD-1 inhibitors. A total of 44 participants were included in the final analysis, of which 41 patients with stage III. The median follow-up time, median relapse-free survival (RFS), and median distance metastasis-free survival (DMFS) for all patients were 18.4 months, 21.6 months, and 30.6 months, respectively. 21 (47.7%) and 20 (45.5%) patients were intravenously administered pembrolizumab and toripalimab, respectively. There were no significant differences in RFS (24.4 months vs. 18.9 months, p = 0.432) or DMFS (30.6 months vs. not reached, p = 0.865) between the pembrolizumab and toripalimab groups, respectively. The median DMFS (41.1 months vs. 9.0 months, p < 0.001) in the wild-type NRAS group was significantly longer than that in the NRAS mutation group. Overall, different levels of TLSs infiltration did not significantly affect patient survival. Only three people discontinued treatment due to adverse events. No treatment-related death occurred during the study period. CONCLUSION: Our study suggests that adjuvant toripalimab and pembrolizumab therapy have comparable efficacies in patients with AM and are both well tolerated. Adjuvant monotherapy with PD-1 inhibitors may not be appropriate for AM with NRAS mutations.

Siponimod Inhibits the Formation of Meningeal Ectopic Lymphoid Tissue in Experimental Autoimmune Encephalomyelitis.

BACKGROUND AND OBJECTIVES: To investigate whether the formation or retention of meningeal ectopic lymphoid tissue (mELT) can be inhibited by the sphingosine 1-phosphate receptor 1,5 modulator siponimod (BAF312) in a murine model of multiple sclerosis (MS). METHODS: A murine spontaneous chronic experimental autoimmune encephalomyelitis (EAE) model, featuring meningeal inflammatory infiltrates resembling those in MS, was used. To prevent or treat EAE, siponimod was administered daily starting either before EAE onset or at peak of disease. The extent and cellular composition of mELT, the spinal cord parenchyma, and the spleen was assessed by histology and immunohistochemistry. RESULTS: Siponimod, when applied before disease onset, ameliorated EAE. This effect was also present, although less prominent, when treatment started at peak of disease. Treatment with siponimod resulted in a strong reduction of the extent of mELT in both treatment paradigms. Both B and T cells were diminished in the meningeal compartment. DISCUSSION: Beneficial effects on the disease course correlated with a reduction in mELT, suggesting that inhibition of mELT may be an additional mechanism of action of siponimod in the treatment of EAE. Further studies are needed to establish causality and confirm this observation in MS.

Clinical efficacy of nivolumab is associated with tertiary lymphoid structures in surgically resected primary tumors of recurrent gastric cancer.

Nivolumab, an immune checkpoint blocker, has been approved for advanced gastric cancer (GC), but predictive factors of nivolumab's efficacy in patients with GC, especially immune cells such as tissue-resident memory T cells or those forming tertiary lymphoid structures (TLS), remain unclear. Tissue samples were obtained from surgically resected specimens of patients with GC who were treated with nivolumab as third-line or later treatment. Immunohistochemical staining was performed to detect the presence of TLS and CD103+ T cells and assess the association between TLSs and response to nivolumab treatment. A total of 19 patients were analyzed. In patients with partial response (PR) to nivolumab, numerous TLS were observed, and CD103+ T cells were found in and around TLS. Patients with many TLS experienced immune-related adverse events more often than those with few TLS (p = 0.018). The prognosis of patients with TLS high was better than those with TLS low. Patients with a combination of TLS high and CD103 high tended to have a better prognosis than other groups. Our results suggested that TLS status might be a predictor of nivolumab effectiveness.

Pharmacology of HIV Cure: Site of Action.

Despite significant advances in HIV treatment over the past 30 years, critical barriers to an HIV cure persist. The HIV reservoir, defined at both the cellular and anatomical level, constitutes the main barrier to cure. While the mechanisms underlying the reservoir are not yet well understood, one theory to explain persistence at the anatomical level is that subtherapeutic exposure to antiretroviral therapy (ART) within certain tissue compartments permits ongoing replication. Characterizing ART pharmacology throughout the body is important in the context of these potential pharmacologic sanctuaries and for maximizing the probability of success with forthcoming cure strategies that rely on latency reversal and require ART to prevent reseeding the reservoir. In this review, we provide a comprehensive overview of ART and latency reversal agent distribution at the site of action for HIV cure (i.e., anatomical sites commonly associated with HIV persistence, such as lymphoid organs and the central nervous system). We also discuss methodologic approaches that provide insight into HIV cure pharmacology, including experimental design and advances within the computational, pharmaceutical, and analytical chemistry fields. The information discussed in this review will assist in streamlining the development of investigational cure strategies by providing a roadmap to ensure therapeutic exposure within the site of action for HIV cure.

Anti-CD20 Depletes Meningeal B Cells but Does Not Halt the Formation of Meningeal Ectopic Lymphoid Tissue.

OBJECTIVE: To investigate whether anti-CD20 B-cell-depleting monoclonal antibodies (ɑCD20 mAbs) inhibit the formation or retention of meningeal ectopic lymphoid tissue (mELT) in a murine model of multiple sclerosis (MS). METHODS: We used a spontaneous chronic experimental autoimmune encephalomyelitis (EAE) model of mice with mutant T-cell and B-cell receptors specific for myelin oligodendrocyte glycoprotein (MOG), which develop meningeal inflammatory infiltrates resembling those described in MS. ɑCD20 mAbs were administered in either a preventive or a treatment regimen. The extent and cellular composition of mELT was assessed by histology and immunohistochemistry. RESULTS: ɑCD20 mAb, applied in a paradigm to either prevent or treat EAE, did not alter the disease course in either condition. However, ɑCD20 mAb depleted virtually all B cells from the meningeal compartment but failed to prevent the formation of mELT altogether. Because of the absence of B cells, mELT was less densely populated with immune cells and the cellular composition was changed, with increased neutrophil granulocytes. CONCLUSIONS: These results demonstrate that, in CNS autoimmune disease, meningeal inflammatory infiltrates may form and persist in the absence of B cells. Together with the finding that ɑCD20 mAb does not ameliorate spontaneous chronic EAE with mELT, our data suggest that mELT may have yet unknown capacities that are independent of B cells and contribute to CNS autoimmunity.

Silica Induction of Diverse Inflammatory Proteome in Lungs of Lupus-Prone Mice Quelled by Dietary Docosahexaenoic Acid Supplementation.

Repeated short-term intranasal instillation of lupus-prone mice with crystalline silica (cSiO2) induces inflammatory gene expression and ectopic lymphoid neogenesis in the lung, leading to early onset of systemic autoimmunity and rapid progression to glomerulonephritis. These responses are suppressed by dietary supplementation with the ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA). Here, we tested the hypothesis that dietary DHA supplementation suppresses cSiO2-induced inflammatory proteins in bronchoalveolar alveolar lavage fluid (BALF) and plasma of lupus-prone mice. Archived tissue fluid samples were used from a prior investigation in which 6 wk-old lupus-prone female NZBWF1 mice were fed isocaloric diets containing 0 or 10 g/kg DHA for 2 wks and then intranasally instilled with 1 mg cSiO2 or vehicle once weekly for 4 wks. Cohorts were terminated at 1, 5, 9 or 13 wk post-instillation (PI). BALF and plasma from each cohort were analyzed by high density multiplex array profiling of 200 inflammatory proteins. cSiO2 time-dependently induced increases in the BALF protein signatures that were highly reflective of unresolved lung inflammation, although responses in the plasma were much less robust. Induced proteins in BALF included chemokines (e.g., MIP-2, MCP-5), enzymes (e.g., MMP-10, granzyme B), adhesion molecules (e.g., sE-selectin, sVCAM-1), co-stimulatory molecules (e.g., sCD40L, sCD48), TNF superfamily proteins (e.g., sTNFRI, sBAFF-R), growth factors (e.g., IGF-1, IGFBP-3), and signal transduction proteins (e.g., MFG-E8, FcgRIIB), many of which were blocked or delayed by DHA supplementation. The BALF inflammatory proteome correlated positively with prior measurements of gene expression, pulmonary ectopic lymphoid tissue neogenesis, and induction of autoantibodies in the lungs of the control and treatment groups. Ingenuity Pathway Analysis (IPA) revealed that IL-1ß, TNF-α, and IL-6 were among the top upstream regulators of the cSiO2-induced protein response. Furthermore, DHA's effects were associated with downregulation of cSiO2-induced pathways involving i) inhibition of ARE-mediated mRNA decay, ii) bacterial and viral pattern recognition receptor activation, or iii) TREM1, STAT3, NF-κB, and VEGF signaling and with upregulation of PPAR, LXR/RXR and PPARα/RXRα signaling. Altogether, these preclinical findings further support the contention that dietary DHA supplementation could be applicable as an intervention against inflammation-driven autoimmune triggering by cSiO2 or potentially other environmental agents.

Pancreatic Cancer Chemotherapy Is Potentiated by Induction of Tertiary Lymphoid Structures in Mice.

BACKGROUND AND AIMS: The presence of tertiary lymphoid structures (TLSs) may confer survival benefit to patients with pancreatic ductal adenocarcinoma (PDAC), in an otherwise immunologically inert malignancy. Yet, the precise role in PDAC has not been elucidated. Here, we aim to investigate the structure and role of TLSs in human and murine pancreatic cancer. METHODS: Multicolor immunofluorescence and immunohistochemistry were used to fully characterize TLSs in human and murine (transgenic [KPC (KrasG12D, p53R172H, Pdx-1-Cre)] and orthotopic) pancreatic cancer. An orthotopic murine model was developed to study the development of TLSs and the effect of the combined chemotherapy and immunotherapy on tumor growth. RESULTS: Mature, functional TLSs are not ubiquitous in human PDAC and KPC murine cancers and are absent in the orthotopic murine model. TLS formation can be induced in the orthotopic model of PDAC after intratumoral injection of lymphoid chemokines (CXCL13/CCL21). Coadministration of systemic chemotherapy (gemcitabine) and intratumoral lymphoid chemokines into orthotopic tumors altered immune cell infiltration ,facilitating TLS induction and potentiating antitumor activity of chemotherapy. This resulted in significant tumor reduction, an effect not achieved by either treatment alone. Antitumor activity seen after TLS induction is associated with B cell-mediated dendritic cell activation. CONCLUSIONS: This study provides supportive evidence that TLS induction may potentiate the antitumor activity of chemotherapy in a murine model of PDAC. A detailed understanding of TLS kinetics and their induction, owing to multiple host and tumor factors, may help design personalized therapies harnessing the potential of immune-oncology.

89Zr-pembrolizumab biodistribution is influenced by PD-1-mediated uptake in lymphoid organs.

BACKGROUND: To better predict response to immune checkpoint therapy and toxicity in healthy tissues, insight in the in vivo behavior of immune checkpoint targeting monoclonal antibodies is essential. Therefore, we aimed to study in vivo pharmacokinetics and whole-body distribution of zirconium-89 (89Zr) labeled programmed cell death protein-1 (PD-1) targeting pembrolizumab with positron-emission tomography (PET) in humanized mice. METHODS: Humanized (huNOG) and non-humanized NOG mice were xenografted with human A375M melanoma cells. PET imaging was performed on day 7 post 89Zr-pembrolizumab (10 µg, 2.5 MBq) administration, followed by ex vivo biodistribution studies. Other huNOG mice bearing A375M tumors received a co-injection of excess (90 µg) unlabeled pembrolizumab or 89Zr-IgG4 control (10 µg, 2.5 MBq). Tumor and spleen tissue were studied with autoradiography and immunohistochemically including PD-1. RESULTS: PET imaging and biodistribution studies showed high 89Zr-pembrolizumab uptake in tissues containing human immune cells, including spleen, lymph nodes and bone marrow. Tumor uptake of 89Zr-pembrolizumab was lower than uptake in lymphoid tissues, but higher than uptake in other organs. High uptake in lymphoid tissues could be reduced by excess unlabeled pembrolizumab. Tracer activity in blood pool was increased by addition of unlabeled pembrolizumab, but tumor uptake was not affected. Autoradiography supported PET findings and immunohistochemical staining on spleen and lymph node tissue showed PD-1 positive cells, whereas tumor tissue was PD-1 negative. CONCLUSION: 89Zr-pembrolizumab whole-body biodistribution showed high PD-1-mediated uptake in lymphoid tissues, such as spleen, lymph nodes and bone marrow, and modest tumor uptake. Our data may enable evaluation of 89Zr-pembrolizumab whole-body distribution in patients.