Genetic aberrations of NLRC5 are associated with downregulated MHC‐I antigen presentation and impaired T‐cell immunity in follicular lymphoma

Follicular lymphoma (FL) is the most common indolent non‐Hodgkin lymphoma. Twenty to twenty‐five percent of FL patients have progression of disease within 24 months. These patients may benefit from immunotherapy if intact antigen presentation is present. Molecular mechanisms impairing major histocompatibility complex class‐I (MHC‐I) in FL remain undefined. Here, by sequencing of 172 FL tumours, we found the MHC‐I transactivator NLRC5 was the most frequent gene abnormality in the MHC‐I pathway. Pyrosequencing showed that epigenetic silencing of the\ua0NLRC5\ua0promoter occurred in 30% of cases and was mutually exclusive to copy number loss (CNL) in\ua0NLRC5\ua0(∼6% of cases). Hypermethylation and CNLs (“NLRC5\ua0aberrant”) had reduced\ua0NLRC5\ua0gene expression compared to wild‐type (WT) cases. By NanoString, there was reduced gene expression of the MHC‐I pathway in aberrant tissues, including immunoproteasome components (PSMB8\ua0and\ua0PSMB9), peptide transporters of antigen processing (TAP1), and MHC‐I (HLA‐A), compared to WT. By immunofluorescent microscopy, fewer NLRC5 protein‐expressing malignant B‐cells were observed in NLRC5 aberrant tissue sections compared to NLRC5 WT (P\ua0\ua00.7;\ua0P\ua

Intratumoral T-cell receptor repertoire is predictive of interim PET scan results in patients with diffuse large B-cell lymphoma treated with rituximab/cyclophosphamide/doxorubicin/prednisolone/vincristine (R-CHOP) chemoimmunotherapy

Objectives: A diverse intratumoral T-cell receptor (TCR) repertoire is associated with improved survival in diffuse large B-cell lymphoma (DLBCL) treated with rituximab/cyclophosphamide/doxorubicin/prednisolone/vincristine (R-CHOP) chemoimmunotherapy. We explored the impact of intratumoral TCR repertoire on interim PET (iPET) done after four cycles of R-CHOP, the relationships between intratumoral and circulating repertoire, and the phenotypes of expanded clonotypes. Methods: We sequenced the third complementarity-determining region of TCRβ in tumor samples, blood at pre-therapy and after four cycles of R-CHOP in 35 patients enrolled in ALLGNHL21 trial in high-risk DLBCL. We correlated the TCR diversity metrics with iPET status, gene expression profiles and HLA-class I genotypes. We then sequenced the FACS-sorted peripheral blood T cells in six patients, and pentamer-sorted EBV-specific CD8+ T cells in one patient from this cohort. Results: Compared with iPET− patients, the intratumoral TCR repertoire in iPET+ patients was characterised by higher cumulative frequency of abundant clonotypes and higher productive clonality. There was a variable overlap between circulating and intratumoral repertoires, with the dominant intratumoral clonotypes more likely to be detected in the blood. The majority of shared clonotypes were CD8+ PD-1HI T cells, and CD8+ T cells had the largest clonal expansions in tumor and blood. In a patient with EBV+ DLBCL, EBV-specific intratumoral clonotypes were trackable in the blood. Conclusion: This study demonstrates that clonally expanded intratumoral TCR repertoires are associated with iPET+ and that the blood can be used to track tumor-associated antigen-specific clonotypes. These findings assist the rationale design and therapeutic monitoring of immunotherapeutic strategies in DLBCL.</p

Successful treatment of Epstein–Barr virus–associated primary central nervous system lymphoma due to post-transplantation lymphoproliferative disorder, with ibrutinib and third-party Epstein–Barr virus–specific T cells

Primary central nervous system lymphoma (PCNSL) occurring following organ transplantation (post-transplantation lymphoproliferative disorder [PTLD]) is a highly aggressive non-Hodgkin lymphoma. It is typically treated with high-dose methotrexate-based regimens. Outcomes are dismal and clinical trials are lacking. It is almost always Epstein–Barr virus (EBV) associated. Two patients (CA1-2) presented with EBV-associated PCNSL after renal transplant. CA1 was on hemodialysis and had prior disseminated cryptococcus and pseudomonas bronchiectasis, precluding treatment with methotrexate. CA2 was refractory to methotrexate. Both were treated off-label with the first-generation Bruton's tyrosine kinase inhibitor ibrutinib for 12 months. Cerebrospinal fluid penetration at therapeutic levels was confirmed in CA1 despite hemodialysis. Both patients entered remission by 2 months. Sequencing confirmed absence of genetic aberrations in human leukocyte antigen (HLA) class I/II and antigen-presentation/processing genes, indicating retention of the ability to present EBV-antigens. Between Weeks 10 and 13, they received third-party EBV-specific T cells for consolidation with no adverse effects. They remain in remission ≥34 months since therapy began. The strength of these findings led to an ongoing phase I study (ACTRN12618001541291).</p

LAG3: a novel immune checkpoint expressed by multiple lymphocyte subsets in diffuse large B-cell lymphoma

Blockade of\ua0the PD-1 axis has modest efficacy in diffuse large B-cell lymphoma (DLBCL), but data regarding LAG3 are sparse. The impact of LAG3 digital gene expression was tested in 309 patients with DLBCL treated with standard chemoimmunotherapy. Cellular distribution of LAG3 protein was determined by immunohistochemistry and flow cytometry. In tumor-infiltrating lymphocytes (TILs), LAG3 expression was highest on CD4+\ua0regulatory T cells (Tregs) and was also highly expressed on CD8+\ua0T cells compared with CD4+\ua0non-Tregs (both\ua0P\ua0= .008). LAG3high\ua0TILs were enriched in PD-1 and TIM-3. LAG3 was also expressed on a proportion of malignant B cells, and these patients had significantly higher LAG3 messenger RNA in their biopsies (P\ua0= .03). LAG3high\ua0gene expression was\ua0associated with inferior survival in discovery/validation cohorts, independent of cell of origin and the international prognostic index. Patients who were PD-L1high\ua0were fivefold more likely to be LAG3high\ua0(P\ua0< .0001). Patients who were LAG3high/PD-L1high\ua0had an inferior progression-free survival (P\ua0= .011) and overall survival (P\ua0= .005) compared with patients who were LAG3low/PD-L1high. Digital spatial protein analysis confirms LAG3 expression on T cells and, surprisingly, tumor-associated macrophages (TAMs) at higher levels than found on CD20+\ua0B cells in the tumor microenvironment. LAG3 is frequently expressed on CD4+\ua0Tregs and CD8+\ua0TILs, typically with other immune checkpoints, and is also present in a proportion of malignant B cells in DLBCL and in areas enriched for TAMs. LAG3high\ua0expression is associated with poor outcome independent of conventional prognosticators

Cathepsin S alterations induce a tumor-promoting immune microenvironment in follicular lymphoma

Bararia et al. discover and functionally characterize a clinically relevant mechanism of tumor and immune cell interaction in follicular lymphoma, a prototypical type of blood cancer. Cathepsin S alterations result in aberrant hyperactivity of this lysosomal cysteine protease and induce a tumor-promoting CD4 T cell enriched immune microenvironment.Tumor cells orchestrate their microenvironment. Here, we provide biochemical, structural, functional, and clinical evidence that Cathepsin S (CTSS) alterations induce a tumor-promoting immune microenvironment in follicular lymphoma (FL). We found CTSS mutations at Y132 in 6% of FL (19/305). Another 13% (37/286) had CTSS amplification, which was associated with higher CTSS expression. CTSS Y132 mutations lead to accelerated autocatalytic conversion from an enzymatically inactive profrom to active CTSS and increased substrate cleavage, including CD74, which regulates major histocompatibility complex class II (MHC class II)-restricted antigen presentation. Lymphoma cells with hyperactive CTSS more efficiently activated antigen-specific CD4 T cells in vitro. Tumors with hyperactive CTSS showed increased CD4 T cell infiltration and proinflammatory cytokine perturbation in a mouse model and in human FLs. In mice, this CTSS-induced immune microenvironment promoted tumor growth. Clinically, patients with CTSS-hyperactive FL had better treatment outcomes with standard immunochemotherapies, indicating that these immunosuppressive regimens target both the lymphoma cells and the tumor-promoting immune microenvironment