The neo-open reading frame peptides that comprise the tumor framome are a rich source of neoantigens for cancer immunotherapy.

Identification of immunogenic cancer neoantigens as targets for therapy is challenging. Here, we integrate cancer whole genome and long-read transcript sequencing to identify the collection of novel open reading frame peptides (NOPs) expressed in tumors, termed the framome. NOPs represent tumor-specific peptides that are different from wild-type proteins and may be strongly immunogenic. We describe an uncharacterized class of hidden NOPs, which derive from structural genomic variants involving an upstream protein coding gene driving expression and translation of non-coding regions of the genome downstream of a rearrangement breakpoint. NOPs represent a vast amount of possible neoantigens particularly in tumors with many (complex) structural genomic variants and a low number of missense mutations. We show that NOPs are immunogenic and epitopes derived from NOPs can bind to MHC class I molecules. Finally, we provide evidence for the presence of memory T-cells specific for hidden NOPs in lung cancer patient peripheral blood.

Selective emergence of antibody-secreting cells in the multiple sclerosis brain.

BACKGROUND: Although distinct brain-homing B cells have been identified in multiple sclerosis (MS), it is unknown how these further evolve to contribute to local pathology. We explored B-cell maturation in the central nervous system (CNS) of MS patients and determined their association with immunoglobulin (Ig) production, T-cell presence, and lesion formation. METHODS: Ex vivo flow cytometry was performed on post-mortem blood, cerebrospinal fluid (CSF), meninges and white matter from 28 MS and 10 control brain donors to characterize B cells and antibody-secreting cells (ASCs). MS brain tissue sections were analysed with immunostainings and microarrays. IgG index and CSF oligoclonal bands were measured with nephelometry, isoelectric focusing, and immunoblotting. Blood-derived B cells were cocultured under T follicular helper-like conditions to evaluate their ASC-differentiating capacity in vitro. FINDINGS: ASC versus B-cell ratios were increased in post-mortem CNS compartments of MS but not control donors. Local presence of ASCs associated with a mature CD45low phenotype, focal MS lesional activity, lesional Ig gene expression, and CSF IgG levels as well as clonality. In vitro B-cell maturation into ASCs did not differ between MS and control donors. Notably, lesional CD4+ memory T cells positively correlated with ASC presence, reflected by local interplay with T cells. INTERPRETATION: These findings provide evidence that local B cells at least in late-stage MS preferentially mature into ASCs, which are largely responsible for intrathecal and local Ig production. This is especially seen in active MS white matter lesions and likely depends on the interaction with CD4+ memory T cells. FUNDING: Stichting MS Research (19-1057 MS; 20-490f MS), National MS Fonds (OZ2018-003).

Human T-bet+ B cell development is associated with BTK activity and suppressed by evobrutinib.

Recent clinical trials have shown promising results for the next-generation Bruton's tyrosine kinase (BTK) inhibitor evobrutinib in the treatment of multiple sclerosis (MS). BTK has a central role in signaling pathways that govern the development of B cells. Whether and how BTK activity shapes B cells as key drivers of MS is currently unclear. Compared with levels of BTK protein, we found higher levels of phospho-BTK in ex vivo blood memory B cells from patients with relapsing-remitting MS and secondary progressive MS compared with controls. In these MS groups, BTK activity was induced to a lesser extent after anti-IgM stimulation. BTK positively correlated with CXCR3 expression, both of which were increased in blood B cells from clinical responders to natalizumab (anti-VLA-4 antibody) treatment. Under in vitro T follicular helper-like conditions, BTK phosphorylation was enhanced by T-bet-inducing stimuli, IFN-γ and CpG-ODN, while the expression of T-bet and T-bet-associated molecules CXCR3, CD21, and CD11c was affected by evobrutinib. Furthermore, evobrutinib interfered with in vitro class switching, as well as memory recall responses, and disturbed CXCL10-mediated migration of CXCR3+ switched B cells through human brain endothelial monolayers. These findings demonstrate a functional link between BTK activity and disease-relevant B cells and offer valuable insights into how next-generation BTK inhibitors could modulate the clinical course of patients with MS.

Improving Glucocorticoid Sensitivity of Brain-Homing CD4+ T Helper Cells by Steroid Hormone Crosstalk.

In early multiple sclerosis (MS), an IFN-γhighGM-CSFhighIL-17low CD4+ T-cell subset termed T helper 17.1 (Th17.1) reveals enhanced capacity to infiltrate the central nervous system. Th17.1 cells express high levels of multidrug resistance protein 1 (MDR1), which contributes to their poor glucocorticoid responsiveness. In this study, we explored whether glucocorticoid sensitivity of Th17.1 cells can generically be improved through synergy between steroid hormones, including calcitriol (1,25(OH)2D3), estradiol (E2) and progesterone (P4). We showed that human blood Th17.1 cells were less sensitive to 1,25(OH)2D3 than Th17 cells, as reflected by lower vitamin D receptor (VDR) levels and reduced modulation of MDR1, IFN-γ and GM-CSF expression after 1,25(OH)2D3 exposure. Upon T-cell activation, VDR levels were increased, but still lower in Th17.1 versus Th17 cells, which was accompanied by a 1,25(OH)2D3-mediated decline in MDR1 surface expression as well as secretion of IFN-γ and GM-CSF. In activated Th17.1 cells, 1,25(OH)2D3 amplified the suppressive effects of methylprednisolone (MP) on proliferation, MDR1 surface levels, secretion of IFN-γ and granzyme B, as well as expression of brain-homing markers CCR6 and VLA-4. The addition of P4 to 1,25(OH)2D3 further enhanced MP-mediated reduction in proliferation, CD25, CCR6 and CXCR3. Overall, this study indicates that glucocorticoid sensitivity of Th17.1 cells can be enhanced by treatment with 1,25(OH)2D3 and further improved with P4. Our observations implicate steroid hormone crosstalk as a therapeutic avenue in Th17.1-associated inflammatory diseases including MS.

Distinct Effector Programs of Brain-Homing CD8+ T Cells in Multiple Sclerosis.

The effector programs of CD8+ memory T cells are influenced by the transcription factors RUNX3, EOMES and T-bet. How these factors define brain-homing CD8+ memory T cells in multiple sclerosis (MS) remains unknown. To address this, we analyzed blood, CSF and brain tissues from MS patients for the impact of differential RUNX3, EOMES and T-bet expression on CD8+ T cell effector phenotypes. The frequencies of RUNX3- and EOMES-, but not T-bet-expressing CD8+ memory T cells were reduced in the blood of treatment-naïve MS patients as compared to healthy controls. Such reductions were not seen in MS patients treated with natalizumab (anti-VLA-4 Ab). We found an additional loss of T-bet in RUNX3-expressing cells, which was associated with the presence of MS risk SNP rs6672420 (RUNX3). RUNX3+EOMES+T-bet- CD8+ memory T cells were enriched for the brain residency-associated markers CCR5, granzyme K, CD20 and CD69 and selectively dominated the MS CSF. In MS brain tissues, T-bet coexpression was recovered in CD20dim and CD69+ CD8+ T cells, and was accompanied by increased coproduction of granzyme K and B. These results indicate that coexpression of RUNX3 and EOMES, but not T-bet, defines CD8+ memory T cells with a pre-existing brain residency-associated phenotype such that they are prone to enter the CNS in MS.

Pregnancy-induced effects on memory B-cell development in multiple sclerosis.

In MS, pathogenic memory B cells infiltrate the brain and develop into antibody-secreting cells. Chemokine receptors not only define their brain-infiltrating capacity, but also assist in their maturation in germinal centers. How this corresponds to pregnancy, as a naturally occurring modifier of MS, is underexplored. Here, we aimed to study the impact of pregnancy on both ex vivo and in vitro B-cell differentiation in MS. The composition and outgrowth of peripheral B cells were compared between 19 MS pregnant patients and 12 healthy controls during the third trimester of pregnancy (low relapse risk) and postpartum (high relapse risk). Transitional, and not naive mature, B-cell frequencies were found to drop in the third trimester, which was most prominent in patients who experienced a pre-pregnancy relapse. Early after delivery, these frequencies raised again, while memory B -cell frequencies modestly declined. CXCR4 was downregulated and CXCR5, CXCR3 and CCR6 were upregulated on postpartum memory B cells, implying enhanced recruitment into germinal center light zones for interaction with T follicular helper (TFH) cells. Postpartum memory B cells of MS patients expressed higher levels of CCR6 and preferentially developed into plasma cells under TFH-like in vitro conditions. These findings imply that memory B- cell differentiation contributes to postpartum relapse risk in MS.

The association of Epstein-Barr virus infection with CXCR3+ B-cell development in multiple sclerosis: impact of immunotherapies.

Epstein-Barr virus (EBV) infection of B cells is associated with increased multiple sclerosis (MS) susceptibility. Recently, we found that CXCR3-expressing B cells preferentially infiltrate the CNS of MS patients. In chronic virus-infected mice, these types of B cells are sustained and show increased antiviral responsiveness. How EBV persistence in B cells influences their development remains unclear. First, we analyzed ex vivo B-cell subsets from MS patients who received autologous bone marrow transplantation (n = 9), which is often accompanied by EBV reactivation. The frequencies of nonclass-switched and class-switched memory B cells were reduced at 3-7 months, while only class-switched B cells returned back to baseline at 24-36 months posttransplantation. At these time points, EBV DNA load positively correlated to the frequency of CXCR3+ , and not CXCR4+ or CXCR5+ , class-switched B cells. Second, for CXCR3+ memory B cells trapped within the blood of MS patients treated with natalizumab (anti-VLA-4 antibody n = 15), latent EBV infection corresponded to enhanced in vitro formation of anti-EBNA1 IgG-secreting plasma cells under GC-like conditions. These findings imply that EBV persistence in B cells potentiates brain-homing and antibody-producing CXCR3+ subsets in MS.

Naive B cells in neuromyelitis optica spectrum disorders: impact of steroid use and relapses.

Neuromyelitis optica spectrum disorders are a group of rare, but severe autoimmune diseases characterized by inflammation of the optic nerve(s) and/or spinal cord. Although naive B cells are considered key players by escaping central tolerance checkpoints, it remains unclear how their composition and outgrowth differ in patients with neuromyelitis optica spectrum disorders. Under complete treatment-naive circumstances, we found that naive mature/transitional B-cell ratios were reduced in the blood of 10 patients with aquaporin-4 immunoglobulin G-positive disease (neuromyelitis optica spectrum disorders) as compared to 11 both age- and gender-matched healthy controls, eight patients with myelin oligodendrocyte glycoprotein-immunoglobulin G-associated disorders and 10 patients with multiple sclerosis. This was the result of increased proportions of transitional B cells, which were the highest in patients with neuromyelitis optica spectrum disorders with relapses and strongly diminished in a separate group of nine patients with neuromyelitis optica spectrum disorders and myelin oligodendrocyte glycoprotein-immunoglobulin G-associated disorders who received corticosteroid treatment. These findings need to be confirmed in longitudinal studies. For purified naive mature B cells of seven patients with neuromyelitis optica spectrum disorders and myelin oligodendrocyte glycoprotein-immunoglobulin G-associated disorders with relapses, Toll-like receptor 9 ligand synergized with interferon-γ to enhance plasmablast formation during germinal centre-like cultures. This was not seen for 11 patients without relapses and nine healthy controls. In the neuromyelitis optica spectrum disorders group, in vitro plasmablast formation corresponded to total and anti-aquaporin-4 immunoglobulin G secretion, of which the latter was found only for relapsing cases. These data indicate that naive B-cell homoeostasis is different and selectively targeted by corticosteroids in patients with neuromyelitis optica spectrum disorders. This also supports further exploration of naive B cells for their use in Toll-like receptor 9-dependent in vitro platforms in order to predict the activity of neuromyelitis optica spectrum disorders.

Brain-homing CD4+ T cells display glucocorticoid-resistant features in MS.

OBJECTIVE: To study whether glucocorticoid (GC) resistance delineates disease-relevant T helper (Th) subsets that home to the CNS of patients with early MS. METHODS: The expression of key determinants of GC sensitivity, multidrug resistance protein 1 (MDR1/ABCB1) and glucocorticoid receptor (GR/NR3C1), was investigated in proinflammatory Th subsets and compared between natalizumab-treated patients with MS and healthy individuals. Blood, CSF, and brain compartments from patients with MS were assessed for the recruitment of GC-resistant Th subsets using fluorescence-activated cell sorting (FACS), quantitative polymerase chain reaction (qPCR), immunohistochemistry, and immunofluorescence. RESULTS: An MS-associated Th subset termed Th17.1 showed a distinct GC-resistant phenotype as reflected by high MDR1 and low GR expression. This expression ratio was further elevated in Th17.1 cells that accumulated in the blood of patients with MS treated with natalizumab, a drug that prevents their entry into the CNS. Proinflammatory markers C-C chemokine receptor 6, IL-23R, IFN-γ, and GM-CSF were increased in MDR1-expressing Th17.1 cells. This subset predominated the CSF of patients with early MS, which was not seen in the paired blood or in the CSF from patients with other inflammatory and noninflammatory neurologic disorders. The potential of MDR1-expressing Th17.1 cells to infiltrate brain tissue was confirmed by their presence in MS white matter lesions. CONCLUSION: This study reveals that GC resistance coincides with preferential CNS recruitment of pathogenic Th17.1 cells, which may hamper the long-term efficacy of GCs in early MS.

The Role of Autoimmunity-Related Gene CLEC16A in the B Cell Receptor-Mediated HLA Class II Pathway.

C-type lectin CLEC16A is located next to CIITA, the master transcription factor of HLA class II (HLA-II), at a susceptibility locus for several autoimmune diseases, including multiple sclerosis (MS). We previously found that CLEC16A promotes the biogenesis of HLA-II peptide-loading compartments (MIICs) in myeloid cells. Given the emerging role of B cells as APCs in these diseases, in this study, we addressed whether and how CLEC16A is involved in the BCR-dependent HLA-II pathway. CLEC16A was coexpressed with surface class II-associated invariant chain peptides (CLIP) in human EBV-positive and not EBV-negative B cell lines. Stable knockdown of CLEC16A in EBV-positive Raji B cells resulted in an upregulation of surface HLA-DR and CD74 (invariant chain), whereas CLIP was slightly but significantly reduced. In addition, IgM-mediated Salmonella uptake was decreased, and MIICs were less clustered in CLEC16A-silenced Raji cells, implying that CLEC16A controls both HLA-DR/CD74 and BCR/Ag processing in MIICs. In primary B cells, CLEC16A was only induced under CLIP-stimulating conditions in vitro and was predominantly expressed in CLIPhigh naive populations. Finally, CLIP-loaded HLA-DR molecules were abnormally enriched, and coregulation with CLEC16A was abolished in blood B cells of patients who rapidly develop MS. These findings demonstrate that CLEC16A participates in the BCR-dependent HLA-II pathway in human B cells and that this regulation is impaired during MS disease onset. The abundance of CLIP already on naive B cells of MS patients may point to a chronically induced stage and a new mechanism underlying B cell-mediated autoimmune diseases such as MS.

B and T Cells Driving Multiple Sclerosis: Identity, Mechanisms and Potential Triggers.

Historically, multiple sclerosis (MS) has been viewed as being primarily driven by T cells. However, the effective use of anti-CD20 treatment now also reveals an important role for B cells in MS patients. The results from this treatment put forward T-cell activation rather than antibody production by B cells as a driving force behind MS. The main question of how their interaction provokes both B and T cells to infiltrate the CNS and cause local pathology remains to be answered. In this review, we highlight key pathogenic events involving B and T cells that most likely contribute to the pathogenesis of MS. These include (1) peripheral escape of B cells from T cell-mediated control, (2) interaction of pathogenic B and T cells in secondary lymph nodes, and (3) reactivation of B and T cells accumulating in the CNS. We will focus on the functional programs of CNS-infiltrating lymphocyte subsets in MS patients and discuss how these are defined by mechanisms such as antigen presentation, co-stimulation and cytokine production in the periphery. Furthermore, the potential impact of genetic variants and viral triggers on candidate subsets will be debated in the context of MS.

Induction of brain-infiltrating T-bet-expressing B cells in multiple sclerosis.

OBJECTIVE: Results from anti-CD20 therapies demonstrate that B- and T-cell interaction is a major driver of multiple sclerosis (MS). The local presence of B-cell follicle-like structures and oligoclonal bands in MS patients indicates that certain B cells infiltrate the central nervous system (CNS) to mediate pathology. Which peripheral triggers underlie the development of CNS-infiltrating B cells is not fully understood. METHODS: Ex vivo flow cytometry was used to assess chemokine receptor profiles of B cells in blood, cerebrospinal fluid, meningeal, and brain tissues of MS patients (n = 10). Similar analyses were performed for distinct memory subsets in the blood of untreated and natalizumab-treated MS patients (n = 38). To assess T-bet(CXCR3)+ B-cell differentiation, we cultured B cells from MS patients (n = 21) and healthy individuals (n = 34) under T helper 1- and TLR9-inducing conditions. Their CNS transmigration capacity was confirmed using brain endothelial monolayers. RESULTS: CXC chemokine receptor 3 (CXCR3)-expressing B cells were enriched in different CNS compartments of MS patients. Treatment with the clinically effective drug natalizumab prevented the recruitment of CXCR3high IgG1+ subsets, corresponding to their increased ability to cross CNS barriers in vitro. Blocking of interferon-γ (IFNγ) reduced the transmigration potential and antigen-presenting function of these cells. IFNγ-induced B cells from MS patients showed increased T-bet expression and plasmablast development. Additional TLR9 triggering further upregulated T-bet and CXCR3, and was essential for IgG1 switching. INTERPRETATION: This study demonstrates that T-bethigh IgG1+ B cells are triggered by IFNγ and TLR9 signals, likely contributing to enhanced CXCR3-mediated recruitment and local reactivity in the CNS of MS patients. ANN NEUROL 2019;86:264-278.

The macrophage migration inhibitory factor pathway in human B cells is tightly controlled and dysregulated in multiple sclerosis.

In MS, B cells survive peripheral tolerance checkpoints to mediate local inflammation, but the underlying molecular mechanisms are relatively underexplored. In mice, the MIF pathway controls B-cell development and the induction of EAE. Here, we found that MIF and MIF receptor CD74 are downregulated, while MIF receptor CXCR4 is upregulated in B cells from early onset MS patients. B cells were identified as the main immune subset in blood expressing MIF. Blocking of MIF and CD74 signaling in B cells triggered CXCR4 expression, and vice versa, with separate effects on their proinflammatory activity, proliferation, and sensitivity to Fas-mediated apoptosis. This study reveals a new reciprocal negative regulation loop between CD74 and CXCR4 in human B cells. The disturbance of this loop during MS onset provides further insights into how pathogenic B cells survive peripheral tolerance checkpoints to mediate disease activity in MS.

T helper 17.1 cells associate with multiple sclerosis disease activity: perspectives for early intervention.

Interleukin-17-expressing CD4+ T helper 17 (Th17) cells are considered as critical regulators of multiple sclerosis disease activity. However, depending on the species and pro-inflammatory milieu, Th17 cells are functionally heterogeneous, consisting of subpopulations that differentially produce interleukin-17, interferon-gamma and granulocyte macrophage colony-stimulating factor. In the current study, we studied distinct effector phenotypes of human Th17 cells and their correlation with disease activity in multiple sclerosis patients. T helper memory populations single- and double-positive for C-C chemokine receptor 6 (CCR6) and CXC chemokine receptor 3 (CXCR3) were functionally assessed in blood and/or cerebrospinal fluid from a total of 59 patients with clinically isolated syndrome, 35 untreated patients and 24 natalizumab-treated patients with relapsing-remitting multiple sclerosis, and nine patients with end-stage multiple sclerosis. Within the clinically isolated syndrome group, 23 patients had a second attack within 1 year and 26 patients did not experience subsequent attacks during a follow-up of >5 years. Low frequencies of T helper 1 (Th1)-like Th17 (CCR6+CXCR3+), and not Th17 (CCR6+CXCR3-) effector memory populations in blood strongly associated with a rapid diagnosis of clinically definite multiple sclerosis. In cerebrospinal fluid of clinically isolated syndrome and relapsing-remitting multiple sclerosis patients, Th1-like Th17 effector memory cells were abundant and showed increased production of interferon-gamma and granulocyte macrophage colony-stimulating factor compared to paired CCR6+ and CCR6-CD8+ T cell populations and their blood equivalents after short-term culturing. Their local enrichment was confirmed ex vivo using cerebrospinal fluid and brain single-cell suspensions. Across all pro-inflammatory T helper cells analysed in relapsing-remitting multiple sclerosis blood, Th1-like Th17 subpopulation T helper 17.1 (Th17.1; CCR6+CXCR3+CCR4-) expressed the highest very late antigen-4 levels and selectively accumulated in natalizumab-treated patients who remained free of clinical relapses. This was not found in patients who experienced relapses during natalizumab treatment. The enhanced potential of Th17.1 cells to infiltrate the central nervous system was supported by their predominance in cerebrospinal fluid of early multiple sclerosis patients and their preferential transmigration across human brain endothelial layers. These findings reveal a dominant contribution of Th1-like Th17 subpopulations, in particular Th17.1 cells, to clinical disease activity and provide a strong rationale for more specific and earlier use of T cell-targeted therapy in multiple sclerosis.