HLA-DP on Epithelial Cells Enables Tissue Damage by NKp44+ Natural Killer Cells in Ulcerative Colitis.

BACKGROUND & AIMS: Ulcerative colitis (UC) is characterized by severe inflammation and destruction of the intestinal epithelium, and is associated with specific risk single nucleotide polymorphisms in HLA class II. Given the recently discovered interactions between subsets of HLA-DP molecules and the activating natural killer (NK) cell receptor NKp44, genetic associations of UC and HLA-DP haplotypes and their functional implications were investigated. METHODS: HLA-DP haplotype and UC risk association analyses were performed (UC: n = 13,927; control: n = 26,764). Expression levels of HLA-DP on intestinal epithelial cells (IECs) in individuals with and without UC were quantified. Human intestinal 3-dimensional (3D) organoid cocultures with human NK cells were used to determine functional consequences of interactions between HLA-DP and NKp44. RESULTS: These studies identified HLA-DPA1∗01:03-DPB1∗04:01 (HLA-DP401) as a risk haplotype and HLA-DPA1∗01:03-DPB1∗03:01 (HLA-DP301) as a protective haplotype for UC in European populations. HLA-DP expression was significantly higher on IECs of individuals with UC compared with controls. IECs in human intestinal 3D organoids derived from HLA-DP401pos individuals showed significantly stronger binding of NKp44 compared with HLA-DP301pos IECs. HLA-DP401pos IECs in organoids triggered increased degranulation and tumor necrosis factor production by NKp44+ NK cells in cocultures, resulting in enhanced epithelial cell death compared with HLA-DP301pos organoids. Blocking of HLA-DP401-NKp44 interactions (anti-NKp44) abrogated NK cell activity in cocultures. CONCLUSIONS: We identified an UC risk HLA-DP haplotype that engages NKp44 and activates NKp44+ NK cells, mediating damage to intestinal epithelial cells in an HLA-DP haplotype-dependent manner. The molecular interaction between NKp44 and HLA-DP401 in UC can be targeted by therapeutic interventions to reduce NKp44+ NK cell-mediated destruction of the intestinal epithelium in UC.

Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma, Version 2.2024, NCCN Clinical Practice Guidelines in Oncology

Chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) are essentially different manifestations of the same disease that are similarly managed. A number of molecular and cytogenetic variables with prognostic implications have been identified. Undetectable minimal residual disease at the end of treatment with chemoimmunotherapy or venetoclax-based combination regimens is an independent predictor of improved survival among patients with previously untreated or relapsed/refractory CLL/SLL. The selection of treatment is based on the disease stage, presence or absence of del(17p) or TP53 mutation, immunoglobulin heavy chain variable region mutation status, patient age, performance status, comorbid conditions, and the agent's toxicity profile. This manuscript discusses the recommendations outlined in the NCCN Guidelines for the diagnosis and management of patients with CLL/SLL.

Exposure of progressive immune dysfunction by SARS-CoV-2 mRNA vaccination in patients with chronic lymphocytic leukemia: A prospective cohort study.

BACKGROUND: Patients with chronic lymphocytic leukemia (CLL) have reduced seroconversion rates and lower binding antibody (Ab) and neutralizing antibody (NAb) titers than healthy individuals following Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) mRNA vaccination. Here, we dissected vaccine-mediated humoral and cellular responses to understand the mechanisms underlying CLL-induced immune dysfunction. METHODS AND FINDINGS: We performed a prospective observational study in SARS-CoV-2 infection-naïve CLL patients (n = 95) and healthy controls (n = 30) who were vaccinated between December 2020 and June 2021. Sixty-one CLL patients and 27 healthy controls received 2 doses of the Pfizer-BioNTech BNT162b2 vaccine, while 34 CLL patients and 3 healthy controls received 2 doses of the Moderna mRNA-1273 vaccine. The median time to analysis was 38 days (IQR, 27 to 83) for CLL patients and 36 days (IQR, 28 to 57) for healthy controls. Testing plasma samples for SARS-CoV-2 anti-spike and receptor-binding domain Abs by enzyme-linked immunosorbent assay (ELISA), we found that all healthy controls seroconverted to both antigens, while CLL patients had lower response rates (68% and 54%) as well as lower median titers (23-fold and 30-fold; both p < 0.001). Similarly, NAb responses against the then prevalent D614G and Delta SARS-CoV-2 variants were detected in 97% and 93% of controls, respectively, but in only 42% and 38% of CLL patients, who also exhibited >23-fold and >17-fold lower median NAb titers (both p < 0.001). Interestingly, 26% of CLL patients failed to develop NAbs but had high-titer binding Abs that preferentially reacted with the S2 subunit of the SARS-CoV-2 spike. Since these patients were also seropositive for endemic human coronaviruses (HCoVs), these responses likely reflect cross-reactive HCoV Abs rather than vaccine-induced de novo responses. CLL disease status, advanced Rai stage (III-IV), elevated serum beta-2 microglobulin levels (ß2m >2.4 mg/L), prior therapy, anti-CD20 immunotherapy (<12 months), and intravenous immunoglobulin (IVIg) prophylaxis were all predictive of an inability to mount SARS-CoV-2 NAbs (all p ≤ 0.03). T cell response rates determined for a subset of participants were 2.8-fold lower for CLL patients compared to healthy controls (0.05, 95% CI 0.01 to 0.27, p < 0.001), with reduced intracellular IFNγ staining (p = 0.03) and effector polyfunctionality (p < 0.001) observed in CD4+ but not in CD8+ T cells. Surprisingly, in treatment-naïve CLL patients, BNT162b2 vaccination was identified as an independent negative risk factor for NAb generation (5.8, 95% CI 1.6 to 27, p = 0.006). CLL patients who received mRNA-1273 had 12-fold higher (p < 0.001) NAb titers and 1.7-fold higher (6.5, 95% CI 1.3 to 32, p = 0.02) response rates than BNT162b2 vaccinees despite similar disease characteristics. The absence of detectable NAbs in CLL patients was associated with reduced naïve CD4+ T cells (p = 0.03) and increased CD8+ effector memory T cells (p = 0.006). Limitations of the study were that not all participants were subjected to the same immune analyses and that pre-vaccination samples were not available. CONCLUSIONS: CLL pathogenesis is characterized by a progressive loss of adaptive immune functions, including in most treatment-naïve patients, with preexisting memory being preserved longer than the capacity to mount responses to new antigens. In addition, higher NAb titers and response rates identify mRNA-1273 as a superior vaccine for CLL patients.

Evidence for Extensive Duplication and Subfunctionalization of FCRL6 in Armadillo (Dasypus novemcinctus).

The control of infections by the vertebrate adaptive immune system requires careful modulation to optimize defense and minimize harm to the host. The Fc receptor-like (FCRL) genes encode immunoregulatory molecules homologous to the receptors for the Fc portion of immunoglobulin (FCR). To date, nine different genes (FCRL1-6, FCRLA, FCRLB and FCRLS) have been identified in mammalian organisms. FCRL6 is located at a separate chromosomal position from the FCRL1-5 locus, has conserved synteny in mammals and is situated between the SLAMF8 and DUSP23 genes. Here, we show that this three gene block underwent repeated duplication in Dasypus novemcinctus (nine-banded armadillo) resulting in six FCRL6 copies, of which five appear functional. Among 21 mammalian genomes analyzed, this expansion was unique to D. novemcinctus. Ig-like domains that derive from the five clustered FCRL6 functional gene copies show high structural conservation and sequence identity. However, the presence of multiple non-synonymous amino acid changes that would diversify individual receptor function has led to the hypothesis that FCRL6 endured subfunctionalization during evolution in D. novemcinctus. Interestingly, D. novemcinctus is noteworthy for its natural resistance to the Mycobacterium leprae pathogen that causes leprosy. Because FCRL6 is chiefly expressed by cytotoxic T and NK cells, which are important in cellular defense responses against M. leprae, we speculate that FCRL6 subfunctionalization could be relevant for the adaptation of D. novemcinctus to leprosy. These findings highlight the species-specific diversification of FCRL family members and the genetic complexity underlying evolving multigene families critical for modulating adaptive immune protection.

NCCN Guidelines® Insights: Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma, Version 3.2022.

The treatment landscape of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) has significantly evolved in recent years. Targeted therapy with Bruton's tyrosine kinase (BTK) inhibitors and BCL-2 inhibitors has emerged as an effective chemotherapy-free option for patients with previously untreated or relapsed/refractory CLL/SLL. Undetectable minimal residual disease after the end of treatment is emerging as an important predictor of progression-free and overall survival for patients treated with fixed-duration BCL-2 inhibitor-based treatment. These NCCN Guidelines Insights discuss the updates to the NCCN Guidelines for CLL/SLL specific to the use of chemotherapy-free treatment options for patients with treatment-naïve and relapsed/refractory disease.

Predictors of Nonseroconversion after SARS-CoV-2 Infection.

Not all persons recovering from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection develop SARS-CoV-2-specific antibodies. We show that nonseroconversion is associated with younger age and higher reverse transcription PCR cycle threshold values and identify SARS-CoV-2 viral loads in the nasopharynx as a major correlate of the systemic antibody response.

Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma, Version 4.2020, NCCN Clinical Practice Guidelines in Oncology.

Chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) are characterized by a progressive accumulation of leukemic cells in the peripheral blood, bone marrow, and lymphoid tissues. Treatment of CLL/SLL has evolved significantly in recent years because of the improved understanding of the disease biology and the development of novel targeted therapies. In patients with indications for initiating treatment, the selection of treatment should be based on the disease stage, patient's age and overall fitness (performance status and comorbid conditions), and cytogenetic abnormalities. This manuscript discusses the recommendations outlined in the NCCN Guidelines for the diagnosis and management of patients with CLL/SLL.

NCCN Guidelines Insights: Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma, Version 2.2019.

Chronic lymphocytic leukemia (CLL) is generally characterized by an indolent disease course. Histologic transformation (also known as Richter's transformation) to more aggressive lymphomas, such as diffuse large B-cell lymphoma or Hodgkin lymphoma, occurs in approximately 2% to 10% of patients and is associated with a poor prognosis. These NCCN Guidelines Insights discuss the recommendations for the diagnosis and management of patients with histologic transformation.

Hairy Cell Leukemia, Version 2.2018, NCCN Clinical Practice Guidelines in Oncology.

Hairy cell leukemia (HCL) is a rare type of indolent B-cell leukemia, characterized by symptoms of fatigue and weakness, organomegaly, pancytopenia, and recurrent opportunistic infections. Classic HCL should be considered a distinct clinical entity separate from HCLvariant (HCLv), which is associated with a more aggressive disease course and may not respond to standard HCL therapies. Somatic hypermutation in the IGHV gene is present in most patients with HCL. The BRAF V600E mutation has been reported in most patients with classic HCL but not in those with other B-cell leukemias or lymphomas. Therefore, it is necessary to distinguish HCLv from classic HCL. This manuscript discusses the recommendations outlined in the NCCN Guidelines for the diagnosis and management of classic HCL.

FCRL5 exerts binary and compartment-specific influence on innate-like B-cell receptor signaling.

Innate-like splenic marginal zone (MZ) and peritoneal cavity B1 B lymphocytes share critical responsibilities in humoral responses but have divergent B-cell receptor (BCR) signaling features. A discrete marker of these subsets with tyrosine-based dual regulatory potential termed "Fc receptor-like 5" (FCRL5) was investigated to explore this discrepancy. Although FCRL5 repressed the robust BCR activity that is characteristic of MZ B cells, it had no influence on antigen receptor stimulation that is blunted in peritoneal cavity-derived B1 B cells. The molecular basis for the receptor's inhibitory function derived from recruitment of the Src homology-2 domain-containing tyrosine phosphatase 1 (SHP-1) to a cytoplasmic immunoreceptor tyrosine-based inhibitory motif. Surprisingly, mutagenesis of this docking site unearthed coactivation properties for FCRL5 that were orchestrated by independent association of the Lyn Src-family kinase with an intracellular immunoreceptor tyrosine-based activation motif-like sequence. FCRL5's unique binary regulation directly correlated with SHP-1 and Lyn activity, which, like BCR function, differed between MZ and B1 B cells. These findings collectively imply a specialized counterregulatory role for FCRL molecules at the intersection of innate and adaptive immunity.

FCRL3 promotes TLR9-induced B-cell activation and suppresses plasma cell differentiation.

Fc receptor-like (FCRL) molecules are preferentially expressed by B lymphocytes and possess tyrosine-based immunoregulatory function. Although they generally inhibit B-cell receptor signaling, their influence on other activation pathways remains largely unexplored. In humans, FCRL3 encodes a type I transmembrane protein harboring both cytoplasmic ITAM and ITIM elements that can repress B-cell receptor activation. Despite this inhibitory property, mounting associations for FCRL3 with autoimmune and lympho-proliferative disorders imply a role for it in promoting B-cell pathogenesis. Here, we explore the influence of FCRL3 on B-cell responses to innate TLR9 stimulation. A detailed survey of blood B-cell populations found that FCRL3 expression increased as a function of differentiation and was higher among memory subsets with innate-like features. FCRL3 ligation augmented CpG oligodeoxynucleotide TLR9-mediated B-cell proliferation, activation, and survival, but surprisingly, abrogated plasma cell differentiation and antibody production. Although FCRL3 amplified the NF-κB and mitogen-activated protein kinase signaling cascades, it halted CpG triggered BLIMP1 induction in an ERK-dependent fashion. These findings indicate that FCRL3 differentially modulates innate signaling in B cells and provide new insight into the potential of this disease-associated receptor to counter-regulate adaptive and innate immunity.

FcRL4 acts as an adaptive to innate molecular switch dampening BCR signaling and enhancing TLR signaling.

Fc receptor-like 4 (FcRL4) is expressed on the surface of a subset of memory B cells (MBCs) located at sites of invading pathogens in mucosal lymphoid tissues in healthy individuals. Recently, FcRL4(+) MBCs were shown to be greatly increased in number in the peripheral blood of HIV-infected viremic individuals, in whom they are associated with B-cell exhaustion, and in individuals chronically reinfected with malaria. In the present study, we provide evidence that the expression of FcRL4 in human B-cell lines disrupts immune synapse formation and blocks antigen-induced BCR signaling at the point of Syk phosphorylation, blocking downstream activation of PLC-γ2 and Vav and the induction of calcium responses and CD69 expression. FcRL4 functions by ligation-independent mechanisms that require the 3 tyrosine residues in its cytoplasmic domain and involves its phosphorylation and association with the tyrosine phosphatases SHP-1 and SHP-2. Remarkably, FcRL4 is concentrated in endosomes after treatment with the TLR9 agonist CpG and enhances signaling through TLR9, as measured by increased expression of CD23. These findings suggest that FcRL4 may act as a molecular switch in B cells to dampen adaptive immune signaling and enhance innate signaling in response to chronic antigenic stimulation.

Enhanced levels of both the membrane-bound and soluble forms of IgM Fc receptor (FcµR) in patients with chronic lymphocytic leukemia.

The association of an IgM-Fc receptor (FcµR) with chronic lymphocytic leukemia (CLL) was suggested more than 30 years ago, but its authenticity has never been formally addressed. We examined the expression of the recently identified FcµR by B and T cells in CLL patients using receptor-specific monoclonal antibodies. CLL B cells (CD5(+)/CD19(+)) expressed much higher levels of FcµR on their cell surface than B cells from healthy donors. Such enhanced expression was more evident in immunoglobulin heavy chain variable region (IGHV)-mutated, CD38(-) or early Rai-stage CLL than in IGHV-unmutated, CD38(+), or advanced Rai-stage CLL. Intriguingly, surface FcµR levels also were significantly elevated in the non-CLL B cells (CD5(-)/CD19(+)) and T cells (CD5(+)/CD19(-)), especially in IGHV-mutated CLL. CLL patients also had high serum titers of FcµR compared with healthy donors, and serum FcµR levels correlated significantly with circulating lymphocyte numbers but not with the IGHV mutation status or Rai stage. The serum FcµR was resolved as an ∼ 40-kDa protein, distinct from the cell surface FcµR of ∼ 60 kDa, and it was produced by both CLL B and non-CLL B cells. Mass spectrometric analysis revealed that the serum FcµR is a soluble form of the receptor encoded by an alternatively spliced FcµR transcript. These findings indicate enhanced levels of both membrane-bound and soluble forms of FcµR in CLL patients.

FCRL1 immunoregulation in B cell development and malignancy.

Immunotherapeutic targeting of surface regulatory proteins and pharmacologic inhibition of critical signaling pathways has dramatically shifted our approach to the care of individuals with B cell malignancies. This evolution in therapy reflects the central role of the B cell receptor (BCR) signaling complex and its co-receptors in the pathogenesis of B lineage leukemias and lymphomas. Members of the Fc receptor-like gene family (FCRL1-6) encode cell surface receptors with complex tyrosine-based regulation that are preferentially expressed by B cells. Among them, FCRL1 expression peaks on naïve and memory B cells and is unique in terms of its intracellular co-activation potential. Recent studies in human and mouse models indicate that FCRL1 contributes to the formation of the BCR signalosome, modulates B cell signaling, and promotes humoral responses. Progress in understanding its regulatory properties, along with evidence for its over-expression by mature B cell leukemias and lymphomas, collectively imply important yet unmet opportunities for FCRL1 in B cell development and transformation. Here we review recent advances in FCRL1 biology and highlight its emerging significance as a promising biomarker and therapeutic target in B cell lymphoproliferative disorders.

FCRLA is a resident endoplasmic reticulum protein that associates with intracellular Igs, IgM, IgG and IgA.

Fc receptor-like A (FCRLA) is an unusual member of the extended Fc receptor family. FCRLA has homology to receptors for the Fc portion of Ig (FCR) and to other FCRL proteins. However, unlike these other family representatives, which are typically transmembrane receptors with extracellular ligand-binding domains, FCRLA has no predicted transmembrane domain or N-linked glycosylation sites and is an intracellular protein. We show by confocal microscopy and biochemical assays that FCRLA is a soluble resident endoplasmic reticulum (ER) protein, but it does not possess the amino acid sequence KDEL as an ER retention motif in its C-terminus. Using a series of deletion mutants, we found that its ER retention is most likely mediated by the amino terminal partial Ig-like domain. We have identified ER-localized Ig as the FCRLA ligand. FCRLA is unique among the large family of Fc receptors, in that it is capable of associating with multiple Ig isotypes, IgM, IgG and IgA. Among hemopoietic cells, FCRLA expression is restricted to the B lineage and is most abundant in germinal center B lymphocytes. The studies reported here demonstrate that FCRLA is more broadly expressed among human B lineage cells than originally reported; it is found at significant levels in resting blood B cells and at varying levels in all B-cell subsets in tonsil.

Cutting edge: FcR-like 6 is an MHC class II receptor.

Receptors for the Fc portion of Ig have been extensively characterized and are known to regulate humoral responses, but members of the closely related FcR-like (FCRL) family have not been found to bind Ig, and to date, no ligand has been identified for any FCRL. Using a cell-based GFP reporter system and a recombinant Fc chimeric protein, we show that human FCRL6, a receptor selectively expressed by cytotoxic T and NK cells, directly binds HLA-DR, an MHC class II molecule. Given the similarity among constant regions of Ig and MHC molecules, these findings suggest that representatives of the FcR and FCRL multigene families may have independently evolved to engage two ancestral elements fundamental to adaptive immunity. This discovery may offer new insight into the interaction between cytotoxic lymphocytes and APCs and may have important implications for better understanding HLA disease susceptibility and pathogenesis.

FcR-like 2 Inhibition of B cell receptor-mediated activation of B cells.

FcR-like (FCRL) 2 is a transmembrane protein with immunomodulatory potential that is preferentially expressed by memory B cells in humans. It has two consensus ITIMs in addition to a putative ITAM sequence in its cytoplasmic domain. We have confirmed the cellular distribution of FCRL2 and analyzed its functional potential to show that coligation with the BCR leads to tyrosine phosphorylation of its ITIM motifs and subsequent Src homology region 2 domain-containing phosphatase-1 recruitment to facilitate inhibition of BCR signaling. Mutational analysis indicates that the tyrosine residues in both inhibitory motifs of FCRL2 are required for complete inhibition of BCR signaling, whereas tyrosines in the putative activation motif are dispensable for signal modulation. These findings suggest a negative immunomodulatory function for FCRL2 in the regulation of memory B cells.

Cutting edge: FcR-like 5 on innate B cells is targeted by a poxvirus MHC class I-like immunoevasin.

Under selective pressure from host immunity, viruses have retained genes encoding immunoevasins, molecules interfering with host viral recognition and clearance. Due to their binding specificities, immunoevasins can be exploited as affinity labels to identify host-encoded molecules of previously unsuspected importance in defense against the relevant class of virus. We previously described an orthopoxvirus MHC class I-like protein (OMCP) that binds with high affinity to the activating receptor NKG2D on NK and T cell subsets, implicating NKG2D in antiorthopoxvirus immunity. In this study, we report that OMCP also binds in an NKG2D-independent manner to B cells and monocytes/macrophages. We identify murine FcR-like 5 (FCRL5), an orphan immunoregulatory protein highly expressed by innate B lymphocytes, as a specific receptor for OMCP. The three N-terminal Ig domains of FCRL5 are required for OMCP binding. The targeting of FCRL5 by an orthopoxvirus immunoevasin strongly implicates it in contributing to host defense against zoonotic orthopoxviruses.

Comprehensive mapping of SARS-CoV-2 peptide epitopes for development of a highly sensitive serological test for total and neutralizing antibodies.

Quantification of the anti-SARS-CoV-2 antibody response has proven to be a prominent diagnostic tool during the COVID-19 pandemic. Antibody measurements have aided in the determination of humoral protection following infection or vaccination and will likely be essential for predicting the prevalence of population level immunity over the next several years. Despite widespread use, current tests remain limited in part, because antibody capture is accomplished through the use of complete spike and nucleocapsid proteins that contain significant regions of overlap with common circulating coronaviruses. To address this limitation, a unique epitope display platform utilizing monovalent display and protease-driven capture of peptide epitopes was used to select high affinity peptides. A single round of selection using this strategy with COVID-19 positive patient plasma samples revealed surprising differences and specific patterns in the antigenicity of SARS-CoV-2 proteins, especially the spike protein. Putative epitopes were assayed for specificity with convalescent and control samples, and the individual binding kinetics of peptides were also determined. A subset of prioritized peptides was used to develop an antibody diagnostic assay that showed low cross reactivity while detecting 37% more positive antibody cases than a gold standard FDA EUA test. Finally, a subset of peptides were compared with serum neutralization activity to establish a 2 peptide assay that strongly correlates with neutralization. Together, these data demonstrate a novel phage display method that is capable of comprehensively and rapidly mapping patient viral antibody responses and selecting high affinity public epitopes for the diagnosis of humoral immunity.

SARS-CoV-2 mRNA vaccination exposes progressive adaptive immune dysfunction in patients with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) patients have lower seroconversion rates and antibody titers following SARS-CoV-2 vaccination, but the reasons for this diminished response are poorly understood. Here, we studied humoral and cellular responses in 95 CLL patients and 30 healthy controls after two BNT162b2 or mRNA-2173 mRNA immunizations. We found that 42% of CLL vaccinees developed SARS-CoV-2-specific binding and neutralizing antibodies (NAbs), while 32% had no response. Interestingly, 26% were seropositive, but had no detectable NAbs, suggesting the maintenance of pre-existing endemic human coronavirus-specific antibodies that cross-react with the S2 domain of the SARS-CoV-2 spike. These individuals had more advanced disease. In treatment-naïve CLL patients, mRNA-2173 induced 12-fold higher NAb titers and 1.7-fold higher response rates than BNT162b2. These data reveal a graded loss of immune function, with pre-existing memory being preserved longer than the capacity to respond to new antigens, and identify mRNA-2173 as a superior vaccine for CLL patients.