Cell Immunoglobulin Research Articles

Dynamics of the inhibitory immune checkpoint TIM-3 in mouse pulmonary phagocytes after silica exposure

Long-term inhalation of silica particles in the workplace causes silicosis, which is incurable and seriously endangers the health of workers. It is believed that silicosis is caused by an imbalance of the pulmonary immune microenvironment, in which pulmonary phagocytes play a crucial role. As an emerging immunomodulatory factor, it is unclear whether T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) participate in silicosis by modulating pulmonary phagocytes function. The purpose of this study was to investigate the dynamic changes of the TIM-3 in pulmonary macrophages, dendritic cells (DCs), and monocytes during the development of silicosis in mice. The plasma levels of soluble TIM-3 in silicosis patients were also examined. Flow cytometry was used to identify alveolar macrophages (AMs), interstitial macrophages (IMs), CD11b+ DC, CD103+ DC, Ly6C+, and Ly6C- monocytes in mouse lung tissues, and further analyses were conducted on the expression of TIM-3. Results showed that soluble TIM-3 was significantly elevated in plasma of silicosis patients, and the level of which was higher in stage II and III patients than that in stage I. In silicosis mice, the protein and mRNA levels of TIM-3 and Galectin9 were significantly upregulated in lung tissues. Specific to pulmonary phagocytes, silica exposure affected TIM-3 expression in a cell-specific and dynamic manner. In macrophages, TIM-3 expression upregulated in AM after 28 days and 56 days of silica instillation, while the expression of TIM-3 in IM decreased at all observation time points. In DCs, silica exposure only caused a decrease of TIM-3 expression in CD11b+ DCs. In monocytes, TIM-3 dynamics in Ly6C+ and Ly6C- monocytes were generally consistent during silicosis development, which significant decrease after 7 and 28 days of silica exposure. In conclusion, TIM-3 may mediate the development of silicosis by regulating pulmonary phagocytes.

TIGIT is a key inhibitory checkpoint receptor in lymphoma

BackgroundPD-1 checkpoint blockade therapy (CBT) has greatly benefited patients with select solid tumors and lymphomas but has limited efficacy against diffuse large B-cell lymphoma (DLBCL). Because numerous inhibitory checkpoint receptors...

ARC-10: A phase 3 study to evaluate zimberelimab + domvanalimab versus pembrolizumab in front-line, PD-L1-high, locally advanced or metastatic non–small-cell lung cancer.

TPS9148 Background: Despite improvements in the management of locally advanced or metastatic non–small cell lung cancer (NSCLC) from programmed cell death/ligand protein 1 (PD-[L]1) inhibition, there remains an unmet need for improved treatment options. PD-L1 expression is a known biomarker of response to anti–PD-L1 therapies in metastatic NSCLC. The combination of PD-1 inhibition with inhibition of the immunosuppressive T cell Immunoglobulin and ITIM domain (TIGIT) pathway is associated with improved response and prolonged progression-free survival in patients with advanced NSCLC. Domvanalimab (dom) is an Fc-silent humanized IgG1 monoclonal antibody (mAb) that blocks TIGIT, thereby reducing immunosuppression of T/natural killer (NK) cells and promoting antitumor activity. Zimberelimab (zim) is an mAb that binds to PD-1 on T/NK cells, preventing PD-L1-mediated immunosuppressive effects and resulting in tumor cell death. This phase 3 study will investigate the efficacy and safety of combination therapy with dom + zim compared with pembrolizumab monotherapy in patients with PD-L1-high NSCLC. Methods: ARC-10 (NCT04736173) is a global, phase 3, randomized, multicenter, open-label study. Eligible patients are adults with histologically confirmed, treatment-naive, locally advanced or metastatic, squamous or non-squamous NSCLC with ≥1 measurable lesion(s) per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1, high expression of PD-L1 (tumor cells [TC] ≥50%), and an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Patients must not have genomic tumor aberrations for which targeted therapies are approved and available (eg, EGFR, ALK, ROS, BRAF, NTRK). Approximately 600 patients will be randomized 1:1 to receive combination therapy with dom + zim (dom 1200 mg intravenous [IV] once every 3 weeks [Q3W] + zim 360 mg IV Q3W) or pembrolizumab monotherapy (200 mg IV Q3W) for 21-day cycles until disease progression, intolerance, or a maximum of 35 cycles. Randomization will be stratified by ECOG performance status (0 vs 1), geographical region (Asia vs non-Asia), and histology (squamous vs non-squamous). The primary efficacy endpoint is overall survival. Secondary efficacy endpoints include blinded independent central review of progression-free survival and confirmed overall response rate according to RECIST v1.1. Safety endpoints include the presence of treatment-emergent adverse events and laboratory parameters. Health-related quality of life will be assessed by measuring the time to first symptom deterioration in the NSCLC-Symptom Assessment Questionnaire total score. Study recruitment is planned in Asia, North and South America, Africa, and Europe. Clinical trial information: NCT04736173 .

STAR-221: A randomized, open-label, multicenter, phase 3 trial of domvanalimab, zimberelimab, and chemotherapy versus nivolumab and chemotherapy in previously untreated, locally advanced, unresectable or metastatic gastric, gastroesophageal junction, and esophageal adenocarcinoma.

TPS4206 Background: The addition of programmed cell death/ligand protein 1 (PD-[L]1) inhibitors to standard chemotherapy has improved outcomes in patients with HER2-negative unresectable or metastatic esophago-gastric adenocarcinomas. Current first-line (1L) treatment for these patients comprises FOLFOX (oxaliplatin, leucovorin, and fluorouracil) and CAPOX (capecitabine and oxaliplatin) chemotherapy, with or without the addition of a PD-1 inhibitor. Domvanalimab (dom) is an Fc-silent humanized IgG1 monoclonal antibody (mAb) that blocks T cell Immunoglobulin and ITIM domains (TIGIT), reducing immunosuppression of T/natural killer (NK) cells and promoting antitumor activity. Zimberelimab (zim) and nivolumab are mAbs that bind to PD-1 on T/NK cells, preventing the immunosuppressive effects of PD-L1 and leading to enhanced immune-mediated tumor cell death. Prior studies (including ARC-7) have demonstrated that the combination of dom + zim is safe, tolerable, and has promising activity in patients with lung cancer and esophago-gastric cancers. This study will investigate whether adding anti-TIGIT therapy to the standard combination of anti-PD-1 therapy and chemotherapy in patients with locally advanced unresectable or metastatic gastric, GEJ, and esophageal adenocarcinoma provides additional clinical benefit. Methods: STAR-221 (NCT05568095) is a global, phase 3, randomized, open-label study. Eligible patients are adults with locally advanced unresectable or metastatic gastric, GEJ, or esophageal adenocarcinoma treated in the 1L setting with ≥1 measurable lesion(s) per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 and an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Patients with known HER2-positive tumors are not eligible. Approximately 970 patients will be randomized 1:1 into 1 of 2 treatment arms. Patients randomized to Arm A will receive either dom 1600 mg + zim 480 mg every 4 weeks (Q4W) in addition to FOLFOX chemotherapy once every 2 weeks (Q2W) or dom 1200 mg + zim 360 mg once every 3 weeks (Q3W) in addition to CAPOX chemotherapy Q3W. Patients randomized to Arm B will receive either nivolumab 240 mg Q2W + FOLFOX Q2W or nivolumab 360 mg Q3W + CAPOX Q3W. Randomization will be stratified by PD-L1 expression (tumor area positivity [TAP] ≥ 5% vs TAP < 5%), ECOG performance status (0 vs 1), and region (USA, Canada, and EU5 countries vs Asia vs rest of world). The dual primary endpoints are overall survival (OS) in the intent-to-treat population and OS in patients with high PD-L1 expression (TAP ≥ 5%). Secondary endpoints include progression-free survival, objective response rate, duration of response, and safety and tolerability. STAR-221 is currently enrolling globally. Clinical trial information: NCT05568095 .

Artificial intelligence (AI) –powered H&E whole-slide image (WSI) analysis of tertiary lymphoid structure (TLS) correlates with immune phenotype and related molecular signatures in non–small-cell lung cancer.

e20520 Background: Recent data suggested immune infiltrates in TLS around tumor bed may be a favorable predictive factor for immunotherapy in various cancers. TLS have been suggested to be correlated with inflamed immune phenotype and relevant inflammatory gene signatures of adaptive immunity. To analyze this relationship, here, we developed an AI model to assess TLS objectively in H&E WSI, and assessed its correlation with immune phenotype and immunologic signatures. Methods: H&E images, relevant gene expression profiles and clinical data from The Cancer Genome Atlas (TCGA) lung cancer dataset (LUAD and LUSC, N = 913) were used for the analysis. Lunit SCOPE TLS, an AI-powered H&E WSI analyzer, was developed with 3.59 x 109 μm2 annotated area and 1,439 H&E stained WSI of 18 cancer types to segment TLS within tumor microenvironment (TME). Based on spatial tumor-infiltrating lymphocyte (TIL) density, IPs were classified into inflamed IP (IIP) as high intratumoral TIL (iTIL), immune-excluded IP (IEP) as low iTIL and high sTIL, and immune-desert IP (IDP) as low TIL overall. The infiltration of immune cells, the activity of related pathways and biological processes, and the signature scores of interest gene sets were analyzed by using the CIBERSORT, DESeq2 and GSEA tools. Results: Of 913 samples, 69.7% (636/913) contain TLS. The median value of the proportion of TLS area within TME was 0.12%, which was applied for the cut point of TLS-high vs -low group. The proportion of TLS-high was not significantly different according to EGFR mutation (mutation vs wild type: 45.5% vs 50.2%), KRAS mutation (49.3% vs 50%), but numerically decreased in the merging set of other driver mutations including ALK, ROS1, RET, MET, NTRK1-3 (36% vs 50.3%, p = 0.223). Interestingly, TLS-high proportion was significantly different according to immune phenotype, as TLS were present in 57.6% of IIP (186/323), 49.8% of IEP (241/484), and 28.3% of IDP (30/106, p < 0.001). TLS-high group was positively correlated with memory B cells (fold change [fc] 1.93, p < 0.001), CD8+ T cells (fc 1.20, p < 0.001), and M1 Macrophages (fc 1.17, p < 0.001), and negatively correlated with neutrophils (fc 0.59, p < 0.001) and M2 macrophages (fc 0.91, p = 0.006). This result was additionally supported by GSEA analysis of GO:BP or Hallmark gene sets which showed TLS-high was associated with B cell receptor signaling pathway (normalized enrichment score [NES] 3.16, p = 0.001), immunoglobulin production (NES 3.07, p = 0.002), and interferon-gamma response (NES 2.20, p = 0.002). Epithelial-mesenchymal transition was negatively associated with TLS-high (NES -1.54, p < 0.001). Conclusions: TLS is associated with inflamed immune phenotype and infiltration of memory B cells as well as CD8+ T cells in non-small cell lung cancer.

B cell subsets reconstitution and immunoglobulin levels in children and adolescents with B non-Hodgkin lymphoma after treatment with single anti CD20 agent dose included in chemotherapeutic protocols: single center experience and review of the literature.

RTX, an anti-CD20 monoclonal antibody, added to chemotherapy has proven to be effective in children and adolescents with high-grade, high-risk and matured non-Hodgkin lymphoma. RTX leads to prompt CD19+ B lymphocyte depletion. However, despite preserved immunoglobulin production by long-lived plasmablasts after treatment, patients remain at risk of prolonged hypogammaglobulinemia. Further, there are few general guidelines for immunology laboratories and clinical feature monitoring after B cell-targeted therapies. The aim of this paper is to describe B cell reconstitution and immunoglobulin levels after pediatric B-NHL protocols, that included a single RTX dose and to review the literature. A retrospective single-center study on the impact of a single RTX dose included in a chemotherapeutic pediatric B Non-Hodgkin Lymphoma (B-NHL) treatment protocols. Immunology laboratory and clinical features were evaluated over an eight hundred days follow-up (FU) period, after completing B-NHL treatment. Nineteen patients (fifteen Burkitt lymphoma, three Diffuse large B cell lymphoma, and one Marginal zone B cell lymphoma) fulfilled the inclusion criteria. Initiation of B cell subset reconstitution occurred a median of three months after B-NHL treatment. Naïve and transitional B cells declined over the FU in contrast to the marginal zone and the switched memory B cell increase. The percentage of patients with IgG, IgA, and IgM hypogammaglobulinemia declined consistently over the FU. Prolonged IgG hypogammaglobulinemia was detectable in 9%, IgM in 13%, and IgA in 25%. All revaccinated patients responded to protein-based vaccines by specific IgG antibody production increase. Following antibiotic prophylaxes, none of the patients with hypogammaglobulinemia manifested with either a severe or opportunistic infection course. The addition of a single RTX dose to the chemotherapeutic treatment protocols was not shown to increase the risk of developing secondary antibody deficiency in B-NHL pediatric patients. Observed prolonged hypogammaglobulinemia remained clinically silent. However interdisciplinary agreement on regular long-term immunology FU after anti-CD20 agent treatment is required.

TIM-3 increases the abundance of type-2 dendritic cells during Leishmania donovani infection by enhancing IL-10 production via STAT3

The outcome of the disease visceral leishmaniasis (VL), caused by Leishmania donovani (LD), largely relies on the relative dominance of host-protective type-1 T helper (Th1) cell response versus disease-promoting type-2 T helper (Th2) cell response. The Th1 and Th2 responses, in turn, are believed to be elicited by type-1 conventional dendritic cells (cDC1) and type-2 conventional DCs (cDC2), respectively. However, it is still unknown which DC subtype (cDC1 or cDC2) predominates during chronic LD infection and the molecular mechanism governing such occurrence. Here we report that in chronically infected mice, the splenic cDC1-cDC2 balance shifted toward the cDC2 subtype and that the receptor T cell immunoglobulin and mucin protein-3 (TIM-3) expressed by DCs played a key role in mediating this effect. Transfer of TIM-3-silenced DCs in fact prevented the predominance of the cDC2 subtype in mice with chronic LD infection. We also found that LD actually upregulated TIM-3 expression on DCs by triggering a TIM-3-mediated signaling pathway STAT3 (signal transducer and activator of transcription 3)→interleukin (IL)-10→c-Src→transcription factors Ets1, Ets2, USF1, and USF2. Notably, TIM-3 promoted STAT3 activation via a non-receptor tyrosine kinase Btk. Adoptive transfer experiments further demonstrated a critical role for STAT3-driven TIM-3 upregulation on DCs in increasing cDC2 abundance in chronically infected mice, which ultimately aided disease pathogenesis by augmenting Th2 responses. These findings document a new immunoregulatory mechanism contributing to disease pathology during LD infection and define TIM-3 as a key mediator of this process.

TIM-3 Qualifies as a Potential Immunotherapeutic Target in Specific Subsets of Patients with High-Risk Soft Tissue Sarcomas (HR-STS).

(1) Background: The expression of T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), an immune checkpoint receptor on T cells, has been associated with dismal outcomes and advanced tumor stages in various solid tumors. The blockade of TIM-3 is currently under examination in several clinical trials. This study examines TIM-3 expression in high-risk soft tissue sarcomas (HR-STS). (2) Methods: Tumor cell expression of TIM-3 on protein level was analyzed in pre-treatment biopsies of patients with HR-STS. TIM-3 expression was correlated with clinicopathological parameters including tumor-infiltrating lymphocyte (TIL) counts, programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PDL-1) expression in patients with HR-STS. Survival dependent on the expression of TIM-3 was analyzed. (3) Results: TIM-3 expression was observed in 101 (56%) out of 179 pre-treatment biopsies of patients with HR-STS. TIM-3 expression was significantly more often observed in undifferentiated pleomorphic sarcomas (UPS) compared to other histological subtypes (p < 0.001), high TIL counts (p < 0.001), and high PD-1 (p < 0.001) and PD-L1 expression (p < 0.001). TIM-3 expression did not have a prognostic impact on survival in patients with HR-STS. (4) Conclusions: This is the first study to demonstrate a significant tumor cell expression of TIM-3 in specific subsets of patients with HR-STS. TIM-3 qualifies as a potential immunotherapeutic target in HR-STS.

Efficient and accurate KIR and HLA genotyping with massively parallel sequencing data.

Killer cell immunoglobulin like receptor (KIR) genes and human leukocyte antigen (HLA) genes play important roles in innate and adaptive immunity. They are highly polymorphic and cannot be genotyped with standard variant calling pipelines. Compared with HLA genes, many KIR genes are similar to each other in sequences and may be absent in the chromosomes. Therefore, although many tools have been developed to genotype HLA genes using common sequencing data, none of them work for KIR genes. Even specialized KIR genotypers could not resolve all the KIR genes. Here we describe T1K, a novel computational method for the efficient and accurate inference of KIR or HLA alleles from RNA-seq, whole-genome sequencing, or whole-exome sequencing data. T1K jointly considers alleles across all genotyped genes, so it can reliably identify present genes and distinguish homologous genes, including the challenging KIR2DL5A/KIR2DL5B genes. This model also benefits HLA genotyping, where T1K achieves high accuracy in benchmarks. Moreover, T1K can call novel single-nucleotide variants and process single-cell data. Applying T1K to tumor single-cell RNA-seq data, we found that KIR2DL4 expression was enriched in tumor-specific CD8+ T cells. T1K may open the opportunity for HLA and KIR genotyping across various sequencing applications.

Identification of a novel small-molecule inhibitor targeting TIM-3 for cancer immunotherapy

PD-1/PD-L1 blockade has achieved substantial clinical results in cancer treatment. However, the expression of other immune checkpoints leads to resistance and hinders the efficacy of PD-1/PD-L1 blockade. T cell immunoglobulin and mucin domain 3 (TIM-3), a non-redundant immune checkpoint, synergizes with PD-1 to mediate T cell dysfunction in tumor microenvironment. Development of small molecules targeting TIM-3 is a promising strategy for cancer immunotherapy. Here, to identify small molecule inhibitors targeting TIM-3, the docking pocket in TIM-3 was analyzed by Molecular Operating Environment (MOE) and the Chemdiv compound database was screened. The small molecule SMI402 could bind to TIM-3 with high affinity and prevent the ligation of PtdSer, HMGB1, and CEACAM1. SMI402 reinvigorated T cell function in vitro. In the MC38-bearing mouse model, SMI402 inhibited tumor growth by increasing CD8+ T and natural killing (NK) cells infiltration at the tumor site, as well as restoring the function of CD8+ T and NK cells. In conclusions, the small molecule SMI402 shows promise as a leading compound which targets TIM-3 for cancer immunotherapy.

TIGIT limits anti-bacterial immunity and is a potential target for immunotherapy

Abstract Inhibitory receptors called “immune checkpoints” restrict overzealous immune responses during inflammatory events such as infection or cancer. Blocking the function of these immune checkpoints using monoclonal antibodies can tip the balance towards immune activation and disease clearance, which has been a revelation in the treatment of various cancers. Considering recent advances have shown that immune dysregulation and evasion are shared hallmarks of cancer and persistent bacterial infection, a better understanding of immune checkpoints during bacterial infection may uncover novel therapeutic targets to help direct host immune responses towards clearing pathogens. In this study, we use a murine model of SalmonellaTyphimurium infection to investigate how immune checkpoints regulate immunity to bacterial infection. We found lymphocytes from infected mice exhibit a phenotype suggestive of dysfunction, characterised by upregulation of multiple co-inhibitory checkpoints. Most strikingly, T cell immunoglobulin and ITIM domain (TIGIT) was upregulated on the majority of CD4 +T cells. We used RNA-sequencing and ex vivostimulation of CD4 +T cells to show that ligation of TIGIT leads to significant functional deficiencies, including reductions in proliferation and pro-inflammatory cytokine production. Importantly, blockade of this immune checkpoint in vivousing anti-TIGIT antibodies was able to enhance functional immunity leading to improved bacterial clearance. Taken together, this research suggests targeting immune checkpoints such as TIGIT has potential to restore host immune responses towards clearing bacteria and managing serious infection. F.S.F.G is funded by a UQ Frazer Institute Laboratory Start-Up Package, and T.R.M. is supported by a UQ HDR PhD Scholarship.

Abstract 443: BAFF Neutralization Impairs The Regression Of Insulin Resistance In Diabetic Mice By Modulating The Innate Immune Response

Adults with type 2 diabetes have a two-to-three-fold increased risk of cardiovascular diseases. B cell activating factor (BAFF), a tumor necrosis factor superfamily member, is implicated both in diabetes and cardiovascular diseases, and hence a therapeutic target of interest. High levels of BAFF are known to promote autoantibody synthesis and autoimmune diseases. We examined if BAFF neutralization with an anti-BAFF antibody improves glucose intolerance in a model of regression of insulin resistance in diabetic mice. Male C57BL/6J mice were fed a high-fat diet (HFD) for 12 weeks and segregated into two groups based on their glucose intolerance determined by a glucose tolerance test. Each group was simultaneously switched from HFD to a normal chow diet and received injections of anti-BAFF or an isotype control antibody every 2 nd week for a total of 6 weeks. As expected, body weights decreased in both groups parallel with the increase in insulin sensitivity. Unexpectedly, the BAFF-neutralized mice had impaired regression of insulin resistance compared to the control mice. Extensive cellular phenotyping of immune cells indicated systemic depletion of only the B2 cells and lower levels of circulating autoantibodies in the BAFF-neutralized mice. Gonadal white adipose tissue (gWAT) was the primary source of BAFF, and no differences in the M1 and M2 macrophages, eosinophils, natural killer cells, helper T cells, and cytotoxic T cell populations were found. RNA sequencing analysis of stromal vascular fraction from gWAT revealed impaired B cell receptor signaling, immunoglobulin production, complement activation, and phagocytosis pathways in the BAFF-neutralized mice. In this line, (i) an increased number of dead adipocytes, (ii) decreased levels of IgG2b and IgM, (iii) increased citrullinated histone H3, neutrophils, and DNA extracellular traps were found in the gWAT of BAFF-neutralized mice. These results suggest attenuated humoral response and a dominant and harmful role of innate response in the gWAT following BAFF neutralization in late-stage obesity. Since anti-BAFF biologics are approved for the treatment of lupus patients, our results highlight the need for further studies on the role of BAFF in late-stage obesity and diabetes.

Delayed B cell reconstitution and maturation with hypogammaglobulinemia in the setting of in utero exposure to rituximab

A four-month-old female presented to the Immunology Clinic for second opinion of immune globulin replacement. She was born at 36 weeks gestation and was small for gestational age. Pregnancy was complicated by in utero exposure to Rituximab. Mom was diagnosed with Stage IV Diffuse large B cell lymphoma at approximately 12 weeks of pregnancy. Treatment for Stage IV Diffuse large B cell lymphoma included rituximab, etoposide phosphate, prednisone, vincristine sulfate, cyclophosphamide, and doxorubicin hydrochloride. Rituximab was continued until 34 weeks gestation. Newborn SCID screen was negative. Flow cytometry on day of life 1 (Table 1) revealed normal T cell populations with undetectable CD19 B cells. IgG was 915 mg/dL, IgA < 5, and IgM < 5. Repeat flow cytometry and immunoglobulins at one month of life revealed: undetectable CD19 B cells, IgG 402 mg/dL. Following birth, she developed recurrent respiratory tract infections including PICU admission secondary to respiratory failure in the setting of adenovirus and human rhinovirus/enterovirus bronchiolitis. At two months of age, IgG decreased to 299 mg/dL and B cells were still undetectable. Prophylactic IVIG was started at 3 months of age. Repeat laboratory evaluation at 4 months of age revealed the following: IgG 510 mg/dL, IgA < 5, and IgM 39 mg/dL, and recovery of CD19 B cells at 1482/mm3. At 5 months of age, B cell phenotyping was performed which revealed low class switched memory B cells. In the setting of low class switched memory B cells, IVIG therapy was continued. Patient did well clinically while on IVIG therapy. Class switched memory B cells normalized at 16 months of age and IVIG was discontinued. Patient did well off IVIG therapy, and at 30-month follow-up, continued to do well without need for IVIG replacement.Table 1Laboratory Study TrendTestDay of life 11 month old2 months oldIVIG started at 3 months old4 months old5 months old16 months old30 months oldAbsolute CD3 cells/mcL Reference Range (1484–5327 cells/mcL)4,183NA5,6436,8895,2744,6323,030Absolute CD4 cells/mcL Reference Range (733–3181 cells/mcL)3,204NA4,4805,0583,8213,1051,850Absolute CD8 cells/mcL Reference Range (370–2555 cells/mcL)979NA1,1051,6571,3461,310999Absolute CD19 cells/mcL Reference Range (370–2306 cells/mcL)<1NA<11,4821,5761,603958Absolute CD16CD56 cells/mcL Reference Range (43–526 cells/mcL)223NA175262187434601IgG mg/dl Reference Range (295–1156 mg/dL)915402299510886626717IgA mg/dL Reference Range (27–246 mg/dL)<5<5<5<515<1<1IgM mg/dL Reference Range (37–184 mg/dL)<5<5<539555397CD27+IgM-IgDcells/mcL Reference Range (5.2–74.2 cells/mcL)NANANANA1.011.113.5In utero exposure of Rituximab can lead to temporary absence ofC19 B cells and transient hypogammaglobulinemia. Despite numeric B cell reconstitution, the maturation of B cells may lag behind. Comprehensive B cell immunophenotyping will help decipher the optimal duration of supplemental immunoglobulin therapy. Frequent monitoring of B cell numbers and immunoglobulins are important and can help guide decision in immune globulin replacement.

Immunogram: a Generalized Framework to Measure a Person’s Immune System.

Abstract “How is my immune system?” This is a simple question, but there is no metric to assess the overall performance of the human immune system. Current clinical methods that evaluate a person’s immune system only provide limited information, like cell counts or immunoglobulin levels, and fail to capture the global immune state. This project aims to develop Immunogram, a generalized framework to measure a person’s immune system. Immunogram is a Julia-based software that links cells, proteins, and other molecules in a knowledge graph. Immunogram delineates subgraph patterns that describe human immune states and uses them to characterize a person’s immune system. Immunogram can be used to identify immune cell types, predict vaccine response, and determine if a person has an ongoing infection. Kwat Medetgul-Ernar was supported by Stanford University Medical Scientist Training Program grant T32-GM007365 and T32-GM145402.

P011 Rituximab and COVID-19 vaccines; are they fit for purpose?

Abstract Background/Aims Since the COVID-19 outbreak the rheumatology community have been concerned about the risk of SARS-CoV-2 infection in patients prescribed immunosuppressing medications. Data suggests that patients receiving Rrtuximab are at increased risk of developing severe outcomes from COVID-19 (1). In our unit all patients receiving rituximab were selected to receive a targeted vaccination and booster programme with all patients receiving at least 2 vaccinations and up to 3 booster vaccinations. We studied the efficacy of the COVID-19 vaccines in rituximab patients, by checking the the Roche Elecsys Anti-SARS-CoV-2-S (Spike) IgG/IgM total antibody levels post vaccination. Our aim was to assess the vaccination response in patients receiving rituximab and to offer advice on continued shielding or alternatively passive immunization with tixagevimab/cilgavimab in those patients who did not mount a response. Methods Taking 39 patients currently on rituximab therapy, we measured Anti-SARS-CoV-2-S (Spike) antibody levels post vaccination. We recorded whether the test was positive or negative, and the numerical result. We recorded rituximab dates of administration and dates of vaccines. We also recorded diagnosis, co-prescribed DMARDs, immunoglobulin levels, white cell and lymphocyte counts. We took record of whether or not the patient subsequently contracted COVID-19, required a hospital admission, ICU or died. Results Of our 39 patients, 21 had Anti-SARS-CoV-2-S (Spike) antibody levels checked. Of these patients, 7 (33%) had a negative spike protein result. Of the patients with a positive result, 8 (38%) had an antibody level between 0-250U/ML, and only 6 (28.6%) had a level &amp;gt;250U/ML (The manufacturer advises that a level above 0.8U/ML is a positive result). Of patients with a negative result, 1 patient had received 3 vaccines, 5 patients had received 4, and 1 patient had 5. All of the patients had received a vaccine &amp;gt;4 weeks prior to receiving the drug. Two patients were co-prescribed Belimumab, 3 were co-prescribed low-dose methotrexate and 2 were not on additional disease modifying agents. The diagnoses of these patients were, 2 patients with SLE, 4 with SPRA, and 1 MPO Vasculitis. There were no significant findings in lymphocyte count, white cell count or immunoglobulin levels. Conclusion These findings suggest that our current COVID-19 vaccination and booster programme may not provide adequate response in patients receiving rituximab therapy. Despite this being a small cohort, these results show that 33% of patients have not mounted a vaccine response and this is concerning. We suggest that vaccine response should be checked in all patients receiving rituximab therapy and those patients who do not mount a vaccine response should be offered passive immunity and advised of possible additional risks regarding COVID-19 exposure. Disclosure S.K. Stewart: None. A. Pendleton: None.

Povetacicept, an Enhanced Dual APRIL/BAFF Antagonist That Modulates B Lymphocytes and Pathogenic Autoantibodies for the Treatment of Lupus and Other B Cell-Related Autoimmune Diseases.

Dysregulated APRIL/BAFF signaling is implicated in the pathogenesis of multiple autoimmune diseases, including systemic lupus erythematosus and lupus nephritis. We undertook this study to develop and evaluate a high-affinity APRIL/BAFF antagonist to overcome the clinical limitations of existing B cell inhibitors. A variant of TACI-Fc generated by directed evolution showed enhanced binding for both APRIL and BAFF and was designated povetacicept (ALPN-303). Povetacicept was compared to wild-type (WT) TACI-Fc and related molecules in vitro and in vivo. Povetacicept inhibited APRIL and BAFF more effectively than all evaluated forms of WT TACI-Fc and selective APRIL and BAFF inhibitors in cell-based reporter assays and primary human B cell assays, mediating potent suppression of B cell proliferation, differentiation, and immunoglobulin (Ig) secretion. In mouse immunization models, povetacicept significantly reduced serum immunoglobulin titers and antibody-secreting cells more effectively than anti-CD20 monoclonal antibodies, WT TACI-Fc, or APRIL and BAFF inhibitors. In the NZB × NZW mouse lupus nephritis model, povetacicept significantly enhanced survival and suppressed proteinuria, anti-double-stranded DNA antibody titers, blood urea nitrogen, glomerulonephritis, and renal immunoglobulin deposition. In the bm12 mouse lupus model, povetacicept significantly reduced splenic plasmablasts, follicular helper T cells, and germinal center B cells. In non-human primates, povetacicept was well tolerated, exhibited high serum exposure, and significantly decreased serum IgM, IgA, and IgG levels after a single dose. Enhanced APRIL and BAFF inhibition by povetacicept led to greater inhibition of B cell populations critical for autoantibody production compared to WT TACI-Fc and CD20-, APRIL-, or BAFF-selective inhibitors. Potent, dual inhibition by povetacicept has the potential to significantly improve clinical outcomes in autoantibody-related autoimmune diseases.

Competitive binding of CD226/TIGIT with poliovirus receptor regulates macrophage polarization and is involved in vascularized skin graft rejection

End-stage organ failure often requires solid organ transplantation. Nevertheless, transplant rejection remains an unresolved issue. The induction of donor-specific tolerance is the ultimate goal in transplantation research. In this study, an allograft vascularized skin rejection model using BALB/c-C57/BL6 mice was established to evaluate the regulation of the poliovirus receptor signaling pathway using CD226 knockout or T cell immunoglobulin and ITIM domain (TIGIT)–crystallizable fragment (Fc) recombinant protein treatment. In the TIGIT-Fc–treated and CD226 knockout groups, graft survival time prolonged significantly, with a regulatory T cell proportion increase and M2-type macrophage polarization. Donor-reactive recipient T cells became hyporesponsive while responding normally after a third-party antigen challenge. In both groups, serum interleukin (IL)-1β, IL-6, IL-12p70, IL-17A, tumor necrosis factor–α, interferon gamma, and monocyte chemoattractant protein-1 levels decreased, and the IL-10 level increased. In vitro, M2 markers, such as Arg1 and IL-10, were markedly increased by TIGIT-Fc, whereas iNOS, IL-1β, IL-6, IL-12p70, tumor necrosis factor–α, and interferon gamma levels decreased. CD226-Fc exerted the opposite effect. TIGIT suppressed TH1 and TH17 differentiation by inhibiting macrophage SHP-1 phosphorylation and enhanced ERK1/2-MSK1 phosphorylation and nuclear translocation of CREB. In conclusion, CD226 and TIGIT competitively bind to poliovirus receptor with activating and inhibitory functions, respectively. Mechanistically, TIGIT promotes IL-10 transcription from macrophages by activating the ERK1/2-MSK1-CREB pathway and enhancing M2-type polarization. CD226/TIGIT–poliovirus receptor are crucial regulatory molecules of allograft rejection.

Development and validation of an m6A RNA methylation regulator-based signature for the prediction of prognosis and immunotherapy in cutaneous melanoma.

Development and validation of an m6A RNA methylation regulator-based signature for the prediction of prognosis and immunotherapy in cutaneous melanoma.

Cottonseed hydrolysate supplementation alters metabolic and proteomics responses in Chinese hamster ovary cell cultures.

Hydrolysates are used as media supplements although their role is not well characterized. In this study, cottonseed hydrolysates, which contained peptides and galactose as supplemental substrates, were added to Chinese hamster ovary (CHO) batch cultures, enhancing cell growth, immunoglobulin (IgG) titers, and productivities. Extracellular metabolomics coupled with tandem mass tag (TMT) proteomics revealed metabolic and proteomic changes in cottonseed-supplemented cultures. Shifts in production and consumption dynamics of glucose, glutamine, lactate, pyruvate, serine, glycine, glutamate, and aspartate suggest changes in tricarboxylic acid (TCA) and glycolysis metabolism following hydrolysate inputs. Quantitative proteomics revealed 5521 proteins and numerous changes in relative abundance of proteins related to growth, metabolism, oxidative stress, protein productivity, and apoptosis/cell death at day 5 and day 6. Differential abundance of amino acid transporter proteins and catabolism enzymes such as branched-chain-amino-acid aminotransferase (BCAT)1 and fumarylacetoacetase (FAH) can alter availability and utilization of several amino acids. Also, pathways involved in growth including the polyamine biosynthesis through higher ornithine decarboxylase (ODC1) abundance and hippo signaling were upregulated and downregulated, respectively. Central metabolism rewiring was indicated by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) downregulation, which corresponded with re-uptake of secreted lactate in the cottonseed-supplemented cultures. Overall, cottonseed hydrolysate supplementation modified culture performance by altering cellular activities critical to growth and protein productivity including metabolism, transport, mitosis, transcription, translation, protein processing, and apoptosis. HIGHLIGHTS: Cottonseed hydrolysate, as a medium additive, enhances Chinese hamster ovary (CHO) cell culture performance. Metabolite profiling and tandem mass tag (TMT) proteomics characterize its impact on CHO cells. Rewired nutrient utilization is observed via glycolysis, amino acid, and polyamine metabolism. Hippo signaling pathway impacts cell growth in the presence of cottonseed hydrolysate.

The effects of KIR2DL4 stimulated NK-92 cells on the apoptotic pathways of HER2 + /HER-breast cancer cells.

Natural killer (NK) cells are immune cells that have attracted significant attention due to their cytotoxic properties. They are believed to be highly effective in cancer therapy. In this study, anti-KIR2DL4 (Killer cell Immunoglobulin like Receptor, 2 Ig Domains and Long cytoplasmic tail 4) was used to stimulate the NK-92 activator receptor to increase their cytotoxicity on breast cancer cell lines. Unstimulated and stimulated NK-92 cells (sNK-92) were cocultured with breast cancer (MCF-7 and SK-BR-3) and normal breast (MCF-12A) cell lines at 1:1, 1:5, and 1:10 (Target:Effector) ratios. The most effective cell cytotoxicity ratio (1:10) was used in the immunostaining and western blot assays to evaluate apoptosis pathway proteins. The sNK-92 cells showed higher cytotoxic activity on breast cancer cells than NK-92 cells. sNK-92 cells had a selective significant cytotoxicity effect on MCF-7 and SK-BR-3 cells but not MCF-12A cells. While sNK-92 cells were effective at all cell concentrations, they were most effective at a 1:10 ratio. Immunostaining and western blots showed significantly higher BAX, caspase 3, and caspase 9 protein levels in all breast cancer cell groups cocultured with sNK-92 than with NK-92 cells. NK-92 cells stimulated with KIR2DL4 showed elevated cytotoxic activity. The cytotoxic activity of sNK-92 cells on breast cancer cells is via apoptosis pathways. However, their effect on normal breast cells is limited. While the obtained data contains only basic information, additional clinical studies are needed to provide a basis for a new treatment model.