HLA-DR Expression Research Articles

High Expression of Tumor HLA-DR Predicts Better Prognosis and Response to Anti-PD-1 Therapy in Laryngeal Squamous Cell Carcinoma

BackgroundHLA-DR is expressed in epithelial and several types of tumor cells. However, the correlation between tumor-expressed HLA-DR (teHLA-DR) and patient outcome as well as its regulation on the tumor microenvironment (TME) of laryngeal squamous cell carcinoma (LSCC) are yet to be elucidated. MethodsHematoxylin and eosin (HE) staining were performed to define the tumor nest and stroma of LSCC tissue microarrays. teHLA-DR tumor cell, CD4+ and CD8+ tumor-infiltrating T lymphocytes (TITLs) were obtained and analyzed through double-labeling immunofluorescence and immunohistochemical staining. The recurrence-free (RFS) and overall survival (OS) curves were plotted using the Kaplan-Meier method and tested by the log-rank test method. Expression of teHLA-DR+ tumor cells and infiltration of T lymphocytes and their corresponding subgroups were analyzed by flow cytometry using fresh LSCC tissue samples. ResultsOur research discovered elevated expressions of multiple MHC-II-related genes in tumor compared to the adjacent normal tissue samples of LSCC patients. We also found that patients in the teHLA-DR high-expression group (teHLA-DRhigh) tend to have less tumor recurrence and better survival outcomes compared to those in the teHLA-DRlow group. Intriguingly, teHLA-DR+ tumor cells had significantly higher PD-L1 and PD-L2 expression and their TME showed increased infiltrated T lymphocytes (TITLs). Flow cytometry analysis and IHC staining indicated that CD4+ TITLs but not CD3+ total TITLs or CD8+ TITLs were significantly enriched in teHLA-DR+ tumors. ConclusionsteHLA-DR may be a predictive marker for favorable prognosis and response to anti-PD-1/PD-L1 therapy of LSCC, possibly due to the increased CD4+ TITLs in the TME.

CD4+ T cell counts and soluble programmed death-1 at baseline correlated with hepatitis B surface antigen decline in HIV/HBV coinfection during combined antiretroviral therapy.

Several studies have described the rapid decline and clearance of hepatitis B surface antigen (HBsAg) in human immunodeficiency virus (HIV)/hepatitis B virus (HBV) coinfection after initiating combined antiretroviral therapy (cART). Early decline of HBsAg levels is associated with HBsAg seroclearance in the treatment of chronic HBV infection. This study aims to evaluate the HBsAg kinetics and the determinants of early HBsAg decline in patients with HIV/HBV coinfection during cART. A total of 51 patients with HIV/HBV coinfection were enrolled from a previously established HIV/AIDS cohort and followed for a median of 59.5 months after cART initiation. Biochemical tests, virology and immunology assessments were measured longitudinally. The kinetics of HBsAg during cART were analyzed. Soluble programmed death-1 (sPD-1) levels and immune activation markers (CD38 and HLA-DR) were measured at baseline, 1-year and 3-year during treatment. HBsAg response was defined as a decline of more than 0.5 log10 IU/ml at 6 months from the baseline after initiation of cART. HBsAg declined faster (0.47 log10 IU/mL) in the first six months and attained a decrease of 1.39 log10 IU/mL after 5-year therapy. Seventeen (33.3%) participants achieved a decline of more than 0.5 log10 IU/ml at the first 6 months of cART(HBsAg response) of which five patients achieved HBsAg clearance at a median of 11 months (range: 6-51 months). Multivariate logistic analysis showed the lower baseline CD4+ T cell levels (OR=6.633, P=0.012) and sPD-1 level (OR=5.389, P=0.038) were independently associated with HBsAg response after cART initiation. The alanine aminotransferase abnormality rate and HLA-DR expression were significantly higher in patients who achieved HBsAg response than in those who did not achieve HBsAg response after cART initiation. Lower CD4 + T cells, sPD-1, and immune activation were related to a rapid HBsAg decline in patients with HIV/HBV-coinfection after the initiation of cART. These findings imply that immune disorders induced by HIV infection may disrupt immune tolerance to HBV, leading to a faster decline in HBsAg levels during coinfection.

Abstract 106: Single Cell High Dimensional Analysis of Human Peripheral Blood Mononuclear Cells Reveals Unique Intermediate Monocyte Subsets Associated with Sex Differences in Coronary Heart Disease

Monocytes are innate immune cells that are defined by three subsets: classical (cMo), intermediate (iMo) and non-classical (nMo). How these subsets become more heterogeneous in phenotype and function in CVD progression remains elusive. We employed single-cell transcriptome and protein analysis to define how human monocytes differ in subjects with moderate to severe CVD. Peripheral blood mononuclear cells (PBMC) were obtained from 61 human subjects, undergoing coronary angiography as part of the Coronary Assessment of Virginia (CAVA) Cohort. Coronary atherosclerosis severity was quantified using Gensini Score (GS). We analyzed 487 immune-related genes and 49 surface proteins at the single cell level in PBMC from each subject using the BD-Rhapsody platform. WNN Seurat 4.0 was used for high dimensional analysis. Data was normalized using GLMM to control for covariates (age, sex, diabetes, and statin use). We identified cMo, iMo, nMo, plasmacytoid DC, and classical DC. We found a significant increase only in the frequencies of intermediate (iMo) monocytes in subjects with high CVD (GS>30) compared to subjects with low CVD (GS<6) ( p =0.004). Spearman correlation analysis with GS from each subject revealed a positive correlation with iMo frequencies (r=0.314, p =0.014) and further showed sex-dependent positive correlation in female patients (r=0.663, p =0.0037). Interestingly, cMo, thought to be potently atherogenic, did not correlate with CVD severity. Further analysis identified 3 iMo subsets, distinguished by expression of HLA-DR, CD86, CXCR3, and PD-L1. We found that frequencies of two of them, iMo_CXCR3 + PDL1 lo ( p =0.0003) and iMo_HLA-DR + CD86 int ( p =0.019), were associated with CVD severity. The iMo_CXCR3 + PDL1 lo immunoregulatory subset was positively correlated with GS in both females (r=0.66, p =0.004) and males (r=0.315, p =0.037). However, the iMo_HLA-DR + CD86 int subset was positively correlated only in females (r=0.55, p =0.022), illustrating substantial heterogeneity among monocyte subsets in a sex-dependent manner. In sum, we identified novel sex- and CVD-dependent iMo subsets in subjects with CVD. Future functional analysis of iMo populations should provide a deeper understanding of long-known sex-differences in CVD progression.

Role of complement activation in cellular activation by systemic lupus erythematosus (SLE) immune complexes (ICs)

Abstract Complement (C’) activation is a prominent feature of SLE ICs, but the mechanism by which C’ mediates cellular activation by ICs is not well understood. To investigate this question, we formed SLE ICs in situ in healthy donor (HD) whole blood (WB) by adding SLE plasma (n=1–3;10% v/v) or HD plasma (as a control) along with apoptotic bodies (generated from RAW 264.7 cells) or RNP-Sm antigen (Arotec Diagnostics, New Zealand) with and without C’ inhibitors (CI). For cellular binding studies, WB was incubated for 45 minutes at 37°C and deposition of C3c and human IgG (ICs) on blood cells was examined by flow cytometry. In these studies, we noted that C’ inhibition significantly reduced deposition of C3c and ICs on B cells {% reduction: C3c (97%) and IgG (97%)}, monocytes {% reduction: C3c (40%) and IgG (49%)} and neutrophils {% reduction: C3c (69%) and IgG (70%)}. To investigate cellular activation, WB was incubated for 22 hours and cellular activation was measured by HLA-DR expression on monocytes by flow cytometry and cytokine release (IL-8, IL-6 and TNF) by ELISA. Incubation of WB with SLE ICs, but not ICs made with HD plasma (control ICs), resulted in increased HLA-DR expression on monocytes (MFI control ICs: 1609 V SLE ICs: 11096), which was susceptible to C’ inhibition (MFI SLE ICs with CI: 4975). Similarly, inflammatory cytokine release by SLE ICs was also significantly inhibited with C’ inhibition {% inhibition: IL-8 (75 %), IL-6 (99%) and TNF (99 %)}. Taken together, our results show significant C’-dependent effects of SLE ICs on cellular binding and activation. Additional studies are underway to investigate the prevalence of C’ activating ICs in SLE patient and correlate their presence with clinical disease. Wendell F. Rosse Complement Research Award, Duke University Medical Center (SK) and Duke Scholars Award, Duke University Medical Center (GMA)

High-dimensional profiling of circulating dendritic cells and monocytes in atopic dermatitis patients by mass cytometry

Abstract Atopic dermatitis (AD) is a chronic inflammatory skin disorder, with a multifactorial pathophysiology, including barrier dysfunction, dysbiosis and immune dysregulation. Although AD has been characterized by a T helper type 2 cell response, the role of the myeloid populations in the pathogenesis of AD still remains unclear. Here, we used mass cytometry, a high-dimensional analysis tool, primarily focusing on these cell populations to identify differences in the immune phenotypes of circulating dendritic cells and monocytes in AD. Peripheral blood mononuclear cells (PBMCs) from 48 Korean AD patients and 48 age-, sex-matched healthy controls were profiled. We report significant differences in the frequency and phenotypic markers of dendritic cells and monocytes. The frequency of CD141 +CLEC9A +cDC1 was significantly decreased in AD, and its frequency negatively correlated with disease severity and serum IgE levels. This suggests that cDC1 may have preferentially migrated to the lesional skin to control the inflammation, especially in severe AD. CLEC9A expression was significantly reduced, while FceRIa expression was significantly increased in cDC1. The expression of FceRIa was also elevated in cDC2, pDC, and Axl +DC. In cDC2, HLA-DR expression was decreased, and CD163 expression was increased. These results indicate changes in the immunophenotypes of circulating dendritic cells in AD compared with healthy controls. Although further investigation is needed, our results suggest a potential role of dendritic cells in modulating AD pathophysiology.

A Case of Pneumocystis jirovecii Pneumonia in an Infant with Ataxia-Pancytopenia Syndrome

BackgroundAtaxia-pancytopenia syndrome (ATXPC), a rare autosomal dominant disorder caused by pathogenic variants in the SAMD9L gene, is characterized by variable onset and severity of cerebellar ataxia, hematologic cytopenias, and immunodeficiency of myeloid, B-, and NK-cells. ATXPC has been associated with viral and bacterial infections, but no cases of opportunistic PJP have been reported. CaseAt 6 weeks of age, the patient was admitted to the hospital for acute hypoxemic respiratory failure due to rhino/enterovirus bronchiolitis, requiring supplemental oxygen. The increased oxygen requirement later led to intubation. A chest x-ray showed diffuse bilateral opacities concerning for pediatric acute respiratory distress syndrome (PARDS). Bronchoalveolar lavage and silver stain revealed organisms morphologically suggestive of Pneumocystis jirovecii pneumonia (PJP), which was confirmed with a positive serum beta-glucan. Based on the diagnosis of PJP, the patient was started on steroids and Bactrim. Immunology was consulted for further evaluation for possible underlying immunodeficiency. The patient had a normal newborn screening. HIV PCR was negative. Repeated TRECs, CD3+, CD8+, CD19+, and immunoglobulin levels (IgG, IgE) were within normal limits, but modest reduction of CD45 RA/RO ratio and IgA were observed. Additionally, natural killer cell function was low. T-cell proliferation to mitogens were normal, but significantly decreased to candida and tetanus antigens. A significant reduction in the expression of HLA-ABC and HLA-DR was observed in the lymphocytes and monocytes of the patient compared to the control. Interestingly, HLA-DR expression on monocytes was particularly affected. A next-generation sequencing of primary immunodeficiency genes identified likely pathogenic, maternally inherited variants: a heterozygous SAMD9L variant (ataxia-pancytopenia syndrome) and NLRP12 variant (familial cold autoinflammatory syndrome 2). DiscussionATXPC has been associated with variable cellular immunodeficiencies. While prior studies have reported recurrent bacterial and viral infections in ATXPC patients, this case is the first to document a PJP opportunistic infection in an infant. It is possible that the NLRP12 variant found in this patient could have also contributed to this clinical presentation. Early diagnosis of ATXPC through clinical recognition and genetic testing may improve prognosis.

20P Spatial analysis of residual tumor tissue provides new clues for post-neoadjuvant treatment approaches in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a very heterogeneous disease with high metastatic recurrence risk. We showed (PMID 34535679) that TNBCs relapsing rapidly (RR) after NACT (in ≤ 12 months) are highly proliferative, immune-cold, often with large residual cancer burden (RCB) and fatal outcome. No specific biomarkers or therapeutic targets are known for this clinical category. In this study we performed spatial molecular analysis of the RR and the RCB3 TNBCs, with the aim to help improving treatment of the TNBC patients with the poorest prognosis. We analyzed residual tumor tissue samples of TNBC patients treated by standard NACT from 01/01/2009 to 31/12/2021. The cases were classified into RR, standardly-relapsing (SR, 1-5 years post-NACT) and without recurrences (NR). The amount of tumor-infiltrating lymphocytes (TILs) was determined by optical microscopy (H&E). Tissue protein expression was spatially resolved by GeoMx® Digital Spatial Profiler (NanoString Technology). Sixteen, 17 and 47 RR, SR and NR cases were analyzed, respectively. GeoMx analysis included 216 Region-of-Interest (ROIs) of tumor microenvironment (TME) and 207 of tumor cells (TC), with balanced (∼50/50) numbers of peripheral (P) and central (C) ROIs. Tumors and ROIs were classified into TIL-rich (LR) and TIL-poor (LP) using cut-off of 10% TILs. The RR TNBCs showed higher Ki67, cleaved caspase 9, granzyme B (GZMB), FoxP3 and fibronectin (FBNCT) in TME-P-LR ROIs but higher GADPH, TIM-3, PD-L1 and IDO in TME-P-LP ROIs. Compared to TNBC-SR, the RR had higher GZMB and CD34 in TME-P-LR ROIs. In TME-C-LR ROIs of the RRs, CD8 expression was low, as well as the one of CD45, CD3 and CD8 in TME-C-LP. The RCB3 category contained 9, 8 and 10 RR, SR and NR cases, respectively. Although most RCB3 cases were TIL-poor, the NR cases showed significantly higher expression of CD3, CD8, GZMA, CD4, CD56, CD95, LAG3, HLA-DR, BIM and BCL6 than the RR cases. By this study we revealed several potential therapeutic targets (fibronectin, IDO, TIM-3, CD34 /angiogenesis) in the RR TNBCs. Interestingly, we also showed that some RCB3 cases have immune-activated TME and good prognosis. Spatial tissue analysis was crucial for these discoveries.

NK Cell Phenotype Is Associated With Response and Resistance to Daratumumab in Relapsed/Refractory Multiple Myeloma.

The CD38-targeting antibody daratumumab has marked activity in multiple myeloma (MM). Natural killer (NK) cells play an important role during daratumumab therapy by mediating antibody-dependent cellular cytotoxicity via their FcγRIII receptor (CD16), but they are also rapidly decreased following initiation of daratumumab treatment. We characterized the NK cell phenotype at baseline and during daratumumab monotherapy by flow cytometry and cytometry by time of flight to assess its impact on response and development of resistance (DARA-ATRA study; NCT02751255). At baseline, nonresponding patients had a significantly lower proportion of CD16+ and granzyme B+ NK cells, and higher frequency of TIM-3+ and HLA-DR+ NK cells, consistent with a more activated/exhausted phenotype. These NK cell characteristics were also predictive of inferior progression-free survival and overall survival. Upon initiation of daratumumab treatment, NK cells were rapidly depleted. Persisting NK cells exhibited an activated and exhausted phenotype with reduced expression of CD16 and granzyme B, and increased expression of TIM-3 and HLA-DR. We observed that addition of healthy donor-derived purified NK cells to BM samples from patients with either primary or acquired daratumumab-resistance improved daratumumab-mediated MM cell killing. In conclusion, NK cell dysfunction plays a role in primary and acquired daratumumab resistance. This study supports the clinical evaluation of daratumumab combined with adoptive transfer of NK cells.

Brief IL-12, IL-15, IL-18 activation drives the acquisition of two distinct memory-like NK cell states

Abstract IL-12/15/18 activation induces natural killer (NK) cell differentiation into cytokine-induced memory-like (ML) NK cells with enhanced IFNγ and killing of tumor targets that is antigen independent, rendering them distinct from conventional (cNK) and CMV-induced adaptive NK cells. In leukemia patients, ML NK cells have an excellent safety profile and 47% CR rate (PMID: 32826231). Despite their clinical promise, little is known about the biology underlying ML NK cells. Based on individual variability of human ML NK cell function, we hypothesized that ML NK cells manifest molecular heterogeneity. To this end, we applied Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) to profile RNA and protein expression on the single-cell level, and evaluated NK cell function. Overnight IL-12/15/18 stimulation induced broad activation with distinct transcriptional programs evident in CD56 brightcompared to CD56 dimNK cells. In contrast, following in vitrodifferentiation for 7 days, IL-12/15/18 activated NK cells acquired a unique transcriptional and protein signature, and manifest one of two ML NK cell states (ML-1, ML-2), or remained similar to cNK cells. ML-1 was marked by increased activating receptor expression (NKp46, NKp30, NKp44) while ML-2 had increased expression of inhibitory receptors, CD25, HLA-DR, MKI67, and IFNG. In functional assays, ML-2 cells produced more IFNγ following cytokine stimulation than ML-1 cells. Further, ML-1 and ML-2 had divergent transcription factor profiles suggesting distinct mechanisms of regulation. Collectively, our findings reveal molecular and functional heterogeneity of ML NK cells, and investigations into ML subsets in patients receiving ML NK cell therapy are ongoing. AAI Intersect Fellowship for Computational Scientists & Immunologists (JAF, TAF, AAP), T32GM139799 (JAF), P50CA171963 (TAF, MMB-E), R01CA205239 (TAF), P30CA91842 (TAF)

Inhibited MHC Class I and MHC Class II antigen processing and presentation upon Sars-CoV-2 infection

Abstract Major histocompatibility (MHC) bound viral peptides are the immune signatures to initiate the selective and cross-reactive T cell responses. Identifying the antigenic targets of adaptive immunity to SARS-CoV-2 is a key towards understanding the pathogenesis of Sars-CoV-2 and the rapidly evolving mutants. We isolated the immunopeptidomes to identify naturally processed and presented MHC-II (HLA-DR, HLA-DP) and MHC-I (HLA-ABC) bound canonical and out-of-frame viral peptides. We identified 5 HLA-DR canonical epitopes (13 peptides) from spike protein, 2 HLA-DP canonical epitopes (4 peptides) from spike and membrane proteins, 1 canonical MHC-Class I peptides and 5 out-of-frame MHC-I peptides from different ORF proteins. The viral peptides were presented in lower abundances as compared to host peptides obscuring the identification of the MHC bound viral peptides. Some of these peptides were shown to recall T cell responses in COVID-19 donors in other reported studies. We observed the downregulation of surface and total HLA-ABC, HLA-DR and HLA-DP expression upon Sars-CoV-2 infection possibly restricting the presentation of viral peptides and therefore delaying the immune response. The mechanisms by which SARS-CoV-2 evades adaptive immune responses mediated by MHC-I are studied in detail by different groups. Hence, we further investigating the modulators of the MHC-II pathway using proteomics-based approach. We observed significant downregulation of CD74 protein, CIITA protein and cathepsins in infected cells affecting the processing and presentation of viral peptides. These results may help in understanding significantly altered antigen presentation in Sars-CoV-2 infected cells.

Localized immune dysregulation is associated with lung injury in severe pediatric COVID-19

Abstract Acute respiratory distress syndrome (ARDS) is a common and devastating consequence of SARS-CoV-2 infection in adults, however severe lung injury is a rare manifestation of COVID-19 in children. Aberrant local immune responses are known to drive lung pathology in adults, but little is known about localized immune responses in children with respiratory failure due to COVID-19. We obtained blood and respiratory tract samples from children (n=35, aged 1 month to 18 years) admitted to the hospital for COVID-19, stratifying by presence of respiratory failure (n=15). We defined the cytokine and chemokine profile of severe disease using a 71 analyte Luminex panel. Children with respiratory failure were found to have increased circulating levels of leukemia inhibitory factor (LIF), a growth factor upregulated during pulmonary inflammation. The local environment revealed significant inflammation with increased levels of pro-inflammatory cytokines including IL-6, IL-8, and IL-33 among others. Application of high dimensional flow cytometry to define immune cell subsets revealed elevated proportions of monocytes in both circulating and local environments. Monocytes demonstrated decreased expression of HLA-DR and CD86, suggesting an immunoparalysis phenotype, previously associated with severe COVID-19 in adults. Further, effector memory T cells in the airway exhibited an exhausted phenotype with high levels of PD-1 expression. Together this analysis reveals a highly inflammatory and dysregulated immune response underlies SARS-CoV-2 induced pediatric lung injury. These findings suggest a shared pathway with adult COVID-19 ARDS and provide new insights into the immune mechanisms associated with severe COVID-19 in children. Supported by grants from NIH (K23AI141686)

HIV DNA positively correlates with HLA-DR+CD8+ T lymphocytes over 8-year suppressive ART.

Despite long-term suppressive cART, the activation level of T lymphocytes remains significantly high HIV-infected individuals. This study aims to unravel the relationship between CD8+ T cell activation and HIV DNA reservoir. In this retrospective study, 82 HIV-infected patients receiving suppressive cART for ≥8 years were included. Total HIV-1 DNA and expression of CD38 and HLA-DR in CD8+ T cell were quantified repeatedly during long-term follow-up. Longitudinal correlation between HIV-1 DNA and CD8+ T cell activation level was analysed using generalized estimating equation (GEE) model. Significant decrease of both total HIV-1 DNA and CD8+ T cell activation level were observed after combined antiretroviral therapy (cART) initiation. However, the expression level of HLA-DR in CD8+ T cells remained abnormally higher than normal range. GEE analysis revealed that HLA-DR expression was positively correlated with total HIV-1 DNA over long-term suppressive cART. HIV DNA reservoir may be closely related to CD8+ T cell activation and the inflammatory state in HIV-infected patients despite long-term cART.

Acute myeloid leukemia with RAM immunophenotype: A new underdiagnosed entity.

Acute myeloid leukemia (AML) with RAM immunophenotype is a distinct subtype of AML, as described by the Children's Oncology Group (COG), with characteristic morphological and immunophenotypic properties. It is characterized by strong CD56 expression with dim to negative CD45, HLA-DR, and CD38 expression. It is an aggressive leukemia with a poor response to induction chemotherapy and/or frequent relapses. Seven cases with the characteristic RAM immunophenotype were identified in this retrospective analysis of newly diagnosed pediatric AML cases from January 2019 to December 2021. Herein, we have critically analyzed their clinical, morphological, cytochemical, immunophenotyping, cytogenetic, and molecular profiles. The patients were traced and followed for their current disease and treatment status. Of 302 cases of pediatric AML (age <18 years), seven cases (2.3%) with the distinct RAM phenotype were observed, with age ranging from 9 months to 5 years. Two patients were misdiagnosed earlier as small round cell tumor because of the strong CD56 positivity and the absence of leukocyte common antigen (LCA), but they were later correctly identified as granulocytic sarcoma. The bone marrow aspirate showed blasts with unusual cohesiveness and clumping with nuclear moulding, mimicking non-hematologic malignancies. Flow cytometry revealed blasts with low side scatter, dim to negative CD45 and CD38, negative cMPO, CD36, and CD11b; moderate to bright CD33, CD117, and bright CD56. The Mean fluorescence intensity (MFI) of CD13 expression was significantly lower as compared to the internal controls. Cytogenetic and molecular studies did not show any recurrent abnormalities. Reverse transcription polymerase chain reaction for CBFA2T3-GLIS2 fusion was performed in 5/7 cases, with one positive result. On clinical follow-up, two patients were refractory to chemotherapy. Six of the seven cases had succumbed to death (duration of survival: 3-343 days after initial diagnosis). AML with RAM immunophenotype, a distinct form of pediatric AML with a poor prognosis, may pose a diagnostic challenge if presented as a soft tissue mass. A comprehensive immunophenotypic evaluation, including stem cell and myeloid markers, is critical for an accurate diagnosis of myeloid sarcoma with the RAM-immunophenotype. Our data demonstrated weak CD13 expression as an additional immunophenotypic finding.

Features of the Immune Response in COVID-19

BACKGROUND This review is devoted to the analysis of the features of the immune response in COVID-19. The review indicates the clinical manifestations of COVID-19, modern data on the immunopathogenesis of the disease and its complications are considered.Aim of STUDY To clarify some pathogenetic mechanisms of the immune response in COVID-19, which can help in creating an algorithm for examining patients for early prognosis and prevention of severe course and complications of the disease.MATERIAL AND METHODS To achieve this goal, the results of domestic and foreign scientific studies on the pathogenesis, diagnosis and treatment of COVID-19 were analyzed. The literature search was carried out in electronic search engines Scopus and PubMed. For the analysis, scientific articles published in the period from 2019 to 2021 were selected; 88% of analyzed works are not older than 5 years.CONCLUSION The late production of type I IFN, an increase in the level of pro-inflammatory monocytes, a decrease in the expression of HLA-DR on monocytes, violation of the presentation of the virus and the formation of specific lymphocytes, the death of T-lymphocytes and profound immunosuppression are of greatest importance for the development of a severe form of COVID-19.

The ability of Interleukin–10 to negate haemozoin-related pro-inflammatory effects has the potential to restore impaired macrophage function associated with malaria infection

BackgroundAlthough pro-inflammatory cytokines are involved in the clearance of Plasmodium falciparum during the early stages of the infection, increased levels of these cytokines have been implicated in the pathogenesis of severe malaria. Amongst various parasite-derived inducers of inflammation, the malarial pigment haemozoin (Hz), which accumulates in monocytes, macrophages and other immune cells during infection, has been shown to significantly contribute to dysregulation of the normal inflammatory cascades.MethodsThe direct effect of Hz-loading on cytokine production by monocytes and the indirect effect of Hz on cytokine production by myeloid cells was investigated during acute malaria and convalescence using archived plasma samples from studies investigating P. falciparum malaria pathogenesis in Malawian subjects. Further, the possible inhibitory effect of IL-10 on Hz-loaded cells was examined, and the proportion of cytokine-producing T-cells and monocytes during acute malaria and in convalescence was characterized.ResultsHz contributed towards an increase in the production of inflammatory cytokines, such as Interferon Gamma (IFN-γ), Tumor Necrosis Factor (TNF) and Interleukin 2 (IL-2) by various cells. In contrast, the cytokine IL-10 was observed to have a dose-dependent suppressive effect on the production of TNF among other cytokines. Cerebral malaria (CM) was characterized by impaired monocyte functions, which normalized in convalescence. CM was also characterized by reduced levels of IFN-γ-producing T cell subsets, and reduced expression of immune recognition receptors HLA-DR and CD 86, which also normalized in convalescence. However, CM and other clinical malaria groups were characterized by significantly higher plasma levels of pro-inflammatory cytokines than healthy controls, implicating anti-inflammatory cytokines in balancing the immune response.ConclusionsAcute CM was characterized by elevated plasma levels of pro-inflammatory cytokines and chemokines but lower proportions of cytokine-producing T-cells and monocytes that normalize during convalescence. IL-10 is also shown to have the potential to indirectly prevent excessive inflammation. Cytokine production dysregulated by the accumulation of Hz appears to impair the balance of the immune response to malaria and exacerbates pathology.

Evaluation of HLA-DR and HLA-DQ expression in gastric cancer tissues.

Despite recent advances in understanding the gastric cancer (GC) biology, the precise molecular mechanism of gastric carcinogenesis and role of deregulated immune responses in GC progression are still not well understood. In this study, mRNA levels of human leukocyte antigen (HLA)-DRA and -DQA1 were assessed in GC patients to find a potential association between expression of these HLA-II molecules and gastric carcinogenesis. Using quantitative real-time (qRT)-PCR, mRNA levels of HLA-DRA and -DQA1 were assessed in 20 pairs of matched GC and normal tissues. Our results showed that overall mRNA level of HLA-DRA was decreased in the tumor samples relative to control tissues (median fold change [FC] = 0.693; P = 0.445). Overall HLA-DQA1 level was increased in the tumor samples relative to control tissues (median FC = 1.659; P = 0.5117). However, the mentioned data were not statistically significant. Meanwhile, using a ≥ 2.5 FC as the cutoff to determine upregulation or downregulation, 35% of patients showed a downregulated expression of HLA-DRA, while 10% of those showed upregulation in HLA-DRA expression. Upregulation and downregulation of HLA-DQA1 expression were detected, respectively, in 35% and 25% of samples. A strong positive correlation was determined between HLA-DRA and HLA-DQA1 levels in tumor tissues (r = 0.7298; P = 0.0003). The results reported here along with future studies can be useful to understand the interplay between immune system and GC, therefore, may be helpful to design an effective immune-based therapy.

Abstract 6384: ATG-034, an LILRB4 antagonist antibody, reinvigorates dendritic cells and prevents tumor progression

Abstract Background: Dendritic cells (DCs) are vital for initiating antigen-specific T cell-dependent antitumor immune responses. However, emerging evidence reveals that local DCs within the tumor microenvironment (TME) are tolerized to facilitate immune evasion. Reprogramming of DCs is recognized as a promising strategy for tumor immunotherapy. Leukocyte immunoglobulin-like receptor B4 (LILRB4) is an inhibitory receptor belonging to the LILR family which is mainly expressed on normal myeloid cells and myeloid-derived malignant cells. It is upregulated on tolerogenic DCs (tolDCs), which exhibit low expression levels of costimulatory molecules and resistance to DC maturation. Targeting LILRB4 to reprogram tolDCs has been reported to be a promising strategy for cancer treatment. Here we report the preclinical development of an LILRB4 antagonist antibody, ATG-034. Method: The protein-based and cell-based binding affinity of ATG-034 were measured using SPR, ELISA and FACS analysis. Fc receptor (FcR) stimulation assay was used to evaluate the ability of ATG-034 to block the interaction of LILRB4 with its ligand fibronectin. ATG-034-mediated enhancement of antigen presentation and co-stimulation ability of DCs was determined by FACS analysis for the change of surface expression of HLA-DR, HLA-ABC, CD86, and CD206. The mixed lymphocyte reaction (MLR) assay was used to assess the immunomodulatory potential of ATG-034. The in vivo antitumor efficacy of ATG-034 was evaluated in a radiation therapy-resistant murine Lewis lung carcinoma (LLC) syngeneic model. Results: ATG-034 binds to LILRB4 protein with a single-digit nM affinity. It potently reversed the fibronectin-mediated inhibition of FcR-driven TNF-α cytokine production. TolDCs were reprogrammed by ATG-034 to immunogenic DCs with significant upregulation of the expression of HLA-DR, HLA-ABC, CD86 and downregulation of the expression of CD206. Furthermore, ATG-034 remarkably reinvigorated tolDCs for priming T cell activation with noticeable upregulation of the expression of CD25 and the secretion of IFN-γ, enhancing antitumor immunity. In addition, 10 mg/kg ATG-034 significantly inhibited the LLC tumor growth in vivo with a TGI of 40.28%, while a benchmark antibody only demonstrated a 24.43% TGI. Conclusion: Our data show that ATG-034 reprograms tolDC, enhancing anti-tumor immunity, and demonstrates potent in vivo anti-tumor efficacy. Therefore, ATG-034 may be a promising strategy for the treatment of cancer. Citation Format: Ao Sun, Enlin Zheng, Huili Cao, Mengshi Cao, Suya Bai, Peng Chen, Linjie Tian, Jay Mei, Bo Shan, Bing Hou. ATG-034, an LILRB4 antagonist antibody, reinvigorates dendritic cells and prevents tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6384.

Primitive haematopoiesis in the human placenta gives rise to macrophages with epigenetically silenced HLA-DR

The earliest macrophages are generated during embryonic development from erythro-myeloid progenitors (EMPs) via primitive haematopoiesis. Although this process is thought to be spatially restricted to the yolk sac in the mouse, in humans, it remains poorly understood. Human foetal placental macrophages, or Hofbauer cells (HBC), arise during the primitive haematopoietic wave ~18 days post conception and lack expression of human leukocyte antigen (HLA) class II. Here, we identify a population of placental erythro-myeloid progenitors (PEMPs) in the early human placenta that have conserved features of primitive yolk sac EMPs, including the lack of HLF expression. Using in vitro culture experiments we demonstrate that PEMP generate HBC-like cells lacking HLA-DR expression. We find the absence of HLA-DR in primitive macrophages is mediated via epigenetic silencing of class II transactivator, CIITA, the master regulator of HLA class II gene expression. These findings establish the human placenta as an additional site of primitive haematopoiesis.

Multivariate indicators of disease severity in COVID-19

The novel coronavirus pandemic continues to cause significant morbidity and mortality around the world. Diverse clinical presentations prompted numerous attempts to predict disease severity to improve care and patient outcomes. Equally important is understanding the mechanisms underlying such divergent disease outcomes. Multivariate modeling was used here to define the most distinctive features that separate COVID-19 from healthy controls and severe from moderate disease. Using discriminant analysis and binary logistic regression models we could distinguish between severe disease, moderate disease, and control with rates of correct classifications ranging from 71 to 100%. The distinction of severe and moderate disease was most reliant on the depletion of natural killer cells and activated class-switched memory B cells, increased frequency of neutrophils, and decreased expression of the activation marker HLA-DR on monocytes in patients with severe disease. An increased frequency of activated class-switched memory B cells and activated neutrophils was seen in moderate compared to severe disease and control. Our results suggest that natural killer cells, activated class-switched memory B cells, and activated neutrophils are important for protection against severe disease. We show that binary logistic regression was superior to discriminant analysis by attaining higher rates of correct classification based on immune profiles. We discuss the utility of these multivariate techniques in biomedical sciences, contrast their mathematical basis and limitations, and propose strategies to overcome such limitations.

Kinetics of CD169, HLA-DR, and CD64 expression as predictive biomarkers of SARS-CoV2 outcome

IntroductionDiscriminating between virus-induced fever from superimposed bacterial infections is a common challenge in intensive care units. Superimposed bacterial infections can be detected in severe SARS-CoV2-infected patients, suggesting the important role of the bacteria in COVID-19 evolution. However, indicators of patients’ immune status may be of help in the management of critically ill subjects.Monocyte CD169 is a type I interferon-inducible receptor that is up-regulated during viral infections, including COVID-19. Monocyte HLA-DR expression is an immunologic status marker, that decreases during immune exhaustion. This condition is an unfavorable prognostic biomarker in septic patients. Neutrophil CD64 upregulation is an established indicator of sepsis.MethodsIn this study, we evaluated by flow cytometry the expression of cellular markers monocyte CD169, neutrophil CD64, and monocyte HLA-DR in 36 hospitalized patients with severe COVID-19, as possible indicators of ongoing progression of disease and of patients’ immune status. Blood testings started at ICU admission and were carried on throughout the ICU stay and extended in case of transfer to other units, when applicable.The marker expression in mean fluorescence intensity (MFI) and their kinetics with time were correlated to the clinical outcome.ResultsPatients with short hospital stay (≤15 days) and good outcome showed higher values of monocyte HLA-DR (median 17,478 MFI) than long hospital stay patients (>15 days, median 9590 MFI, p= 0.04) and than patients who died (median 5437 MFI, p= 0.05). In most cases, the recovery of the SARS-CoV2 infection-related signs was associated with the downregulation of monocyte CD169 within 17 days from disease onset. However in three surviving long hospital stay patients, a persistent upregulation of monocyte CD169 was observed. An increased neutrophil CD64 expression was found in two cases with a superimposed bacterial sepsis.ConclusionMonocyte CD169, neutrophil CD64, and monocyte HLA-DR expression can be used as predictive biomarkers of SARS-CoV2 outcome in acutely infected patients. The combined analysis of these indicators can offer a real-time evaluation of patients’ immune status and of viral disease progression versus superimposed bacterial infections. This approach allows to better define the patients’ clinical status and outcome and may be useful to guide clinicians’ decisions. Our study focused on the discrimination between the activity of viral and bacterial infections and on the detection of the development of anergic states that may correlate with an unfavorable prognosis.