Role of immune mediators in predicting hospitalization of SARS-CoV-2 positive patients.

BACKGROUND: Several immune mediators (IM) including cytokines, chemokines, and their receptors have been suggested to play a role in COVID-19 pathophysiology and severity. AIM: To determine if early IM profiles are predictive of clinical outcome and which of the IMs tested possess the most clinical utility. METHODS: A custom bead-based multiplex assay was used to measure IM concentrations in a cohort of SARS-CoV-2 PCR positive patients (n = 326) with varying disease severities as determined by hospitalization status, length of hospital stay, and survival. Patient groups were compared, and clinical utility was assessed. Correlation plots were constructed to determine if significant relationships exist between the IMs in the setting of COVID-19. RESULTS: In PCR positive SARS-CoV-2 patients, IL-6 was the best predictor of the need for hospitalization and length of stay. Additionally, MCP-1 and sIL-2Rα were moderate predictors of the need for hospitalization. Hospitalized PCR positive SARS-CoV-2 patients displayed a notable correlation between sIL-2Rα and IL-18 (Spearman's ρ = 0.48, P=<0.0001). CONCLUSIONS: IM profiles between non-hospitalized and hospitalized patients were distinct. IL-6 was the best predictor of COVID-19 severity among all the IMs tested.

HLA genotyping using the Illumina HLA TruSight next-generation sequencing kits: A comparison.

Illumina first introduced their TruSight human leucocyte antigen (HLA) next-generation sequencing (NGS) typing kit in 2015 and subsequently followed up with a new version in 2016. Here we report on our experience comparing the two versions of the Illumina HLA NGS kits.

HLA genotyping in the clinical laboratory: comparison of next-generation sequencing methods.

Implementation of human leukocyte antigen (HLA) genotyping by next-generation sequencing (NGS) in the clinical lab brings new challenges to the laboratories performing this testing. With the advent of commercially available HLA-NGS typing kits, labs must make numerous decisions concerning capital equipment and address labor considerations. Therefore, careful and unbiased evaluation of available methods is imperative. In this report, we compared our in-house developed HLA NGS typing with two commercially available kits from Illumina and Omixon using 10 International Histocompatibility Working Group (IHWG) and 36 clinical samples. Although all three methods employ long range polymerase chain reaction (PCR) and have been developed on the Illumina MiSeq platform, the methodologies for library preparation show significant variations. There was 100% typing concordance between all three methods at the first field when a HLA type could be assigned. Overall, HLA typing by NGS using in-house or commercially available methods is now feasible in clinical laboratories. However, technical variables such as hands-on time and indexing strategies are sufficiently different among these approaches to impact the workflow of the clinical laboratory.

Loss of B Cells in Patients with Heterozygous Mutations in IKAROS.

BACKGROUND: Common variable immunodeficiency (CVID) is characterized by late-onset hypogammaglobulinemia in the absence of predisposing factors. The genetic cause is unknown in the majority of cases, and less than 10% of patients have a family history of the disease. Most patients have normal numbers of B cells but lack plasma cells. METHODS: We used whole-exome sequencing and array-based comparative genomic hybridization to evaluate a subset of patients with CVID and low B-cell numbers. Mutant proteins were analyzed for DNA binding with the use of an electrophoretic mobility-shift assay (EMSA) and confocal microscopy. Flow cytometry was used to analyze peripheral-blood lymphocytes and bone marrow aspirates. RESULTS: Six different heterozygous mutations in IKZF1, the gene encoding the transcription factor IKAROS, were identified in 29 persons from six families. In two families, the mutation was a de novo event in the proband. All the mutations, four amino acid substitutions, an intragenic deletion, and a 4.7-Mb multigene deletion involved the DNA-binding domain of IKAROS. The proteins bearing missense mutations failed to bind target DNA sequences on EMSA and confocal microscopy; however, they did not inhibit the binding of wild-type IKAROS. Studies in family members showed progressive loss of B cells and serum immunoglobulins. Bone marrow aspirates in two patients had markedly decreased early B-cell precursors, but plasma cells were present. Acute lymphoblastic leukemia developed in 2 of the 29 patients. CONCLUSIONS: Heterozygous mutations in the transcription factor IKAROS caused an autosomal dominant form of CVID that is associated with a striking decrease in B-cell numbers. (Funded by the National Institutes of Health and others.).

Report on the effects of fragment size, indexing, and read length on HLA sequencing on the Illumina MiSeq.

Single-molecule sequencing should allow for unambiguous, accurate, and high-throughput HLA typing. In this proof of principle study, we investigated the effects of fragment size for library preparation, indexing strategy, and read length on HLA typing. Whole gene amplicons of HLA-A, B, C, DRB1, and DQB1 were obtained by long-range PCR. For library preparation, two fragment sizes were evaluated: 100-300bp and 300-600bp. For sample multiplexing, two indexing strategies were compared: indexing-by-amplicon, where each individual amplicon is barcoded, and indexing-by-patient, where each patient's five loci are equimolarly pooled after PCR and indexed with the same barcode. Sequencing was performed on an Illumina MiSeq instrument using paired-end 150bp and 250bp read lengths. Our results revealed that the 300-600bp fragments in the 2×250 MiSeq group gave the most accurate sequencing results. There was no difference in HLA typing results between the two indexing strategies, suggesting that indexing-by-patient, which is much simpler, is a viable option. In conclusion, enzymatic fragmentation of pooled whole gene amplicons is a suitable strategy for HLA typing by next-generation sequencing on the Illumina MiSeq.

The mouse major histocompatibility complex: some assembly required.

We have assembled a contig of 81 yeast artificial chromosome clones that spans 8 Mb and contains the entire major histocompatibility complex (Mhc) from mouse strain C57BL/6 (H2b), and we are in the process of assembling an Mhc contig of bacterial artificial chromosome (BAC) clones from strain 129 (H2bc), which differs from C57BL/6 in the H2-Q and H2-T regions. The current BAC contig extends from Tapasin to D17Leh89 with gaps in the class II, H2-Q, and distal H2-M regions. Only four BAC clones were required to link the class I genes of the H2-Q and H2-T regions, and no new class I gene was found in the previous gap. The proximal 1 Mb of the H2-M region has been analyzed in detail and is ready for sequencing; it includes 21 class I genes or fragments, at least 14 olfactory receptor-like genes, and a number of non-class I genes that clearly establish a conserved synteny with the class I regions of the human and rat Mhc.

Mhc class I and non-class I gene organization in the proximal H2-M region of the mouse.

A bacterial artificial chromosome (BAC) contig was constructed across the proximal part of the H2-M region from the major histocompatibility complex (Mhc) of mouse strain 129 (H2bc). The contig is composed of 28 clones that span approximately 1 megabasepair (Mb), from H2-T1 to Mog, and contains three H2-T genes and 18 H2-M genes. We report the fine mapping of the H2-M class I gene cluster, which includes the previously reported M4-M6, the M1 family, the M10 family, and four additional class I genes. All but two of the H2-M class I genes are conserved among haplotypes H2k, H2b, and H2bc, and only two genes are found in polymorphic HindIII fragments. Six evolutionarily conserved non-class I genes were mapped to a 180 kilobase interval in the distal part of the class I region in mouse, and their order Znf173-Rfb30-Tctex5-Tctex6- Tctex4-Mog was found conserved between human and mouse. In this Znf173-Mog interval, three mouse class I genes, M6, M4, and M5, which are conserved among haplotypes, occupy the same map position as the human HLA-A class I cluster, which varies among haplotypes and is diverged in sequence from the mouse genes. These results further support the view that class I gene diverge and evolve independently between species.

Studies on the tissue-related phenotypic heterogeneity of murine B cells.

The development of B cells is accompanied by their ability to specifically enter the peripheral lymphoid tissues. Recently, we described a novel rat monoclonal antibody (IBL-2; IgG2b/kappa) reacting with a 26/29-kD heterodimeric structure of the cell surface. This mAb has been found to recognize differentially the peripheral B cells of mice depending on their tissue origin. The majority of splenic B cells as well as the mature B cells in the bone marrow were stained with this mAb, whereas the B lymphocytes isolated from LN or Peyer's patches displayed only negligible reactivity. We extended these observations by analyzing the relationship between the expression of IBL-2 antigen and L-selection on the surface of B-cell precursors in the bone marrow by multiparameter flow cytometry. Within the B220 positive compartment, a significant difference of L-selectin expression could be observed between the various IBL-2-reactive subsets. Furthermore, we investigated whether evidences for the establishment of tissue-associated phenotypic heterogeneity similar to that found in normal mice could be found upon the adoptive transfer of normal unselected splenic lymphocytes into SCID recipients (Spl-SCID). It has been found that a large part of the splenic B cells preserved their IBL-2 reactivity, whereas the LN B cells had lost the IBL-2 antigen in Spl-SCID. These data indicate that the phenotypic difference within the SCID mice may be the result of the migration of B lymphocytes from the spleen toward the lymph nodes, and the altered expression of the IBL-2 antigen correlates with this process.

Production and flow cytometric application of a monoclonal anti-glucocorticoid receptor antibody.

Detection and monitoring the expression and level of intracellular glucocorticoid receptor (GCR) is necessary in many clinical and experimental situations. Binding of radioactive steroids (3H dexamethasone) to the cytosolic fractions of cells has been recently used. However, it is an expensive, time-consuming technique difficult to use in routine diagnostics. In this article we describe a novel, simple method for GCR detection, using a FITC-conjugated anti-GCR monoclonal antibody (mAb) for flow cytometric measurements in permeabilized cells. The monoclonal antibody was raised against a conserved sequence (150-176 amino acids) of the regulatory part of the receptor. Synthetic peptide (called APTEK-26) fragment of the receptor conjugated to different carriers (TG, BSA) was used for immunization and screening of the hybridomas. The a-GCR 8E9, 3C8 and 5E4 clones (IgG1) were further characterized by immunoserological methods for their reactivity against overlapping synthetic peptide fragments of the receptor and by Western blot technique on cytosolic fraction of HEP G2 cells (containing the GCR). Furthermore the mAbs could be used for the FACS based detection of GCR, despite its low number of antigen structure within the cells. Solving the problem of nonspecific binding of the secondary antibodies we used our high affinity IgG1 a-GCR mAbs directly labeled with the fluorescent dye FITC. The fluorescent labeling of the GCRs in HEP G2 cell line and human peripheral blood mononuclear cells (PBMC) were demonstrated by flow cytometric analysis after fixation with 4% paraformaldehyde and permeabilization with saponin. Competition with molar excess of unlabelled antibodies and with the GCR peptide fragment confirmed the specific binding of the 8E9 and 5E4 mAbs to the GCRs. Monitoring the GCR level by flow cytometry would be useful in clinical diagnostics, e.g., in steroid-treated patients and in steroid-resistant states.

Genomic evolution of the distal Mhc class I region on mouse Chr 17.

A 5-Mb YAC contig, partly supplemented with BAC contigs, was created from the distal Mhc class I region on mouse Chr 17. The gene order of Znf173-Tctex5-Mog-D17Tu42-D17Leh 89 is conserved between mouse and human but not the physical distance, supporting the independent expansion of Mhc class I genes in the so-called accordion model of Mhc evolution. The distal H2-M region includes the breakpoint of conserved synteny between mouse and human as well as the In(17)4 t-inversion. The H2-M region is rich in L1 repeats, implying that the insertion of L1 repeats may be associated with the evolutionary flexibility to break a chromosome.

Tissue-associated phenotypic heterogeneity of peripheral B cells in mice.

After their primary differentiation and selection in the bone marrow, the cells of B lineage are distributed to the peripheral lymphoid system. Here we report that, with the use of a novel rat monoclonal antibody (IBL-2), a tissue-related phenotypic difference could be observed in the peripheral B-cell compartment in mouse. The antigen recognized by this antibody is a 25,000/29,000 MW heterodimeric cell surface molecule which is resistant to phosphatidylinositol-phospholipase C treatment, but is sensitive to proteases. The antigen was found to be expressed by the majority of B cells from the spleen, whereas the B cells from other peripheral sources (lymph nodes and Peyer's patches) proved to be negative. The staining pattern of splenic B cells was heterogeneous, containing a substantial dim population (IBL-2lo), and a smaller, intensely stained fraction (IBL-2hi) within the positive subset. Unlike the B cells, the T cells were negative in every peripheral lymphoid tissue analysed. In addition, the ratios between the various IBL-2-reactive B cells (positive to negative and, within the positive population, the IBL-2lo to IBL-2hi, respectively) in the spleen were quite similar to that of B cells in the bone marrow. Furthermore, the levels of L-selectin expressed by the various IBL-2-reactive subpopulations were found to be heterogeneous both in the bone marrow and in the spleen. The bone marrow cells could be resolved into double negative, L-selectin +/-/IBL-2lo, L-selectin--IBL-2lo, and L-selectin-/IBL-2hi populations, respectively. In the spleen, an additional fraction with L-selectin+/IBL-2- phenotype could be detected. In both tissues, the overwhelming majority of IBL-2hi cells were found at the MEL-14- compartment. We conclude that either these findings may reflect a heterogeneous development state within the peripheral B-cell pool, with a substantial fraction of splenic B cells being less differentiated than those in other peripheral lymphoid tissues, or alternatively, the differential reactivity of murine B cells with the IBL-2 monoclonal antibody is due to their tissue location.