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Antigen processing in the class II MHC pathway depends on conventional proteolytic enzymes, potentially acting on antigens in native-like conformational states. CD4+ epitope dominance arises from a competition between antigen folding, proteolysis, and MHCII binding. Protease-sensitive sites, linear antibody epitopes, and CD4+ T-cell epitopes were mapped in the plague vaccine candidate F1-V to evaluate the various contributions to CD4+ epitope dominance. Using X-ray crystal structures, antigen processing likelihood (APL) predicts CD4+ epitopes with significant accuracy without considering peptide-MHCII binding affinity. The profiles of conformational flexibility derived from the X-ray crystal structures of the F1-V proteins, Caf1 and LcrV, were similar to the biochemical profiles of linear antibody epitope reactivity and protease-sensitivity, suggesting that the role of structure in proteolysis was captured by the analysis of the crystal structures. The patterns of CD4+ T-cell epitope dominance in C57BL/6, CBA, and BALB/c mice were compared to epitope predictions based on APL, peptide binding to MHCII proteins, or both. For a sample of 13 diverse antigens larger than 200 residues, accuracy of epitope prediction by the combination of APL and I-Ab-MHCII-peptide affinity approached 40%. When MHCII allele specificity is also diverse, such as in human immunity, prediction of dominant epitopes by APL alone approached 40%. Since dominant CD4+ epitopes tend to occur in conformationally stable antigen domains, crystal structures typically are available for analysis by APL; and thus, the requirement for a crystal structure is not a severe limitation.

Supplementary Figure from Distinct Patterns of Clonal Evolution Drive Myelodysplastic Syndrome Progression to Secondary Acute Myeloid Leukemia

Supplementary Table from Distinct Patterns of Clonal Evolution Drive Myelodysplastic Syndrome Progression to Secondary Acute Myeloid Leukemia

Clonal evolution in myelodysplastic syndrome (MDS) can result in clinical progression and secondary acute myeloid leukemia (sAML). To dissect changes in clonal architecture associated with this progression, we performed single-cell genotyping of paired MDS and sAML samples from 18 patients. Analysis of single-cell genotypes revealed patient-specific clonal evolution and enabled the assessment of single-cell mutational cooccurrence. We discovered that changes in clonal architecture proceed via distinct patterns, classified as static or dynamic, with dynamic clonal architectures having a more proliferative phenotype by blast count fold change. Proteogenomic analysis of a subset of patients confirmed that pathogenic mutations were primarily confined to primitive and mature myeloid cells, though we also identify rare but present mutations in lymphocyte subsets. Single-cell transcriptomic analysis of paired sample sets further identified gene sets and signaling pathways involved in two cases of progression. Together, these data define serial changes in the MDS clonal landscape with clinical and therapeutic implications.Significance:Precise clonal trajectories in MDS progression are made possible by single-cell genomic sequencing. Here we use this technology to uncover the patterns of clonal architecture and clonal evolution that drive the transformation to secondary AML. We further define the phenotypic and transcriptional changes of disease progression at the single-cell level.See related article by Menssen et al., p. 330 (31).See related commentary by Romine and van Galen, p. 270.This article is highlighted in the In This Issue feature, p. 265

Tuberculosis (TB) is a communicable disease that is a major cause of ill health. The hilly region of Uttarakhand has difficult terrain and is one of the least accessible regions of the country. This present study was to evaluate the Clinico-pathological and demographic profile of patients of cervical tubercular lymphadenitis in hilly region of Uttarakhand, India. Detailed History and physical examination was to done and all previous investigations were compiled. Routine investigations like HIV, RBS and Chest X-Ray PA view, if not done earlier, were done of every patient. Lymph nodes were assessed according to system adopted by the American Academy of Otolaryngology Head and Neck Surgery. The age of the patients ranged from 14 to 64 years with a mean age of 35 years (SD=12.27). The commonest age group was 16-30 years (42.73%). Male: Female ratio was 1:1.34. Among 110 patients, 88 (80%) presented with solid lymph nodes, 20 (18.2%) with abscess and 2 (1.8%) with discharging sinus. FNAC was done in all 110 cases and found AFB positive in 45 (40.9%) cases. Disease is relatively more common in younger patients. Constitutional symptoms were absent in some of the patients.

Clonal hematopoiesis (CH) is an age-associated phenomenon that increases risk for hematologic malignancy and cardiovascular disease. CH is thought to increase cardiovascular disease risk through inflammation in the peripheral blood1. Here, we profile peripheral blood gene expression in 104,566 single cells from a cohort of 17 CH patients and 6 controls. We discovered that patients harboringDNMT3AandTET2CH mutations at baseline and in response to IL-6 stimulation confer a pro-inflammatory profile to CD14+ monocytes through previously unrecognized pathways including Macrophage Inhibitory Factor (MIF). A germline genetic variant in MIF modifiesTET2CH cardiovascular disease risk. Using mitochondrial lineage tracing, we used a novel method to compare gene expression between mutated and non-mutated cells within individual CH patients. We found that the mutated CH monocytes, but not non-mutated monocytes are pro-inflammatory, explaining why patients with larger CH clones have increased cardiovascular disease risk.

Clonal evolution in myelodysplastic syndrome (MDS) can result in clinical progression and secondary acute myeloid leukemia (sAML). To dissect changes in clonal architecture associated with this progression, we performed single-cell genotyping of paired MDS and sAML samples from 18 patients. Analysis of single-cell genotypes revealed patient-specific clonal evolution and enabled the assessment of single-cell mutational cooccurrence. We discovered that changes in clonal architecture proceed via distinct patterns, classified as static or dynamic, with dynamic clonal architectures having a more proliferative phenotype by blast count fold change. Proteogenomic analysis of a subset of patients confirmed that pathogenic mutations were primarily confined to primitive and mature myeloid cells, though we also identify rare but present mutations in lymphocyte subsets. Single-cell transcriptomic analysis of paired sample sets further identified gene sets and signaling pathways involved in two cases of progression. Together, these data define serial changes in the MDS clonal landscape with clinical and therapeutic implications.Significance:Precise clonal trajectories in MDS progression are made possible by single-cell genomic sequencing. Here we use this technology to uncover the patterns of clonal architecture and clonal evolution that drive the transformation to secondary AML. We further define the phenotypic and transcriptional changes of disease progression at the single-cell level.See related article by Menssen et al., p. 330 (31).See related commentary by Romine and van Galen, p. 270.This article is highlighted in the In This Issue feature, p. 265