Supporting Information for Reconstructing dynamic molecular states from single-cell time series

Details of mathematical derivations and computation for the proposed method

Relatively Low Level of Antigen-specific Monocytes Detected in Blood from Untreated Tuberculosis Patients Using CD4⁺ T-cell Receptor Tetramers

<div><p>The <em>in vivo</em> kinetics of antigen-presenting cells (APCs) in patients with advanced and convalescent tuberculosis (TB) is not well characterized. In order to target <em>Mycobacterium tuberculosis</em> (MTB) peptides- and HLA-DR-holding monocytes and macrophages, 2 MTB peptide-specific CD4⁺ T-cell receptor (TCR) tetramers eu and hu were successfully constructed. Peripheral blood (PBL) samples from inpatients with advanced pulmonary TB (PTB) were analyzed using flow cytometry, and the percentages of tetramer-bound CD14⁺ monocytes ranged from 0.26–1.44% and 0.21–0.95%, respectively; significantly higher than those measured in PBL samples obtained from non-TB patients, healthy donors, and umbilical cords. These tetramers were also able to specifically detect macrophages <em>in situ via</em> immunofluorescent staining. The results of the continuous time-point tracking of the tetramer-positive rates in PBL samples from active PTB outpatients undergoing treatment show that the median percentages were at first low before treatment, increased to their highest levels during the first month, and then began to decrease during the second month until finally reaching and maintaining a relatively low level after 3–6 months. These results suggest that there is a relatively low level of MTB-specific monocytes in advanced and untreated patients. Further experiments show that MTB induces apoptosis in CD14⁺ cells, and the percentage of apoptotic monocytes dramatically decreases after treatment. Therefore, the relatively low level of MTB-specific monocytes is probably related to the apoptosis or necrosis of APCs due to live bacteria and their growth. The bactericidal effects of anti-TB drugs, as well as other unknown factors, would induce a peak value during the first month of treatment, and a relatively low level would be subsequently reached and maintained until all of the involved factors reached equilibrium. These tetramers have diagnostic potential and can provide valuable insights into the mechanisms of antigen presentation and its relationship with TB infection and latent TB infection.</p> </div

SNPs associated with serum ferritin concentrations.

a<p>Genomic position is based on NCBI build 36.</p>b<p>m, minor allele, M, major allele, MAF indicates the minor allele frequency for allele m; MM indicates serum ferritin concentrations for homozygous carriers of major alleles, Mm indicates heterozygous carriers and mm indicates homozygous carriers of minor alleles.</p>c<p>P-values were calculated based on multivariate linear regression analysis adjusted for population stratification, age and BMI assuming an additive model.</p><p>SNPs associated with serum ferritin concentrations.</p

Quantile-Quantile plot of genome-wide quantitative trait loci mapping for log-transformed serum ferritin concentrations.

<p>Quantile-Quantile plot of genome-wide quantitative trait loci mapping for log-transformed serum ferritin concentrations.</p

General characteristics of participants in the two-stage GWAS stratified by the rs5742933.

a<p>Ferritin levels were log-transformed and the values presented were back-transformed.</p>b<p>One-way ANOVA was used to compare means of the continuous variables, while chi-square test was used to compare the differences for categorical variables in subgroups stratified by rs5742933.</p><p>General characteristics of participants in the two-stage GWAS stratified by the rs5742933.</p

Association of serum ferritin concentrations with SNPs at chromosome 2.

<p>X-axis shows base positions from 190,414 Kb to 190,357 Kb. Y-axis shows –log10 P-values from linear regression adjusting for population stratification, age and BMI and stage information. Ferritin concentrations were log-transformed and fit for a normal distribution. The bottom panels describe all genes in the region.</p

HLA-DRB1 alleles of 9 active PTB outpatients.

<p>HLA-DRB1 alleles of 9 active PTB outpatients.</p

Percentage of early-apoptosis CD14⁺ cells from healthy donors or the continuously treated PTB patients.

<p>Apoptotic CD14⁺ cells were analyzed by flow cytometry using Annexin V plus PI staining <i>in vitro</i>. Annexin V⁺PI⁻ cells represent the early apoptotic populations. Healthy, healthy donors; untreated, untreated PTB patients; within 5 days, PTB patients who received regular anti-TB treatment within 5 days; 15 to 30 days, PTB patients received regular treatment for 15–30 days; More than 30 days: PTB patients received regular treatment for >30 days. The presented data indicate that the percentage of apoptotic cells dramatically decreased after treatment.</p

Statistical analyses of the tetramer-bound CD14⁺ monocytes detected in PBL samples from active PTB inpatients.

<p>The percentage of tetramer-bound CD14⁺ monocytes was detected by flow cytometry. (A) The clustered bar graph shows that the median percentages of tetramer-bound CD14⁺ monocytes in the PBL samples from PTB inpatients (second bar; 0.6% and 0.45%) were significantly higher than those of the control groups (Mann-Whitney U test, <i>p</i><0.01). N1 and N2 indicate the number of samples stained with the eu- and hu-tetramer in each group, respectively. (B) The scatter graphs show that a high percentage of tetramer-bound CD14⁺ monocytes was only observed in PBL samples from PTB inpatients following staining with both tetramers.</p

HLA-DRB1 alleles of active PTB patients and the percentages of TCR tetramer-bound CD14⁺ monocytes in PBL samples.

a<p>The positivity rate indicates the median percentage of double-stained-positive monocytes in PBL samples from active PTB patients.</p