Circulating T cell-monocyte complexes are markers of immune perturbations
- La Jolla Institute for Immunology, United States
- University of Colombo, Sri Lanka
- North Colombo Teaching Hospital, Sri Lanka
- National Institute of Infectious Diseases, Sri Lanka
- National Tuberculosis Reference Laboratory, Sri Lanka
- National Hospital for Respiratory Diseases, Sri Lanka
- Genetech Research Institute, Sri Lanka
- Johns Hopkins School of Public Health, United States
- Universidad Peruana Cayetano Heredia, Peru
- Tohoku University Graduate School of Medicine, Japan
- University of California, San Diego, United States
Figures
Figure 1 with 2 supplements
Two cell populations expressing both T cell (CD3) and monocyte (CD14) surface markers exist in the live singlet cell population of PBMC from human subjects.
(A) The top 100 most variable genes in memory CD4+ T cells across TB uninfected (TBneg) and LTBI infected subjects. (B) Immune cell type specific expression of the 22-var genes identified in A). …
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Figure 1—source data 1
Non-parametric spearman correlation between the 22-var genes in memory CD4+ T cells from human subjects.
Memory CD4+ T cells were sorted from 30 LTBI subjects and 29 TB uninfected control subjects (gating strategy Figure 1—figure supplement 1A) and their transcriptomic profile defined by RNA sequencing. The 22-var genes were identified as presented in Figure 1A.
- https://doi.org/10.7554/eLife.46045.005
Figure 1—figure supplement 1
Identification of a CD14 +population within memory CD4 T cells.
(A) Gating strategy to isolate bulk memory CD4+ T cells. (B) Backgating of CD14+ cells within sorted memory CD4+ T cells.
Figure 1—figure supplement 2
Gating strategy to identify CD3+CD14+ cells.
https://doi.org/10.7554/eLife.46045.004
Figure 2 with 3 supplements
CD3+CD14+ cells are tightly bound T cell:monocyte complexes that represent in vivo association.
(A) Gating strategy and (B) random gallery of events for monocytes (CD14+CD3), T cells (CD3+CD14-), CD3+CD14hi cells and CD3+CD14mid cells determined by imaging flow cytometry (ImageStreamX, MkII …
Figure 2—figure supplement 1
Technical variations in sample preparation do not impact the frequency of T cell:monocyte complexes.
(A) Non-parametric spearman correlation between T cell:monocyte complexes frequencies in whole blood versus fresh PBMC. Red blood cells were magnetically depleted from fresh whole blood using the …
Figure 2—figure supplement 2
Accumulation of CD3, LFA1 and ICAM1 at the interface of a T cell:monocyte complex.
Single z-plan (0μm) images (left) and z-plane stacks (right) of the region marked (dashed rectangle) from one sorted CD3+CD14+ Tcell:monocyte complex displaying accumulation of LFA1 and ICAM1 at the …
Figure 2—figure supplement 3
Effect of physical and chemical sample manipulation on the frequency of T cell:monocyte complexes.
The frequency of T cell:monocyte complexes was assessed with or without various treatments (as outlined in the table) from frozen PBMC of three healthy individuals. T cell:monocyte complexes were …
Figure 3 with 1 supplement
Conventional flow cytometry parameters and expression of T cell/monocyte canonical markers cannot differentiate between T cells and monocytes in a complex vs. not in a complex.
(A) 2D density plots of A, H and W from FSC and SSC parameters for CD3-CD14+ Monocytes (red), CD3+CD14- T cells (green) and CD3+CD14+ T cell:monocyte complexes (T:M, blue). Representative staining …
Figure 3—figure supplement 1
Gating strategy to identify CD3+CD14+ cells with or without a CD45-SSC gate filtering.
https://doi.org/10.7554/eLife.46045.011
Figure 4 with 4 supplements
The constant of association Ka between monocytes and T cells (and T cell subsets) varies with the presence and nature of immune perturbations.
(A) Non-parametric spearman correlation between the frequency of T cell:monocyte complexes and the product of singlet T cells and monocyte frequencies in healthy subjects (n = 59). (B) Formula for …
Figure 4—figure supplement 1
T cell:monocyte constant of association Ka in subjects with active TB, latent TB or TB uninfected individuals.
T cell:monocyte constant of association Ka was calculated as explained in Figure 4B from active TB samples (n = 15) collected at diagnosis from Sri Lanka, latent TB samples collected from subjects …
Figure 4—figure supplement 2
T cell:monocyte constant of association Ka fluctuates as a function of time following Tdap boost administration.
Previously vaccinated healthy subjects (n = 16) were re-immunized with Tdap and blood collected before, one day, three days, seven days and fourteen days post boost. Plots represent the median and …
Figure 4—figure supplement 3
Comparison of constant of association Ka between monocytes and T cell subsets across different immune perturbations.
Constant of association Ka for each T cell subset and monocytes was calculated as explained in Figure 4B from active TB subjects at diagnosis (n = 25), individuals with acute dengue hemorrhagic …
Figure 4—figure supplement 4
Frequencies of T cell:monocyte complexes in different immune perturbation models.
Frequencies of T cell:monocyte complexes (and T cell subsets:monocyte complexes) expressed as percent of live cells were determined in active TB subjects at diagnosis (n = 25) and two months post …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | CCR7-PerCpCy5.5; clone G043H7; mouse monoclonal | Biolegend | Cat# 353220 | 1:50 (4 µl per test) |
| Antibody | CD2-BV421; clone RPA-2.10; mouse monoclonal | Biolegend | Cat# 300229 | 1:66 (3 µl per test) |
| Antibody | CD3-AF700; clone UCHT1; mouse monoclonal | BD pharmigen | Cat# 557943 | 1:66 (3 µl per test) |
| Antibody | CD3-AF488; clone UCHT1; mouse monoclonal | Biolegend | Cat# 300415 | 1:200 (1 µl per test) |
| Antibody | CD4-APCeF780; clone RPA-T4; mouse monoclonal | eBiosciences | Cat# 47-0049-42 | 1:200 (1 µl per test) |
| Antibody | CD5-APCCy7; clone L17F12; mouse monoclonal | Biolegend | Cat# 364009 | 1:66 (3 µl per test) |
| Antibody | CD7-APC; clone CD7-6B7; mouse monoclonal | Biolegend | Cat# 343107 | 1:66 (3 µl per test) |
| Antibody | CD8a-BV650; clone RPA-T8; mouse monoclonal | Biolegend | Cat# 301042 | 1:200 (1 µl per test) |
| Antibody | CD14-APC; clone 61D3; mouse monoclonal | Tonbo biosciences | Cat# 20–0149 T100 | 1:200 (1 µl per test) |
| Antibody | CD14-AF594; clone HCD14 | Biolegend | Cat# 325630 | 1:200 (1 µl per test) |
| Antibody | CD14-AF647; clone 63D3 | Biolegend | Cat# 367128 | 1:200 (1 µl per test) |
| Antibody | CD14-BV421; clone HCD14 | Biolegend | Cat# 325628 | 1:200 (1 µl per test) |
| Antibody | CD14-PE; clone 61D3; mouse monoclonal | eBioscience | Cat# 12-0149-42 | 1:200 (1 µl per test) |
| Antibody | CD27-BV650; clone O323 | Biolegend | Cat# 302827 | 1:100 (2 µl per test) |
| Antibody | CD33-APC; clone WM53 | Biolegend | Cat# 303407 | 1:200 (1 µl per test) |
| Antibody | CD36-APCCy7; clone 5–271 | Biolegend | Cat# 336213 | 1:100 (2 µl per test) |
| Antibody | CD45-PerCpCy5.5; clone HI30; mouse monoclonal | Tonbo biosciences | Cat# 65–0459 T100 | 1:66 (3 µl per test) |
| Antibody | CD45RA-eF450; clone HI100; mouse monoclonal | eBiosciences | Cat# 48-0458-42 | 1:200 (1 µl per test) |
| Antibody | CD64-AF488; clone 10.1; mouse monoclonal | Biolegend | Cat# 305010 | 1:200 (1 µl per test) |
| Antibody | CD163-PECy7; clone GHI/61; mouse monoclonal | Biolegend | Cat# 333613 | 1:100 (2 µl per test) |
| Antibody | ICAM1(CD54); unconjugated; clone HCD54; mouse monoclonal | Biolegend | Cat# 322704 | 1:40 (5 µl per test) |
| Antibody | LFA1(CD11a); unconjugated; clone TS2/4; mouse monoclonal | Biolegend | Cat# 350602 | 1:40 (5 µl per test) |
| Antibody | LFA1(CD11a/CD18)-AF647; clone m24; mouse monoclonal | Biolegend | Cat# 363412 | 1:40 (5 µl per test) |
Additional files
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Supplementary file 1
Fluorochrome-conjugated antibodies used in the study.
- https://doi.org/10.7554/eLife.46045.017
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Transparent reporting form
- https://doi.org/10.7554/eLife.46045.018
