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Quantification of T-cell dynamics from telomeres to DNA labelling

Quantification of T-cell dynamics from telomeres to DNA labelling. Jos é Borghans Dpt. Immunology University Medical Center Utrecht. How long does a T cell live?. Estimates vary widely How is T-cell turnover disturbed in HIV infection, leukemia,

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Quantification of T-cell dynamics from telomeres to DNA labelling

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  1. Quantification of T-cell dynamics from telomeres to DNA labelling José Borghans Dpt. Immunology University Medical Center Utrecht

  2. How long does a T cell live? • Estimates vary widely • How is T-cell turnover disturbed in HIV infection, leukemia, or after stem cell transplantation?

  3. What is the difficulty? • How to follow a lymphocyte from its birth to its death? • Extrapolation from mice to men v.v.

  4. Experiments of nature (1): T-cell reconstitution after chemotherapy 3 years of age 23 years of age Underestimate?: Cells may also die during reconstitution Overestimate?: Cells undergo little competition Mackall et al. 1995

  5. CD45RA (“naive”) CD45RO (“memory”) Experiments of nature (2): Loss of T cells with chromosome damage Immunological memory resides in a population with rapid turnover: Memory T cells have a shorter lifespan (~250 d) than naive T cells (~1000 d) Michie et al. Nature 1992 Caution: Cells have DNA damage, and cell numbers are low

  6. Static versus dynamic markers of T-lymphocyte turnover Static • Ki67-expression (protein expressed in G1,S,G2,M phase) • Annexin V staining (stains phosphatidylserine translocation) Dynamic Natural markers: • T-cell telomere lengths • T-cell receptor excision circles Labelling: • (CFSE labelling) • BrdU labelling • Stable isotope labelling

  7. Changes in telomere lengths are no direct measure of T-cell division Weng et al. PNAS 1995

  8. Changes in TRECs do not directly reflect thymus output Douek et al. Nature 1998

  9. Static versus dynamic markers of T-lymphocyte turnover Static • Ki67-expression (protein expressed in G1,S,G2,M phase) • Annexin V staining (stains phosphatidylserine translocation) Dynamic Natural markers: • T-cell telomere lengths • T-cell receptor excision circles Labelling: • (CFSE labelling) • BrdU labelling • Stable isotope labelling

  10. Dynamic markers (1) BrdU Labelling BrdU = 5-bromo-2’-deoxyuridine Nucleoside analog incorporated instead of thymidine

  11. Determine percentage BrdU+ cells by FACS analysis Kovacs et al. 2001 How to quantify leukocyte turnover?

  12. Model for BrdU labelling up-labelling: determined by p+d down-labelling determined by p-d

  13. Expected changes in the percentage of BrdU+ cells p+d Kovacs et al. 2001 p-d See today’s exercise But… possible toxicity, almost only done in mice, only short-term labelling

  14. Dynamic markers (2) Stable isotope labelling Deuterium (2H) replaces hydrogen in DNA of proliferating cells Measure deuterium-enrichment using GC/MS Main advantage: no interference with cell dynamics

  15. Long-term administration Hellerstein et al. 2003 Vrisekoop et al. 2008 Stable isotope labelling 2H2-glucose 2H2O Intravenously Hellerstein et al. 1999, 2003 McCune et al. 2000 Mohri et al. 2001 Ribeiro et al. 2002 Macallan et al. 2003,2004 Wallace et al. 2004

  16. U(t) Label p - dL - dL Enrichment in DNA of cells Fraction labeled DNA (!) L changes by: Yields two parameters: p and d p from upslope, d from up- and downslope 2H2O labeling does not distinguish between production in thymus and periphery!

  17. d p Typically: p < d Conclusion: d is no good measure of average turnover rate It represents the turnover of labelled cells Paradox: d is typically larger than p Asquith et al. 2002

  18. Short labelling periods give higher estimate of d Asquith et al. 2002

  19. Average turnover (p) needs data during up-labeling period D-glucose D2O Advantages of heavy water: long-term labeling possible many data points during labeling

  20. BrdU Potentially toxic Measures labeled cells Up: p+d Down: p-d Deuterium labeling Non-toxic (non-radioactive) Measures labeled DNA strands Up: p (and d) Down: -d

  21. NaiveT-cell dynamics of mice and men

  22. From mice to men...(How) can we extrapolate? • How to scale? mice 2 years men 80 years mice 2 years men 80 years • Effect of thymic involution during aging? • Young/old mouse = young/old human?

  23. Of mice...Naive T-cell dynamics using 2H2O H-O-H Label acquisition: - proliferation - thymic output Loss of label: - cell death - differentiation/migration H-O-H 4w 2H2O C57Bl/6 % labeled time

  24. Expected lifespan: Naive CD4: 6 weeks Naive CD8: 11 weeks Naive CD4 p = 0.023 d* = 0.022 60 40 20 0 Percentage labeled Naive CD8 p = 0.013 d* = 0.011 60 40 20 0   200 100 0 150 50 Time (days) Young mice (12 week old) Short lifespan of naive T cells in mice

  25. And men...T-cell dynamics using 2H2O H-O-H H-O-H % labeled ~ 400 ml D2O on day 0 ~ 50 ml D2O daily during 9 weeks time

  26. T-cell turnover in healthy individuals Vrisekoop et al. PNAS 2008 Lifespan 6.0 9.40.6 1.0 year

  27. T-cell turnover in young healthy men Naive CD4 60 40 20 0 Percentage labeled Naive CD8 60 40 20 0 200 0 100 150 50 Time (days) Young adult mice (12 week old) Lifespan 6.0 9.40.6 1.0 year Vrisekoop et al. PNAS 2008

  28. Labeled naive T cells present 3 years after stop of label For all fits of naive T cells: p = d All naive T-cells are very long-lived in humans

  29. Scaling from mice to men... mice 2 years 80 years men Mice live 80 weeks naive CD4 T cells 6 weeks, CD8 11 weeks Humans live 80 years naive CD4 T cells 6 years, CD8 10 years

  30. From mice to men...(How) can we extrapolate? • How to scale? mice 2 years men 80 years • Effect of thymus involution during aging? • Young/old mouse = young/old human?

  31. Thymus involution in mice and men Mice Men Steinman et al. 1985

  32. Proliferation Differentiation Naïve Thymic output Death Fraction labeled DNA Time in days 2H2O labeling does not distinguish between production in thymus and periphery Label acquisition: - thymic output - proliferation Loss of label: - cell death - differentiation/migration

  33. Distinguishing between T-cell proliferation and thymic output C57BL/6 ATx or ShamTx at 7 weeks of age • Isolation of spleen, PLNs and thymus • FACS analysis • Mathematical modelling

  34. Naive T-cell dynamics after thymectomy N’ = ε f(t) + rnN – dnN2 Naive T cells: Naive CD4 (spleen) Naive CD8 (spleen) Control Cells (x106) Control Tx Tx Mouse age (weeks) ε = 2.8 10-2 rn = – 3.0 10-3 dn = 1.7 10-9 ε = 2.8 10-2 rn = – 2.0 10-3 dn = 6.8 10-10 0.9% of thymocytes per day

  35. Thymic output Thymectomy Loss Thymic output: CD4 thymic emigrants  0.35 106 / day CD8 thymic emigrants  0.12 106 / day Peripheral proliferation Contribution of thymic output and T-cell proliferation in mice Thymic output Peripheral proliferation Total Naive T-cell production Naive CD4  p = 0.28 106 / day Naive CD8  p = 0.13 106 /day Heavy water Loss Naive T-cell production in mice almost completely due to thymic output!

  36. p = 0.023 p = 0.024 p = 0.013 p = 0.011 Time (days)‏ If everything depends on thymic output, what happens when thymic output declines? Young mice:12 weeks Old mice: 85 weeks 42 days 43 days ? 91 days 77 days

  37. Naive T-cell dynamics in mice hardly change with age p = 0.023 p = 0.024 p = 0.013 p = 0.011 Time (days)‏ Young mice:12 weeks Old mice: 85 weeks 43 days 42 days 91 days 77 days The naive pool of a mouse is a big homogeneous pool of thymus emigrants Naive T-cell proliferation hardly occurs at any age in mice!

  38. This is completely different in humans! Evidence for naive T-cell proliferation in men…

  39. TREC decline with age originally interpreted to reflect thymus decline Child Adult Douek et al. Nature 1998 Rodewald Nature 1998

  40. If naive T-cells proliferate homeostatically, TREC contents do decline Naive TREC decline in humans suggests that naive T-cell proliferation contributes to the naive T-cell pool in humans Hazenberg et al. 2000, Dutilh et al. 2003

  41. CD31 proposed as marker for non-divided naive CD4 T cells Naive Memory thymus CD31+ CD31- CD31+ naive T cells have consistently higher TREC content than CD31- Suggestion: CD31+ naive T cells represent (non-divided) thymic emigrants CD31- naive T cells have divided Kimmig et al J. Exp Med 2002

  42. CD4 SP 1 CD31+ 0.1 Sj TREC content/ cell 0.01 CD31- 0.001 0.0001 0 10 20 30 40 50 60 70 80 Age (years) However, even CD31+ naive T cells divide in humans! TREC content per cell Age (in years) See also Kilpatrick et al. 2008

  43. Fewer thymus emigrants (TE) than CD31+ naive T cells Naive Memory thymus CD31+ CD31- Naive Memory thymus CD31+ CD31- TE non-TE

  44. s(t) A/c = pN + s(t) Which part of T-cell production comes from the thymus? T cells: TRECs: TREC content: Adapted from Hazenberg et al. 2000

  45. CD4 SP 1 CD31+ 0.1 Sj TREC content/ cell 0.01 CD31- 0.001 s(t) 0.0001 A/c = 0 10 20 30 40 50 60 70 80 pN + s(t) Age (years) Use TREC content of cord blood or SP thymocytes to measure c TREC content per cell Age (in years) See also Kilpatrick et al. 2008

  46. 1.1 1.0 0.9 0.8 0.7 0.6 Fraction RTE 0.5 0.4 0.3 0.2 0.1 0.0 0 10 20 30 40 50 60 70 80 90 100 Age (years) In human adults <10% of naive T cells is produced by the thymus Based on CD31 Fraction naive cells produced in thymus Based on TREC contents Age (in years)

  47. Extrapolating from mice to men mice 2 years 80 years men Proliferation RTE RTE RTE RTE Men Proliferation Prediction: without proliferation there should be no TREC dilution in mice…

  48. And indeed... no TREC decline in naive T cells from mice

  49. NaiveT-cell dynamics of mice and men Quantification of T-cell dynamicsfrom telomeres to DNA labelling “The best-laid schemes o' mice an' men, Gang aft agley” The most carefully prepared plans may go wrong

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