Compositions and methods for assessing cytotoxicity of single cells

Patent metadata

Field	Value
Publication number	US-9244071-B2
Application number	US-200913145300-A
Country	US
Kind code	B2
Filing date	Jul 13, 2009
Priority date	Jan 21, 2009
Publication date	Jan 26, 2016
Grant date	Jan 26, 2016

Abstract

The invention provides a method of analyzing interactions between pairs of target and effector cells utilizing high-throughput screenings methods for profiling large numbers of single cells in microarrays.

First claim

What is claimed is:

1. A method of assessing cytotoxicity of single cells using arrays of microwells, the method comprising steps of:
monitoring lysis of target cells in microwells that contain a single effector cell and at least one target cell; and
simultaneously profiling activation markers or secreted soluble mediators,
wherein the monitoring comprises a step of detecting lysis of said target cells by said single effector cell using a fluorescent indicator; and
wherein the profiling comprises a step of contacting the microwells with a substrate, wherein a surface of the substrate contains thereon agents that bind to said activation markers or secreted soluble mediators.

2. The method of claim 1 , wherein the monitoring step comprises:
culturing the at least one target cell and the single effector cell under conditions conducive to lysis of the target cell by the effector cell.

3. The method of claim 2 , further comprising a step of:
recovering the single effector cell from at least one of the microwells.

4. The method of claim 2 , wherein the monitoring comprises detecting loss of target cell fluorescence over time.

5. The method of claim 3 , further comprising culturing the recovered single effector cell to obtain a clonal amplification of the recovered single effector cell.

6. The method of claim 3 , further comprising characterizing sequence or expression of one or more genes in the recovered single effector cell.

7. The method of claim 1 , wherein the single effector cell and the target cells are human cells.

8. The method of claim 1 , wherein said profiling further comprises a step of
determining on the surface of the substrate the locations of agent binding to said activation markers or secreted soluble mediators.

9. The method of claim 1 , wherein the single effector cell is a cytotoxic T lymphocyte (CTL).

10. The method of claim 1 , wherein the activation markers or secreted soluble mediators comprise one or more cytokines.

11. The method of claim 1 , 2 , 3 , or 8 wherein the target cells are HIV-infected cells.

12. The method of claim 1 , wherein the depth of the microwells is less than 100 μM.

13. The method of claim 1 , wherein dyes are used to label both the target cells and the single effector cell.

14. The method of claim 1 , further comprising using software to recognize where killing took place and to locate those microwells for a micromanipulator.

15. The method of claim 1 , 2 , or 9 , wherein secreted soluble mediators are simultaneously profiled.

16. The method of claim 11 , wherein secreted soluble mediators are simultaneously profiled.

17. The method of claim 9 , wherein the activation markers or secreted soluble mediators comprise one or more cytokines.

18. The method of claim 1 , wherein the microwells containing the target cells and single effector cells are held in physical contact with said substrate, and wherein said substrate is a glass slide pre-functionalized with said agents to capture the activation markers or secreted soluble mediators.

19. The method of claim 15 , wherein the microwells containing the target cells and single effector cells are held in physical contact with said substrate, and wherein said substrate is a glass slide pre-functionalized with said agents to capture the activation markers or secreted soluble mediators.

Assignees

• Love J Christopher
• Walker Bruce
• Varadarajan Navin
• Julg Boris
• Massachusetts Inst Technology
• Gen Hospital Corp

Inventors

• Love J Christopher
• Walker Bruce
• Varadarajan Navin
• Julg Boris

Classifications

• G01N2333/16

  HIV-1, HIV-2 · CPC title

• G01N33/56972 Primary

  White blood cells · CPC title

• G01N33/5047

  Cells of the immune system · CPC title

• C40B30/06

  by measuring effects on living organisms, tissues or cells · CPC title

• C12Q1/02 Primary

  involving viable microorganisms · CPC title