Method for obtaining regulatory t cells derived from thymic tissue and use of said cells as cell immunotherapy in immune system disorders
a technology of thymic tissue and thymic cells, which is applied in the field of clinical immunology and cell immunotherapy, can solve the problems of reducing the quality of cells, limiting the use of thymic cells, and reducing the number of tregs, and achieving unheard-of thymic cell yields
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Benefits of technology
Problems solved by technology
Method used
Image
Examples
example 1
for Obtaining and Purifying thyTreg Cells from Human Thymic Tissue
[0180]The protocol herein described was developed in the IISGM Immune-Regulation Laboratory for purifying Treg cells from thymic tissue (thyTreg cells) removed from paediatric patients during heart surgeries. The protocol makes it possible to obtain a massive number of thyTreg cells with very high purity and optimal phenotype. The protocol is also GMP-compatible and fulfils the requirements for the subsequent use of the cells as immunotherapy in humans.
[0181]The phases of the protocol are as follows:
[0182]1. Removal and transport of the thymic tissue.
[0183]2. Disaggregation of the tissue and obtainment of thymocytes.
[0184]3. Purification of the Treg cells.
[0185]4. Cultivation and activation of the thyTreg cells.
1.1. Removal and Transport of the Thymic Tissue
[0186]The thymic tissue is usually removed to access the heart during paediatric heart transplants and other congenital heart disease surgeries. Given the large si...
example 2
of the Suppressive Capacity of thyTreg Cells Obtained with the Protocol Described in Example 1
[0197]The suppressive capacity of the thyTreg cells produced in the previous example was analysed by measuring the capacity thereof to inhibit in vitro the cell proliferation of TCD4 and TCD8 lymphocytes, which are the primary mediators in cellular rejection to grafts and also autoimmune diseases. For such purpose, thyTreg cells were co-cultivated with allogeneic peripheral blood mononuclear cells (PBMC) dyed with CFSE (CellTrace™ CFSE proliferation kit, Invitrogen) in the ratio thyTreg:PBMC 1:2. The PBMCs were previously activated with PMA and lonomycin (Sigma) in order to trigger the proliferation of T CD4+ and T CD8+ lymphocytes, comparing the proliferation of these cells with cultures in which the thyTregs were not included. Proliferation of responder T cells was measured by flow cytometry as the reduction in the fluorescence intensity of CFSE staining. CD3, CD4 and CD8 labeled antibodi...
example 3
ting the CD8+Positive Cell Population Provides a Higher Yield of Treq Cells
[0199]The vast majority (80%) of the total thymocytes that are isolated from the thymus are double positive (CD4+CD8+) cells (FIG. 4A). The inventors have unexpectedly shown that a very high proportion of Treg cells are inside this DP fraction. Indeed, it has been shown by the inventors that around 41% of the thyTreg cells population obtained as final product when following the protocol described in Example 1 (at day 7 of culture) are DP cells (FIG. 4B, 4C). Thus, a previous depletion of the CD8+ cells as described by Dijke I. E., et al., 2016 or MacDonald et al. 2017, where a CD4+CD8-CD25+ was selected prior to Treg cells stimulation and expansion, will eliminate completely the DP fraction, and consequently the Foxp3+DP population will not be present in the final product, the recovery of CD25+Foxp3+ cells decreasing dramatically. Moreover, without willing to be bound by theory, due to its less-differentiated...
PUM
| Property | Measurement | Unit |
|---|---|---|
| pore diameter | aaaaa | aaaaa |
| concentrations | aaaaa | aaaaa |
| concentrations | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More