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Using microbiota metabolites to differentiate naïve t-cells and related methods to induce or prevent inflammatory conditions

a technology of nave t cells and microbiota metabolites, which is applied in the field of using microbiota metabolites to differentiate nave t cells and related methods to induce or prevent inflammatory conditions, can solve the problems of major hurdles still existing in the translation of this therapeutic strategy, and achieve the effect of increasing the stability of a treg cell

Inactive Publication Date: 2019-01-31
TEXAS A&M UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent describes a method for increasing the stability of a type of immune cell called Treg cells. This is achieved by contacting the Treg cells or their precursors with a specific type of molecule called a TDMM. The TDMM can be administered to a subject either as a precursor, prodrug, or acceptable salt. The method can be used to create induced Treg cells in the laboratory or to treat a subject with an endogenous nTreg population. The technical effect of this patent is to provide a way to improve the stability and function of Treg cells for immunotherapy applications.

Problems solved by technology

Although the adoptive transfer of iTregs has the promise to be safe in the clinic, major hurdles still exist in the translation of this therapeutic strategy from lab bench to bedside.

Method used

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  • Using microbiota metabolites to differentiate naïve t-cells and related methods to induce or prevent inflammatory conditions
  • Using microbiota metabolites to differentiate naïve t-cells and related methods to induce or prevent inflammatory conditions
  • Using microbiota metabolites to differentiate naïve t-cells and related methods to induce or prevent inflammatory conditions

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0150]The following is a description of methodology to isolate naïve T cells and assay the effect of any TDMM on the potential differentiation into Treg (or other) cell type.

[0151]Mesenteric lymph node (MLN) and spleen are harvested from WT or FOXP3EGFP C57Bl / 6 mice and homogenized. Spleen tissue is subjected to red blood cell lysis and pooled into a single cell suspension for counting. Counted cells are resuspended in sterile FACS buffer (PBS++ with 05% bovine serum albumin) at a concentration of ˜5×107 cells / mL and stained for 45 minutes at 4° C. with anti-CD4 and anti-CD25 fluorophore-conjugated monoclonal antibodies (mAb). After two washes, the stained cells are resuspended at ˜6×106 cells / mL in FACS buffer and immediately sorted. High purity (>95%) CD4+ CD25− naïve T cells (1×106 cells / mL) are cultured in anti-CD3 mAb (5 μg / mL) and anti-CD28 mAb (2 μg / mL) coated plates in the presence of TGF-β (5 ng / mL), IL-2 (100 U / mL), and desired TDMMs or a solvent control for at least 72 ho...

example 2

[0153]The following is an additional description of an exemplary procedure that was used to isolate naïve T cells and induce differentiation in vitro.

[0154]Cell Isolation

[0155]CD4+ CD25− T cells were isolated to high purity (>98%) from the pooled spleen and mesenteric lymph nodes of C57BL / 6 mice (Jackson Labs) with a BD FACS Aria II flow cytometer. Fc Block (BD), αCD4-efluor450 (eBioscience clone GK1.5) and αCD25-PECy7 (eBioscience clone PC61.5) antibodies were used for staining before sorting.

[0156]In Vitro T Cell Differentiation

[0157]Isolated cells were cultured at an initial concentration of 1×105 cells / well in RPMI 1640 supplemented with 2-mercaptoethanol, gentamicin, penicillin, streptomycin, and 10% FCS (all from Life Technologies) in a 96-well round bottom plate (Falcon) coated with 5 μg / mL αCD3 (BioXcell clone 145-2C11) and 2 μg / mL αCD28 (BioXcell clone 37.51) for 72 hours. For Th1-skew: 5 ng / mL IL-12 (Peprotech cat. #210-12) and 10 g / mL αIL-4 (BioXCell clone 11B11) were add...

example 3

[0158]The following is a description of methodology to characterize the subset of stimulation conditions and resulting differentiated iTreg cells.

[0159]Naïve T cells are cultured for 3 days using concentrations of cytokines and TDMMs that resulting in a significant increase in the levels of iTreg cells. After 72 h, cells are stimulated with PMA / ionomycin (10 ng / mL PMA, 1 mM ionomycin). For flow cytometry, cells are treated with Golgi plug (Brefeldin A) for four hours and then stained with fluorophore-conjugated mAbs against both inflammatory (e.g., IL-6, IL-12, IL-17, TNF-α) and anti-inflammatory (e.g., IL-10, IL-35, TGF-β) cytokines. Samples are stained with the maximum number of antibodies. Furthermore, expression of established Treg markers (FOXP3, CD25hi, CD103, CD62L, GITR, PD-1, CD152, CD127, ICOS, Granzyme-B) [Vignali, D. A., L. W. Collison, and C. J. Workman, Nat Rev Immunol 8(7):523-32. (2008); Chen, J., et al., Int Immunopharmacol 11(5):610-7 (2011)] and markers of an infl...

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Abstract

Disclosed are compositions derived from the commensal microbiota and related methods for inducing and differentiating naive T cells. In some embodiments, the compositions and methods can be selectively used to generate stable regulatory T-cells (Tregs or iTregs) or stabilize such Tregs to prevent inflammatory responses and instead promote antigen tolerance. Alternatively, in other aspects, select compositions can be used to promote pro-inflammatory T cell development, such as through the induced development and stabilization of Th1 and Th7 cells.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS[0001]This application claims the benefit of Provisional Application No. 62 / 310,643, filed Mar. 18, 2016, Provisional Application No. 62 / 310,648, filed Mar. 18, 2016, Provisional Application No. 62 / 310,606, filed Mar. 18, 2016, and Provisional Application No. 62 / 310,630, filed Mar. 18, 2016, each of which is expressly incorporated herein by reference in its entirety.STATEMENT OF GOVERNMENT LICENSE RIGHTS[0002]This invention was made with Government support under GM106251 awarded by The National Institutes of Health, National Institute of General Medical Sciences; AI110642 awarded by The National Institutes of Health, National Institute of Allergy and Infectious Disease; A095788 awarded by The National Institutes of Health, National Institute of Allergy and Infectious Disease; and MCB-1120827, awarded by the National Science Foundation. The Government has certain rights in the invention.FIELD OF THE INVENTION[0003]This disclosure relates to com...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/0783A61K31/405A61K35/17A61K31/475A61P29/00
CPCC12N5/0637A61K31/405A61K35/17A61K31/475A61P29/00A61K31/7004A61K31/404C12N2501/999A61P1/00A61K2035/122A61K2039/577C12N2502/1121A61P37/06C12N2501/2302C12N2501/51C12N2501/515C12N2501/15C12N2501/24C12N2501/2306C12N2501/2323C12N2501/2304C12N5/0636C12N2500/30C12N2500/36A61K2039/57G16B40/20G16B20/00G16B5/00A61K2239/38A61K39/4615A61K2239/31C12N5/064A61K39/4644A61K39/4622A61K39/4611C12N2501/2301C12N2501/2321C12N2501/2312C12N2502/1157A61K45/06
Inventor ALANIZ, ROBERT C.JAYARAMAN, ARULLEE, KYONGBUM
Owner TEXAS A&M UNIVERSITY
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