Analysis of Immune Repertoire with Fluorogenic Probes

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​Rapid analysis of T cells for the diagnosis of diseases in which the T cell repertoire is altered.  Cells are distributed in aqueous droplets and their T cell receptor mRNAs are amplified in presence of “sloppy” molecular beacons.  The melt profiles of molecular beacons in thousands of droplets reveals the T cell repertoire of the individual.

Invention Summary:

In our immune system, B cells produce antibodies, and T cells express T-cell receptors.  To recognize a diverse array of antigens that an individual will encounter in their life, immune cells recombine disparate gene segments called the variable (V), diversity (D), and joining (J) regions into unique combinations during cell maturation.  The resulting T cell repertoire (TCR) potentially contains trillions of specificities.  The immense TCR diversity shrinks during many disease conditions due to the expansion of some T cell clones.  This occurs particularly during autoimmune diseases, such as Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), T-cell lymphomas, and in diverse infections, ranging from Tuberculosis to COVID-19.  These changes in the T cell repertoire can be used as hallmarks for the diagnostics and therapy management of these diseases.  Tracking changes in the T cell repertoire is a powerful but currently poorly accessible way to diagnose and monitor autoimmune diseases and infections.

Rutgers researchers and collaborators have invented a rapid and low-cost diagnostic procedure that relies on polymerase chain reactions carried out on single T cells enclosed in aqueous droplets suspended in oil.  mRNAs encoding T cell receptors are amplified in the presence of “sloppy” molecular beacons that can recognize all gene segments.  The gene segments used in each hundred of thousands of T cells are determined from information-rich melt profiles of the molecular beacons using a machine learning algorithm.  Unlike the current bulk RNA sequencing approaches, this procedure also provides information about which combination of the two chains of the T cell receptor (alpha and beta) is present in each T cell, resolving one of approximately three million possible gene-segment combinations per cell.  This procedure can be implemented on a simple device where T cells are introduced as input and after a brief PCR and analysis, a measure of T cell repertoire depth is obtained, making it a suitable tool for rapid diagnostics and therapy management.

Market Applications:

• Determine T and B cell immune cell repertoires in autoimmune conditions, T-cell lymphomas, and infections.
• Allows analysis of T cell repertoires of hundreds of thousands of cells simultaneously and enables faster, more affordable diagnosis.  It can be used by physicians and medical personnel to guide treatment plans without special expertise.

Advantages:

Currently, deep sequencing is the mainstay of T cell repertoire analysis, which is very expensive, requires specialized expertise, takes a long time, and suffers from amplification biases.

• Technology is less expensive and simpler.
• Allows diagnosis-driven therapy management of diverse autoimmune diseases such as lupus, arthritis, psoriasis, ulcerative colitis, and hematological cancers.
• Reduction of amplification bias and reporting of alpha and beta pairing in thousands of single T cells.

Publications:

•    Low-cost

Intellectual Property & Development Status: PCT application published No. WO2025/058951 and is available for licensing and/or research collaboration. For any business development and other collaborative partnerships, contact:  marketingbd@research.rutgers.edu