Detection and measurement of tissue-infiltrating lymphocytes
First Claim
1. A method for identifying lymphocytes belonging to a functional subset that have infiltrated a solid tissue comprising:
- sorting a sample of lymphocytes from an accessible tissue of an individual into at least one functional subset;
generating a clonotype profile for each functional subset of lymphocytes from the accessible tissue by amplifying recombined nucleic acid molecules obtained from said at least one subset to obtain a plurality of amplicons and performing high throughput sequencing of the resulting plurality of amplicons to provide a list of clonotype sequences that identify individual lymphocytes of each functional subset;
generating at least one clonotype profile from at least one sample of the solid tissue by amplifying recombined nucleic acid molecules obtained from said at least one sample of solid tissue to obtain a plurality of amplicons and performing high throughput sequencing of the resulting plurality of amplicons to provide a list of clonotype sequences in each sample; and
identifying lymphocytes belonging to a functional subset that have infiltrated from the accessible tissue into the solid tissue by identifying a clonotype sequence from the solid tissue that is present in the list of clonotype sequences of a functional subset from the accessible tissue, wherein said step of identifying lymphocytes further includes determining numbers of lymphocytes of each of said at least one functional subset,wherein one or more of the clonotype profiles for one or more of the functional subsets comprise at least 1000 clonotypes of at least 30 nucleotides.
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Abstract
The present invention is drawn to methods for measuring numbers, levels, and/or ratios of cells, such as lymphocytes, infiltrated into a solid tissue, such as a tumor or a tissue affected by an autoimmune disease, and to methods for making patient prognoses based on such measurements. In one aspect, methods of the invention comprise sorting lymphocytes from an accessible tissue, such as peripheral blood, into functional subsets, such as cytotoxic T cells and regulatory T cells, and generating clonotype profiles of each subset. An inaccessible disease-affected tissue is sampled and one or more clonotype profiles are generated. From the latter clonotype profiles, levels lymphocytes in each of the functional subsets are determined in the disease-affected tissue by their clonotypes, which are identified from lymphocytes sorted into subsets from the accessible tissue.
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Citations
29 Claims
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1. A method for identifying lymphocytes belonging to a functional subset that have infiltrated a solid tissue comprising:
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sorting a sample of lymphocytes from an accessible tissue of an individual into at least one functional subset; generating a clonotype profile for each functional subset of lymphocytes from the accessible tissue by amplifying recombined nucleic acid molecules obtained from said at least one subset to obtain a plurality of amplicons and performing high throughput sequencing of the resulting plurality of amplicons to provide a list of clonotype sequences that identify individual lymphocytes of each functional subset; generating at least one clonotype profile from at least one sample of the solid tissue by amplifying recombined nucleic acid molecules obtained from said at least one sample of solid tissue to obtain a plurality of amplicons and performing high throughput sequencing of the resulting plurality of amplicons to provide a list of clonotype sequences in each sample; and identifying lymphocytes belonging to a functional subset that have infiltrated from the accessible tissue into the solid tissue by identifying a clonotype sequence from the solid tissue that is present in the list of clonotype sequences of a functional subset from the accessible tissue, wherein said step of identifying lymphocytes further includes determining numbers of lymphocytes of each of said at least one functional subset, wherein one or more of the clonotype profiles for one or more of the functional subsets comprise at least 1000 clonotypes of at least 30 nucleotides. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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Specification