Method for Studying V(D)J Combinatory Diversity

Abstract

The present invention pertains to a method of visualizing an immunological status of an individual, by combining the degree of immunoglobulin and/or TCR diversity and another biological marker linked to the immunological status of said individual in a two- or three-dimensional graph.

1 . A method of visualizing an immunological status of an individual, comprising the following steps:
 A) using a biological sample from said individual, measuring a first biological marker linked to the immunological status;   B) using the same sample or another sample taken from the same individual at the same time, determining the degree of immunoglobulin and/or TCR diversity of said individual;   C) combining the data obtained in steps A) and B) and representing the result (i) in a two-dimensional graph showing the degree of immunoglobulin or TCR diversity on one axis and the first biological marker on the second axis, or (ii) in a three-dimensional graph showing the degree of immunoglobulin diversity on one axis, the degree of TCR diversity on another axis, and the first biological marker measured in step A) on a third axis.   
     
     
         2 . The method of  claim 1 , wherein said first biological marker is selected from the group consisting of lymphocytes count, acute lymphocyte count and the count of one or several cell populations selected from the group consisting of Treg, Th17, NKT, natural killers, granulocytes, dendritic cells, macrophages, myeloid-derived suppressor cells (MDSC), basophil cells, mast cells, neutrophil cells and eosinophil cells. 
     
     
         3 . A method of visualizing an evolution of an immunological status of an individual, comprising visualizing, in a same graph and by the method of  claim 1 , the immunological status of the same patient, at two or more time points, wherein the points of the graph representing two consecutive immunological statuses of said patient are linked by an arrow. 
     
     
         4 . The method of  claim 3 , wherein for each arrow:
 if the arrow points to a point representing an immunological status which is better than its starting point, the arrow is drawn in a first color symbolizing an improvement of the patient's immunological status;   if the arrow points to a point representing an immunological status which is worst than its starting point, the arrow is drawn in a second color symbolizing a degradation of the patient's immunological status; and   in intermediate cases, the arrow is drawn in a third color.   
     
     
         5 . The method of  claim 4 , wherein the first color is green, the second color is red and the third color is yellow or orange. 
     
     
         6 . The method according to  claim 1 , wherein in step C), two two-dimensional graphs are used, wherein one graph shows the degree of immunoglobulin diversity on one axis and the lymphocyte count on the second axis and the other graph shows the degree of TCR diversity on one axis and the lymphocyte count on the second axis. 
     
     
         7 . The method according to  claim 1 , wherein in step C), the data are examined by means of a three-dimensional graph showing the degree of immunoglobulin diversity on one axis, the degree of TCR diversity on another axis, and the lymphocyte count on a third axis. 
     
     
         8 . The method according to  claim 1 , wherein the immunoglobulin and/or TCR diversity which is determined is the combinatorial diversity. 
     
     
         9 . The method according to  claim 1 , wherein immunoglobulin and/or TCR diversity is determined from genomic DNA originating from a biological sample from said individual, by performing the following steps:
 A) amplifying fragments of said genomic DNA by multiplex polymerase chain reactions (PCRs), at least one of which is a multi-n-plex PCR with n≧2 using, in a single reaction, n different pairs of primers, each allowing the amplification of at least two DNA fragments characteristic of at least two different chromosomal rearrangements, carried out with a combination of at least 3 primers, constituting at least 2 different pairs of primers having the following characteristics:
 (i) each pair of primers is constituted of a sense primer which specifically hybridizes upstream of and/or in a given V or D gene and of an antisense primer which specifically hybridizes downstream of and/or in a given J gene, so as to allow the amplification of at least two fragments characteristic of two distinct V(D)J or D-J rearrangements; 
 (ii) the primers are thermodynamically compatible; 
 (iii) the primers are chosen in such a way that the fragments amplified with the first pair of primers can be distinguished from the fragments amplified with the second pair of primers; 
   B) detecting the amplification products obtained in step A;   C) analyzing the diversity of the repertoire of T and/or B lymphocytes of said individual by analyzing the diversity of the amplification products detected in step B),   wherein the diversity of the amplification products detected in step B) corresponds to the diversity of the repertoire of T and/or B lymphocytes of said individual.   
     
     
         10 . The method according to  claim 9 , wherein in step C), a level of risk is assigned at least to the following 4 zones:
 (i) low count (<1000 Ly/μL) and low V-J combinatorial diversity (<70% for TCR and <60% for immunoglobulins): high infectious risk and a high risk of mortality;   (ii) low count (<1000 Ly/μL) but normal V-J combinatorial diversity (>70% for TCR and >60% for immunoglobulins): low infectious risk;   (iii) normal count (1000-3500 Ly/μL) and low V-J combinatorial diversity (<70% for TCR and <60% for immunoglobulins): medium infectious risk;   (iv) normal count (1000-3500 Ly/μL) and normal V-J combinatorial diversity (>70% for TCR and >60% for immunoglobulins): the immune repertoire corresponds to that of the general population.   
     
     
         11 . The method as claimed in  claim 9 , in which the graph also comprises the following 2 zones:
 (v) count above normal (>3500 Ly/μL) and low V-J combinatorial diversity (<70% for TCR and <60% for immunoglobulins): high lymphoproliferative risk;   (vi) count above normal (>3500 Ly/μL) and normal V-J combinatorial diversity (>70% for TCR and >60% for immunoglobulins): medium lymphoproliferative risk.   
     
     
         12 . The method according to  claim 9 , wherein in step C), a level of risk is assigned at least to the following 8 zones:
 (i) low count (<1000 Ly/μL) and low V-J combinatorial diversity (<70% for TCR diversity and <60% for immunoglobulins diversity): high risk of infection (including viral reactivation) and/or cancer development, possibly leading to death;   (ii) low count (<1000 Ly/μL) and low TCR V-J combinatorial diversity, with normal immunoglobulins diversity (<70% for TCR diversity and >60% for immunoglobulins diversity): high risk of infection (including viral reactivation), and/or cancer development, possibly leading to death;   (iii) low count (<1000 Ly/μL) and low BCR V-J combinatorial diversity, with normal TCR diversity (>70% for TCR diversity and <60% for immunoglobulins diversity): medium risk of infection (including viral reactivation) and/or cancer development, possibly leading to death;   (iv) low count (<1000 Ly/μL) but normal V-J combinatorial diversity (>70% for TCR diversity and >60% for immunoglobulins diversity): low risk of infection (including viral reactivation) and of cancer development;   (v) normal count (1000-3500 Ly/μL) and low V-J combinatorial diversity (<70% for TCR diversity and <60% for immunoglobulins diversity): medium risk of infection (including viral reactivation) and/or cancer development, possibly leading to death;   (vi) normal count (1000-3500 Ly/μL) and low TCR V-J combinatorial diversity, with normal BCR V-J combinatorial diversity (<70% for TCR diversity and >60% for immunoglobulins diversity): medium risk of infection (including viral reactivation), and/or cancer development, possibly leading to death;   (vii) normal count (1000-3500 Ly/μL) and low BCR V-J combinatorial diversity with normal TCR V-J combinatorial diversity (>70% for TCR diversity and <60% for immunoglobulins diversity): medium risk of infection (including viral reactivation), and/or cancer development, possibly leading to death;   (viii) normal count (1000-3500 Ly/μL) and normal V-J combinatorial diversity (>70% for TCR diversity and >60% for immunoglobulins diversity): the immune repertoire is similar to the one of the general population and can be considered as “healthy”.   
     
     
         13 . The method as claimed in  claim 12 , in which the three-dimensional graph also comprises the following 4 zones:
 (ix) count above normal (>3500 Ly/μl) and low V-J combinatorial diversity (<70% for TCR diversity and <60% for immunoglobulins diversity):lymphoproliferative risk;   (x) count above normal (>3500 Ly/μL) and low TCR V-J combinatorial diversity with normal BCR V-J combinatorial diversity (<70% for TCR diversity and >60% for immunoglobulins diversity): T cell lymphoproliferative risk;   (xi) count above normal (>3500 Ly/μL) and low BCR V-J combinatorial diversity with normal TCR V-J combinatorial diversity (>70% for TCR diversity and <60% for immunoglobulins diversity): B cell lymphoproliferative risk;   (xii) count above normal (>3500 Ly/μl) and normal V-J combinatorial diversity (>70% for TCR diversity and >60% for immunoglobulins diversity): medium lymphoproliferative risk.   
     
     
         14 . The method according to  claim 1 , wherein said biological sample is selected from the group consisting of a whole blood sample, a blood clot, PBMCs and a tissue biopsy.