Immunorepertoire normality assessment method and its use
The present disclosure generally pertains to a method for comparing the immunorepertoire diversity of a patient to the shared immunorepertoire of a group of individuals to identify whether the immunorepertoire diversity of the patient is normal or abnormal relative to the shared immunorepertoire of the group of individuals.
1. A method for determining the immunorepertoire diversity in a patient, the method comprising:
(a) amplifying polynucleotides from a population of white blood cells from a human or animal subject in a reaction mix comprising target-specific nested primers directed to variable regions of the cells, to produce a set of first amplicons, at least a portion of the target-specific nested primers comprising additional nucleotides which, during amplification, serve as a template for incorporating into the first amplicons a binding site for at least one common primer;
(b) transferring a portion of the first reaction mix containing the first amplicons to a second reaction mix comprising at least one common primer;
(c) amplifying, using the at least one common primer, the first amplicons to produce a set of second amplicons;
(d) sequencing the second amplicons to identify CDR3 sequences in the subpopulation of white blood cells;
(e) using the identified CDR3 sequences to quantify the percentage of pCDR3 represented by the sample to provide a normality index, wherein pCDR3 sequences are shared by and identified from a reference pool of individuals and wherein the pCDR3 includes 100 or more of the group consisting of SEQ ID NO:1 through SEQ ID NO: 4014; and
(f) identifying whether the normality index is normal or abnormal, wherein a normal state is characterized by the normality index meeting or exceeding a minimum percentage of pCDR3 and an abnormal state is characterized by the normality index failing to meet or exceed a minimum percentage of pCDR3.
2. The method of claim 1 , wherein the population of cells is selected from the group consisting of all T cells [panT], CD8+ T cells [cytotoxic T (Ta)], CD4+ T cells and their subsets [TH1, TH2, TH17, regulatory T (Treg), follicular T (TFH)], developmental subsets of T cells, such as naive T (Tn), activated T (Ta), and memory T (Tm).
3. The method of claim 1 , wherein the population of white blood cells is about 100,000 randomly-selected cells.
4. The method of claim 1 , wherein the method is repeated about 10 to 100 times, each time with a random selection of cells, to determine the average percentage of pCDR3 expressed by the patient.
5. The method of claim 1 , wherein the minimum percentage of pCDR3 is a percentage number from about 25 to about 75.
6. The method of claim 1 , wherein the minimum percentage of pCDR3 is about 25 percent.
7. The method of claim 1 , wherein the pCDR3 is composed of at least the 1000 most-frequently-shared CDR3 from the reference pool of individuals.
8. The method of claim 1 , wherein the pCDR3 includes SEQ ID NO:1 through SEQ ID NO: 4014.
9. The method of claim 1 , wherein the pCDR3 is composed of CDR3 shared by at least 50 percent of the pool of individuals.
10. The method of claim 9 , wherein the pool contains at least 100 individuals.
11. The method of claim 9 , wherein the pool contains about 1000 individuals.
12. The method of claim 9 , wherein the pool is composed of healthy control individuals.
13. The method of claim 9 , wherein the pool is composed of individuals with one or more specific disease states.
14. The method of claim 9 , wherein the pool of individuals is approximately age-matched to the patient.
15. The method of claim 9 , wherein the pool of individuals is gender-matched to the patient.