Method of measuring adaptive immunity
View Patent ↗A method of measuring immunocompetence is described. This method provides a means for assessing the effects of diseases or conditions that compromise the immune system and of therapies aimed to reconstitute it. This method is based on quantifying T-cell diversity by calculating the number of diverse T-cell receptor (TCR) beta chain variable regions from blood cells.
1. A composition, comprising:
(a) a plurality of V-segment primers, and
(b) a plurality of J-segment primers,
each of said plurality of V-segment primers and said plurality of J-segment primers consisting of 15 to 50 nucleotides,
wherein each of said V-segment primers comprises a first sequence and a second sequence, wherein said first sequence is complementary to a portion of a first region of at least one V-segment, said first region located immediately 5′ to a second region of said V-segment where untemplated deletions occur during TCR or IG gene rearrangement, wherein said second region of said V-segment is adjacent to and 5′ to a V-recombination signal sequence (V-RSS) of said V-segment, wherein said first sequence is located 3′ to said second sequence on said V-segment primer,
wherein each of said J-segment primers has a first sequence and a second sequence, wherein said first sequence is complementary to a portion of a first region of a J-segment, said first region located immediately 3′ to a second region of said J-segment where untemplated deletions occur during TCR or IG gene rearrangement, wherein said second region of said J-segment is adjacent to and 3′ to a J-recombination signal sequence (J-RSS) of said J-segment, wherein said first sequence is located 3′ to said second sequence on said J-segment primer, and
wherein a single multiplex polymerase chain reaction (PCR) amplification of rearranged nucleic acid molecules from a sample comprising lymphocytes obtained from a mammalian subject, using said V-segment and J-segment primers produces at least 10 4 distinct amplicons representing a diversity of rearranged TCR or IG CDR3 sequences present in said sample.
2. The composition of claim 1 , wherein said first sequence of each V-segment primer comprises a sequence that is complementary to a single Vγ segment or a family of similar Vγ segments, and said first sequence of each J-segment primer comprises a sequence that is complementary to a Jγ segment, and wherein V-segment and J-segment primers permit amplification of a TCRγ CDR3 region.
3. The composition of claim 1 , wherein said first sequence of each V-segment primer comprises a sequence that is complementary to a single Vδ segment or a family of similar Vδ segments, and said first sequence of each J-segment primer comprises a sequence that is complementary to a Jδ segment, and wherein V-segment and J-segment primers permit amplification of a TCRδ CDR3 region.
4. The composition of claim 1 , wherein said first sequence of each V-segment primer comprises a sequence that is complementary to a single Vα segment or a family of similar Vα segments, and said first sequence of each J segment primer comprises a sequence that is complementary to a Jα segment, and wherein V-segment and J-segment primers permit amplification of a TCRα CDR3 region.
5. The composition of claim 1 , wherein said first sequence of each V-segment primer comprises a sequence that is complementary to a single Vβ segment or a family of similar Vβ segments, and said first sequence of each J-segment primer comprises a sequence that is complementary to a Jβ segment, and wherein the plurality of V-segment and J-segment primers are capable of amplifying a TCRβ CDR3 region.
6. The composition of claim 1 , wherein the V-segment primers have similar annealing strength.
7. The composition of claim 1 , wherein all J-segment primers anneal to a same conserved framework region motif.
8. The composition of claim 1 , wherein the amplicon comprises a sequence that starts from a conserved motif, includes a CDR3 junction and extends into the V-segment, and wherein the amplicon is capable of being used to diagnostically identify the J-segment.
9. The composition of claim 1 , further comprising a set of sequencing oligonucleotides, wherein the sequencing oligonucleotides are complementary to a region of the amplicons.
10. The composition of claim 1 , wherein each of the amplicons span a V-D-J junction.
11. The composition of claim 1 , wherein the V-segment primers are each capable of hybridizing to a plurality of V-segments and the J-segment primers are each capable of hybridizing to a plurality of J-segments.
12. The composition of claim 1 , further comprising a universal C segment primer for generating cDNA from mRNA.
13. The composition of claim 5 , wherein the 3′ end of the V-segment primer is anchored at position -43 in the Vβ segment relative to the recombination signal sequence (V-RSS).
14. The composition of claim 5 , wherein the plurality of V-segment primers consist of at least 14 primers specific to 14 different Vβ genes.
15. The composition of claim 5 , wherein said first sequences of said plurality of V-segment primers comprise sequences that are selected from the group consisting of SEQ ID NOS:1-45.
16. The composition of claim 5 , wherein said first sequences of said plurality of V-segment primers comprise sequences that are selected from the group consisting of SEQ ID NOS:58-102.
17. The composition of claim 5 , wherein each V-segment is complementary to at least one V-segment primer.
18. The composition of claim 5 , wherein the primers do not cross an intron/exon boundary.
19. The composition of claim 5 , wherein the plurality of J-segment primers hybridize with a conserved element of the Jβ segment, and have similar annealing strength.
20. The composition of claim 5 , wherein the plurality of J-segment primers consist of at least five primers specific to five different Jβ genes.
21. The composition of claim 5 , wherein the plurality of J-segment primers have sequences that are selected from the group consisting of SEQ ID NOS:46-57 and 483.
22. The composition of claim 5 , wherein the plurality of J-segment primers have sequences that are selected from the group consisting of SEQ ID NOS:103-113, 468 and 484.
23. The composition of claim 5 , wherein there is a J-segment primer for each Jβ segment.
24. The composition of claim 5 , wherein the amplified Jβ gene segments each have a unique four base tag at positions +11 through +14 downstream of the J-RSS site.
25. The composition of claim 24 , further comprising a set of sequencing oligonucleotides that hybridize adjacent to a four base tag within the amplified Jβ gene segments at positions +11 through +14 downstream of the J-RSS site.
26. The composition of claim 24 , wherein the sequencing oligonucleotides are selected from the group consisting of SEG ID NOS:470-482.
27. A composition comprising:
(a) a plurality of V-segment primers, and
(b) a plurality of J-segment primers,
each of said plurality of V-segment primers and said plurality of J-segment primers consisting of 15 to 50 nucleotides,
wherein each of said V-segment primers comprises a first sequence and a second sequence, wherein said first sequence is complementary to a portion of a first region of a first region of said V-segment, said first region located immediately 5′ to a second region of said V-segment where untemplated deletions occur during IGH gene rearrangement, wherein said second region of said V-segment is adjacent to and 5′ to a V-recombination signal sequence (V-RSS) of said V-segment, wherein said first sequence is located 3′ to said second sequence on said V-segment primer,
wherein each of said J-segment primers has a first sequence and a second sequence, wherein said first sequence is complementary to a portion of a first region of a J-segment, said first region located immediately 3 40 to a second region of said J-segment where untemplated deletions occur IGH gene rearrangement, wherein said second region of said J segment is adjacent to and 3′ to a J-recombination signal sequence (J-RSS) of said J-segment, wherein said first sequence is located 3′ to said second sequence on said V-segment primer, and
wherein a single multiplex polymerase chain reaction (PCR) amplification of rearranged nucleic acid molecules of an immunoglobulin heavy chain (IGH) V H region from a sample comprising lymphocytes obtained from a mammalian subject, using said V-segment and J-segment primers produces at least 10 4 distinct amplicons representing a diversity of rearranged IGH sequences present in said sample.
28. A composition comprising:
(a) a plurality of V-segment primers, and
(b) a plurality of J-segment primers,
each of said plurality of V-segment primers and said plurality of J-segment primers consisting of 15 to 50 nucleotides,
wherein each of said V-segment primers has a first sequence and a second sequence, wherein said first sequence is complementary to a portion of a first region of said V-segment, said first region located immediately 5′ to a second region of said V-segment where untemplated deletions occur during IGL gene rearrangement, wherein said second region of said V segment is adjacent to and 5′ to a V-recombination signal sequence (V-RSS), wherein said first sequence is located 3′ to said second sequence on said V-segment primer,
wherein each of said J-segment primers has a first sequence and a second sequence, wherein said first sequence is complementary to a portion of a first region of said J-segment, said first region located immediately 3′ to a second region of said J-segment where untemplated deletions occur during IGL gene rearrangement, wherein said second region of said J segment is adjacent to and 3′ to a J-recombination signal sequence (J-RSS), wherein said first sequence is located 3′ to said second sequence on said J-segment primer, and
wherein a single multiplex polymerase chain reaction (PCR) amplification of rearranged nucleic acid molecules of an immunoglobulin light chain (IGL) V L region from a sample comprising lymphocytes obtained from a mammalian subject, using said V-segment and J-segment primers produces at least 10 4 distinct amplicons representing a diversity of rearranged IGL sequences present in the sample.
29. The composition of claim 1 , wherein the sample comprises genomic DNA obtained from the mammalian subject.
30. The composition of claim 1 , wherein the sample comprises cDNA transcribed from mRNA obtained from the mammalian subject.
31. The composition of claim 1 , wherein the V-segment and J-segment primers are capable of amplifying the TCR or IG CDR3 region in the single multiplex polymerase chain reaction (PCR) to produce at least 10 5 distinct amplicons.
32. The composition of claim 1 , wherein the V-segment and J-segment primers are capable of amplifying the TCR or IG CDR3 region in the single multiplex polymerase chain reaction (PCR) to produce at least 10 6 distinct amplicons.
33. The composition of claim 9 , wherein the sequencing oligonucleotides are selected from the group consisting of SEG ID NOS:470-482.
34. The composition of any one of claim 1 , 27 , or 28 , wherein said second sequence of each of said V-segment primers comprises a sequencing oligonucleotide sequence.
35. The composition of claim 34 , wherein said sequence oligonucleotide is selected from the group consisting of SEQ ID NOS:470-482.
36. The composition of any one of claim 1 , 27 , or 28 , wherein said second sequence of each of said J-segment primers comprises a sequencing oligonucleotide sequence.
37. The composition of claim 36 , wherein said sequencing oligonucleotide sequence is selected from the group consisting of SEQ ID NOS:470-482.
38. The composition of any one of claim 1 , 27 , or 28 , wherein said first region of said V-segment is at least 40 bases from a consensus motif at a 5′ end of the V-RSS.
39. The composition of any one of claims 1 , 27 or 28 , wherein each of said J-segment primers has a 3′ end that is positioned at least 14 base pairs from a consensus motif at a 3′ end of a J-recombination signal sequence (J-RSS).
40. The composition of any one of claim 1 , 27 or 28 , wherein an amplicon produced from said V-segment primers and said J-segment primers is approximately 200 bases in length.
41. The composition of claim 5 , wherein said first sequences of said plurality of V-segment primers consist of SEQ ID NOS: 1-45.
42. The composition of claim 5 , wherein said first sequences of said plurality of V-segment primers consist of SEQ ID NOS: 58-102.
43. A method comprising:
(a) selecting a plurality of V-segment primers of claim 1 ; and
(b) selecting a plurality of J-segment primers of claim 1 ;
(c) combining the V-segment and J-segment primers with a sample of genomic DNA_comprising rearranged nucleic acid molecules of a TCR CDR3 region from lymphocytes obtained from a mammalian subject;
(d) amplifying said rearranged nucleic acid molecules from said sample using said V-segment and J-segment primers, thereby producing at least 10 4 distinct amplicons representing a diversity of TCR genes.
44. The method of claim 43 , wherein said first sequence of each V-segment primer comprises a sequence that is complementary to a single Vβ segment or a family of Vβ segments, and each J-segment primer comprises a sequence that is complementary to a Jβ segment.
45. The method of claim 43 , further comprising sequencing the amplicons.
46. The method of claim 45 , wherein sequencing utilizes a set of sequencing oligonucleotides that hybridize to a defined region within the amplicons.
47. The method of claim 46 , further comprising calculating the total diversity of TCRβ CDR3 sequences among the amplicons.
48. The method of claim 47 , wherein the method shows that the total diversity of a normal human subject is greater than 1*10 6 sequences.
49. The method of claim 47 , wherein the method shows that the total diversity of a normal human subject is greater than 2*10 6 sequences.
50. The method of claim 47 , wherein the method shows that the total diversity of a normal human subject is greater than 3*10 6 sequences.
51. A method of diagnosing immunodeficiency in a human subject, comprising:
(i) measuring the diversity of at least 10 4 rearranged TCR CDR3 sequences of the subject, wherein measuring the diversity of at least 10 4 rearranged TCR CDR3sequences comprises the steps of:
(a) selecting a plurality of V-segment primers and a plurality of J-segment primers of claim 1 ; and
(b) combining the V-segment and J-segment primers with a sample of genomic DNA comprising rearranged nucleic acid molecules of a TCR CDR3 region from lymphocytes obtained from the subject;
(c) amplifying said rearranged nucleic acid molecules from said sample using said V-segment and J-segment primers, thereby producing at least 10 4 distinct amplicons representing a diversity of TCR CDR3 genes in said sample;
(d) sequencing the amplicons;
(e) calculating the total diversity of TCR CDR3 sequences among the amplicons for said subject; and
(ii) comparing the total diversity of TCR CDR3 sequences of said subject to a total diversity of TCR CDR3 sequences obtained from a normal subject.
52. The method of claim 51 , wherein comparing the diversity is determined by calculating using the following equation:
Δ
(
t
)
=
∑
x
E
(
n
x
)
measurement
1
+
2
-
∑
x
E
(
n
x
)
measurement
2
=
S
∫
0
∞
ⅇ
-
λ
(
1
-
ⅇ
-
λ
t
)
ⅆ
G
(
λ
)
wherein G(λ) is the empirical distribution function of the parameters λ l , . . . , λ S , n x is the number of clonotypes sequenced exactly x times, and
E
(
n
x
)
=
S
∫
0
∞
(
ⅇ
-
λ
λ
x
x
!
)
ⅆ
G
(
λ
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.
53. The method of claim 51 , wherein the diversity of at least two samples of genomic DNA are compared.
54. The method of claim 53 , wherein one sample of genomic DNA is from a patient and the other sample is from a normal subject.
55. The method of claim 53 , wherein one sample of genomic DNA is from a patient before a therapeutic treatment and the other sample is from the patient after treatment.
56. The method of claim 53 , wherein the two samples of genomic DNA are from the same patient at different times during treatment.
57. The method of claim 53 , in which a disease is diagnosed based on the comparison of diversity among the samples of genomic DNA.
58. The method of claim 53 , wherein the immunocompetence of a human patient is assessed by the comparison.