Note: Descriptions are shown in the official language in which they were submitted.
WO~1/19816 PCT/US91/04317
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IDENTIFICATION OF CELL SUBPOPULATIONS USING
MODIFIED PCR TO AMPLIFY EXPRESSION INTER~EDIATES
Technical Field
The invention is directed to applying a
modified form of a polymerase chain reacti~n (PC~) to the
expression intermediates in cells where the nature of a
cell subpopulation is characterized by these
intermediates. More specifically, the invention concerns
identifying subpopulations of B or T-lymphocytes by
characterizing the DN~ and mRNA int~rmediates for their
def ining immunoglobulin or T-cell receptor proteins.
This characterization is made possibie by amplification
of these expression intermediates using a modi~ied form
of PCR in which one primer is degenerate and the other is
a perfect match.
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Backaround Art
B-cell and T-cell subpopulations are character-
ized by the nature of the immunoglobulin ~Ig) or T-cell
receptor (TCR) proteins respectively produced by the
~ individual cell. These characteristic proteins are as-
; sembled from germ line DNA sequences present in
unrearranged form in the genomic DNA of the germ line.
Onc~ the segments encoding the complete sequences of
these proteins are assembled during cellular
differentiation, the lineage of the subpopulation is
determined. The rearranged, assembled genes will be
effectively present as an mRNA in the clones of the cell
line.
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W091/19816 PCT~US91/04317
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In the case of T-~ells, for example, the
characteristic proteins are the TCR heterodimers. The
typical TCR heterodim~r is a combination of an ~- and
~-subunit, although a small percentage of T-cells
contain, instead or in addition, a similar pair
designated r ~ . Both types of TCR have substantially
similar features: starting from the N-terminus, the
proteins are composed of a variable (V) sequence
(preceded by a leader) which varies from one T-cell
population to another, fQllowed by a joining (J) segment
in the case of ~- and r-subunits or by both a diversity
(D) and J segment in the case of ~ and ~ proteins, and
then a constant region which is characteristic of each o~
the four TC~ chains and largely invariant.
Characterization of the V, D an~ J regions of these
proteins would establish the identity of t~e particular
subpopulation of interest. The heavy and light chains of
Igs have similar structures. In various vertebrate
species, multitudinous (i.e., 101-1ol3) such
subpopulations have been postulated.
It is of particular interest to characteri2P
individual T-cell populations in a number of contexts.
For example, T-cells which are present in autoimmune
infiltrates are thought to be those responsible for the
undesirable immune response. Thus, for example, in type
I diabetes, which is caused by an autoimmune response to
the pan~reatic islets, infiltrates of ~hese islets should
contain T-cells which are the specific su~type
responsible for the disease. Identification of unique
TCR expressed on these T-cells would permi~ design of
therapeutic measures directed against them specifically.
Similar remarks can be made concerning T-cells which are
malignant lymphomas or associated with organ graft
rejection. By characterization of these subpopulations,
specific immunotherapies can be devised.
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WO 9ttl9816 PCT/US91/04317
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- It is currently thought that foreign antigens
are cleaved enzymatically and the fragments presented to
T-cells in the context of a major histocompatibility
complsx (MHC) encoded glycoprotein. T-cell receptors
have regions which bind to both the antigen fra~ments and
MHC products. Amon~ TCRs, the greatest concentration of
structural variability resides in th~ J or D-J junctional
reyions which seem to make the primary contacts with the
specific an~igenic pep~ide fragment; the variable (V)
regions, which pro~ably make con~act with the MHC
glycoprotein presenting the antigen fragment, show more
limited variability. The genes for many of the variable
regions present in T-cell populations for human and
murine ~- and ~-su~units have been sequenced. Thus, data
are available which permi~ the ascertainment of the pres-
ence of consensus sequences in the Yariable regions.
~See, for example, the determinations referenced in
Davis, M.M., et al., Nature (1988) 334:3950402.)
Theoretically, recovered T-cells ~or B-cells)
. 20 could be characterized by sequencing cDNA molecules
encoding their characteristic TCR ~or Ig). However, the
production of cDNA libraries necessary for applying this
approach to a particular candidate TCR chain, while pos-
sible, is intensely laboriou~. The present invention of-
fers a way to obtain copious quantities o~ the relevant
gene which can then readily be cloned and sequenced.
The invention method utilizes the recently
developed polymerase chain reaction ~PCR) which involves
a highly selective and highly effective amplification of
;~ 30 a desired DNA sequence at the expense of unwanted
sequences. The PCR technique as oriqinally described
relies on the use of completely matched (sensa and
antisense) primers at the 5' and 3' ends of the sequence
to be amplif ied . Polymerization in the pregence of these
primers results in a million or billionfold amplification
WO 91/19~16 PCI/US91/04317
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of the desired sequence in a relatively small number of
polymerase rounds (Saiki, ~.K., et al., Science (1985)
230:1350~1354).
The PCR technique has been used for genomic and
for cDNA cloning (Scharf, S., et al.l Science (1986)
233:1076-1078; Saiki, R.K., e~ al., Science (198~)
239:487-491). It is also known that the primers need not
exactly match the gene sequence perfPctly (Lee, c.c., et
al., Science (1988) 239:1288-1291). Others have also
utilized restriction sites built into the primers to ~s-
sist in the subsequent cloning of the amplif ied sequence.
The use of degenerate primers to amplify genes
where the nucleotide sequence is predicted ~rom a known
amino acid s~quence has been employed for the cloning of
some genes. Generally, knowledge of an amino acid
: seque~ce predicts degenerate coding sequences (see, for
example, Gonzalez, G.A., et ~1., Nature ~1989)
337:749-752; Lee, C.C., et al., Scienc_ ~1988)
239:1288-1291). Others have applied the PCR reaction to
the variable regions of immunoglobulins by using a
32-fold degenerate primer in conjunction with a single
primer wheréin neither first or second primers was a
complete match for the inherently variable regions of the
protein segment sequenced. The variable region amplified
contained all three of th~ Ig complementarity determining
regions (CDR)~ CDR1, CDR2, and CDR3. (For the TCR, by
analogy to immunoglobulins where the V-region-encoded
CDRl and CDR2 regions are less variable, and the CDR3
varies the most, it is believed that the CDR1 and CDR2
regions of TCR associate with the MHC and the CDR3 region
. associates with the antigenic peptide (see abova).) The
PCR technique using the combination of a defined but not
necessarily perfectly matched constant primer in
conjunction with a degenerate primer was also applied to
ganes encoding the variable heavy chain from spleen DNA
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W091/19816 PCT/US91/04317
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of a mouse immun~zed with lysozyme (Ward, E.S., et al.,
Nature (1989) 341:544-546).
For reasons similar to those with respect to
the immunoglobulin variable region, application of the
classical PCR techniques to the gene encoding the T-
cell-characterizing proteins is not directly possible,
since, by definition, the variable region presents an
unXnown sequence to which a completely matched primer
cannot bs designed. The present invention overcomes this
handicap by providing for the design of a consensus
primer which is applicable to a m~ltitude of variable
regions and by modification of the PCR protocol to
accsmmodate the presumptive lac~ of total match between
:~ this primer and substrate.
Disclosure of the Invention
The invention provides a means for selective
:: ampli~ication of the characterizing genetic material in
subpopulations of B-cells and T-cells in any arbitrary
candidate sample. Tha method relies on modification of
the polymerase chain reaction (PCR) using a degenerate
: primer for consensus portion of the variable region of
genes encoding B-cell- or T-cell-characterizing protein.
Accordingly, in one aspect, the invention is
directed to a method to conduct a polymerase chain re-
action to amplify the codi~g region for a protein having
` a variable region and a constant region, such as a T-
cell receptor subunit or an immunoglobulin light or heavy
`~ 30 chain, which method comprises subjecting a cDNA encoding
said subunit to a PCR using as a 3~ (antisense) primer a
~ DNA sequence which is a precise match to the gene
: encoding the constant region of the subunit and as a
second 5' primer a degenerate "match" to the gene
encoding a consensus region in the variable region of
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WO91/19816 PCTtUS91/04317
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said subunit,-or; vice ver~a, a 3' (antisense) primer
which is a degenerate match to the gene encoding a
portion of the variable region and a 5' ~sense) primer
which complements the constant region. The senses re
reversed if the complement to the coding sequence is
used~ In other aspects, the invention is direc~ed to
methods to identify B or T-cell subpopulations in a
sample by applying the modified PCR techniques of the
invention to cDNA prepared from B or T-cell mRNA in said
sample, cloning and sequencing the amplif ied DNA.
Brief Descri~tion of the Drawinas
Figure 1 is a diagram of the assembled genes
encoding typical ~ and ~ chains of the T-cell receptor.
Figure 2 is a diagram of the assembled gPnes
encoding the heavy and light chains of immunoglobulins.
Figure 3 shows the sequences of typical
variable, joinin~, and constant regions of genes encoding
several variants of TCR chains.
Modes of Carrvinq Out the Invention
The invention methods permit the effective
amplification of a DNA sequence which encodes a protein
that has a predictable, constant region along with an un-
known variable region wherein the variable regioncontains a framework sequence sufficient to permit the
design of a degenerate consensus primer which can serve
as the ~atching primer for multiple variable regions.
There are three general steps in the invention
process: isolation or preparation of the B or T-
lymphocytes to be characterized, extraction of mRNA and
preparation of first-strand cDNA, and PCR conducted on
this first-strand cDNA. This cDNA will be the antisense
sequence; there is no reason its complement could not be
prepared and used as the PCR substrate instead.
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WO91/198~6 PC~/U~91/04317
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T and B-cells of interest are obtained from in
vlvo sources such as peripheral blood, lymph nodes,
spleen, and organ infil~rates caused by autoimmune re-
activity, tumor reactivity, or response to tissue
engraftment. Isolation of RNA from the cells of interest
can be performed directly, or subsequent to functional
and/or phenotypic characterization of subpop~lations,
For example, the pancreatic islets of mi~e which are
genetically susceptible to autoimmune diabetes (the NOD
lo strain) can be prepared from surgically removed and dis-
sociated pancreata, and RNA extracted by methods
generally known in the art. Since only T-cells can serve
as sources of TCR RNA, removal of associated cells (such
as islet cells) derived from other lineages is not
necessary for this analysis. Furthermore, the T-cells,
for example, in the population can be increased by
stimulation with cytokines, such as IL-2, or other T-
cell-specific mitogens, such as phorbol myristate acetate
(PMA).
mRNA is extracted and first-strand cDNA
synthesis is conducted using standard procedures.
First-strand cDNA synthesis can also be performed on only
minute quantities of extracted RNA by use o~ the PCR
technigue. For example, priming of the reverse
transcription reaction can be accomplished with oligo
d(T) or with antisense primers specific for a known
upstream seguence. Also, for PCR reactions conducted
with limiting amounts of RNA tsuch as that extraeted from
106 or fewer cells), the number of cycles can be
increased to at least 60 without the requirement for
further purification o~ PCR products and secondary
addition of Taa polymerase (Rapopolee, D.A., et al., J
Cell ~iochem (1989) 39:1-11).
In the classical polymerase chain reaction, a
DNA sequence to be amplified is provided with a sense and
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WO91/19816 PCT/US91/04317
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antisense primer, one of which is designed as a match for
the 5~ end of the sequence to be copied, and the other
for the 3 ' end. As used herein, ~match~ refers either to
the same sequQnce as found i~ the substrate DNA or its
complement, as the case may be. By ~match~ is also meant
both a pr~cise match, in which case ~his will be
specified, or a match which is sufficient to permit PCR
to proceed, even if there is no primer in the degenerate
mixture which in fact exactly matches the substrate DNA.
Typical degenerate primers have at least 17 b~ses in the
matching portion--longer oligomers can be used. The
illustrated variable TCR ~ primer has a length of 39
bases, part of which is additional sequance to provide
restriction sites.
lS The method of the invention is most useful to
characterize cells which are themselves characterized by
the production of a particular protein. ~his situation
is found in, specifically, T and B lymphocytes wherein
the properties are determined by the TCR or Ig subunits
produced. Because the genes encoding these protein
subunits are expressed, a convenient source of the as-
sembled coding sequence is the mRNA which can readily be
isolated from the cells. The mRNA is reverse-transcribed
to obtain a single-strand cDNA, which provides the
initial template ~or the PCR amplification. The
resulting cDNA will be a complement to the sense coding
sequence; hence, the primer matching the sequence on the
N-terminal portion should be the sense primer, and that
matching the sequence close to the C-terminus should be
the antisense primer.
In the case of the TCR subunits, the variable
region containing the consensus sequence is near the
N-terminus, as shown in Figure l. The constant region
comprises the C-terminus. DNA sequences which encode the
constant region and the variable regions of the TCR in
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W091/19816 PCT/U591/04317
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human, murine, and ra~ TCR subunits are compiled in the
GenBank, ~MBL, VecBase, and NBRF data bases. Reference
may be made to these compilations to ascertain the ap-
propriate regions for primer ~onstruction.
As shown in Figure 2, the organization of
immunoglobulin subunit heavy and light chains is similar.
DNA sequences encoding variable, diversity, joining and
constant regions of various immunoglobulins are also
found in the GenBank, EMBL, VecBase and NBRF data bases.
In the method of the invention, one of the
primers in the modif ied PCR is preferably a precise match
to the part of the gene encoding the constant sequence in
the protein. However, because the conditions are
adjusted to account for lack of an exact match in the
lS variable region, it is not required that this be the
case. Mismatches comparable to those to which ~he
; hybridization conditions are adjusted can be employed as
well. In the case of TCR or Ig, the sense primer will be
constructed to the downstream constant region, or a por-
tion thereof when first-strand cDNA is employed as a
template. Use of the co~plement to the first-strand cDNA
would require the converse. The degenerate primer, which
is a "match" for a consensus region, will ~e constructed
on the basis of genes encoding the variable regions in
the case of TCR and Ig. Examination of the published
gene sequences for the variable region will permit the
ascertainment of the appropriate consensus regions and
the design of appropriate sequences.
In the case o~ the T-cell receptor protein ~
and ~ subunits, a consensus region is found between amino
acids 30 and 40, just downstream of what is presumed to
be the "CDRl"-encoding region of the gene. A consensus
region is found in a similar position in the TCR
~ubunit.
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WO91/198~6 P~T/US91~04317
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The polymerase ~hain reaction is conducted
using these primers with a modification to the protocol
to account for the mismatch of the degenerate primer. In
general, this involves lowerrng the temperature in the
initial cycles so as to provide lower hybridization
stringency. This temperature is raised in the later
repeated cycles as the subsequent strands synthesized
will incorp~rate the conse~sus primer and perfectly match
the subs~rate.
Application of the modified PCR of the
invention to the identified identification o~ T-cells is
especially useful. Amplification and sequencing of the
TCR ~xpressed in T-cells inYolved in autoimmune
responses, graft rejection, or lymphomas will make
possible the design of peptides which can be used as
vaccines or monoclonal antibodies directed against the
TCR for use in the treatment of these conditions. See,
for example, Vandenbark, A.A~, et al., Nature (1989)
24l:541-554; Acha-Orbea, H., et al., Cell (1988)
54:263-273.
The following Examples are intended to il-
lustrate, but not to limit, the invention.
ExamDle l
PreParation of T-Cell Samples
To obtain T-cell clones ~or analysis, DBA/Z
mice were immunized at the base of the tail with 100-200
ug sperm whale myoglobin in 50% complete Freund's
adjuvant, and the draining lymph nodes were removed 8
days later. The lymph node cells were cultured with
irradiated syngeneic spleen cells as antigen-presenting
cells ~APC) and the sperm whale myoglobin for-12 days.
The cells were restimulated 3 times with APC and the
56-131 cyanogen bromide fragment of the sperm whale
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WO9l/1981~ PCT/US~ 4317
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myoglobin at 12-13 day intervals, and the bulk cultures
were cloned and subcloned by limiting dilutions.
ExamE~e 2
PreParation of cDNA
Res~ing T-cells prepared as in Example 1 (m~re
than 10 days after last antigen stimulation) were
Ficoll purified and resuspended at 1.5-1 x 106/ml in
medla containing 10 ng/ml PMA, 250 ng/ml ionophore and
10-25 U/ml human recombinant IL-~. After 24 hours, the
cultures were diluted 1:2-1:4 into media containing rIL-2
alone. Optimal RNA levels were shown after 3-5 days.
Total RNA was prepared by homogenization of 5-lo x lo6
cells in guanidine thiocyanate and centrifugation through
cesium chloride, according to the method of Chirgwin,
J.M., et al., Biochemistry (1979) 18:5294_5299.
A first-strand cDNA synthesis was performed on
10 mg of total RNA using oligo-dT priming with reverse
transcriptase, according to the method of Gubler, U., et
al., Genq (1983) 25:263-269. Ten to fifty percent of the
transcription reaction was used as a template for PCR
amplification.
~xam~l~ 3
Conduct of ~CR
A. The oligonucleotide primers for the
constant and consensus regions were determined from
published sequences by Chien, Y., et al., Nature (1984)
312:31-35; Saito, H., et al., ibid, 36-42; Arden, B., et
al., Nature (198S) 316:783-787; Becker, D.M., et al.,
Nature (1985) 317:430-434; McElliot, D.L., et al., J
Immunol (1988) 140:4123-4131; Yague, J., et al., Nucleic
Acids Res (1988) 11355-11i63. The constant region primer
for the ~ chain had the sequence
5'-TCAACTGGACCACAGCCTCAG-3'. The 216-fold degenerate
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WO91/19816 PCT/US91/04317
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oligom~ric primer for the consensus variable region
purchased from Operon Technologies, San Pablo,
Californi~, had the sequence 5~-TAAGCGGCCGCTGGTACZLMCAGC
ATCCXGGMGAAGGCC-3'. In this primer,
Z represents 40%A/40%~/l5%C/5%T;
; L represents AjG/T;
M represents 45%A/50%G/5%C; and
X represents C/T.
The degenerate primer was llsed at 1 uM final
concentration in a 100 ul PCR reaction with 1 uM constant
reg on 3' primer.
Th temperature of annealing segmen~s was 2
cycles at 37C, 1 cycle at 42C, and 27 cycles at 55C.
The amplified product was purified and cloned
by digesting the PCR-amplified material with NotI,
providing a cloning site at the 5' consensus oligo
available for ligation into a polylinXer derivati~e of
M13 bearing an NotI site. The general purification
process and ligation into M13 vectors, with the above
exception for NotI restriction site, was described by
; Acha-Orbea, H., et al., Cell ~1988) 54:267-273.
The results of the amplified sequences of six
recovered TCR ~ chains were sequenced using standard
techniques. The results o~ this sequencing are shown in
Figure 3. The results showed that clone 8.2 is a member
o~ the V-~-4 family and clones 10.3, 12.2, 14.12, and
14.16 are members of the V-~-1 family. Clone 9.4 ap-
pears to define a new-family pro~isionally designated
V-~-15. The clone designations in Figure 3 are on the
left.
A similar protocol and the same consensus
primer is also useful to amplify rat and human TCR
chains and murine TCR B chains.
B. In a manner analogous to that set forth in
paragraph A of this example, PCR amplification of TCR
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WO91/19816 PCT/US91/04317
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ch~ins from in vlvo and in vitro cell sources is
conducted. The consensus primer is designed to a
con~erved region in TC~ V8 sequence similarly located to
that of the ~ sequence. The ~onsensus primer is us~d in
combination with a variety of TCR CB or JB
oligonucleotides to permit amplification of ~CRB chains
which utilize members of the murine VB f~milies
2,5,6,8,lO,12 and 15. The sequence is 5' TAA GCG GCC GCA
; TGS LYT GGT AYW XXC AG 3' where S=G/T, L=A/G/T, Y=C/T,
:~: 10 W=A/C AND X=A/G and contains a NotI restriction site near
the 5' end. The PCR cycling conditions used with this
primer are identical to those used with the TCR V~
consensus primer. This primer can also be used for
amplification of rat TCR ~ chains.
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