Method of producing antibodies to a restricted population of T lymphocytes, antibodies produced therefrom and methods of use thereof

This invention relates to a method of obtaining antibodies specific to a population of T-lymphocytes (T cells) restricted by V.beta. gene usage. The invention also relates to the antibodies obtained and to methods of using them. According to the invention, T cells are incubated with an effective amount of a superantigen under conditions and for a time sufficient to allow division and growth of T-cells reactive to the superantigen. The incubated T-cells are injected into a mammal, and thereafter serum containing antibodies specific to the incubated and injected T-cells is removed from the mammal.

BACKGROUND OF THE INVENTION 
This invention relates to a method of obtaining antibodies specific to a 
population of T lymphocytes (T cells, TCL) restricted by V.beta. gene 
usage, antibodies thus obtained and methods of use thereof. 
More than 95% of all T cells have a cell surface receptor designated the 
"T-cell receptor" (hereinafter, "TCR"). For a review of T cell ontogeny, 
see Moyer and Reinherz, "T Lymphocytes: Ontogeny, Function, and Relevance 
to Clinical Disorders", N. Engl. J. Med., 317:1136-1142 (1987). TCR 
consists of a clonotypic Ti .alpha.-.beta. heterodimer with an apparent 
molecular weight (Mw) of 90 kD and a monomorphic T3 molecule containing 
five subunits (.gamma., .delta., .zeta., .epsilon., and .eta.). The 
.gamma. subunit has an apparent Mw of 25 kD, the .delta. and .epsilon. 
subunits each have an apparent Mw of 20 kD, the .zeta. subunit has an 
apparent Mw of 16 kD, and the .eta. subunit an apparent Mw of 22 kD. All 
five receptor subunits are transmembrane proteins. As is the case with 
antigen-binding heavy and light chains of immunoglobulin proteins, TCR 
.alpha. and .beta. proteins contain both variable (hereinafter "V") and 
constant (hereinafter "C") regions. For a review of immunoglobulin 
proteins and gene usage, see Lewin, Genes III, John Wiley & Sons, Inc. pp. 
642-653 (1987), which is incorporated herein by reference. 
It appears that the Ti subunits form a binding site for antigen and the 
major histocompatibility complex (hereinafter "MHC") through interaction 
of their V domains. Antigen recognition is important for activation of 
both cytotoxic effector T cells and immunoregulatory T cells. Cytotoxic T 
cells lyse specific target cells, including tumor cells and virus-infected 
cells, whereas immunoregulatory T cells induce or suppress the cells of 
the immune system either directly or indirectly through lymphokines. 
Analyses of many of the cDNA nucleotide sequences encoding a variety of 
.beta. chains have led to the recognition of structural similarity between 
genes encoding .beta. chains and genes encoding immunoglobulins. Thus, 
genes encoding .beta. chains contain V, constant (C), joining (J) and 
diversity (D)-like elements The present invention is concerned with the V 
region of the chain, hereinafter termed V.beta.. In humans, approximately 
57 V.beta. genes are known to exist in the Ti .beta. locus on chromosome 7 
at 7q35 Robinson, "The Human T Cell Receptor .beta.-chain Gene Complex 
Contains at Least 57 Variable Gene Segments", J Immunol , 146:4392-4397 
(1991) 
T cell proliferation requires the interaction of the Ti complex with 
antigen and interleukin-2 (hereinafter "IL-2"). Although resting T cells 
express no receptors for IL-2; after the T cell receptors are activated by 
antigen and MHC, induction of IL-2 receptors occurs within hours The 
activation also leads to endogenous induction and secretion of IL-2, DNA 
synthesis and cell mitosis. 
Various disease states and physiological disorders are associated with T 
cell dysfunctions. These disorders, are characterized by a particular 
subset, or restricted population, of T cells which are thought to be 
responsible for the dysfunction The restricted population is recognized by 
its expression of only one or a few related types of TCR and can be 
monitored by the type of V.alpha. and/or V.beta. gene expressed. A 
restricted set of T cells is one in which the T cells express one or a few 
common V genes but are otherwise dissimilar By comparison, a clonal 
population of T cells, such as may be derived from a tumor, is a 
population of T cells that are the progeny of a single cell and are hence 
virtually identical. 
Disorders thought to be caused by T cell dysfunctions include but are not 
limited to various autoimmune diseases such as systemic lupus 
erythematosus multiple sclerosis, myasthenia gravis, diabetes mellitus, 
and various forms of arthritis such as rheumatoid arthritis. These 
dysfunctions are characterized by the expansion of a restricted T cell 
population that expresses one or a few V.beta. genes from a V.beta. 
family. Different patients express a single V.beta. but not necessarily 
the same V.beta. gene as another patient. For instance, expanded T cell 
populations from MS patients express one of the V genes from the related 
group of V.beta.12, V.beta.13, V.beta.14, V.beta.15 and V.beta.17. Several 
methods of therapy have been proposed based on eliminating or blocking the 
T cell population responsible for a dysfunction. For review see Janeway, 
"Immunotherapy by Peptides" Nature 341:482-483 (1989); and Hashim et al., 
"Antibodies for V.beta.8 Receptor Peptide Suppress Experimental Autoimmune 
Encephalomyelitis", J. Immunol., 144:4621-4627 (1990), which are 
incorporated herein by reference. 
There is considerable evidence of selective TCR V.beta. gene usage among 
rodent T cells which mediate a number of experimental autoimmune diseases. 
For example, in experimental allergic encephalomyelitis (EAE), 
V.beta.8.2.sup.+ T cells play a central role. In five different strains of 
rats, encephalitogenic T cell clones and hybridomas, reactive against 
myelin basic protein (MBP) peptide fragments, were found to be uniformly 
V.beta.8.2.sup.+. Burns et al., "Both Rat and Mouse TcRs Specific for the 
Encephalitogenic Determinant of MBP use Similar V.alpha. and V.beta. Chain 
Genes Even Though the MHC and Encephalitogenic Determinants Being 
Recognized are Different", J. Exp. Med., 169:27 (1989). Similarly, 
V.beta.8.2 is expressed on over 85% of T cells reacting to the 
encephalitogenic MBP peptide in strains of mice susceptible to EAE. 
Acha-Orbea et al., "Limited Heterogeneity of T Cell Receptors From 
Lymphocytes Mediating Autoimmune Encephalomyelitis Allows Specific Immune 
Intervention" Cell 54:563 (1988); and Urban et al , "Restricted Use of T 
Cell Receptor Genes in Murine Autoimmune Encephalomyelitis Raises 
Possibilities for Antibody Therapy", Cell, 54:577 (1988). The mouse 
V.beta.8 gene family products are homologous to the human V.beta.12, 
V.beta.13, V.beta.14, V.beta.15 and V.beta.17 gene products. 
The in vivo administration of mAb specific for V.beta.8.2 has been shown to 
both protect mice from the development of EAE induced by a subsequent 
challenge with MBP, and to ameliorate the clinical course of EAE in mice 
already affected. Acha-Orbea et al. (1988). It has also been found that 
EAE can be vaccinated against. In this case, the anti-T cell response is 
mediated by another set of T cells Lohse et al,. "Control of Experimental 
Autoimmune Encephalomyelitis by T Cells Responding to Activated T Cells", 
Science, 244:820-824 (1989). 
In another animal disease model, collagen-induced arthritis, T cells 
reactive against type II collagen which are capable of transferring 
arthritis to naive syngeneic mice are virtually all V.beta.8.2.sup.+ 
Banerjee et al., "Possible Role of V.beta. T Cell Receptor Genes in 
Susceptibility to Collagen-Induced Arthritis in Mice", J Exp Med , 167:832 
(1988). These observations suggest that expression of the V.beta.8.2 gene 
product may be associated with an autoimmune T cell pool in rodents, for 
instance, T cells derived from the CD4.sup.- CD8.sup.- V.beta.8.2 
expressing thymocyte subpopulations. Fowlkes et al., "A Novel Population 
of T Cell Receptor .alpha..beta. Bearing Thymocytes Which Predominantly 
Express a Single V.beta. Gene Family", Nature, 329:251 (1987); Shortman et 
al., "Mouse Strain Differences in Subset Distribution and T Cell Antigen 
Receptor Expression Among CD4.sup.- CD8.sup.- Thymocytes", Immunol. Cell 
Biol., 66:423 (1988); and Takahama et al., "Phenotype, Ontogeny, and 
Repertoire of CD4.sup.- CD8.sup.- T Cell Receptor .alpha..beta..sup.+ 
Thymocytes: Variable Influence of Self-Antigens on T Cell Receptor V.beta. 
Usage", J. Immunol., 146:1134 (1991). 
Recently, it has been possible to determine the TCR gene usage of a 
population of T cells. Bertness et al., "T cell Receptor Gene 
Rearrangements as Clinical Markers of Human T cell Lymphomas", N. Engl. J. 
Med., 313:534-538 (1985). Such determinations have relied on 
anticlonotypic antibodies directed at epitopes found on V domains of TCR 
and cDNA probes that detect clone-specific DNA rearrangements. However, 
the availability of anticlonotypic antibodies and cDNA probes has been 
limited to the availability of naturally occurring clonal populations of T 
cells such as from tumors. This drawback renders these methods less 
clinically applicable than would be the case if a wide variety of 
antibodies were available to the full range of TCR V gene products 
associated with T cell dysfunctions. This is particularly important in T 
cell dysfunctions which are characterized by restricted rather than clonal 
populations of T cells. It would be useful to have a method of obtaining 
antibodies specific for the protein products of the TCR V gene families 
for the purposes of diagnoses and therapeutics of various disorders 
related to T cell dysfunctions. 
SUMMARY OF THE INVENTION 
It has now been found that antibodies specific for a restricted set of T 
cells having common TCR V.beta. gene usage can be obtained by incubating T 
cells with an effective amount of a superantigen (SA) under conditions and 
for a time sufficient to allow division and growth of T cells reactive to 
the SA, injecting the incubated T cells into a mammal and obtaining the 
anti-V.beta. antibodies from the mammal. 
Additional antibodies can be made to T cells expressing other TCR V.beta. 
genes in the restricted set, by selectively removing, or depleting, T 
cells recognized by an antibody produced by the method of the present 
invention. The remaining T cells are injected into a mammal to produce 
antibodies. The depletion step may be followed by another cycle of SA 
expansion. Depletion, expansion and injection can be repeated a number of 
times with a single sample of T cells to provide a panel of antibodies 
specific for a variety of V.beta. gene products each of which recognizes a 
single restricted set of T cells. Such antibodies and methods for their 
use are provided.

DETAILED DESCRIPTION OF THE INVENTION 
SAs are a group of proteins that activate a large proportion of the T cell 
repertoire based on dual avidity for MHC class II antigens and TCR 
epitopes common to products of one or several TCR .beta. chain V gene 
families. Several SA induce massive T cell proliferation and cytokine 
secretion and have been implicated in clinical syndromes, frequently those 
characterized by shock and generalized immunosuppression. SA activation of 
a more restricted T cell response may also have an effect on the immune 
system related to autoimmune disorders. Friedman et al., "A Potential Role 
for Microbial Superantigens in the Pathogenesis of Systemic Autoimmune 
Disease", Arth & Rheum , 34:468-480 (1991). 
SA identified to date include microbial and viral proteins. The microbial 
SA include several staphyloccal enterotoxins, a fragment of the group a 
streptococcus M protein, and MAM, a soluble mitogen produced by Mycoplasma 
arthritidis. Unite et al., "The V specific Superantigen Staphylococcal 
Enterotoxin B: Stimulation of Mature T cells and Clonal Deletion in 
Neonatal Mice", Cell 56:27-35 (1989); Tomai et al., "Superantigenicity of 
Streptococcal M Protein", J. Exp. Med., 172:359-362 (1990); and Atkin et 
al., "Stimulation of Mouse Lymphocytes by a Mitogen Derived from 
Mycoplasma arthritidisV. A Small Basic Protein from Culture Supernatants 
is a Potent T cell Mitogen", J. Immunol., 137:1581-1589 (1986) which are 
incorporated herein by reference. Note that M. arthritidis is a causative 
agent of inflammatory arthritis in rodents. Cole and Ward, "Mycoplasma as 
Arthritogenic Agents", The Mycoplasmas, Vol. IV, New York, Academic Press 
(1979). The microbial toxins that function as SA are among the most potent 
mitogens known. MAM, for example, induces half-maximal T cell 
proliferation at concentrations of less than 1.times.10.sup.-11 M. Atkin 
et al. (1986). Virally encoded SA are typified by those encoded by the 
mouse mammary tumor virus. Choi et al., "A Superantigen Encoding in the 
Open Reading frame of the 3' Long Terminal Repeat of Mouse Mammary Tumour 
Virus", Nature, 350:203-207 (1991). 
In the mouse, MAM has been shown to behave as a classic microbial SA, 
selectively inducing the proliferation of V.beta.8.sup.+ and 
V.beta.6.sup.+ murine T Cells. Cole et al., "Stimulation of Mouse 
Lymphocytes by a Mitogen Derived From M. arthritides. VII. Responsiveness 
is Associated With Expression of a Product(s) of the V.beta.8 Gene Family 
Present on the T cell Receptor .alpha./.beta. for Antigen", J. Immunol., 
142:4131 (1989). While MAM is mitogenic for human T cells, the level of 
proliferation induced is quite modest compared to that triggered by the S. 
aureus-derived SA, and no data exist regarding TCR V gene dependence of 
MAM recognition. It has now been found that MAM-reactive human T cells 
utilize a restricted group of TCR V.beta. gene products. Monoclonal 
antibody (mAb) C1, the mAb described herein which was generated by the 
immunization of mice with a non-clonal MAM-reactive human T cell line, 
recognizes a disulfide linked heterodimer, consistent with the 
.alpha./.beta. TCR, on approximately 3-6% of peripheral T cells, and 
40-60% of MAM reactive T cells. 
As with other mAb specific for TCR V gene products, C1 reacts with a small 
fraction of both the CD4.sup.+ and CD8.sup.+ subsets of all donors tested, 
including cord blood T cells. With respect to SA recognition, it was 
determined that MAM-reactive TCL are greatly enriched in C1.sup.+ cells, 
while TCL responsive to SA with which MAM-reactive TCL are less 
crossreactive, SEE and TSST-1, are depleted of C1.sup.+ T cells. Studies 
employing the polymerase chain reaction to amplify TCR .beta. chain cDNA 
from C1.sup.+ TCL cells demonstrate that C1 identifies an epitope 
expressed on the V.beta.17 gene product. Taken together, these results 
show that MAM recognition by human T cells is restricted by TCR V.beta. 
gene usage and that a major fraction of MAM-reactive human T cells are 
V.beta.17.sup.+. 
The present invention thus includes antibodies produced by the methods 
described herein. In particular, mAb C1 is encompassed by the present 
invention. mAb C1 has been deposited in the American Type Culture 
Collection (ATCC) and been given accession HB 10874. 
It has now been found that mAb C1 is a unique example of a TCR V gene 
specific mAb generated by immunizing with a restricted T cell population. 
As discussed above, MAM is a relatively weak mitogen for human T cells, 
suggesting a restricted population of MAM-reactive T cells. MAM is thus 
the preferred SA for use in the present invention, however, any other SA 
is embodied by the present invention. Staining data demonstrate that the 
TCR epitope recognized by mAb C1 is expressed not only on &gt;60% of the 
immunizing TCL, which had been retriggered repetitively with MAM, but is 
also present on a relatively large fraction of peripheral T cells 
activated by MAM for only several days in primary short term cultures 
(Table III). The successful application of this method, utilizing TCL 
responsive to other microbial SA as immunogens, or MAM reactive T cells 
depleted of C1.sup.+ T cells greatly expands the available panel of mAbs 
against human TCR V gene products. 
Utilizing mAb C1 to determine what percentage of T cells are recognized by 
C1, it was found that on average, C1.sup.+ T cells represent approximately 
3-5% of the peripheral T cell pool. The marked enrichment of C1.sup.+ T 
cells in even short-term cultures of MAM activated PBL, provide strong, 
albeit indirect, evidence that all C1.sup.+ T cells are MAM reactive. 
Additionally, subsets of MAM-reactive T cells express V.beta. genes other 
than V.beta.17, the population recognized by mAb C1. These other subsets 
were shown to exist by several lines of evidence. First, resting 
peripheral blood T cells, depleted of C1.sup.+ cells, show an undiminished 
proliferative response to MAM over a wide range of SA concentrations. 
Second, only 40-60% of T cells which persist in long term cultures 
maintained by repetitive retriggering with MAM plus autologous APC are 
C1.sup.+. Third, MAM reactive TCL which have been depleted of C1.sup.+ T 
cells exhibit potent, SA specific lysis of MAM bearing target cells. The 
method described herein can thus be utilized to identify other V.beta. 
genes used by MAM-reactive T cells and to generate mAbs against their 
products. Such a method is facilitated by depleting the MAM reactive cells 
of C1.sup.+ T cells, re-expanding the remaining T cells with MAM and using 
the resulting cells to induce antibody production. Subsequently produced 
antibodies can then be used in repeated rounds of depletion, expansion and 
antibody production. Depletion of cells recognized by a specific antibody 
is rapid and efficient. For instance, MAM reactive T cells treated with 
C1, exposed to goat anti-mouse IgG antibodies linked to iron and exposed 
to a magnet, are selectively depleted of all C1.sup.+ T cells such that 
they do not reappear even after repeated rounds of expansion with MAM. 
The observation that human MAM-reactive T cells identified by mAb C1 are 
V.beta.17.sup.+ is of particular interest in light of the restricted 
nature of T cells in diseases characterized by T cell dysfunctions. Amino 
acid sequence analysis has demonstrated considerable homology between the 
products of murine V.beta.8 genes which are expressed by MAM-reactive 
murine T cells and several human TCR V.beta. gene families, including 
V.beta.17, V.beta.12, V.beta.13, V.beta.14 and V.beta.15. Cole et al., 
"Stimulation of Mouse Lymphocytes by a Mitogen Derived from M. 
arthritidis. VIII. Selective Activation of T Cells expressing Distinct 
V.beta. T Cell Receptors From Various Strains of Mice by the 
`Superantigen` MAM", J. Immunol., 142:4131 (1989), and Chothia et al., 
"The Outline Structure of the T Cell .alpha./.beta. Receptor", EMBO J., 
7:3745 (1988). Moreover, there are reports implicating selective usage of 
V.beta.17 and several other TCR V.beta. gene families by autoimmune human 
T cells. For example, expanded populations of MBP-reactive T cells in the 
peripheral blood of multiple sclerosis (MS) patients have been reported. 
In this report each individual MS patient used a particular TCR V.beta. 
gene family for TCR in MBP-reactive T cells. Ben-Nun et al., "Restricted 
T-cell Receptor V.beta. Gene Usage by Myelin Basic Protein-Specific 
T-cells Clones in Multiple Sclerosis: Predominant Genes Vary in 
Individuals", Proc. Natl. Acad. Sci. USA, 88:2466-2470 (1991). 
Preferential usage of V.beta.17 as well as V.beta.12, V.beta.14 and 
V.beta.15 among those T cells reactive against an encephalotogenic MBP 
peptide presented in association with DR2 and DR3, two MHC class II genes 
that are over-represented in the MS patient population. Wucherpfennig et 
al., "Shared Human T Cell Receptor V.beta. Usage to Immunodominant Regions 
of Myelin Basic Protein", Science 248:1016 (1990) In addition, 
V.beta.17.sup.+ T cells have been reported to be enriched among activated 
T cells isolated from the synovial tissue of patients with rheumatoid 
arthritis (RA). Howell et al., "Clonal Infiltrates of Activated 
V.beta.17.sup.+ T Cells in Synovial Tissues of Rheumatoid Arthritis 
Patients", J. Cell Biochem. Suppl., 15A:295 (1991). Another study shows 
that V.beta.14.sup.+ T cells are overrepresented among synovial fluid T 
cells from patients with rheumatoid arthritis. Pallard and West, Science, 
253:325-329 (1991). 
According to the method of the present invention, mAb to a non-clonal, but 
restricted, population of T cells are made as described in detail in the 
Examples presented below. Briefly, T cells are isolated from whole blood 
or plasma by any method known in the art. Preferred methods include but 
are not limited to separation of T cells from non-T cells by the formation 
of rosettes with SRBC and centrifugation on a Ficoll Hypaque gradient 
according to standard procedures. The isolated T cells are then incubated 
with any SA at a concentration of SA sufficient to cause T cell 
proliferation. Once the SA is added, the cells express the IL-2 receptor 
such that exogenous IL-2 must be supplied at some point. Generally, IL-2 
is not added initially. This is because a small amount of endogenous Il1-2 
is produced and is sufficient to stimulate cells that have a strong 
response to the SA. Exogenous IL-2 is withheld for about one week to 
prevent proliferation of cells that respond weakly to the SA. After SA 
treatment, the cells are allowed to grow for a suitable amount of time, 
usually about two to three weeks under suitable growth conditions. 
In order to eliminate T cells expressing TCR V.beta. gene products, for 
which antibodies are already available, the T cells are incubated with an 
anti-V.beta. specific antibody, and subsequently exposed to goat 
anti-mouse immunoglobulin anti-body. The immune complexes which form 
between the anti-mouse antibody, the V.beta. specific antibody and the T 
cells recognized by the V.beta. specific antibody, are then removed, for 
instance by magnetic beads and a magnet or fluorescence activated cell 
sorting (FACS) depending on the selective marker attached to the 
anti-mouse antibody. After allowing the remaining cells to grow, an 
optional selection step may be performed utilizing one of the isolation 
methods described above. The whole cells are then injected into the animal 
host under conditions suitable to cause antibody formation. The antibodies 
obtained are screened on the cell line with which the animals were 
immunized. As a negative control, cells derived from the same donor but 
treated with a different, non-cross reactive SA can be used. 
Immunization with the incubated T cells can be effected by methods 
including but not limited to subcutaneously, intraperitoneally, 
intravenously, intramuscularly or directly into lymph nodes. 
As with all immunogenic compositions for eliciting antibodies, the 
immunogenically effective amounts of the T cells must be determined 
empirically. Factors to be considered are the immunogenicity of the T 
cells, whether or not the T cells will be complexed with or covalently 
attached to an adjuvant or carrier protein or other carrier, route of 
administration, and number of immunizing doses to be administered. Such 
factors are known in the vaccine art and it is well within the skill of 
immunologists to make such determinations without undue experimentation. 
The number of T cells needed to stimulate antibody production will vary 
somewhat according to the nature of the T cells (i.e. which V.beta. they 
express) and animal species, in addition to the factors described above. 
As little as 1.times.10.sup.6 cells may be sufficient to elicit an immune 
response and up to about 20-100.times.10.sup.6 cells or more could also be 
used. Preferably, the effective amount to ensure antibody production is 
about 10.times.10.sup.6 cells for mice. The T cells are not mixed with an 
adjuvant or adsorbent. Generally, the cells are merely mixed with a 
physiologically acceptable carrier such as normal saline or a buffering 
compound suitable for administration to mammals. 
The presence of the antibodies of the present invention, either polyclonal 
or monoclonal, can be determined by various assays. Assay techniques 
include but are not limited to immunofluorescence (IF) by 
cytofluorographic analysis or by cell sorting, indirect 
immunofluoroscence, immunoprecipitation, ELISA, agglutination and Western 
blot techniques. Analysis of V.beta. gene usage can be done by DNA 
sequencing, preferably a DNA amplification step is added such as 
polymerase chain reaction (PCR) as described below. 
The preferred technique is IF by cytofluorographic analysis rather than by 
cell sorting. Briefly, about 1.times.10.sup.5 peripheral blood T cells or 
SA reactive TCL cells are mixed with hybridoma culture supernatants, 
washed, counterstained with fluorescein labelled goat anti-mouse IgG, 
washed and examined for immunofluorescence staining on a cytofluorograph, 
for instance an Ortho IIs. Procedures involving the use of agglutination 
assays are well known in the art of blood screening. Western blots are 
performed essentially according to the methods described by Towbin et al., 
Proc. Natl. Acad. Sci. USA, 76:4350 (1979). 
The antibodies obtained by the method of the present invention can be used 
in methods of detection of the presence of particular populations of T 
cells bearing the gene products of a TCR V.beta. chain family and can be 
used to quantitate the percentage of these populations of T cells in the 
total population of T cells. This is useful in diagnosing various diseases 
related to T cell dysfunctions wherein a particular restricted population 
of T cell is over-represented within the entire T cell population. 
Preferably, the methods which use the antibodies to detect the presence of 
particular types of T cells in a sample involve contacting the sample with 
at least one of the antibodies under conditions which allow the formation 
of an immunological complex between the antibody and the specific T cell 
that may be present in the sample. The formation of an immunological 
complex if any, indicating the presence of the specific T cell type in the 
sample, is then detected and measured by suitable means. 
Such methods include, but are not limited to, homogeneous and heterogeneous 
binding immunoassays, such as indirect immunofluorescence, 
radioimmunoassays (RIA), ELISA and Western blot analyses as discussed 
above. The antibodies may be labeled or unlabeled depending on the type of 
assay used. Labels which may be coupled to the antibodies are those known 
in the art and include but are not limited to enzymes, radionucleotides, 
fluorogenic and chromogenic substrates, cofactors, biotin/avidin, 
colloidal gold and magnetic particles. Modification of the antibodies 
allows for coupling by any known means to carrier proteins or peptides or 
to known supports, for example, polystyrene or polyvinyl microtiter 
plates, glass tubes or glass beads and chromatographic supports, such as 
paper, cellulose and cellulose derivatives, and silica. 
Preferred assay techniques, especially for large-scale clinical screening 
of patient T cells include but are not limited to indirect 
immunofluorescence. For instance, the antibodies may be directly bonded to 
T cell specimens in solution or in situ in histological specimens and 
detected by fluorescence microscopy. 
The antibodies are also suitable for use as therapeutic agents. For 
instance, the antibodies may be used unaltered or coupled to toxins 
including but not limited to ricin and diphtheria toxin and administered 
to a patient. Antibodies used alone are capable of fixing complement and 
initiating cytolysis of the target cell. Once the antibodies bind to the 
specific T cell, they cause the death or removal of the T cell and thus 
ameliorate the dysfunction caused by the T cells. The antibodies are 
generally administered with a pharmaceutically acceptable carrier or 
vehicle therefor. A pharmaceutically acceptable carrier is one that does 
not cause an adverse physical reaction upon administration and one in 
which the antibodies are sufficiently soluble and retain their activity to 
deliver a therapeutically effective amount of the compound. The 
therapeutically effective amount and method of administration of the 
antibodies may vary based on the individual patient, the indication being 
treated and other criteria evident to one of ordinary skill in the art. A 
therapeutically effective amount of the antibodies is one sufficient to 
induce death or removal of a sufficient number of the specific T cells to 
ameliorate the dysfunction without causing significant side effects such 
as non-specific T cell lysis or organ damage. The route(s) of 
administration useful in a particular application are apparent to one or 
ordinary skill in the art. 
Routes of administration include, but are not limited to, parenteral, and 
direct injection into an affected site. Parenteral routes of 
administration include but are not limited to intravenous, intramuscular, 
intraperitoneal and subcutaneous. For most T cell dysfunctions, 
intravenous administration is preferred, but where the dysfunction is 
localized, such as in arthritis, direct injection to the affected site 
will result in increased effectiveness and decreased side effects such as 
non-specific organ damage. 
The invention also encompasses antibodies made in response to the SA 
reactive T cells and which recognize TCR V.beta. proteins. Such antibodies 
can be either polyclonal or monoclonal. Methods for making both types of 
antibodies are well known in the art. Methods of immunization and antibody 
production, purification and characterization are known in the art and 
need not be described in detail. The preferred antibodies are monoclonal 
(mAb) and are made by any method known in the art, for instance by the 
method described by Kohler and Milstein, "Continuous Cultures of Fused 
Cells Secreting Antibody of Predefined Specificity", Nature, 256:495-497 
(1975) which is incorporated herein by reference. 
The present invention includes compositions of the antibodies described 
above, suitable for parenteral administration including, but not limited 
to, pharmaceutically acceptable sterile isotonic solutions. Such solutions 
include, but are not limited to, saline and phosphate buffered saline for 
intravenous, intramuscular, intraperitoneal, subcutaneous or direct 
injection into a joint or other affected area. 
Antibodies used in therapeutics suffer from several drawbacks such as a 
limited half-life and propensity to elicit an immune response. Several 
methods have been proposed to overcome these drawbacks. Antibodies made by 
these methods are encompassed by the present invention and are included 
herein. The use of the words herein "antibodies" and "mAb" include the 
specific embodiments discussed below. One such method is the "humanizing" 
of antibodies by cloning the gene segment encoding the antigen binding 
region of the antibody to the human gene segments encoding the remainder 
of the antibody. Only the binding region of the antibody is thus 
recognized as foreign and is much less likely to cause an immune response. 
An article describing such antibodies is Reichmann et al., "Reshaping 
Human Antibodies for Therapy", Nature 332:323-327 (1988), which is 
incorporated herein by reference. Another method to avoid the drawbacks 
found in antibody therapy can be found in the use of peptide analogues 
which mimic the antigen binding region of the antibody but are not 
themselves antibodies. An article describing such antibody mimetics is 
Saragovi et al., "Design and Synthesis of a Mimetic from an Antibody 
Complementarity-Determining Region", Science, 253:792-795 (1991), which is 
incorporated herein by reference. 
The following Examples are meant to illustrate but not limit the present 
invention. 
Example 1 
Reagents Used in Succeeding Examples 
Staphylococcal enterotoxins SEA, SEB, SEC.sub.1, SEC.sub.2, SEC.sub.3 and 
SEE as well as toxic shock syndrome toxin, TSST-1, were obtained from 
Toxin Technology, Madison, WI and used according to the manufacturer's 
instructions. Partially purified MAM was isolated from M. arthritidis 
culture supernatants according to the method described by Atkin et al., 
(1986). All SA were used at a final concentration predetermined to be 
optimal for T cell proliferation, 1:4000 for MAM and 10-25 ng/ml for the 
Staphylococcal-derived SA. 
Example 2 
Isolation and Fractionation of Lymphocytes 
Fresh peripheral blood or tonsil lymphocytes were isolated by 
Ficoll-Hypaque centrifugation according to the manufacturer's 
instructions. T cells were isolated from non-T cells by E-rosette 
formation with neuraminidase-treated sheep red blood cells according to 
the method described by Kaplan and Clark, "An Improved Rosetting Assay for 
Detection of Human T Lymphocytes", J. Immunol. Met., 5:131-135 (1974), and 
a second Ficoll-Hypaque centrifugation. Residual T cells were removed from 
the non-T cell fraction by treatment with anti-T3 antibodies obtained from 
hybridoma ATCC CRL 8001 (OKT3) obtained from ATCC, followed by the 
addition of magnetic beads coated with goat anti-mouse antibody and 
physical separation of the bead-bound T cells utilizing a magnet according 
to the manufacturer's instructions (Dynal, Inc., Great Neck, N.Y.). 
Example 3 
Generation of SA-reactive T helper (T ) cell lines 
CD4.sup.+ peripheral blood T cells were isolated from unselected T cell 
populations obtained as described in Example 2, by incubating the T cells 
with an excess of anti-CD8 mAb followed by washing and physical removal of 
T cells binding antibody to CD8 utilizing magnetic beads coated with goat 
anti-mouse antibody and a magnet according to the manufacturer's 
instructions (Dynal). The CD4.sup.+ -enriched populations were cocultured 
with X-irradiated autologous antigen presenting cells (APC) and either MAM 
or SEE. After 5 days, semi-purified human IL-2 (Electro-Nucleonics, Inc., 
Fairfield, N.J.) was added at a final concentration of 5%. Cultures were 
retriggered weekly with APC, and the relevant SA, and expanded in the 
presence of IL-2. Cell lines were maintained in culture media consisting 
of RPMI 1640 (Gibco Laboratories, Grand Island, N.Y.) containing 10% fetal 
bovine serum (fbs) (Whittaker, Mass. Bioproducts, Walkersville, Md.), 
penicillin and streptomycin (50 .mu.g/ml, Gibco), and 2 mM glutamine 
(Gibco). 
Example 4 
CD23 Induction assay 
The specific interaction of SA-reactive CD4.sup.+ human T cells and 
SA-bearing B cells results in the rapid expression of the CD23 activation 
antigen on a fraction of the resting B cell pool. Friedman et al., "A 
Potential Role for Microbial Superantigens in the Pathogenesis of Systemic 
Autoimmune Disease" Arthr & Rheum., 34:468 (1991). 
The induction of B cell surface CD23 expression by T.sub.h cells has been 
detailed previously. Crow et al., (1986). Briefly, 5.times.10.sup.5 
purified tonsillar B cells were cultured in final medium with 
1.5.times.10.sup.5 X-irradiated CD4.sup.+ MAM- or SEE-reactive TCL cells. 
Cultures were supplemented with final medium alone or medium containing an 
optimal concentration of the various SA (MAM was used at a 1:4,000 
dilution, whereas the other SA were used at 100 ng/ml). After 16 hours, B 
cells were assayed for CD23 expression by indirect immunofluorescence 
staining utilizing mAb EBVCS.sub.2 (generously donated by Dr. Bill Sugden 
and Stan Metzenberg, Madison, Wis.) and counterstained with 
fluorescein-conjugated F(ab').sup.2 fragments of goat anti-mouse IgG 
according to the manufacturer's instructions (Tago, Inc., Burlingame, 
Calif.). The percentage of positively staining cells was determined by 
analysis on an Ortho IIs cytofluorograph (Ortho Diagnostic Systems, Inc., 
Westwood, Mass.). 
The results obtained show that CD4.sup.+ SEE-reactive TCL cells induce 
optimal CD23 expression on B cells bearing TSST-1 or any of the SEs, but, 
as shown in Example 7, trigger little CD23 expression by MAM bearing B 
cells. This functional evidence of crossreactivity by SA-activated TCL 
cells is consistent with reports that activated human T cells are somewhat 
promiscuous in their proliferative responses to the staphylococcal-derived 
SA. Fleischer et al. (1991). Importantly, however, the patterns of CD23 
expression observed suggest that MAM and SEE-specific human T cells show 
little cross-reactivity and may therefore utilize different TCR V.beta. 
gene products. Thus, the SEE-reactive TCL provide an excellent comparison 
for screening MAM-specific TCR mAbs. 
Example 5 
Generation of Monoclonal Antibodies 
In order to generate mAb to a clonal population of T cells, Balb/c mice 
were immunized on 4 occasions with 1.times.10.sup.7 MAM-reactive TCL cells 
in 100 .mu.l phosphate buffered saline (PBS, 10 mM NAPO.sub.4, 150 mM 
NaCl, pH 7.2) which had been expanded in long-term culture (7 weeks) by 
weekly restimulation with autologous APC and MAM as described in Example 
2. Essentially, the method described by Kohler and Milstein (1975) was 
used to produce the mAb. Briefly, three days after the final immunization, 
the mice were sacrificed and their splenocytes fused with the HGPRT 
deficient myeloma cell line SP2/0 or NS-1. Hybridomas which demonstrated 
reactivity with a small fraction of freshly isolated (resting) peripheral 
T cells were screened against the MAM-reactive TCL used for immunization 
and an SEE-reactive TCL-derived from the same donor. The presence of 
antibodies was detected by indirect immunofluorescence. Briefly, two color 
immunofluorescence was prepared by first incubating 5.times.10.sup.5 cells 
with various mAb for 30 min. at room temperature. This was followed by 3 
washes in PBS-BSA 1%, azide 0.2% and goat anti-mouse Ig-FITC (GAM-FITC) 
for 30 min. at room temperature. The cells were washed 3 times and 
incubated with a negative control IgG1 mAb for 30 min. at room temperature 
to quench free GAM-FITC binding sites. The cells were washed 3 times and 
incubated with phycoerythrin (PE) labeled anti-CD4 or anti-CD8 mAb 
(UBI/Olympus, Lake Success, NY) for 30 min. at room temperature. The cells 
were finally washed 3 times and analyzed on an ORTHO cytofluorograph. The 
results shown in Table I represent the ratio of double positive (FITC +PE) 
cells over total CD4 or CD8 positive cells expressed as a percentage. In 
this manner, a mAb termed Cl, was identified. 
As shown in FIG. 3, Cl stains between 3-6% of peripheral T cells, &gt;60% of 
the MAM-reactive TCL used as immunogen, and virtually no SEE-reactive TCL 
cells. Immunoprecipitation studies (FIG. 4) showed that mAb C1 recognizes 
a disulfide-linked heterodimer consistent with the .alpha./.beta. TCR. 
Finally, as with other TCR mAb specific for V gene products, C1 recognizes 
a small subset of peripheral T cells from all donors tested, including 
samples of cord blood T cells. While C1.sup.+ cells are found among both 
CD4.sup.+ and CD8.sup.+ T cells, some donors show selective enrichment of 
C1.sup.+ T cells in one or the other T cell subset (Table I). 
mAb C1 was utilized to screen a number of SA-reactive TCL propagated in 
vitro. PBL were activated weekly with X-irradiated autologous APC and the 
indicated SA. The percentage of T cells staining with each of the anti-TCR 
mAbs was assessed each week 6 days after retriggering with X-irradiated 
APCs and SA. 
Cord blood lymphocytes and PBL obtained from normal adult donors were 
analyzed by two-color immunofluorescence staining for distribution of 
C1.sup.+ T cells in the CD4.sup.+ and CD8.sup.+ T cell subsets. Single 
color immunofluorescence was performed according to the method described 
by Crow et al. (1986). 
TABLE I 
______________________________________ 
Percentage of C1.sup.+ T cells in 
CD4.sup.+ and CD8.sup.+ subpopulations 
C1.sup.+ CD4.sup.+ 
C1.sup.+ CD8.sup.+ 
______________________________________ 
Adult blood 
1 14.00 3.77 
2 6.40 2.30 
3 7.98 5.56 
4 5.65 7.87 
5 6.63 11.32 
Cord bloods 
1 7.69 3.76 
2 4.75 3.98 
3 3.99 3.20 
4 3.45 4.48 
5 5.65 7.87 
Mean .+-. SD 6.62 .+-. 2.83 
5.41 .+-. 2.64 
______________________________________ 
TABLE II 
______________________________________ 
Peripheral Blood T-cells Triggered 
With MAM are Highly Enriched in C1 
Percentage of T-cells Staining 
Superantigen Positively With Anti-TCR mAbs 
Stimulus C1 C37 OT145 S511 Ti3a 
______________________________________ 
Primary MAM 55.6 0.0 2.5 2.5 5.6 
culture TSST 0.6 1.3 2.8 0.8 1.9 
SEA 5.1 0.6 2.9 2.9 5.9 
SEB 26.5 0.4 2.7 9.0 1.5 
SEE 3.0 3.4 1.2 0.2 12.5 
SEC1 17.8 4.8 3.0 7.3 5.2 
SEC2 15.1 1.8 0.0 7.1 1.7 
SEC3 9.2 4.1 1.9 4.5 6.2 
Secondary 
MAM 39.4 1.2 0.2 2.8 2.7 
culture TSST 0.2 0.0 1.1 0.0 0.0 
SEA 1.2 0.0 0.0 0.0 0.0 
SEB 10.1 0.6 2.2 6.4 0.7 
SEE 0.6 1.2 0.5 0.1 15.3 
SEC1 9.4 1.0 1.4 2.0 3.1 
SEC2 15.5 1.6 0.0 5.0 0.4 
SEC3 6.9 0.0 0.0 0.8 0.0 
Tertiary 
MAM 32.4 0.2 0.0 1.5 2.7 
culture TSST 0.0 0.1 0.3 2.6 1.6 
SEA 0.7 0.3 0.4 0.7 0.9 
SEB 7.3 0.5 0.0 3.3 3.3 
SEE 0.1 0.3 2.1 1.3 11.9 
SEC1 8.7 0.6 0.7 2.5 0.9 
SEC2 17.0 0.0 0.0 7.4 0.0 
SEC3 6.2 0.0 0.1 1.8 0.2 
______________________________________ 
As shown (Table II) short term activation of peripheral T cells with a 
panel of SA demonstrates a clear enrichment of C1.sup.+ cells among the T 
cells activated by MAM and several of the SE with which MAM-specific TCL 
cells crossreact in the CD23 induction assay, in particular, SEB, SEC1, 
SEC2, SEC3. In contrast, C1.sup.+ T cells are not well represented among 
non-crossreactive TSST-1 or SEE-activated T cells. T cells expanded by 
weekly retriggering with SEB or SEC 1 and autologous APC show a marked 
fall off in the percentage of C1.sup.+ T cells (Table II). 
These results indicate that SA such as SEB are recognized by T cells 
expressing several TCR V.beta. gene family products among which C1.sup.+ T 
cells are a minor component with a relatively low binding affinity for 
SEB. In contrast, C1.sup.+ T cells are greatly expanded in short-term 
cultures of MAM-activated T cells and remain well represented. In the 
experiment presented in Table 3, (described in Example 8) the percentage 
of C1.sup.+ T cells decreases somewhat over time in the culture stimulated 
weekly by MAM. However, in most experiments, C1.sup.+ T cells represent 
between 50 and 60% of TCL repetitively triggered with MAM (FIG. 3). In 
FIG. 3, PBL shown in the upper right; the CD4.sup.+ MAM-reactive T cell 
line used for immunization shown in the lower right; or a CD4.sup.+ 
SEE-reactive T cell derived from the same donor shown in the lower left 
were analyzed by indirect immunofluorescence staining for reactivity with 
C1. Background staining of PBL with PBS and fluoresceinated anti-mouse Ig 
is shown in the upper left. 
The results obtained indicate that C1.sup.+ T cells comprise a stable 
population of 15-20% of SEC.sub.2 reactive TCL cells. These results 
suggest that C1.sup.+ T cells represent the major population of human T 
cells reactive with MAM, and a significant fraction of the SEC.sub.2 
-responsive T cell pool. 
Example 6 
Generation of TCL enriched for TCR V gene usage 
Tonsil T cell aliquots were incubated at room temperature with saturating 
concentrations of non-cross-reactive TCR V gene specific mAb: C37 (V.beta. 
5.2/5.3) Wang et al., "A Monoclonal Antibody Detecting a Shared 
Determinant on the Human T Cell Antigen Receptor Molecule", Hybridoma, 
5:179 (1986), OT145 (V.beta. 6.7a) Posnett et al., "Inherited Polymorphism 
of the Human T Cell Antigen Receptor Detected by a Monoclonal Antibody" 
Proc Natl Acad Sci USA, 83:7888 (1986); and Li et al., "Allelic Variations 
in the Human T Cell Receptor V.beta. 6.7 Gene Products", J. Exp. Med., 
171:221 (1990) and C1. After 30 minutes, cells were washed 3 times, 
resuspended in final medium and cultured at a final concentration of 
0.5.times.10.sup.6 /ml in the presence of goat anti-mouse antibody-coated 
magnetic beads according to the manufacturer's instructions (Dynal). Beads 
were added at a ratio of 20 beads to 1 target T cell. After 5 days, 
magnetic beads were removed, the T cells washed and recultured with IL-2 
alone for 48 hours. Cultures were maintained with IL-2 and weekly feeding 
with periodate-treated allogeneic non-T feeder cells. 
These cultures become highly enriched in T cells expressing the relevant 
V.beta. gene, depending on the initial mAb used for stimulation. Usually, 
this occurs over a 6-day period. Occasionally, a second cycle of 
stimulation was required to achieve greater than 95% specific V.beta. 
expression. At the time these TCL were utilized as effectors in the 
cytolytic assay or for RNA isolation as described in Example 10, each was 
virtually 100% for T cells expressing the appropriate TCR V.beta. gene 
products. 
Example 7 
Assay of SA-dependent Cytolysis 
In order to show that the MAM-reactive T cell population contains both a 
C1.sup.+ and a C1- population of T cells, a functional assay was 
performed. Anti-TCR mAbs are mitogenic; this characteristic was used to 
formally prove that C1.sup.+ T cells are MAM-reactive by allowing the 
selective activation and expansion of T cells expressing the relevant TCR 
epitope. Aliquots of tonsillar T cells were treated with saturating 
concentrations of C1 or either of two non-cross-reactive TCR V.beta. gene 
product specific mAb: C37 (V.beta. 5.2/5.3, and OT145 (V.beta.6.7a). TCL 
were generated as described in Example 2. These lines are virtually pure 
with respect to reactivity with the relevant anti-TCR mAb (FIG. 4). 
TCL cells were assayed for cytolytic activity in a 4-hour [.sup.51 Cr] 
release assay according to the method described by Friedman et al., 
"Amplification of Altered Self-Reactive Cytolytic T Lymphocyte Responses 
by Cloned Allospecific Human T Helper Cells", J. Clin. Invest., 82:1722 
(1988) which is incorporated herein by reference, utilizing an MHC class 
II antigen-bearing target cell line, B cell lymphoblastoid cell line 8866. 
Briefly, 8866 cells were incubated for 2 hours at 37.degree. C. with 0.1 
mCi [.sup.51 Cr] in the presence of final medium alone, or the indicated 
SA. 
Peripheral blood T cells were expanded in culture by weekly activation with 
mAb C1, goat anti-mouse Ig-coated magnetic beads, and IL-2 in order to 
generate a C1.sup.+ TCL. In addition, an aliquot of the peripheral T cell 
population was depleted of C1.sup.+ cells utilizing the magnetic beads, 
and activated with MAM, autologous X-irradiated APC's, and IL-2. This 
MAM-reactive C1.sup.- TCL was retreated weekly to insure complete 
depletion of residual C1.sup.+ T cells. At the time these TCL cells were 
used as effectors in the cytolytic assay (after 4 weeks of culture), less 
than 0.5% of the TCLs stained with C1. Both the MAM-reactive C.sup.- TCL 
and the C1.sup.+ TCL were utilized as effector cells in a cytolytic assay 
against [.sup.51 Cr]-labeled 8866 target cells, either untreated or 
"pulsed" with SA as described above. Both TCL efficiently and specifically 
lyse MAM bearing target cells, suggesting a MAM reactive C1.sup.- T cell 
population. 
As shown in FIG. 5, both the MAM-reactive TCL and the C1.sup.+ TCL 
specifically and efficiently lyse the MAM bearing 8866 target cells. Data 
are presented as mean percent lysis of target cells at each effector to 
target cell ratio. These data confirm that C1.sup.+ T cells are MAM 
reactive. Taken together, these findings support the existence of a 
MAM-reactive human T cell population distinct from that which expresses 
the C1 epitope. 
FIG. 4 shows the results obtained when tonsillar T cells, expanded in 
culture by weekly activation with an anti-TCR mAb, goat anti-Ig-coated 
magnetic beads, and IL-2, in triplicate at the effector to target ratios 
indicated in FIG. 4 for cytotoxic activity against [.sup.51 Cr] release 
assay. Target cells consisted of a lymphoblastoid B cell line 8866 either 
untreated (8866) or "pulsed" for one hour at 37.degree. C. with MAM 
(8866.sub.MAM) or TSST-1 (8866.sub.TSST-1). Briefly, purified tonsillar B 
cells were cultured with medium alone or the indicated SA at 100 ng/ml, 
except MAM, which was used at 1/4000 dilution. CD4.sup.+ MAM-reactive TCL 
cells or CD4.sup.+ SEE-reactive TCL cells were added, cultures incubated 
for 16 hours, then analyzed for CD23 expression by indirect 
immunofluorescence staining. Depicted are the cytotoxic activities of C37 
activated T cells; mAb 0T145 activated T cells; and C1 activated T cells. 
The phenotype of these three cell lines at the time of assay are as 
follows: 
C37 activated=99% C37.sup.+, 22% CD4.sup.+, 77% CD8.sup.+ 
OT145 activated=100% OT145.sup.+; 16% CD4.sup.+; 85% CD8.sup.+; 
C1 activated=99% C1.sup.+ 43% CD4.sup.+ 61% CD8.sup.+ 
While each line contained a CD4.sup.+ T cell fraction, CD8.sup.+ T cells 
predominated, comprising 60-80% of the TCL population. A CD8.sup.+ T 
cell-dependent function was therefore assessed, by determining if these 
TCL cells could lyse MHC class II positive target cells in a SA dependent 
manner. In the experiment depicted in FIG. 4, no significant lysis of 
untreated 8866 target cells by any of the TCL is observed. However, the 
C1.sup.+ TCL selectively lyse MAM-bearing 8866 cells, while both the 
OT145.sup.+ and C37.sup.+ TCL cells effectively lyse TSST-1 bearing, but 
not MAM-bearing, targets. It should be noted that the proliferative 
response of human T cells to TSST-1 is reportedly dominated by the V.beta. 
2.sup.+ fraction. Choi et al., "Selective Expansion of T Cells Expressing 
V.beta. 2 in Toxic Shock Syndrome", J. Exp. Med., 172:981 (1990). The 
lysis of TSST-1 bearing targets by V.beta..beta.6.7a.sup.+ and 
V.beta.5.2/5.3.sup.+ TCL cells therefore represents another example of the 
cross-reactivity of activated T cell responses to S. aureus-derived SA. 
Fleischer et al., "An Evolutionarily Conserved Mechanism of T Cell 
Activation by Microbial Toxins: Evidence for Different Affinities of T 
Cell Receptor Toxin Interaction" J. Immunol , 146:11 (1991) . 
The experiment depicted in FIG. 4 has been performed on three separate 
occasions utilizing C1.sup.+ TCL independently derived from different 
donors. In all studies, the results are similar to those shown in FIG. 5. 
Thus, C1.sup.+ T cells, activated and expanded with C1, demonstrate 
functional specificity for MAM. 
Example 8 
Immunoprecipitation of TCR Utilizing C1 
A MAM-reactive TCL with 60% C1.sup.+ T cells was radio-iodinated with 
lactoperoxidase and peroxide, using 25.times.10.sup.6 cells and 2.5 mCi 
[.sup.125 ]. Cell lysis and immunoprecipitations with SPA-Sepharose and 
monoclonal antibodies were performed as previously described by Posnett et 
al., "A Novel Method for Producing Antipeptide Antibodies. Production of 
Site-Specific Antibodies to the T Cell Antigen Receptor .beta. Chain", J. 
Biol. Chem., 263:1719 (1988). 
The expansion of C1.sup.+ cells in short-term cultures of SE-activated T 
cells (Table II) indicates that C1.sup.+ T cells can account for the 
pattern of SA responsiveness associated with MAM-reactive TCL (FIG. 2). 
However, these data do not rule out the existence of C1.sup.- MAM-reactive 
T cells. Indeed, the observation that repetitive triggering of 
MAM-reactive T cells with MAM results in a TCL that is maximally 50-60% 
C1.sup.+ provides indirect evidence that a C1.sup.- MAM-reactive T cell 
population exists. To address this point, aliquots of fresh peripheral T 
cells were depleted of C1.sup.+, C37.sup.+ or OT145.sup.+ T cells by 
treatment with the relevant mAb followed by physical removal of the 
reactive T cells utilizing magnetic beads bearing goat anti-mouse IgG. 
Peripheral blood T lymphocytes were depleted of T cells reacting with 
anti-TCRmAbs C1 or C37 utilizing magnetic beads coated with anti-mouse 
IgG. Untreated or mAb-depleted T cells were assayed, in triplicate, for 
proliferative responses against medium alone, autologous APC, or the 
indicated SA in the presence of autologous APC. The percentage of C1.sup.+ 
T cells present in each responder T cell population was detected by 
immunofluorescence staining. In Table III experiments 1 and 2 describe the 
results of separate studies involving two different normal donors. 
TABLE III 
__________________________________________________________________________ 
C1 Depleted T-cells Proliferate in Response to MAM 
[.sup.3 H]-Tdr incorporation 
Description (cpm) induced by: 
of responder MAM SEE TSST-1 
population Media 
APC.sub.xr 
APC.sub.xr 
APC.sub.xr 
APC.sub.xr 
% C1.sup.+ 
__________________________________________________________________________ 
Exp. 1 
E.sup.+ 
3.5 
85 6,681 
37,978 
39,313 
69,033 
E.sup.+ C1 
0.3 
53 1,159 
24,226 
25,667 
55,629 
E.sup.+ C37 
2.8 
2,969 
1,837 
25,509 
23,847 
59,855 
Exp. 2 
E.sup.+ 
4.6 
82 1,170 
26,748 
122,655 
54,603 
E.sup.+ C1 
0.9 
267 1,348 
27,721 
99,509 
46,956 
E.sup.+ C37 
4.1 
239 1,184 
29,513 
77,742 
58,827 
__________________________________________________________________________ 
AS shown in Table III, while this procedure efficiently reduces or 
eliminates the C1.sup.+ T cell pool, as detected by immunofluorescence 
staining, the proliferative response to MAM was not affected. It should be 
pointed out that T cell populations depleted of C1.sup.+ cells maintain 
strong proliferative responses over a wide range of MAM concentrations (6 
log dilutions). In additional studies, the cytolytic activity of a 
C1.sup.+ TCL and a MAM-reactive TCL depleted of C1.sup.+ cells, both 
derived from the same donor were compared. 
Example 9 
Analysis of TCR V.beta. Gene by Polymerase Chain Reaction (PCR) 
Three T cell lines were prepared by stimulating normal peripheral blood T 
cells with either OT145 (V.beta.6.7), C37 (V.beta.5.2/5.3) or C1 mAb as 
described in Example 8. Total cellular RNA was isolated from each cell 
line by the acid guanidinium thiocyanate-phenol-chloroform method. 
Chomczynski and Sacchi, "Single-step Method of RNA Isolation by 
Guanidinium Thiocyanate-Phenol-Chloroform Extraction", Anal. Biochem., 
165:156 (1987). 
cDNA was synthesized with reverse transcriptase, using an anti-sense 
C.beta. primer the sequence of which is described below, according to the 
method described by Li et al., (1990). 
The PCR was performed with a panel of V.beta. specific sense primers, in 
parallel reactions where each V.beta. primer was matched with the C.beta. 
anti-sense primer situated 55 bp from the 5' end of the C region. Two 
paired C.beta. primers were used as a positive control. Each cDNA 
preparation was tested for the optimal dilution. PCR conditions included 
primers at 0.5 .mu., Replinase (Dupont) 2 U, buffer containing 3.0 mM 
MgCl.sub.2 (20 buffer, Dupont), [.sup.32 P]-dCTP 20 .mu.Ci, cold dNTPs at 
0.2 mM each in a final volume of 20 .mu.l . Amplification was done for 1 
min. at 94.degree. C., 1 min at 51.degree. C. and 1 min at 72.degree. C. 
for 25 cycles. 
PCR products were analyzed using polyacrylamide gel electrophoresis on a 5% 
polyacrylamide gel. The gel was dried and exposed to film. 
__________________________________________________________________________ 
C.beta. 
(anti-sense) 
5' 
CTTCTGATGGCTCAAACAC 
3' (SEQ ID NO: 1) 
C.alpha. 
5' (sense) 
5' 
GAACCCTGACCCTGCCGT 
3' (SEQ ID NO: 2) 
C.alpha. 
3' (anti-sense) 
5' 
TCATAAATTCGGGTAGGATC 
3' (SEQ ID NO: 3) 
V.beta. 
2 (sense) 
5' 
GTTTCTCATCAACCATGCAA 
3' (SEQ ID NO: 4) 
V.beta. 
6 (sense) 
5' 
TCAGGTGTGATCCAATTTC 
3' (SEQ ID NO: 5) 
V.beta. 
5.3/5.2 
(sense) 
5' 
GTCAGGGGCCCCAGTTTAT 
3' (SEQ ID NO: 6) 
V.beta. 
17 (sense) 
5' 
ACAGCGTCTCTCGGGAGA 
3' (SEQ ID NO: 7) 
__________________________________________________________________________ 
Specific PCR amplification of V.beta.17 gene products from a C1.sup.+ TCL 
was performed. The PCR amplified cDNA from three cell lines (OT145.sup.+, 
C37.sup.+, C1.sup.+) were obtained with C.alpha. primer (positive 
control), C.beta.-V.beta.2 primers (negative control), 
C.beta.-V.beta.5.2/5.3 primers, C.beta.-V.beta.6 primers or 
C.beta.-V.beta.17 primers. specific bands are indicated with arrows. In 
each case, the bands migrated as expected based on the estimated size of 
the amplified segment. 
TCL were prepared with three mAb: OT145 (V.beta.6.7.alpha.), C37 
(V.beta.5.2/5.3), and C1, as described above. Each of these polyclonal T 
cell lines contained &gt;98% cells positive with the relevant mAb. RNA was 
isolated and cDNA synthesized with reverse transcriptase. Aliquots of cDNA 
were PCR amplified with different primer combinations. The results 
obtained showed that each cell line expressed a specific V.beta. . As 
expected, the OT145.sup.+ cells expressed V.beta.6, and the C37.sup.+ 
cells expressed V.beta.5.2/5.3. The C1.sup.+ cells expressed V.beta.17. 
None of these TCL expressed V.beta.2 and all of them expressed C.alpha.. 
In other experiments the C1.sup.+ cell line was analyzed with primers 
specific for V.beta.1-V.beta.20. No primer combinations other than 
V.beta.17-C.beta. amplified a .beta.-chain product. Thus, V.beta.17 
appears to represent the sole V.beta. gene product recognized by C1. 
V.beta.17 is thought to represent a V.beta. family with a single gene copy 
based on counting bands on Southern blots. Robinson, "The Human T Cell 
Receptor .beta. Chain Gene Complex Contains at Least 57 Variable Gene 
Segments: Identification of Six V.beta. Genes in Four New Gene Families", 
J. Immunol., 146:4392 (1991); Concannon et al., "Diversity and Structure 
of Human T Cell Receptor .beta. Chain Variable Region Genes", Proc. Natl. 
Acad. Sci. USA, 83:6598 (1986); and Kimura et al , "Sequences and 
Repertoire of the Human T Cell Receptor .alpha. and .beta. Chain Variable 
Region Genes in Thymocytes", Eur. J. Immunol., 17:375 (1987). 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 7 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CTTCTGATGGCTCAAACAC19 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GAACCCTGACCCTGCCGT18 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
TCATAAATTCGGGTAGGATC20 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GTTTCTCATCAACCATGCAA 20 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
TCAGGTGTGAT CCAATTTC19 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GTCAGGGGCCCCAGTTTAT19 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
ACAGCGTCTCTCGGGAGA18