rev:  February 11, 2003

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CD CLUSTERED ANTIBODIES  

(anti-Human and others as indicated)

RDI Divison of researchd Industries Intl  offers a wide line of  antibodies. Since no one antibody works best for all applications (flow cytometry, neutralization, blotting, histochemistry, ELISA, etc), we offer many different types of antibodies to help solve this problem. Please inquire for other applications or types of antibodies not listed below. All products are for in vitro research use only-not for use in or on humans or animals-not for use in diagnostics. Price/availability/specifications subject to change without notice.

T CELL STIMULATION Antibodies

Mouse anti-human T Cell Stimulation Antibodies

The following antibodies are provided as purified antibody without preservative (sterile filtered)

They have specifcially tested for T cell stimulation applications by CLB Reagents:

see new information below on these antibodies!

Monoclonals For T Cell Stimulation Experiments: for in vitro research use only

The following CD antibodies have been tested and found to specifically stimulate T cells in cell culture. Each antibody is provided as 100 microliters per vial. The researcher is advised to review the below references for particular applications of these products. Please also contact us for additional information and for assistance from the authors.

In one researchers lab, these antibodies have been used in T cell cloning experiments in the absence of accessory cells. The best accessory cell free system employed either the CLB-T3/T4.E (* an IgE subclass CD3E monoclonal) or mitogenic combinations of CD2 mAb, combined with CD28 antibodies and 10U/ml IL-2 (fresh mAb and IL-2 added every week). Cloning efficiency of approx 50% can be achieved (when cells are seeded at 1 cell/well). However cultures can not be maintained for more than 3 weeks. At that time an essential (but unknown) signal has to be given by feeder cells.

-from the lab of Dr. Rene A.W. van Lier-Dept of Clinical Viro Immunology-CLB Reagents          

 PRICE:  $375.00/vial of 100 microliters (azide free)  $344.00/vial  3 or more any combination

                   (approx 150-200ug/100ul)   Bulk qunatities quoted on request

CATALOG#           CD#        CLONE         CLB-CODE

RDI-M1651clb        CD2        6G4               CLB-T11.1/1

RDI-M1652clb        CD2        4B2               CLB-T11.2/1

RDI-M1650clb        CD28      15E8 (402)    CLB-CD28/1

RDI-M1654clb        CD3        1XE               CLB-T3/4.E

ref: 1) R. de Jong, et al. Regulation of T-Cell differentiation by CD2 and CD28 accessory molecules, IMMUNOLOGY, 74, 175, 1991.

2) R.A. Gruters, et al., Non-mitogenic T cell activation signals are sufficient for induction of human immunodeficiency virus transcription, EUR. J. IMMUNOLOGY, 21, 67, 1991.

3) R.A.W. Van Lier, et al, Signals involved in T cell activation. T Cell proliferation induced through synergistic action of anti-CD28 and anti-CD2 monoclonal antibodies, EUR. J. IMMUNOLOGY, 18, 167, 1988.

4) R.A.W. Van Lier, et al, Induction of T cell Proliferation with CD3 switch-variant monoclonal antibodies:effects of heavy chain isotype in monocyte-dependent systems, EUR. J. IMMUNOLOGY, 1987, 17:1599-1604.

Among the more than 130 CD markers on human lymphocytes known today (1), those directly involved in lymphocyte stimulation are particularly important: these are the target molecules to study the immune system at the cellular level. Using lymphocyte-stimulating monoclonal antibodies, the actual stimulatory machinery can be taken apart in its separate components, especially when combinations of different antibodies are used. Some monoclonals can directly stimulate T cells into proliferation, while others require a 'second signal' to do so. The CLB range of monoclonals contains antibodies of both categories.

PRACTICAL APPLICATIONS

Induction of T lymphocyte proliferation The T cell receptor (TCR), the antigen receptor on the surface of T cells, is closely associated with a series of polypeptides designated as the CD3 (T3) complex. Signals generated through binding of antigen to the TCR are transduced to the intracellular compartment by means of the chains of the CD3 complex.Mimicking the activation by antigens and lectins, the binding of CD3 antibodies to T cells results in the activation of tyrosine kinase, in a rise in the intracellular calcium concentration, generation of diacylglycerol and activation of protein kinase C. Both calcium and protein kinase C serve as intracellular messengers for the induction of gene activation (2).

However, binding of CD3 antibody as such is not sufficient to initiate proliferation, because additional signals from e.g. monocytes or other accessory cells are required as well. Firstly, monocytes induce cross-linking of the CD3/TCR complex by binding through their Fc receptors. This cross-linking is an essential step for T cell activation. Secondly, accessory cells are required for the production of lymphokines, such as Interleukin-1 (IL-1) (2).

The presentation of CD3 antibodies in an insoluble form, as well as the addition of IL-1 and IL-2 have been shown to circumvent the monocyte requirement (2). Some authors have observed that in the presence of IL-2, soluble CD3 monoclonal antibodies can induce T cell proliferation, whereas others did not.This variability is caused by difference in avidity or epitope specificity of the monoclonal antibodies used. The heavy chain isotype of the antibody plays a role as well. It has been found that a variant CD3 monoclonal antibody, expressing an epsilon heavy chain is capable to induce T cell proliferation in the absence of monocytes (3). CLB sells this antibody, the Cat.No. is M1654.Some CD monoclonal antibodies are extremely strong stimulants of human T-lymphocytes, i.e. they can induce and sustain the proliferation of virtually every single T-lymphocyte, regardless of its antigen-recognition specificity, while no accessory cells are needed (3).

Multiple proliferation signals

Apart from the CD3 molecule, other membrane molecules also play a role in T cell activation. An alternative pathway of triggering involves the CD2 molecule, formerly known as T11 (the sheep erythrocyte receptor) (4). Binding of CD2 monoclonal antibodies may result in the elevation of intracellular Ca-levels as a first trigger to proliferation. However, a second signal is required, which may be administered via the CD28 molecule, a disulfide-linked homodimer with chains of 44 kD. While CD28 monoclonal antibodies by themselves are unable to induce T cell proliferation, they can do so in combination with CD2 and CD3 monoclonal antibodies, as well as upon addition of the phorbol ester PMA,the latter being a stimulant of protein kinase C (4,5).

By using combinations of monoclonal antibodies directed against CD3,CD2 and CD28, the different activation pathways can be analyzed. Applications include not only immunological research, but clinical diagnostics as well, since imbalance in a distinct stimulation pathway may lead to impaired immunity.

TESTING OF CELLULAR IMMUNOCOMPETENCE

The proliferative capacity of T lymphocytes is an important parameter of cellular immunocompetence in patients with inborn or acquired immunodeficiencies, including AIDS. The T cell-stimulating monoclonals from CLB have been proven very suitable to this end. An important example of a diagnostic application is the longitudinal follow-up of patients infected with the Human Immunodeficiency Virus (HIV).

Thus, in a longitudinal study of 300 individuals infected with HIV-1, it was found that the decline of T cell stimulation as induced by CD3 monoclonal antibodies was the first parameter to decline, even when the numbers of CD4+ cells were still unaltered (see fig.1) (6). Thus, low T cell responsiveness to monoclonal CLB-CD3 epsilon antibody is a useful early marker of progression towards AIDS (7).

LYMPHOCYTE REACTIVITY IN MULTIPLE SCLEROSIS

Another example of T cell-reactivity studies involves the diminished response in Multiple Sclerosis. Using combinations of lymphocyte-stimulating monoclonal antibodies mentioned above, it was shown that a reduced T cell reactivity only occurred in accessory -cell dependent responses, e.g. to soluble CD3 monoclonal antibody: in an accessory-cell independent system (e.g. upon stimulation with insoluble CD3 antibody or combination with CD28 antibody, MS patients showed normal T cell reactivity (8).

WHOLE-BLOOD LYMPHOCYTE CULTURES

TO perform a lymphocyte stimulation test, considerable amounts of blood are required for the isolation of lymphocytes. With immunodeficient patients, this is often not possible. At CLB, a whole-blood lymphocyte culture method was developed, which is particularly suitable for the stimulation assay using CD3 monoclonal antibodies (see above), (9). This test is very reproducible and is useful in longitudinal studies when the lymphocyte counts fluctuate.

INDUCTION OF T CELL CYTOTOXICITY AND HELPER T CELL FUNCTION

Cytotoxic T lymphocytes (CTL) have the capacity to lyse target cells, an important effector activity of T lymphocytes. Certain stimulating anti lymphocyte monoclonals can activate T lymphocytes to cytotoxic activity (10,11). This is an important tool in both diagnosis and research. T cell stimulation by monoclonal antibodies can also induce helper activity in human T lymphocytes for the activation and immunoglobulin production of human B lymphocytes (11).

RECOVERY OF TUMOUR-SPECIFIC CYTOTOXIC T CELLS

Stimulation of human T cells by monoclonal antibodies and combinations of monoclonal antibodies is a powerful method to obtain populations of T cells that are cytotoxic towards autologous tumour cells. Thus, it was shown that tumour-infiltrating lymphocytes (TIL) can be efficiently expanded from tumor tissue,in the presence of both CD3 and CD28 monoclonal antibodies (12,13).

REFERENCES:

1. Leucocyte typing V,(S.F.Schlossman et al.,ed).Oxford University Press, 1995

2. R.A.W. van Lier et al.:'Immobilized anti-CD3 monoclonal anti bodies induce accessory cell independent lymphokine production , proliferation and helper activity in human T lymphocytes. Immunology 68:45 (1989).

3. R.A.W. van Lier et al.: 'Functional studies with anti-CD3 heavy chain isotype switch-variant monoclonal antibodies. Accessory cell-independent induction of Interleukin-2 respons iveness in T cells by epsilon anti-CD3. J.Immunol.139:2873

(1987).

4. R.A. W. van Lier et al.:'Signals involved in T cell activation.T cell proliferation induced through the synergistic action of anti-CD28 and anti-CD2 monoclonal antibodies'. Eur.J.Immunol. 18:167 (1988).

5. H.M. Kuiper et al.:'Influenc of CD28 costimulation on cytokine production is mainly regulated via IL-2'. Immunology 83:38 (1994).

6. F.Miedema:'Immunological abnormalities in the natural history of HIV infection: mechanisms and clinical relevance'.Immunodeficiency reviews 3: 173 (1992).

7. M.T.L. Roos et al.:'Cellular and humoral immunity in various cohorts of male homosexuals in relation to infection with human immunodeficiency virus'. Neth.J.Med. 34: 132 (1989).

8. M.H.G. Rep et al.: 'Functional defects in peripheral blood T cells of multiple sclerosis patients. Diminished in vitro responsiveness in accessory cell-dependent activation systems' J.Neuroimmunol. 52:139 (1994).

9. E.Bloemena et al.:'Whole-blood lymphocyte cultures.J.Immunol. Methods 122:161 (1989).

10.G.A.van Seventer et al.:'Regulatory role of the CD8 antigen in both CD3 and CD2 monoclonal antibody-induced monspecific cyto toxicity of class I and class II-allospecific cytotoxic T cell clones'. Eur.J.Immunol. 18:1973 (1988).

11.R. de Jong et al.:'Regulation of T cell differentiation by CD2 and CD28 accessory molecules'. Immunology 74:175 1991).

12.M.J. Flens et al.:'Efficient expansion of tumor-infiltrating lymphocytes from solid tumors with combined CD3 and CD28 mono clonal antibodies'. Cancer Immunol.Immunother. 37:323(1993).

13.W.M.C. Mulder et al.:'Culture of tumour-infiltrating lymphocytes from melanoma and colon carcinoma: removal of tumour cells does not affect tumour-specificity'. Cancer Immunol. Immunother. 41:293(1995).

The Use of the CD3-E in an HIV Progression assay (please call for additional information)

The CD3E-IgE subclass antibody is being used in a novel research assay to follow the progression of HIV infected patients. Studies have indicated that the low T-cell responsiveness to CLB-CD3E is an early marker of progression towards AIDS in HIV-I infected individuals. The CD3E ab is provided in ascites (100 microliters) under cat#RDI-M1654clb $438.00 =approx 1000 tests in duplicate. *The use of the IgE subclass variant of CD3E has been found to react better than other subclasses

ref: 1) Schellekens, P.Th.A. et al, LOW T-cell responsiveness to activation via CD3/TCR is a prognostic marker for AIDS in HIV-1 infected men. J. CLIN. IMMUNOL. 1990, 10:121-127

2) Bloema, E. et al, 1989. Whole-Blood lymphocyte cultures. JIM 122:161-167.

3) Van Lier, R.A.W., 1987. Functional studies with anti-CD3 heavy chain isotype switch variant monoclonal antibodies. J. IMMUNOL. 139:2873-2879.

4) Koot, M. et al, Virus phenotype and T-cell reactivity associated with clinical progression in HIV-1infected asymptomatic individuals treated with zidovudine. J. INFEC DIS. in press (1993)

5) Gruters, R.A. et al. (1991) AIDS 5:43 -47 (1991)

Application:

-all samples should be run in triplicate

-an unstimulated lymphocyte control should also be assayed in each run

-Healthy donors of both sexes and between 18-62 years of age serve as controls. These donors will be referred to as reference population.

1. 10ml of venous blood is collected in evacuated blood collection tubes containing preservative free sodium heparin (143 USP units). Samples should be kept at room temperature and used within 24 hours.

2. Whole blood lymphocyte culture is performed in round bottom microplate plates by diluting the heparinized venous blood 1:10 (v/v) with Iscove's Modified Dulbecco's Medium (IMDM) suplemented with penicillin (100U/ml), streptomycin (100ug/ml), gentamycin (80ug/ml, effective concentration 54ug/ml) and 2-mercaptoethanol (5 X 10 -EO5 M).

3. To each well add 150ul of the diluted blood.

4. To each well add 20ul of the prediluted CD3 (CLB-T3/4.E) which has been diluted 1:500 in the above medium.

5. The cultures are incubated at 37 DEG C in a humidified atmosphere of 5% CO2 in air for 4 days.

6. 24 hours before harvesting, 20ul, containing 0.4uCi of 3H-Thymidine (specific activity of 200 Ci/mmol) is added to each well.

7. For harvesting, the contents of each well are rmeoved by suction (Titertek TM Cell Harvester, SKATRON), collected on glass fibre filters (Printed Filtermat A, size 102 X258mm, Wallac), Pharmacia, Finland) and dried. After addition of scintillation fluid, counting is performed in a liquid scintillation counter (1205 betaplate, LKB Wallac) (efficiency +_ 40%).

8.  Record the counts as cpm/15ul whole blood.

RDI Divison of researchd Industries Intl

San Jose, 95123 CA Snell ave 658

USA

or 408-780-0908

EMAIL:margaret@cellular-research.com

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