APPARATUS, SUBSTANCE AND METHOD FOR TREATMENT FOR THE IMMUNE SYSTEM

Abstract: The present invention is based on the discovery that a sub-set of human T8<+> cells in blood selectively bind to the lectin vicia villosa and that this sub-set includes contrasuppressor cells, i.e. cells that prevent suppressor cells in the immune system inhibiting helper cells or convert tolerogenic to immunogenic signals in contact sensitivity. Thus, by removing contrasuppressor cells from blood by passing the blood over vicia villosa, it is possible to allow the suppressor cells to act to suppress the immune reaction in patients suffering from diseases of the immune system or who have undergone transplant surgery. The present invention provides an apparatus, a method and a substance for treatment of human beings suffering from a disease of the immune system or who have undergone transplant surgery.

Description: Apparatus, Substance and Method for Treatment for the Iantune System The present invention relates to the use of the lectin vicia villosa in the treatment of diseases of the immune system and also in the suppression of the rejection of transplanted organs. _ Vicia villosa is a known lectin and is obtained from a vetch; it is commercially available from Sera-Lab Limited of Crawley Down, Sussex, RH10 4FF, United Kingdom and has been described in a paper in The Journal of Biological Chemistry, Vol 258, 8, pp. 5165. Vicia villosa is known to bind T-cells obtained from mice in in vitro experiments. It has, however, never been used in vivo and in particular has not been used thera- peutically. The mammal immune system involves not only antibody- forming cells but also helper-inducer cells that help (or induce) the antibody-forming cells to form antibodies, suppressor cells that inhibit helper cells and contrasuppressor cells that prevent suppressor cells inhibiting helper cells or convert tolerogenic to immunogenic signals in contact sensitivity. The present invention is based on the discovery that a sub-set of human T8 cells acts as a contrasuppressor and that these cells are preferentially bound by the lectin vicia villosa. Using this discovery, it is possible to treat patients suffering from defects in their immunity system, e.g. autoimmune diseases or patients that have undergone organ transplant surgery by removing from their blood T8 contrasuppressor cells. This removes the curb on the patients' suppressor cells, which will then be free to suppress the formation of antibodies and delayed hypersensiti¬ vity and thereby reduce an autoimmune reaction or prevent rejection of transplanted tissue. According to a first aspect of the present invention there is provided vicia villosa optionally supported on a carrier, e.g. sepharose beads or on a plastic sheet or membrane, for use in treating diseases of the immune system and also rejection of transplanted tissue. The invention also provides a blood- treatment apparatus comprising vicia villosa supported on a carrier, means for conducting blood from a patient to the carrier and means for conducting blood that has passed over the vicia villosa back to the patient. The advantage of using a plastic meftnbrane as the carrier for vicia villosa is that such a membrane can be inserted into a blood dialysis machine in the place of the normal diffusion membranes used for blood dialysis of kidney patients. Blood can then be passed through the machine and treated therein in accordance with the present invention which means that no special apparatus need be developed. In the course of treatment, blood may be removed from a patient and passed over the carrier coated with vicia villosa, which selectively binds the contra¬ suppressor T8 cells and so removes them from the blood, following which the blood may be passed directly back to the patient. According to a second aspect of the invention, there is provided a process of treating human blood, the process comprising passing the blood over vicia villosa supported on a carrier. Another aspect of the present invention is a method of treating patients who have undergone transplant surgery, rhe method comprising removing blood from the patient, passing it over vicia villosa supported on a carrier and returning it to the patient. As well as binding contrasuppressor cells, vicia villosa also binds the sugar N-acetyl-D-glactosamine. This suggests a practical method of regenerating used devices contain¬ ing v-cia villosa coated on a carrier, the method comprising passing a solution of N-acetyl-D-glactosamine through the used device; the sugar displaces the contrasuppressor cells bound to the vicia villosa, thereby regenerating the device for further use. The following experiments are described purely by way of example: Experiment 1 The helper and suppressor activities of various cell reconstitutions were investigated. To investigate the helper function, cell reconstitu- tions were each pulsed with a predetermined dose of 1 jαg of streptococcal antigen for 24 hours in Marbrook flasks. Any helper factor generated and released into the supernatent liquor during this period was assayed by adding it to Costar culture plate (Cambridge, Massachusetts) at a concentration of 10 ml per culture plate, which had previously been determined as the optimum dose, in the presence of 10 x 10 unprimed spleen cells and 100 ng/ml of dinitrophenylated (DNP) streptococcal antigen. The number of anti-DNP antibody forming cells (AFC) were determined on day 4 using the modified Cunningham assay described in Infec. I un. 6, 903 with DNP coated and unseated sheep red cells. All tests were carried out in tripli¬ cate. The assay without any added helper factor, i.e. without any supernatent liquor being added, was also performed and the number of antibody forming cells produced were counted. The help generated by each cell reconstitution was calculated according to the following formula: Help (%) = AFC(Help) - AFC (No HF) AFC (No HF) X 1UU where AFC(Help) is the assay of antibody forming cells from the various cell reconstitutions and AFC (No HF) is the number of antibody forming cells formed without helper factor being added to the culture. Suppressor factor was prepared from putative suppressor cells present in cell reconstitutions by culturing the cells in the presence of Ing of strepto¬ coccal antigen for 24 hours and the supemascent liquor was then assayed for its ability to suppress helper cells. Helper cells were induced from CBA mouse spleen cells by culturing them in vitro with 100 ng of strept¬ ococcal antigen for four days. The supernatent liquor 5 6 was then added to 3 x 10 helper cells, 10 x 10 u n primed CBA spleen cells and 100 Ω g of dinitrophenylated strepto¬ coccal antigen. The number of antibody forming cells on day 4 were assayed in a manner similar to that described above in connection with the calculation of help. The number of antibody forming cells formed in the above assay without the addition of the super- natent liquor was alsocounted as was the number of antibody forming cells produced without any helper cells or liquor. The suppression of the various cell reconstitutions was then calculated according to the following formula: s TO _onα) . .00 - feg pp).- %< g g) x 10 0 where AFC (Supp) is the assay obtained from the various cell reconstitutions; AFC (No SF/HC) is the assay without helper cells or suppressor factor from supernatent liquor, and AFC (No SF) is the assay with helper cells but without suppressor factor from supernatent liquor. In connection with the above tests, it is pointed out that it is accepted from previous experiments (Lehner T, 1982, J. Immunology 129, 1936) that mouse helper cells are similar to their human counterparts and that tests conducted with mouse cells hold good for human as if the tests had been conducted with human cells; appropriate human cells cannot ethically be obtained in sufficient numbers. In the cell reconstitutions, specimens of venous blood were withdrawn from 10 healthy subjects. All subjects were HLA-DRwβ " , 6 had DR4 and 4 had DR2, 3 or 5. Mononuclear cells were separated by the Ficoll triosil (FT) method (Scand. J. Clin. Lab. Invest. _21, 77). Adherent cells, predominantly monocytes, were prepared by adherence of FT separated cells to plastic plates (Falcon 3002). The FT separated and plastic non-adherent cells were passed through a nylon wool column to remove + + B cells and any adherent cells. T8 and T4 cells were separated in some experiments by killing T cells either with monoclonal T4 or T8 antibodies (obtained from Ortho Laboratories) and rabbit complement (obtained by Buxted Rabbit Co., Ltd.,) as described previously in J.Immunol.129,1936. The resulting T cell populations were characterised by indirect im unofluorescence. ' T8 cells were panned with the lectin ' vicia villosa according to a method described in Proc.Nath.Acad.Sci 15_, 2844. Falcon plates (3002; 6 cm diameter) were incubated with 2.5 ml of Vicia villosa at a concentration of 0.5 mg per ml in Hank's solution (pH 6.5), for 2 hours at room temperature. After removal of the -unbound lectin, * the plates were washed twice with Hank's solution. T8 cells were then added to the plates at a concentration of about 20 x 10 cells in 3 ml Hank's solution, for 30 minutes at 38 C. The non-adherent cells (hereinafter referred to as T8 W-N C) were removed and the cells adherent to the plate (hereinafter referred to as T8 W-AC) were washed twice with Hank's solution. The adherent cells were then recovered from the plate by adding 3 ml N-acetyl-D-galactosamine, at a concentration of 2 mg per ml in Hank's solution, for 10 minutes at 37 C. The help and suppression of various cell reconstit- utions were carried out and the results are given in Table 1. TABLE 1 AFC count Cell Reconstitution % Cell Ratio Help Suppression Help Suppression (x 30 6 ) (a) T4 + HC + T8 + C + T8 + VV-AC 3 : 1.5 90 130 309 (b) T4 + HC + T8 + C + T8 + VV-NAC 3 : 1.5 30 33 36 95 In Table 1, the abbreviations M T4 + HC" stands + + for T4 helper cells, "T8 C" stands for unseparated T8 cells, "AFC count" stands for the assay of antibody forming cells found in the assay described above, and "Help (%)" and "Suppression (%)" are the help and suppression calculated according to the formulae ^ given above. In calculatingthe Help (%) and Suppression (%) the following were the values measured for the terms in the formulae: AFC (No HF) (a) 33 (b) 22 AFC (No SF/HC) 33 27 AFC (No SF) 163 137 Similar tests were also conducted on the reconstituted cell population shown in Table 2 and the results were as shown in that Table. Table 2 Cell Reconsti u ion Cell Ratio AFC % ( lO 6 ) Help Suppression Help Suppression T8 + VV-NAC 5 37 87 23 56 T4 + HC + T8 + VV-AC 4 : 1 133 143 343 11 T4 + HC + T8 + VV- AC 4 : 1 2 133 0 19 The Help (%) and Suppression (%) figures were calculated from the formulae given above from the following measured terms: AFC (No HF): 30 AFC (No SF/HC) : 33 AFC (No SF) : 157 From Tables 1 and 2, it can be seen that T8 W-AC and T4 HC generate significant helper activity. Contrasuppression by T8 W-AC cells is demonstrated by the T4 + HC + T8 + C + T8 + W-AC test which induced helper but no suppressor function whereas substitution of T8 + W-NAC for TS + W-AC resulted in a reduction in ielp and greater than 90% suppression. From these results, it can be seen that the T8 W-AC cells act as con rasuppressors, i.e. preventing suppressor cells from inhibiting the helper cells. Removal of these cells from the human body will therefore result in the body's natural suppression system being given free rein to suppress helper cells and thus reduce the amount of antibodies produced. The low amount of suppression in the reconstitution of T4 + HC + T8 W-NAC is not due to the fact that T8 + W 7 -NAC is acting as a contrasuppressor but rather because the amount of suppressor cells in the reconstitution is low. Suppressor cells are present in the T8 C and hence the suppression increases markedly when these cells are added to the reconstitution system. Experiment 2 Ψ -AC T cells bind antigens as is demonstrated by- radio labelling streptococcal antigen (SA) according to the chloramine-T method, (Hunter and Greenwood, Nature 194 : 495) using 40 μC± of 125 per l^ig of SA. 10 T cells were incubated with the predetermined dose of 1 ug of 125_ labelled SA for 30 minutes at 4 C with azide. The cell's were then washed, smears prepared on slides, dipped into photographic emulsion and kept in the dark at 4 C for 12 days. The slides were then developed, fixed and stained with methyl green pyron- in. The slides were examined under the microscope, using a x 100 oil immersion objective and the total number of silver grains were counted in about 500 cells, as well as the number of grains in adjacent cell-free zones, corresponding to an area of 500 cells. The results were expressed as the net grain count, that is the number of grains counted on 500 cells from which the number of grains in the cell free area was subtracted. The tests were carried out on the following cell populations with the following results: TABLE 3 Cell Population Mean Grain Count Untreated T cells 247 T W-AC 453 T W 7 -NAC 32 where "T W-AC" means T cells that adhered to vicia villosa when subjected to the separation with vicia villosa described above and "T W 7 -NAC" means the non- adherent cells obtained from the same separation. From these results it is clear that T W-AC cells bind much more strongly to streptococcal antigen than T W-NAC. It can be postulated that the T W 7 -AC cells bind with the antigens and present them directly to helper cells, thereby increasing the immune response to the antigen. Thus, the removal from the body of T " W 7 -AC will reduce the immune response of a patient. The present invention can be performed by coating vicia on a carrier, e.g. sepharose beads and passing blood through a bed of such beads, or by coating vicia villosa on a membrane, fitting the membrane into a kidney dialysis machine and passing blood from the patient, through the machine and back to the patient again.

Claims: CLAIMS 1. An apparatus for continuously treating blood, which apparatus comprises vicia villosa supported on a carrier, means for conducting blood from a patient to the vicia villosa and means for conducting blood that has passed over the vicia villosa back to the patient. 2. An apparatus as claimed in claim 1, wherein the carrier is a sheet of plastics material or sepharose beads. 3. Vicia villosa optionally supported on a carrier for use in treating diseases of the immune system or the rejection of transplanted tissue. 4. Vicia villosa as claimed in claim 3, wherein the carrier is in the form of a plastic sheet or sepharose beads. 5. The use of vicia villosa for the manufacture of a medicament or composition for treating diseases of the immune system or the rejection of transplanted tissue. 6. A method of treating patients who have undergone transplant surgery or who are suffering from a disease of the immune system, which method comprises removing blood from the patient, passing it over vicia villosa supported on a carrier and returning it to the patient. 7. A method as claimed in claim 6, which includes stopping the flow of blood and passing a solution of N-acetyl- D-glactosamine over the vicia villosa to regenerate the vicia villosa. 8. A method of treating human blood which comprises passing the blood over vicia villosa supported on a carrier. 9. A method as claimed in claim 8, which includes passing a solution of N-acetyl-D-glactosamine over the vicia villosa after the blood.

Inventors: LEHNER THOMAS, LEHNER, THOMAS
Applicants: SERA LAB LTD, ST THOMAS HOSP MED SCHOOL, COUNCIL OF GOVERNORS OF THE UNITED MEDICAL AND DEN, SERA-LAB LIMITED
Application year: 1985
Main classification: A61M1
IPC codes: A61K35, A61K38, A61M1