Cellular proliferation T cell suppressor factor

The invention relates to factor(s) which suppress cellular proliferation and antibody production and would be useful to control disorders such as those involving abnormal cellular proliferation.
Previous suppressor factors have been reported in the literature [See Grillot-Courvalin, Catherine et al., (1981) Nature 292:844; Greene, Warner C., et al. (1982) J. Immunol, 129:1986; M. J. Taussign, et al. (1979) Nature 277: at 305 and 308; Sirkka Kontianen, et al. (1978) Nature 274:477; Eisenthal, A., et al. (1979) Ann N.Y. Acad. Sci. 332:367; Smith, R. T., et al. (1970) Am. J. Pathol. 60:495; Namba, Y., et al. (1975) Inflammation 1:5; Lee, S. C., et al. (1977) J. Immunol. 118:88; Jegosothy, B. V., et al. (1979) J. Exp. Med. 150:622; Namba, Y., et al. (1977) J. lmmunol. 118:1379; Jegosathy, B. V., et al. (1976) Science 193:1260; and Waksman, B. H., et al. (1978) Cell Immunol. 36:180].
The suppressor factor(s) described in the invention and different from all these factors because they: (a) exhibit different functional properties, (b) exhibit different molecular weight; (c) are produced constitutively and in substantially higher quantities.
SUMMARY OF THE INVENTION
The invention provides for a human suppressor factor(s) (SF) isolated from human lymphoblastoid tumor cell supernatant where it is constitutively found. SF is characterized by suppression of mitogen, antigen or alloantigen driven cellular proliferation of human peripheral blood leukocytes as well as suppression of antibody production, synthesis and secretion.
SF has a molecular weight range of 55,000 to 70,000 daltons.
SF suppresses the cellular proliferative response of T and B cells. This suppression of cellular response occurs in the presence of pokeweed mitogen (PWM), concanavalin A (CON A), phytohemagglutinin (PHA) and mixtures thereof.
SF inhibits the proliferative response of human peripheral blood mononuclear leukocytes to allogenic cells in mixed lymphocyte cultures. SF also inhibits the proliferation of tumor cell lines while having no effect on natural killer cell (NK) cytotoxicity.
SF inhibits antibody production in vitro by human peripheral blood mononuclear leukocytes exposed to PWM while not affecting the viability of human peripheral blood mononuclear leukocytes in culture.
SF does not cause lysis of K-562 leukemic cell targets as determined by chromium release assay. SF also does not induce the differentiation of suppressor T-cell.

BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows ACA 44 gel filtration chromatography analysis of a) 153 b) 169 and c) Jurkaat suppressor factors. Suppressor activity of the proliferative response of human peripheral blood mononuclear cells to PHA ( ) was found in the range of 50-70 kd. The 160 suppressor factor was eluded in the same range (data not shown).
FIG. 2 shows fractionation of the 160 suppressor factor by anion exchange DEAE-HPLC. The hollow dot line represents absorbance at 280 nm and the solid dot line represents suppression of proliferative response.
FIG. 3 shows chromatofocusing of the (a) Jurkat b) 160 and (c) 169 suppressor factors. Proliferative response to PHA is plotted against fraction number.

DETAILED DESCRIPTION OF THE INVENTION
In the invention factor(s) have been identified which suppress mitogen or antigen (alloantigen) driven cellular proliferation of human peripheral blood leukocytes and antibody production.
Such factor(s) have potential use for example in the treatment of patients with cancer, graft versus host disease(s), autoimmune disease(s) and lymphoproliferative malignancy disorder(s) such as leukemia.
Factor(s) are described and identified in this application, which inhibit leukocyte proliferative responses. In particular these factor(s), suppressor factor (SF), have been found to be produced by hematopoietic cells such as human lymphoblastoid cell lines--especially T cell lines an others such as B cell lines or erythroleukemic cell lines. Particular T cell lines known to produce suppressor factor(s) are Jurkat, HPB-ALL, TALL-1, HD-MAR, SKW-3, DND 41, HPB-MLT and MOLT-4. Erythroleukemic cell lines such as K-562 can also produce SF. B cell lines such as CESS can also produce SF.
Supernatants from such cells inhibit the proliferative responses of T-lymphocytes to mitogens such as phytohemagglutinin (PHA), concanavalin A (CON A) and pokeweed mitogen (PWM). (Tables I-III).
It also inhibits the proliferative responses of T-cells to allogenic cells in mixed lymphocyte culture (MLC). (Table IV).
Also this factor(s) inhibits antibody production of blood cells, and especially in vitro by human peripheral blood mononuclear leukocytes in the PWM-driven system (Table V). SF may inhibit certain proliferative responses of B-cells.
The action of SF appears to be cytostatic, not cytotoxic, since it:
(1) does not affect the viability of human peripheral blood mononuclear leukocytes in culture after a four day incubation (Table VI); and
(2) does not cause lysis of K562 leukemic cell targets which can be determined by such assays as the chromium release assay.
This factor(s) does not affect natural killer (NK) cell cytotoxicity against K-562 targets. (Table VII). Also this factor inhibits the growth in vitro of cells from certain human tumor cell lines (lung, colon, etc.) (Table VIII).
These SF(s) exhibit a relative molecular weight in the range of 55-70,000, as determined by AcA-44 gel filtration.
Tables I-III shows the inhibitory effect of SF on the peripheral blood mononuclear leukocyte proliferative response to mitogens CON A, PWM and PHA. In these examples SF is produced by Jurkat, Molt-4, K-562 and HPB-ALL cells as well as other cell lines. In addition, hematopoietic cell hybridomas produce SF. Cess B cell line generally is positive for SF.
Administration in vivo of partially purified preparations of the Jurkat suppressor factor of the subject invention and the 160(36(38F3)) suppressor factor inhibited significantly the primary antibody response of C3H mice to sheep erythrocytes when administered intravenously (see Table IX) or intraperitoneally (data not shown). In contrast, purified preparations of the 169(36(38F3)) suppressor factor had no effect in vivo as anticipated by the in vitro experiments.
The dosages administered were as follows: 2.5 suppressor factor units (SFU) per injection given intravenously on days 1, 2, 3 and 4 in a total volume of 50 microliters. A suppressor factor unit was defined as the reciprocal of the maximum dilution that inhibited by 50% the proliferative response of human peripheral blood mononuclear leuckocytes to PHA.
The Jurkat, 153(36(38F3) and 160(36(38F3)) suppressor factors significantly inhibited the proliferative response of highly purified leukemic B cells from patients with chronic lymphocytic leukemia to preparations containing B-cell growth factor (BCGF), anti-immunoglobulin M (anti-M) and/or Staphylococcus aureas Cowans I (SAC) (see Table X).
The Jurkat suppressor factor is different from the 160(36(38F3) suppressor factor because:
(a) the Jurkat suppressor factor significantly inhibits in vitro CFU-C, whereas the 160(36(38F3) suppressor factor does not (see Table XI); and
(b) the Jurkat suppressor factor has an isoelectric point of 5.2, whereas the 160(36(38F3) suppressor factor has two isoelectric points of 5.7 and 4.7 (see FIG. 3).
The Jurkat suppressor factor is different from the 169(36(38F3)) suppressor factor because:
(a) the Jurkat suppressor factor inhibits in vitro CFU-C, whereas the 169(36(38F3)) suppressor factor does not (see Table XI); and
(b) the Jurkat suppressor factor has an isoelectric point of 5.2, whereas the 169(36(38F3)) suppressor factor has an isoelectric point of 4.7 (see FIG. 3).
The Jurkat suppressor factor, the 169(36(38F3)) suppressor factor and the 77(38F3) suppressor factor are different because the Jurkat suppressor factor suppresses in vitro proliferative responses of mouse splenocytes and thymocytes, whereas the 169(36(38F3)) suppressor factor and the 77(38F3) suppressor factor do not (see Tables XII and XIII);
The above inherent characteristics of the suppressor factors clearly demonstrate that:
(a) the Jurkat suppressor factor of the subject application is useful to inhibit immunological responses in vivo; and
(b) the Jurkat suppressor factor of the subject application is different from the T-T cell hybridoma suppressor factors.
The examples illustrated in the tables are for illustrative purposes only and are not meant to limit the invention to the examples shown. It is noted that the invention is not limited to one single suppressor factor but it is believed that SF may be more than one molecule or molecular form or, different cell lines produce different suppressor factors exhibiting similar or partially similar properties.
SF can be useful in treating or preventing graft versus host disease, also in patients who have received heterologous or autologous transplants whether of bone marrow, kidney, heart, etc. Also it would be useful in treating autoimmune diseases such as Systemic Lupus Erythematosus, Myasthenia Gravis, Rheumatoid Arthritis, Multiple Sclerosis and Allergies. SF has applications in the treatment of lympho-proliferative disorders such as leukemia as well as in the treatment of other malignancies, including human solid tumors such as lung, colon, etc.
TABLE IA__________________________________________________________________________Inhibition of proliferative response of peripheralblood mononuclear leukocytes to PHA, by SF producedby the Jurkat, HPB-All and Molt 4 lines PROLIFERATIVE RESPONSES (CPM) Jurkat HPB-All Molt-4 % suppres- % suppres- % suppres-% SF (v/v) CPM sion CPM sion CPM sion__________________________________________________________________________medium 513 .+-. 175 -- 513 .+-. 175 -- 513 .+-. 175 --medium + PHA 11058 .+-. 1883 -- 11058 .+-. 1883 -- 11058 .+-. 1883 --medium + PHA 2351 .+-. 223 79% 6318 .+-. 1689 43% 2399 .+-. 339 78%+0.015% SFmedium + PHA 2612 .+-. 337 76% 2199 .+-. 1792 35% 1118 .+-. 28 89%+0.05% SFmedium + PHA 2861 .+-. 269 74% 8800 .+-. 2172 20% 998 .+-. 105 91% +0.1% SFmedium + PHA 4404 .+-. 390 60% 1778 .+-. 111 84% 1333 .+-. 328 88% +0.5% SFmedium + PHA 3921 .+-. 277 65% 1419 .+-. 234 87% 1127 .+-. 77 89%+0.95% SFmedium + PHA 4156 .+-. 256 62% 1564 .+-. 156 86% 1130 .+-. 138 89% +1.9% SFmedium + PHA 4426 .+-. 354 60% 2186 .+-. 265 80% 1240 .+-. 82 89%+3.75% SFmedium + PHA 3512 .+-. 302 68% 2335 .+-. 319 79% 985 .+-. 36 91% +15% SFmedium + PHA 3538 .+-. 571 68% 2685 .+-. 236 76% 728 .+-. 57 93% +30% SF__________________________________________________________________________
TABLE IB__________________________________________________________________________Inhibition of proliferative response of peripheralblood mononuclear leukocytes to PHA, by SF producedby the K562, HPB-All, Molt 4 and CESS tumor cell linesPROLIFERATIVE RESPONSES (CPM)__________________________________________________________________________ K562 Molt-4 Cess HPB-LL % suppres- % suppres- % suppres- % suppres-% SF (v/v) CPM sion CPM sion CPM sion CPM sion__________________________________________________________________________medium 2279 .+-. 14 -- 2279 .+-. 14 -- 2279 .+-. 14 -- 2279 .+-. 14 --medium + PHA 56211 .+-. 3811 -- 56211 .+-. 3811 -- 56211 .+-. 3811 -- 56211 .+-. 3811 --medium + PHA 1624 .+-. 88 97% 1499 .+-. 248 97% 48729 .+-. 4699 13% 8124 .+-. 399 86%+1.87% SFmedium + PHA 1515 .+-. 190 97% 1287 .+-. 130 98% 51048 .+-. 6139 9% 8109 .+-. 816 86%+3.75% SFmedium + PHA 2091 .+-. 942 96% 1249 .+-. 132 98% 53579 .+-. 3152 5% 6687 .+-. 773 88% +7.5% SFmedium + PHA 1536 .+-. 119 97% 1225 .+-. 143 98% 59898 .+-. 5211 6% 7916 .+-. 1116 86% +15% SFmedium + PHA 2009 .+-. 254 96% 865 .+-. 101 98% 31823 .+-. 1550 43% 5434 .+-. 777 90% +30% SF__________________________________________________________________________
TABLE 1C______________________________________Inhibition of proliferative response of human peripheralblood mononuclear leukocytes to PHA, by SF producedby TALL-1, HD-Mar, SKW-3, DND-41 and HPB-MLT Cell line Supernatant** CPM % Suppression______________________________________MNC* None 4677 .+-. 1367MNC + PHA None 93646 .+-. 9369MNC + PHA TALL-1 18119 .+-. 3822 81MNC + PHA HD-Mar 9914 .+-. 1182 89MNC + PHA SKW-3 9268 .+-. 434 90MNC + PHA DND-41 28633 .+-. 3356 69MNC + PHA HPB-MLT 12136 .+-. 342 87______________________________________ *MNC: Human, peripheral blood mononuclear leukocytes. **Cell line supernatants were employed at concentration of 25% (v/v).
TABLE II__________________________________________________________________________Inhibition of proliferative response of peripheralblood mononuclear leukocytes to CON A, by SF producedby the Jurkat, HPB-All and Molt 4 lines PROLIFERATIVE RESPONSES (CPM) Jurkat HPB-All Molt-4 % suppres- % suppres- % suppres-% SF (v/v) CPM sion CPM sion CPM sion__________________________________________________________________________medium 265 .+-. 47 -- 265 .+-. 47 -- 265 .+-. 47 --medium + CON A 40792 .+-. 7959 -- 40792 .+-. 7454 -- 40792 .+-. 7454 --medium + CON A 3916 .+-. 239 90% 61751 .+-. 13610 -- 14440 .+-. 6966 65%+0.015% SFmedium + CON A 3606 .+-. 379 91% 31713 .+-. 11104 22% 4814 .+-. 580 88%+0.05% SFmedium + CON A 3889 .+-. 413 90% 12399 .+-. 2129 69% 4126 .+-. 681 90% +0.1% SFmedium + CON A 4489 .+-. 649 89% 6414 .+-. 1356 84% 3233 .+-. 688 92% +0.5% SFmedium + CON A 3671 .+-. 294 91% 3886 .+-. 1055 90% 2718 .+-. 910 93% 0.95% SFmedium + CON A 3615 .+-. 211 91% 3678 .+-. 765 91% 2244 .+-. 446 94% +1.9% SFmedium + CON A 3649 .+-. 350 91% 3879 .+-. 734 90% 26622 .+-. 401 93%+3.75% SFmedium + CON A 3605 .+-. 191 91% 3421 .+-. 404 92% 1729 .+-. 108 96% +15% SFmedium + CON A 3862 .+-. 381 91% 3637 .+-. 296 91% 855 .+-. 48 98% +30% SF__________________________________________________________________________
TABLE III______________________________________Inhibition of proliferative response of peripheralblood mononuclear leukocytes to PWM by SF producedby the Jurkat tumor cell line PROLIFERATIVE RESPONSES (CPM) Jurkat% SF (v/v) CPM % suppression______________________________________Medium 703 .+-. 84 --Medium + PWM 13119 .+-. 1792 --Medium + PWM 4631 .+-. 389 65%+1.56% SFMedium + PWM 4560 .+-. 373 65%+3.12% SFMedium + PWM 3624 .+-. 381 72%+6.25% SFMedium + PWM 4474 .+-. 306 66%+12.5% SFMedium + PWM 5095 .+-. 352 61% +25% SF______________________________________
TABLE IV______________________________________Inhibition by SF produced by the Jurkat tumorcell lines, of the proliferative responses of humanperipheral blood mononuclear leukocytes to allogeneic cells inmixed lymphocyte culture PROLIFERATIVE RESPONSES (CPM) Jurkat% SF (v/v) CPM % suppression______________________________________Medium 7382 --Medium + 1.56% SF 3849 48%Medium + 3.12% SF 3229 56%Medium + 6.25% SF 3922 47% Medium + 12.50% SF 3678 50% Medium + 25.00% SF 6159 17%______________________________________
TABLE V__________________________________________________________________________Inhibition of de novo immunoglobulin synthesis and secretion of humanperipheral blood mononuclear leukocytes in the PWM-induceddifferentiation system, by SF produced by theJurkat, HPB-ALL, Molt-4, K562 and CESS CELL LINES. Cell line Supernatants Immunoglobulin* Dilutions IgM (microg/dl) IgA (microg/dl) IgG (microg/dl)__________________________________________________________________________Mononuclear cells + PWM None 243.0 70.5 54.3 JurkatMNC + PWM 1:3 19.0 l.t. 2.0 <2.0MNC + PWM 1:50 20.5 l. t. 2.0 NDMNC + PWM 1:1000 15.0 3.75 NDMNC + PWM 1:10000 19.0 4.5 ND HPB-ALLMNC + PWM 1:3 10.5 l.t. 2.0 l.t. 2.0MNC + PWM 1:50 35.0 5.7 l.t. 2.0MNC + PWM 1:1000 27.3 11.3 l.t. 2.0MNC + PWM 1:10000 22.3 13.0 l.t. 2.0 Molt-4MNC + PWM 1:3 23.5 l.t. 2.0 NDMNC + PWM 1:50 36.7 14.3 l.t. 2.0MNC + PWM 1:1000 35.7 16.0 l.t. 2.0 K562MNC + PWM 1:3 9.9 l.t. 2.0 NDMNC + PWM 1:50 17.8 l.t. 2.0 l.t. 2.0MNC + PWM 1:1000 18.3 9.0 ND CESSMNC + PWM 1:3 12.2 l.t. 2.0 10.3MNC + PWM 1:50 18.0 l.t. 2.0 NDMNC + PWM 1:1000 18.3 11.2 ND__________________________________________________________________________ Determined by ELISA ND not determined l.t. less than
TABLE VI______________________________________The viability of peripheral blood mononuclearleukocytes is not affected by prolongedincubation with SF produced by the Jurkat*tumor cell lines % Viability Duration of treatmentTumor cell lines 20 hrs 44 hrs 68 hrs 88 hrs______________________________________Medium * alone 98% 100% 96% 93%Jurkat 99% 96% 95% 90%______________________________________ * This effect is also seen with HPBALL, K562 and Molt4 supernatants.
TABLE VII______________________________________Suppressor factor(s) preparations produced by the Jurkatcell lines do not affect natural killer cytotoxicity: mediated byperipheral blood mononuclear leukocytes, against K562 targets % Cytotoxicity Effector to target ratio Donor 1 Donor 2SF Source 50:1 25:1 50:1 25:1______________________________________Medium 66 59 58 50Jurkat 68 56 51 40______________________________________
TABLE VIII__________________________________________________________________________Inhibition of the growth of human lung tumor cell lines by SF -containing supernatants from the Jurkat, HPB-ALL, K562 and Molt-4human tumor cell lines Cell Numbers Lines SK-LC-6 SK-LC-14 Hours of Hours of Treatment TreatmentSupernatants* 0 60 90 0 60 90__________________________________________________________________________None 0.5 .times. 10.sup.5 1.4 .times. 10.sup.5 5.7 .times. 10.sup.5 0.5 .times. 10.sup.5 1.7 .times. 10.sup.5 4.2 .times. 10.sup.5Jurkat 0.5 .times. 10.sup.5 0.6 .times. 10.sup.5 1.5 .times. 10.sup.5 0.5 .times. 10.sup.5 1.4 .times. 10.sup.5 1.9 .times. 10.sup.5HPB-All 0.5 .times. 10.sup.5 1.1 .times. 10.sup.5 1.4 .times. 10.sup.5 0.5 .times. 10.sup.5 2.1 .times. 10.sup.5 3.0 .times. 10.sup.5K562 0.5 .times. 10.sup.5 0.6 .times. 10.sup.5 1.2 .times. 10.sup.5 0.5 .times. 10.sup.5 0.7 .times. 10.sup.5 2.0 .times. 10.sup.5Molt-4 0.5 .times. 10.sup.5 0.7 .times. 10.sup.5 2.7 .times. 10.sup.5 ND__________________________________________________________________________ *These were used at a dilution of 30% (v/v).
TABLE IX__________________________________________________________________________INHIBITION OF THE IN VIVO PRIMARY ANTIBODYRESPONSE OF C3H MICE TO SHEEP ERYTHROCYTIESBY THE 160(36(38F3)) SUPRESSOR FACTOR Plaque forming cells per 1 .times. 10.sup.6 splenocytes* Exp. 1 Exp. 2 Exp. 3 Exp. 4__________________________________________________________________________No immunization 17 .+-. 23 29 .+-. 9 13 .+-. 12 35 .+-. 23Sheep erythrocytes 842 .+-. 6 1092 .+-. 86 838 .+-. 59 715 .+-. 14Sheep erythrocytes 408 .+-. 77** 117 .+-. 13** 96 .+-. 7** 216 .+-. 16**plus 160(36(38F3))*Sheep erythrocytes 832 .+-. 56 936 .+-. 71 1010 .+-. 17 811 .+-. 7plus 169(36(38F3))*Sheep erythrocytes ND 93 .+-. 9** 114 .+-. 24** 111 .+-. 3**plus Jurkat*__________________________________________________________________________ *C3H mice were immunized i.p. with sheep erythrocytes and received (i.v.) on day 1 2.5 suppressor factor units (SFU) from a purified suppressor factor preparation, in a total volume of 50 microliters. Suppressor facto injections (2.5 SFU per injection) were also given i.v. on days 2, 3, and 4. The animals were sacrificed on day 5, the spleens were removed, lymphocytes were prepared and plaque forming cells were determined. A suppressor factor unit (SFU) was defined as the reciprocal of the maximum dilution that inhibited by 50% of the proliferative response of human peripheral blood mononuclear leukocytes to PHA. **Statistically significant inhibition was observed (p at least less than 0.005).
TABLE X______________________________________INHIBITION OF PROLIFERATION OF PURIFIEDLEUKEMIC B CELLS FROM PATIENTS WITH CHRONICLYMPHOCYTIC LEUKEMIA BY SUPPRESSOR FACTORSPRODUCED BY HUMAN T-T CELL HYBRIDOMAS ANDTHE JURKAT T-CELL LINEStimulatory Suppressor ProliferationAgents(s) Factor Response (cpm)______________________________________PATIENT 1None None 977None 153(36(38F3)) 99BCGF (1:32)+anti-.mu. None 54,121BCGF (1:32)+anti-.mu. 153(36(38F3)) 194SAC (1:2000) None 855SAC (1:2000) 153(36(38F3)) 126BCGF (1:32)+anti-.mu. +SAC None 64,924(1:2000)BCGF (1:32)+anti-.mu. +SAC 153(36(38F3) 192(10:2000)PATIENT 2None None 3,491None 160(36(38F3)) 206BCGF (1:32)+anti-.mu. None 14,399BCGF (1:32)+anti-.mu. 160(36(38F3)) 177BCGF (1:32)+anti-.mu. Jurkat 187______________________________________
TABLE XI__________________________________________________________________________EFFECT OF SUPPRESSOR FACTORS ON COLONY FORMATION BY HUMANHEMOPOIETIC PROGENITOR CELLS Colonies/Clusters per 10.sup.5 Non-adherent Low Density Normal Human Bone Marrow Cells Day 7 Day 14 Day 14 Day 7 CFU-GM Day 14 BFU-E BFU-E Day 14Suppressor Final CFU-GM Colonies CFU-GM Colonies Colonies CFU-GEMMFactor** Dilution Colonies + Clusters Colonies #1 #2 Colonies__________________________________________________________________________Control Medium 61 .+-. 4 111 .+-. 8 59 .+-. 2 37 .+-. 2 49 .+-. 5 8 .+-. 1160 1/20 50 .+-. 2 107 .+-. 7 56 .+-. 3 35 .+-. 3 47 .+-. 1 7.5 .+-. 0.5 1/100 57 .+-. 6 111 .+-. 14 58 .+-. 1 41 .+-. 2 49 .+-. 1 8 .+-. 1 1/1000 62 .+-. 3 113 .+-. 5 59 .+-. 4 42 .+-. 3 52 .+-. 3 7.5 .+-. 1.5169 1/20 58 .+-. 5 114 .+-. 10 58 .+-. 3 35 .+-. 3 47 .+-. 2 9 .+-. 1 1/100 63 .+-. 6 111 .+-. 3 58 .+-. 6 40 .+-. 5 52 .+-. 2 8 .+-. 1 1/1000 57 .+-. 4 106 .+-. 5 56 .+-. 3 39 .+-. 1 50 .+-. 5 9 .+-. 2Jurkat 1/20 63 .+-. 4 112 .+-. 3 23 .+-. 5 28 .+-. 3 48 .+-. 3 8 .+-. 1 1/100 64 .+-. 7 109 .+-. 5 65 .+-. 5 37 .+-. 3 52 .+-. 3 8 .+-. 0 1/1000 61 .+-. 5 110 .+-. 4 60 .+-. 4 40 .+-. 1 49 .+-. 3 8 .+-. 2160 pI 4.7 1/100 64 .+-. 4 111 .+-. 3 59 .+-. 2 38 .+-. 1 52 .+-. 4 8.5 .+-. 0.5 1/1000 62 .+-. 2 111 .+-. 1 60 .+-. 3 41 .+-. 3 53 .+-. 3 8.5 .+-. 0.5 1/10000 57 .+-. 4 114 .+-. 4 62 .+-. 4 42 .+-. 3 54 .+-. 4 7.5 .+-. 0.5160 pI 5.7 1/100 64 .+-. 5 111 .+-. 4 60 .+-. 7 39 .+-. 2 47 .+-. 2 7.5 .+-. 0.5 1/1000 60 .+-. 3 108 .+-. 1 59 .+-. 2 41 .+-. 5 53 .+-. 3 9 .+-. 1 1/10000 58 .+-. 2 110 .+-. 3 60 .+-. 5 37 .+-. 4 47 .+-. 3 10 .+-. 2__________________________________________________________________________ Results are expressed as mean +/- 1 S.E.M. for 3 plates/pt for CFUGM and plates/pt for BFUE/CFU-GEMM assays. See Broxmeyer et. al., J. Immunol. 135:2502-2506, 1986 for exact details of methods. In Brief: (1) CFUGM assay was performed in 0.3% Agar with 10% v/v 5637 CM. Colonies were 50 cells/aggregate and clusters were 3-50 cells/aggregate. Only colonies wer scored on day 14 since colonies were large and clusters difficult to distinguish. (2) BFUE assays were performed in 0.3% methylecellulose with either 0.5 units Toyoba Erythropoietin plus 0.1 mM hemin (=BFUE #1) or with erythropoietin, hemin and 10% v/v 5637 CM (=BFUE #2) BFUE #1 may be more mature than BFUE #2. CFUGEMM were scored on same plates as BFUE #2. Cultures were incubated at 37.degree. C. in low (5%) oxygen tension. **Partially purified preparations by ammonium sulfate precipitation of th 160, 169 and Jurkat suppressor factors were used. Also highly purified preparations by chromatofocusing of the 160 suppressor factor (two peaks of activity of pI 5.7 and 4.7) were evaluated. These preparations were highly effective in suppressing proliferative responses of human peripheral blood mononuclear leukocytes to PHA dilutions higher than 1:100,000.
TABLE XII__________________________________________________________________________INHIBITION OF PROLIFERATIVE RESPONSES OF BALB/cSPLEEN LYMPHOCYTES TO CON A BY SUPPRESSOR FACTORSPRODUCED BY HUMAN T-T CELL HYBRIDOMAS Counts per minuteSuppressor Con A Con AFactor Medium 3 .mu.g/ml 6 .mu.g/ml__________________________________________________________________________Control 3.451 .+-. 810 116,557 .+-. 20,248 82,024 .+-. 17,853160(36(38F3))2.5% (v/v) 5,615 .+-. 708 8,709 .+-. 573 9,656 .+-. 804 5% 5,835 .+-. 370 8,174 .+-. 922 8,954 .+-. 52010% 5,750 .+-. 1,266 7,312 .+-. 1,565 8,041 .+-. 45820% 5,628 .+-. 814 6,260 .+-. 1,042 5,864 .+-. 669159(36(38F3))2.5% (v/v) 7,624 .+-. 355 151,540 .+-. 4,885 100,230 .+-. 17,377 5% 8,778 .+-. 856 126,152 .+-. 26,677 92,750 .+-. 18,48310% 6,604 .+-. 552 44,436 .+-. 5,503 15,262 .+-. 4,60420% 2,570 .+-. 536 5,107 .+-. 1,392 4,538 .+-. 1,991169(36(38F3))2.5% (v/v) 7,404 .+-. 55 167,288 .+-. 2,362 99,033 .+-. 499 5% 8,502 .+-. 1,566 141,004 .+-. 12,097 84,857 .+-. 6,47510% 8,076 .+-. 883 117,821 .+-. 9,652 82,417 .+-. 6,57120% 3,468 .+-. 431 124,251 .+-. 8,489 104,251 .+-. 8,48977(38F3)2.5% (v/v) 7,120 .+-. 426 109,377 .+-. 4,615 86,640 .+-. 6,361 5% 7,905 .+-. 970 141,647 .+-. 9,880 74,975 .+-. 2,83510% 6,723 .+-. 970 158,123 .+-. 32,110 73,488 .+-. 4,37120% 4,295 .+-. 266 138,389 .+-. 26,418 110,036 .+-. 7,561Jurkat2.5% (v/v) 12,721 .+-. 510 3,215 .+-. 751 3,024 .+-. 1,071 5% 7,012 .+-. 498 3,250 .+-. 138 2,941 .+-. 24610% 3,875 .+-. 802 2,995 .+-. 246 3,390 .+-. 30920% 3,447 .+-. 1,044 4,226 .+-. 387 3,945 .+-. 436__________________________________________________________________________ BALB/c mouse spleen lymphocytes were cultured in RPMI1640 supplemented with 10% fetal calf serum, HEPES buffer, glutamine and antibodies in flat bottomed microtiter plates with Con A and supernatants containing suppressor factors. After three days 37.degree. C., the cultures were pulsed with .sup.3 Hthymidine and harvested 18 hrs. later.
TABLE XIII__________________________________________________________________________INHIBITION OF PROLIFERATIVE RESPONSES OF BALB/cMOUSE THYMOCYTES TO CON A BY SUPPRESSOR FACTORSPRODUCED BY HUMAN T-T CELL HYBRIDOMAS Counts per minuteSuppressor Con A Con AFactor Medium 3 .mu.g/ml 6 .mu.g/ml__________________________________________________________________________Control 2,276 .+-. 129 180,972 .+-. 17,909 221,760 .+-. 27,796160(36(38F3))2.5% (v/v) 3,307 .+-. 971 3,237 .+-. 170 3,755 .+-. 106 5% 3,088 .+-. 1,788 3,957 .+-. 1,092 4,786 .+-. 59310% 2,969 .+-. 940 4,977 .+-. 2,321 5,228 .+-. 32520% 3,571 .+-. 1,994 5,016 .+-. 938 5,536 .+-. 605159(36(38F3))2.5% (v/v) 225 .+-. 19 4,132 .+-. 521 15,435 .+-. 5,486 5% 963 .+-. 128 3,271 .+-. 111 3,553 .+-. 98710% 2,469 .+-. 294 3,050 .+-. 91 2,242 .+-. 45720% 3,000 .+-. 84 8,295 .+-. 2,455 2,700 .+-. 211169(36(38F3))2.5% (v/v) 2,673 .+-. 152 166,300 .+-. 19,495 247,600 .+-. 56,223 5% 2,088 .+-. 467 180,519 .+-. 12,662 302,994 .+-. 71,94910% 3,856 .+-. 428 192,006 .+-. 43,278 286,007 .+-. 57,48920% 4,659 .+-. 1,279 202,671 .+-. 19,523 298,623 .+-. 16,52477(38F3)2.5% (v/v) ND 124,507 .+-. 18,981 234,600 .+-. 21,645 5% 147,000 .+-. 29,060 208,890 .+-. 21,56310% 172,036 .+-. 38,984 293,000 .+-. 57,38420% 157,127 .+-. 40,534 235,660 .+-. 41,730Jurkat2.5% (v/v) 5,568 .+-. 573 5,746 .+-. 708 3,894 .+-. 513 5% 6,705 .+-. 1,583 5,310 .+-. 441 4,039 .+-. 62910% 5,546 .+-. 312 7,089 .+-. 2,124 4,626 .+-. 2,20520% 3,438 .+-. 596 7,335 .+-. 1,100 5,953 .+-. 2,542__________________________________________________________________________ BALB/c thymocytes were cultured in RPMI1640 supplemented with 10% fetal calf serum HEPES buffers glutamine and antibodies in flat bottomed microtiter plates with Con A and supernatants containing suppressor factors (1 .times. 10.sup.6 cells per well in 200 microliters). After three days at 37.degree. C. the cultures were pulsed with .sup.3 Hthymidine and harvested 18 hrs. later.
MATERIALS AND METHODS
Isolation of peripheral blood mononuclear leukocytes and depletion of monocytes. Mononuclear cells from normal donors were isolated by centrifugation on a Ficoll/Hypaque density cushion (Boyum, A. (1968) Scand. Clin, Lab. Invest. 21:(Suppl. 97) 77), at room temperature. The cell were washed three times in Hanks, balanced salt solution (HBSS) and resuspended in RPMI-1640 containing 15% heat-inactivated fetal calf serum at a concentration of 4.times.10.sup.6 cells/ml. Lymphocyte separator reagent (Technicon Instrument Co., Tarrytown, N.Y.) was added to the mononuclear cell suspension at a volume ration of 1:2 and the mixture was incubated at 37.degree. C. on a rotator for 30 min. Phagocytic cells were depleted by subsequent centrifugation at 400.times.g for 20 min on a Ficoll/Hypaque density cushion. Lymphoid cells depleted of phagocytic cells were collected from the interface, washed three time with Hank's balanced salt solution (HBSS) and resuspended at 4.times.10.sup.6 cells/ml.
Preparation of T lymphocytes. T lymphocytes were prepared by rosetting with neuraminidase-treated sheep erythrocytes (SRBC) (25 units/ml of 5% SRBC) followed by centrifugation on Ficoll/Hypaque as previously described [Platsoucas et al. (1980) J. Immunol. 125:1216]. Two-milliliter aliquots of lymphocytes (4.times.10.sup.6 /ml) in HBSS were mixed with 0.5 ml of heat-inactivated and SRBC-absorbed fetal calf serum and 2 ml of 1% neuraminidase treated SRBC. The mixture was incubated for 5 min at 37.degree. C., centrifuged for 5 min at 200.times.G, and incubated at 4.degree. C. for an additional hour. The rosettes were resuspended carefully and incubated on ice for an additional 15 min. The cell suspensions were layered on a Ficoll/Hypaque density cushion and centrifuged at 400.times.G for 20 min at controlled temperature (22.degree. C.). Non-T cells were recovered from the interface and were washed three times with HBSS. Rosetting T cells were recovered from the pellets after lysis of attached SRBC by Tris-buffered 0.83% ammonium chloride (pH 7.2). The T cells were washed three times with HBSS. E-rosetting cells prepared by this method were more than 95% T lymphocytes, as determined by rerosetting with SRBC without nonspecific esterase-positive cells and less than 2% immunoglobulin-bearing cells. E-rosette-negative cells contained more than 70% surface immunoglobulin cells, as determined by immunofluorescence, and less than 1% of E-rosette forming cells or non-specific esterase positive cells. These cells were used as B cells. B cells are used for B cell growth factor assay.
Proliferative response to mitogens. Human peripheral blood mononuclear leukocytes (at a concentration of 1.times.10.sup.6 cells/ml) were cultured in RPMI-1640 containing 10% fetal calf serum and supplemented with 25 mM Hepes, 2 mM L-glutamine and 100 units/ml Penicillin and 100 micrograms/ml streptomycin. One hundred microliters of the cell suspension were stimulated on U-microliter plates (Scientific Products) by various concentration of mitogens (PHA-P, Con A, PWM) at 37.degree. C. in a humidified incubator in a 5% CO.sub.2, 95% air environment. The cultures were pulsed with 25 microliters of tritiated thymidine (specific activity, 6.7 Ci/mmol, New England Nuclear, Boston, Mass.) after 72 hours and harvested using an automatic cell harvester 24 hour after the addition of the isotopes.
Mixed lymphocyte culture. Human peripheral blood mononuclear leukocytes from various donors were prepared as above.
Responding cells (1.times.10.sup.5) were cultured with 1.times.10.sup.5 stimulating cells in round bottom microtiter plates in total volume of 0.2 ml. The stimulating cells were inactivated by x-irradiation (2000 rads). The culture medium is RPMI-1640 supplemented with 10% fetal calf serum, 25 mM Hepes, 2 mM L-glutamine, penicillin (100 units/ml) and streptomycin (100 microgram/ml). The cultures were incubated at 37.degree. C. in a humidified atmosphere with 5% CO.sub.2, pulsed the 5th day with 1 micro Ci/well of .sup.3 H-thymidine (New England Nuclear, Boston, Mass., specific activity 6.7 Ci/mmole) (Skarton, Norway). All cultures were performed in quadruplicate.
Natural killer cytotoxicity. Natural killer cytotoxicity was determined as previously described (Platsoucas, et al. (1980) J. Immunol. 125:1216). Target cells of the K562 and Molt-4 lines, maintained in RPMI-1640 supplemented with 10% fetal calf serum, glutamine and antibiotics as above were labelled with 300 microliters of .sup.51 Cr per 2.times.10.sup.6 cells [sodium (.sup.51 Cr) chromate, New England Nuclear, Boston, Mass.] for 2 hours. The target cells were washed three times and then resuspended in the same medium, at a concentration of 5.times.10.sup.4 cells/ml. Effector lymphocytes were washed three times in RPMI-1640 supplemented with 10% fetal calf serum, arranged at the appropriate concentration and one hundred microliters were added to one hundred microliters of target cells in U-bottom microtiter plates (Nunclon, Denmark), to achieve effector to target ratios 100:1, 50:1, and 25:1 etc. The plates were centrifuged at 40.times.g for 2 min and subsequently incubated at 37.degree. C. in a humidified 5% CO.sub.2 atmosphere. After 4 hours, the plates were centrifuged at 500.times.g for 5 min and 100 microliters of the supernatants were collected and counted for .sup.51 Cr release in a well-type Auto Gamma scintillation counter.
Percent specific lysis is calculated by the formula: ##EQU1## where E=mean cpm released in the presence of effector cells. S=mean cpm spontaneously released by target cells incubated with medium alone, and T=mean cpm released after treating target cells with Triton .times.100 (1:100 dilution).
Cell viability. Peripheral blood mononuclear leukocytes (MNL) were cultured at a concentration of 1.times.10.sup.6 cells/ml in RPMI 1640 supplemented with 10% heat-inactivated FCS, 25 mM herpes buffer, 2 mM L-glutamine, and the antibiotics streptomycin 100 micrograms/ml and penicillin 100 U/ml for up to 88 hrs.
Control samples were incubated in the above medium and test samples are incubated with SF produced by Jurkat et al cell lines. Samples were withdrawn at 20 hrs, 44 hr, 68 hrs and 88 hrs. Cells were washed 2.times. and viability was determined by Trypan blue dye exclusion.
Origin of cells. Cell line used in the specification were established cell lines from patients with various leukemias.
M.W. Determinations. These were carried out by AcA-44 ultrogel (LFB) filtration in isotonic phosphate buffered saline (PSB). Molecular weight markers employed involved:
______________________________________Bovine serum albumin 68,000 M.W.Ovalbumin 43,000 M.W.Cytochrome c 11,700 M.W.______________________________________
Induction of de novo Ig synthesis and secretion by human peripheral blood mononuclear leukocytes in the PWM-induced differentation cysts. Human peripheral blood mononuclear leukocytes were cultured at 1.times.10.sup.6 cells/ml in RPMI-1640 supplemented with 10% heat inactivated fetal calf serum, 2 mM glutamine, Hepes and antibiotics as previously described, in total volume of 2 ml, for 7 days at 37.degree. C. in 5% CO.sub.2 in a humidified incubator. Pokeweed mitogen (10 microgram/ml, optimal concentration; Grand Island Biological Co., Grand Island, N.Y.) was added from the beginning of the culture. After incubation for 7 days the tubes were centrifuged at 400.times.g and supernatants were carefully withdrawn and stored at -20.degree. C. until assayed for immunoglobulin.
Determination of de Novo IgG, IgA, IgM immunoglobulins by enzyme-linked immunoabsorbent assay (ELISA). These determinations were carried out by a modification of the method described by Engvall and Perlmann J. Immunol. 109:129 (1972). Rabbit anti-human immunoglobulin antibody, heavy chain specific (mu, gamma or alpha) (Accurate Chemical) were arranged at a concentration of 5 g/ml in 0.10M Na.sub.2 CO.sub.3, pH 9.6, containing 0.05% sodium azide. Two hundred microliters of antibody solution per well were transferred into 96-well round bottom microliter plates and incubated at 37.degree. C. for 3 hours. The plates were stored at 4.degree. C. until use and were stable for over two weeks. Before use the plates were washed with PBS containing 0.02% Tween 20 three times and were allowed to remain at room temperature for 5 min, between washings. Several dilutions of the unknown immunoglobulin containing supernatants were prepared in PBS containing 0.02% Tween 20 and volumes of 0.2 ml will be transferred to the plates. The plates were incubated for 5 hours at room temperature, on a rocket platform.
Supernatants were removed by aspiration and the tubes were washed three times with PBS, containing 0.02% Tween 20. Alkaline phosphatase conjugated rabbit anti-human immunoglobulin antibody heavy chain specific was obtained from AMP Immunoreagents Inc., Sequin, Tex. (gamma.degree. mu.degree., or alpha.degree. heavy chain specific). Before use the conjugates were absorbed with 1% ovalbumin solution in phosphate buffered saline (1 hour at room temperature), to absorb extra gluteraldehyde. One ml of conjugate diluted 1:500 with PBS - Tween 20, was added to the anti-human Ig-human Ig coated tubes, and the tubes were incubated for 16 hours at room temperature. Subsequently, the unbound conjugate was removed by washing the plates three times with PBS Tween 20. The amount of bound alkaline phosphatase rabbit anti-human heavy chain specific, was determined using p-nitrophenylphosphate (NPP) (Sigma) as a substrate. One ml of 1 mg/1 NPP, in 0.05M sodium carbonate buffer (pH 9.8) containing 10.sup.-3 M MgCl.sub.2 was added to the plates and the released p-nitrophenolate was measured at 405 nm after one hour, using a titertek ELISA reader. Standard curves were constructed using purified IgG, IgA or IgM immunoglobulins for polyclonal immunoglobulin secretion, or purified paraproteins from patients with multiple myeloma for the determination of idiotypic immunoglobulin secretion.
PBS=phosphate buffered saline
Inhibition of the growth of human tumor cell lines by supernatants containing the SF. Cells from human tumor lung lines were arranged at a concentration of 5.times.10.sup.4 cells/ml, in Minimal Essential Medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine and antibiotics (streptomycin 100 microgram/ml and penicillin 100 U/ml). One ml of cell suspension was transferred to 24 well plates. Cultures were set up in triplicate. After the cells were attached, supernatants containing SF(s) were added. After 60 and 90 hours in culture, the cells were detached with EDTA (final concentration 0.28%) and counted. Isolation of suppressor factor(s). Supernatants containing suppressor factors are first concentrated using either ammonium sulfate precipitation (80%) (by the method described by Dixon, Biochem. J. 54:457, 1953) or concentration by Amicon ultrafiltration. Subsequently the concentrated factor preparation is subject to gel filtration or ionic exchange DEAE-high pressure liquid chromatography (HPLC) or preparative chromatofocusing. These isolation procedures can also be applied sequentially.
Gel filtration of the 160, 169, 153 and Jurkat suppressor factors was carried out by standard procedures using ACA-44 ultragel (LKB, Bromma, Sweden). Representative results are shown in FIG. 1. These factors had a molecular weight in the range of 50,000-70,000.
Studies were carried to purify further these factors by ionic exchange HPLC using protein PAK DEAE 5PW columns, by the method of Strahler et al. (J. Chromatography 266:281, 1983). Representative results are shown in FIG. 2. Elution of the suppressive activity was achieved by using a linear gradient of 0.02M. tris, pH 7.8 to 0.02M tris, pH 7.8, 0.5M NaCl. The suppressive activity of the 160 factor was eluted from the column by 0.35M NaCl. The 169 suppressor factor exhibited a similar elution profile.
Chromatofocusing (Sluyterman and Elgersman, J. Chromatography 150:17, 1978); Sluyterman and Wijdenes, J. Chromatography 150:31, 1978) of the suppressor factors was carried out by applying solubilized precipitates obtained from ammonium sulfate concentrated suppressor factor or suppressor factor preparations purified by the methods described above [dialyzed against 25 mM imidazole (Aldrich), pH 7.4 for 24 hr at 4.degree. C.] to Polybuffer Exchanger 94 (PBE 84) ionic exchange media (Pharmacia) equilibrated with imidazole buffer]. The flow rate during loading of the column with suppressor factor and focusing was 50 ml/cm.sup.2 /hr and it was generated using a 2132 microperpex peristaltic pump. Focusing was carried out using polybuffer 74 (PB 74), pH 3.80, to facilitate the formation of a linear pH gradient. The focused material eluted from the column was collected in 5 ml fractions or less using a 2211 superac fraction collector and a 2238 Uvicord SII continuous flow spectrometer to measure eluent optical density at 280 nm (LKB). The pH of each collected fraction was determined and the endpoint of the chromatofocusing run was identified when the pH of the eluent reached the pH of PB 74 (pH 3.8). The fractions were brought to pH of 7.2, and their ability to suppress proliferative responses of PBMC to PHA was tested.
Chromatofocusing analysis of the 160 suppressor factor revealed activity peaks at isoelectric points of 4.7 and 5.7. The 169 suppressor factor revealed one peak of activity at isoelectric point of 4.7. In contrast, the Jurkat-derived suppressor factor exhibited one peak of suppressive activity at isoelectric point of 5.2 (FIG. 3). Inhibition of proliferation (90% or higher) by factor material present in these peaks was evident at a dilution of at least 1:100,000. On the basis of the distinct isoelectric points and their different functional properties (see previous amendments) it was concluded that the hybridoma-derived 160 and 169 factors and the Jurkat-derived factor are different.

Number	Name	Date
4464355	Fabricius et al.	Aug 1984
4468379	Gottlieb	Aug 1984
4486409	Goldfarb	Dec 1984

Cellular proliferation T cell suppressor factor

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