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Depletion of murine T cells by in vivo monoclonal antibody treatment is enhanced by adding an autologous anti-rat κ chain antibody
Journal of Immunological Methods, 111 (1988) 219-226 Elsevier
219
JIM04817
Depletion of murine T cells by in vivo monoclonal antibody treatment is enhanced by adding an autologous anti-rat x chain antibody T.J. Goldschmidt 1,2, R. Holmdahl 1 and L. Klareskog a t Department of Medical and Physiological Chemistry, University of Uppsala, The Biomedical Center, Box 575, S-751 23 Uppsala, Sweden, and 2 Department of Experimental Medicine, Pharmacia A B, S-751 82 Uppsala, Sweden (Received 22 December 1987, accepted 22 February 1988)
In vivo treatment with monoclonal antibodies can be used for elimination of various T lymphocyte subsets from peripheral lymphoid organs and blood and thereby be used both to analyze the role of different T cells in immunoregulation and for the treatment of experimental immunological diseases. However, one problem with this approach has been that not all monoclonal antibodies given in vivo eliminate their target cells. We now show in the murine system that the normally inefficiently depleting H129.19 (anti-CD4) and 53.6.7 (anti-CD8) antibodies can be used for efficient depletion of their respective target cells when combined with injection of a secondary mouse anti-rat x (MAR18.5) antibody. The efficacy of the depletion protocols was ascertained by double staining techniques and cytofluorometric analysis. It is suggested that the presently used sandwich method applying a homologous secondary monoclonal antibody may provide an alternative to class switching or other manipulations of primary antibodies in increasing the efficacy of in vivo antibody treatment. Key words: Monoclonal antibody therapy; CD4 T cell; CD8 T cell
Introduction During recent years techniques have been developed for the depletion of lymphoid cells in vivo by treatment with monoclonal antibodies. Treatment of mice in vivo with monoclonal antibodies directed to surface markers on T lymphocytes has, for example, been used to alter the immune response in various systems. Thus, it has been possible to block humoral responses (Cobbold et al., 1984; Coulie et al., 1985; Wofsy et al., 1985;
Correspondence to: T.J. Goldschmidt, Department of Medical and Physiological Chemistry, The Biomedical Center, University of Uppsala, Box 575, S-751 23 Uppsala, Sweden.
Ranges, 1987) and to induce tolerance to specific antigens (Benjamin et al., 1986; Gutstein et al., 1986; Waldman et al., 1987), to delay allograft rejection (Cobbold et al., 1987) and to retard experimental autoimmune diseases (Holmdahl et al., 1985; Ranges et al., 1985; Waldor et al., 1985; Wofsy, 1986). However, a major shortcoming of using various monoclonal anti-T cell antibodies for the investigation of in vivo immune processes by cell depletion is that some antibodies do not effectively eliminate their targets in vivo. Whether a monoclonal antibody will eliminate their target or not has been suggested to depend both on the structure of the Fc part of the antibodies (Ledbetter et al., 1979; Cobbold et al., 1984; Kaminski et al., 1986) and on the properties of the
0022-1759/88/$03.50 © 1988 Elsevier Science Pubfishers B.V. (Biomedical Division)
220 surface marker recognized by the antibody (Howard et al., 1982). One of the most commonly used antibodies for depletion studies in the mouse is the GK1.5 antibody which is specific for the CD4 receptor on murine T cells (Dialynas et al., 1983). Treatment with this rat IgG2b antibody effectively eliminates most CD4 + cells in vivo (Ranges et al., 1985; Waldor et al., 1985; Wofsy et al., 1985). Another rat monoclonal antibody directed against a spatially related determinant on the CD4 receptor is H129.19 (Pierres et al., 1984) which is of the IgG2a isotype. The results in this study show that this antibody binds to, but does not eliminate, the CD4 + target cells in vivo. Using these antibodies we report the development of a new approach for in vivo depletion of T cells which is based on the additional treatment with a homologous anti-rat antibody, MAR18.5 (Lanier et al., 1982).
Materials and methods Animals
Female D B A / 1 mice were bred and kept at the Biomedical Center in Uppsala. All mice were 8-12 weeks old and had an approximate weight of 20 g. All mice were age-matched at the beginning of the experiment. Cell culture
Hybridomas were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with glutamine 10 mM, penicillin 100 U / m l , streptomycin 100 # g / m l , 2-mercaptoethanol 5 x 10 -5 M and fetal calf serum 6-10% (KC Biological, Lennexa, KS). Some monoclonal antibodies were produced in serum-free medium supplemented by insulin (10 t~g/ml), transferrin (10/zg/ml) (Sigma, St. Louis, MO), ethanolamine (20 /~M) and nonessential amino acids (1%) (Flow Laboratories, U.K.) before being subjected to chromatography purification. Monoclonal antibodies
H y b r i d o m a supernatants were stored at - 2 0 o C. The antibodies were purified either by affinity chromatography on Protein A-Sepharose (Pharmacia, Uppsala, Sweden) (MAR18.5) or with
TABLE I CHARACTERISTICS OF MONOCLONAL ANTIBODIES USED FOR IN VIVO TREATMENT AND FOR IN VITRO ANALYSIS Hybridoma H129.19 MAR18.5 GK1.5 53.6.7 3A7 53.7.3 a
Isotype Rat IgG2a MouseIgG2a Rat IgG2b Rat IgG2a Rat IgG a Rat IgG2a
Target antigen CD4 (L3T4) Rat x chain CD4 (L3T4) CD8 (Ly2) Thyl CD5 (Lyl)
Reference Pierres(1984) Lanier(1982) Dialynas(1983) Ledbetter(1979) L~gdberg (1985) Ledbetter(1979)
The IgG subclass is not known.
cation exchange chromatography and gel filtration (Carlsson et al., 1985) (H129.19, 53.6.7 and GK1.5). In some experiments antibodies were used which had been concentrated by ammonium sulfate precipitation. The specific antibody concentrations were determined by ELISA using the corresponding purified monoclonal antibody as a standard (Holmdahl et al., 1985). All antibody preparations used for in vivo treatments were dialyzed against PBS and sterile filtered. No difference in in vivo depletion effects could be detected between purified antibodies and antibodies only subjected to enrichment and concentration by ammonium sulfate precipitation. Biotinylation of purified antibodies was performed as previously described (Holmdahl et al., 1986). The antibodies denoted 53.6.7 (anti-CD8), 53.7.3. (anti-CD5) and 3A7 (anti-Thyl) were used in the form of hybridoma supernatants. The various monoclonal antibodies that were used for in vivo treatment and for FACS analysis are listed in Table I. In vivo treatment
The mice were thymectomized at the age of 8-12 weeks, (day - 1 4 ) . 2 weeks later, on day - 1 and day 0, the anti-mouse T cell monoclonal antibodies (H129.19, GK1.5 and 53.6.7, respectively) were injected intraperitoneally. In the cases of combined treatment, the MAR18.5 antibody was injected intraperitoneally 1 h after injection of the primary antibodies (H129.19 and 53.6.7 respectively). Immunization
After the monoclonal antibody treatment on day 0, the mice were immunized by intradermal
221 injections of Freund's complete adjuvant, emulsified in phosphate-buffered saline (PBS), in the base of the tail and in both hind foot pads. The immunization was performed in order to enlarge the otherwise small lymph nodes to facilitate further phenotypic analyses of the cells within these lymph nodes. This treatment was accompanied by a relative decrease in the numbers of T ceils in the enlarged lymph nodes which explains the somewhat lower number of lymph node cells staining for the different T cell antibodies in this study as compared to certain other studies.
Fluorescence analysis Popliteal and inguinal lymph nodes were removed on day 4 - 5 and separate single cell suspensions were prepared from each mouse. The staining procedure of the lymph node cells was then performed in 96 well microtitration plates (Nunc, Copenhagen, Denmark). In the case of single staining, a two-step procedure was performed. 50 /~1 of a cell suspension containing approximately I x 106 cells/ml were incubated for 10 min at room temperature (or 30 min at 4 ° C), with 50/~1 of rat monoclonal antibody specific for the mouse T cell surface markers CD4, CD8 or CD5. As a control for non-specific binding, normal rat serum Ig ( N R I G ) was used. After incubation the cells were washed twice in PBS supplemented with 0.5% bovine serum albumin (Sigma, St. Louis, MO) to avoid plastic adherence and with 0.02% sodium azide to prevent capping of the surface
markers (Howard, 1982) and centrifuged 2 - 3 min between the different steps. Cell-bound rat antibody was then visualized by an affinity-purified F I T C (fluorescein isothiocyanate)-conjugated goat anti-rat Ig monoclonal antibody, which had been absorbed with mouse serum (Kirkegaard and Perry Laboratories, Gaithersburg, MD). In the case of double staining a purified biotinylated rat anti-mouse T cell monoclonal antibody was subsequently added to the cells. Binding of biotinylated antibodies was then visualized by a phycoerythrin-conjugated streptavidin (Becton Dickinson Immunocytometry Systems, Mountain View, CA). In order to avoid binding of the biotinylated rat antibody to free sites on the FITCconjugated goat anti-rat antibody, the single stained cells were first saturated with excess of N R I G . The cells were then analyzed by fluorescence-activated flow cytometry in a FACStar (Becton Dickinson). Lymphocytes were selected by forward and right angle light scattering and the viability was checked by exclusion of propidium iodide (Festin et al., 1987). In each sample 4000-8000 cells were counted. Results and discussion
Treatment with a combination of H 1 2 9 . 1 9 + MAR18.5 but not with H129.19 alone efiminates CD4 + cells from peripheral lymph nodes. In an initial experiment thymectomized mice were treated in vivo with GK1.5, with H129.19 or
TABLE II CYTOFLUOROMETRIC ANALYSIS OF CD4÷ LYMPH NODE CELLS AFTER TREATMENT WITH CD4÷ SPECIFIC MONOCLONALANTIBODIES Monoclonal antibody treatment (dose)
% CD4 + lymph node cells at various times after treatment 5 days a 8 weeks b 14 weeks b
% Thyl+, CD8lymph node cells 14 weeks c
Untreated control GK1.5 (2 x 100/~g) H129.19 (2 × 100/~g) H129.19 (2 x 100/Lg)+ MAR18.5 (2 × 100 t~g)
21 3 19 4
n.d. 12 23 9
28 6 n.d. 6
n.d. d 9 17 7
a Each value represents one mouse. Results were confirmed in three additional non-immunized mice. b Each value represents one mouse. c The double stainings at 14 weeks were performed on the same lymph node cells as the single stainings. d Not done.
222 with a combination of H129.19 and MAR18.5 antibodies. 5 days later lymph node cells from the mice were analyzed by cytofluorometry for effects on the CD4 ÷ cell subset (Table II). Both GK1.5 and combined H129.19 + MAR18.5 treatment substantially reduced the numbers of cells stained for CD4. The lymph node cells from the mice treated with H129.19 alone were, however, not different from untreated controls w h e n staining for CD4. This pattern was also seen when the analyses were carried out 8 and 14 weeks after antibody injection which indicated that the target cells had been eliminated rather than the CD4 receptor having been modulated. Thus if the antibodies had only modulated the CD4 receptors, their reappearance could be expected to occur when the added antibodies had been cleared from the circulation (MacDonald et al., 1986; Pedersen et al., 1987). To further confirm that the CD4 ÷ cells were eliminated we performed double staining with 53.6.7 (anti-CD8) and 3A7 (anti-Thyl) antibodies of the remaining cells 14 weeks after treatment (Fig. 1). Double staining for the p a n - T
CD8
cell marker Thyl and for CD8 on lymph node cells from untreated mice showed three distinct populations: (1) Thyl-, C D 8 - non-T cells; (2) CD8 +, Thyl ÷ T cells; (3) Thyl ÷, C D 8 - T cells which are mainly CD4 ÷ cells (Waldor et al., 1985). If the mice were anti-CD4 treated by either GK1.5 or H129.19 + MAR18.5, a reduction of Thyl ÷, C D 8 - lymph node cells was seen, corresponding to the reduction of CD4 ÷ cells detected by single staining by H129.19, indicating that the CD4 ÷ T cells were indeed eliminated (Table II). The elimination of C D 4 ÷ cells after GK1.5 or H129.19 + MAR18.5 was, however, not complete neither at 5 days nor at a longer time after injection. As indicated in Fig. 2 it was not possible to eliminate all cells with increasing amounts of antibody since there was no major difference between a total dose of 2 0 / , g compared with a total dose of 2 0 0 / , g H129.19. The reason for the inability to deplete the remaining 2-5% is unknown. Possible explanations are the existence of inaccessible CD4 + cells in the peripheral tissue, refractory CD4 ÷ non-T cells (Jefferies et al., 1985) or cells
..
-.
.. b ) CD8
"
"
"
Thyl
Fig. 1. Flow cytometric analysis of double stained lymph node cells 4 days after in vivo anti-CD4 monoclonal antibody treatment. The cells were stained by an anti-Thyl pan-T cell marker monoclonal antibody, 3A7 (visualized by FITC-conjugated goat anti-rat Ig) and with biotinylated anti-CD8 monoclonal antibody, 53.6.7 (visualized by phycoerythrin-conjugated streptavidin), a shows lymph node cells from an untreated thymectomizedcontrol mouse and b shows lymph node cells from a thymectomizedmouse treated with H129.19 (200/*g) + MAR18.5 (200 ~tg).
223
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Fig. 2. Results from flow cytometry analyses of single stained l y m p h node cells obtained at 4 days after in vivo monoclonal antibody treatment in thymectomized mice. In a is seen a chequerboard titration of various doses of the monoclonal antibodies H129.19 and MAR18.5 administrated in vivo. The ordinate shows percent l y m p h node cells obtained 4 days after monoclonal antibody treatment and stained with H129.19 + FITC conjugated anti-rat Ig. The abscissa shows the administered dose of MAR18.5. Each point represents one mouse. The experiment shown is one of two experiments which yielded similar results. In b is seen staining on lymph node cells from an untreated mouse and from mice treated with either H129.19 or with H129.19 in combination with MAR18.5. The lymph node cells were stained with either H129.19 or normal rat Ig (NRIG) as primary antibodies and with FITC conjugated goat anti-rat Ig as secondary antibody. Each pair of N R I G or CD4 stained bars represents one mouse. The result shown is one of two experiments.
with a low density of CD4 receptors. Furthermore, the resident fraction of CD4 ÷ cells slightly increased with time, an expansion that was not dependent on the thymus since the mice had been
thymectomized. Several explanations for this expansion are possible and include the proliferation of remaining peripheral mature CD4 + cells or repopulation of the lymph nodes with immature
224 20 -la- 0 ,ug 53.6.7 •.e- 20 ~g 53.6.7 66 )Jg 53.6.7 -0- 200 ug 53.6.
(,9
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200
Fig. 3. Results from flow cytometryanalyses of single stained lymph node cells obtained 4 days after monoclonal antibody treatment of thymectomizedmice with various doses 53.6.7 and MAR18.5 antibodies. The ordinate shows percent lymph node cells stained with 53.6.7 followed by FITC-conjugatedgoat anti-rat Ig. The abscissa shows the administrated dose of MAR18.5. The indicated total doses of monoclonal antibodies represents the sum of two intraperitoneal injections given on 2 consecutive days. Each point represents one mouse. The experiment shownis one of two experiments which gave similar results.
CD4 + T lymphocytes, something that is known to occur in nude mice (Hunig, 1983).
An excess of MAR18.5 versus H129.19 is optimal for in oivo depletion To investigate which concentrations of H129.19 and MAR18.5 were optimal for in vivo depletion a chequerboard titration experiment was performed, the results of which are shown in Fig. 2. The antibodies H129.19 or MAR18.5, when given alone, did not have any detectable depletion effect. In the combined treatment of H129.19 + MAR18.5 the efficacy of depletion was critically dependent on the dose of MAR18.5. The optimal dose was the same or greater than that of H129.19. Essentially the same degree of depletion was obtained with all concentrations of H129.19 (20, 66 and 200/Lg) provided that MAR18.5 was given in an equal amount or in excess. However, a tendency towards an H129.19 dependent effect could be seen when the MAR18.5 was given in high doses (200 #g). The most likely explanation for the observed phenomena is that an excess of H129.19 versus MAR18.5 was ineffective since the administered MAR18.5 appears to bind to free H129.19, instead of to cell bound H129.19. Flow cytometric analysis of lymph node cells obtained from mice treated with H129.19 alone
showed positive staining even when no primary H129.19 antibodies were added in the staining procedure (Fig. 2b). The numbers of cells stained by the secondary antibody only in H129.19 treated mice were similar to the numbers of H129.19 stained cells in lymph nodes from untreated mice. These data suggest that when injected alone the H129.19 antibodies most likely bind to the cell surface neither modulating the CD4 receptors nor eliminating the CD4 ÷ cells.
Efimination of CD8 + target cells using the 53.6. 7 antibody is facilitated by the addition of MAR18.5 antibodies To investigate whether an enhanced depletion effect using a combination of primary antibody and MAR18.5 treatment, could be used for antibodies other than H129.19, we also analyzed the in vivo effects of treatment with the anti-CD8 specific, rat monoclonal IgG2a antibody, 53.6.7. Similar chequerboard titration experiments to those performed with H129.19 (anti-CD4) and MAR18.5 were carried out (Fig. 3) and the results, as analyzed by double staining on lymph node cells, are shown in Fig. 4 (in this case anti-CD5 was used as the pan-T cell marker). The results obtained were similar to those observed with H129.19. One difference, however, was that 53.6.7
225
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-~
CD4
~
~
~
-
~
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CD4
.... ~
:~
PanT (CD5)
( b )
~,
~-. PanT (CD5)
Fig. 4. Flow cytometry analysis of double stained lymph node cells obtained 4 days after in vivo treatment of thymectomizedmice. The cells were stained with an anti-CD5 pan-T cell marker monoclonal antibody, 53.7.3 (visualized by FITC-conjugated goat anti-rat Ig) together with a biotinylated anti-CD4 monoclonal antibody, H129.19 (visualizedby phycoerythrin-conjugated streptavidin). In a is seen the results of staining lymph node cells from an untreated control mouse and in b lymph node cells from a mouse treated with 53.6.7 (66 #g) + MAR18.5 (66/tg) antibodies.
given alone partly eliminated the target CD8 + cells and thus not only bound passively to the cell surface.
Conclusions Both the H129.19 and 53.6.7 antibodies used alone are functionally suboptimal for in vivo depletion of their respective target ceils. The antiC D 4 reactive H129.19 antibody did not initiate any killing of the target cells although the antib o d y did bind to the cell surface in vivo. The anti-CD4 reactive antibody, GK1.5, on the other hand, was effective when used alone in eliminating the CD4 target cells in vivo. It is possible that this difference was dependent on the isotype of the antibodies used since H129.19 is of the rat I g G 2 a and GK1.5 of the IgG2b subclass. It has been suggested that the IgG2b isotype is the most effective of the rat isotypes for in vivo depletion work (Cobbold et al., 1984). The depletion capacity in vivo is, however, not restricted to rat IgG2b isotypes since 53.6.7 which is an I g G 2 a protein, partially eliminates the CD8 target cells. Never-
theless, the present experiment shows that the capacity to deplete target cells in vivo can be facilitated by additional treatment with a mouse anti-rat x monoclonal antibody (MAR18.5). This mouse antibody is of the I g G 2 a isotype, which is the most effective of the mouse immunoglobulin isotypes in eliminating target cells in vivo (Kaminski et al., 1986). It is also possible that the addition of a secondary antibody induces cross-linking of the cell surface molecules which further strengthens the interaction with Fc-receptors on cytotoxic macrophages or enhances complement activation. This method of using autologous secondary antibodies m a y be of value in a number of different situations where monoclonal antibodies are required to eliminate their target cells.
Acknowledgements The technical assistance of Roger Festin is greatly appreciated. The work was supported by the Swedish Medical Research Council, King
226
Gustaf V 80-year foundation and the Swedish Technical Development Agency.
References Carlsson, M., Hedin, H., lngan~is, M., H~irfast, B. and Blomberg, F. (1985) Purification of in vitro produced mouse monoclonal antibodies, A two-step procedure utilizing cation exchange chromatography and gel filtration. J. Immunol. Methods 79, 89. Cobbold, S.P., Jayasuriya, A., Nash, A., Prospero, T.D. and Waldmann, H. (1984) Therapy with monoclonal antibodies by elimination of T-cell subsets in vivo. Nature 312, 548. Coulie, P.G., Coutelier, J.P., Uyttenhove, C., Lambotte, P. and Van Snick, J. (1985) In vivo suppression of T-dependent antibody responses by treatment with monoclonal antiL3T4 antibody. Eur. J. Immunol. 15, 638. Dialynas, D.P., Quan, Z.S., Wall, K.A., Pierres, A., Quintans, J., Loken, M.R., Pierres, M. and Fitch, F.W. (1983) Characterization of the murine T cell surface molecule, designated L3T4, identified by monoclonal antibody GK1.5: Similarity of L3T4 to the human Leu-3/T4 molecule. J. Immunol. 131, 2445. Festin, R., Bj~Srklund, B. and T~Stterman, T.H. (1987) Detection of triple antibody-binding lymphocytes in standard single laser flow cytometry using colloidal gold, fluorescein and phycoerytbrin as labels. J. Immunol. Methods 101, 23. Gutstein, N.L., Seaman, W.E., Scott, J.H. and Wofsy, D. (1986) Induction of immune tolerance by administration of monoclonal antibody to L3T4. J. Immunol. 137, 1127. Holmdahl, R., Olsson, T., Moran, T. and Klareskog, L. (1985) In vivo treatment with monoclonal anti-T cell antibodies. Scand. J. Immunol. 22, 157. Holmdahl, R., Rubin, K., Klareskog, L., Larsson, E. and Wigzell, H. (1986) Characterization of the antibody response in mice with type II collagen-induced arthritis, using monoclonal anti-type II collagen antibodies. Arthritis Rheum. 29, 400. Howard, F.D., Ledbetter, J.A., Carter, D.P., Smith, L.M. and McCormell, H.M. (1982) The lateral mobility and surface distribution of Lyt-1, Lyt-2, and Lyt-3 on mouse thymocytes. Mol. Immunol. 19, 1481. Hunig, T. (1983) T-cell function and specificity in athymic mice. Immunol. Today 4, 84. Jeffries, W.A., Green, J.R. and Williams, A.F. (1985) Authentic T helper CD4 (W3/25) antigen on rat peritoneal macrophages. J. Exp. Med. 162, 117. Kanainski, M.S., Kitamura, K., Maloney, D.G., Campbell, M.J. and Levy, R. (1986) Importance of antibody isotype in monoclonal anti-idiotype therapy of a murine B cell
lymphoma. A study of hybridoma class switch variants. J. Immunol. 136, 1123. Lanier, L.L., Gutman, G.A., Lewis, D.E., Griswold, S.T. and Warner, N.L. (1982) Monoclonal antibodies against rat immunoglobulin kappa chains. Hybridoma 1, 125. Ledbetter, J.A. and Herzenberg, L.A. (1979) Xenogenic monoclonal antibodies to mouse lymphoid differentiation antigens. Immunol. Rev. 47, 63. L0gdberg, L., Gunter, K.C. and Shevach, E.M. (1985) Rapid production of monoclonal antibodies to T lymphocyte functional antigens. J. Immunol. Methods 79, 239. MacDonald, T.T., Calcins, C.E. and Rothermel, A.L. (1986) Modulation in vivo of Lyt 2 antigen expression on T cells by anti-Lyt 2 antibody: effects on Con A-induced unresponsiveness. Immunology 59, 181. Pedersen, B.K., Kharazmi, A., Theander, T.G., Odum, N., Andersen, V. and Bendtzen, K. (1987) Selective modulation of the CD4 and CD16 molecular complexes by Pseudomonas aeruginosa alkaline protease and elastase. Annual meeting, Scandinavian Society for Immunology, 1987 (abstract). Scand. J. Immunol. 26, 324. Pierres, A., Naquet, P., Van Agthoven, A., Bekkhoucha, F., Denizot, F., Mishal, Z., Schrnitt-Verhulst, A.-M. and Pierres, M. (1984) A rat anti mouse T4 monoclonal antibody (H129.19) inhibits the proliferation of Ia-reactive T-cell clones and delineates two phenotypically distinct (T4 positive, Lyt-2,3 negative, and T4 negative, Lyt-2,3 positive) subsets among anti-Ia cytolytic T-cell clones. J. Imrnunol. 132, 2775. Qin, S., Cobbold, S., Tighe, H., Benjamin, R. and Waldmann, H. (1987) CD4 monoclonal antibody pairs for immunosuppression and tolerance induction. Eur. J. Immunol. 17, 1159. Ranges, G.E., Sriram, S. and Cooper, S.M. (1985) Prevention of type Ii collagen-induced arthritis by in vivo treatment with anti-L3T4. J. Exp. Med. 162, 1105. Ranges, G.E., Cooper, S.M. and Sriram, S. (1987) In vivo immunomodulation by monoclonal anti-L3T4. 1. Effects on humoral and cell-mediated immune response. Cell. Immunol. 106, 163. Waldor, M.K., Sriram, S., Hardy, R., Herzenberg, L.A., Herzenberg, L.A., Lanier, L., Lira, M. and Steinman, L. (1985) Reversal of experimental allergic encephalomyelitis with monoclonal antibody to a T-cell subset marker. Science 227, 415. Wofsy, D. (1986) Administration of monoclonal anti-T cell antibodies retards murine lupus in BXSB mice. J. Immunol. 136, 4554. Wofsy, D., Mayes, D.C., Woodcock, J. and Seaman, W.E. (1985) Inhibition of humoral immunity in vivo by monoclonal antibody to L3T4: studies with soluble antigens in intact mice. J. Immunol. 135, 1698.
219
JIM04817
Depletion of murine T cells by in vivo monoclonal antibody treatment is enhanced by adding an autologous anti-rat x chain antibody T.J. Goldschmidt 1,2, R. Holmdahl 1 and L. Klareskog a t Department of Medical and Physiological Chemistry, University of Uppsala, The Biomedical Center, Box 575, S-751 23 Uppsala, Sweden, and 2 Department of Experimental Medicine, Pharmacia A B, S-751 82 Uppsala, Sweden (Received 22 December 1987, accepted 22 February 1988)
In vivo treatment with monoclonal antibodies can be used for elimination of various T lymphocyte subsets from peripheral lymphoid organs and blood and thereby be used both to analyze the role of different T cells in immunoregulation and for the treatment of experimental immunological diseases. However, one problem with this approach has been that not all monoclonal antibodies given in vivo eliminate their target cells. We now show in the murine system that the normally inefficiently depleting H129.19 (anti-CD4) and 53.6.7 (anti-CD8) antibodies can be used for efficient depletion of their respective target cells when combined with injection of a secondary mouse anti-rat x (MAR18.5) antibody. The efficacy of the depletion protocols was ascertained by double staining techniques and cytofluorometric analysis. It is suggested that the presently used sandwich method applying a homologous secondary monoclonal antibody may provide an alternative to class switching or other manipulations of primary antibodies in increasing the efficacy of in vivo antibody treatment. Key words: Monoclonal antibody therapy; CD4 T cell; CD8 T cell
Introduction During recent years techniques have been developed for the depletion of lymphoid cells in vivo by treatment with monoclonal antibodies. Treatment of mice in vivo with monoclonal antibodies directed to surface markers on T lymphocytes has, for example, been used to alter the immune response in various systems. Thus, it has been possible to block humoral responses (Cobbold et al., 1984; Coulie et al., 1985; Wofsy et al., 1985;
Correspondence to: T.J. Goldschmidt, Department of Medical and Physiological Chemistry, The Biomedical Center, University of Uppsala, Box 575, S-751 23 Uppsala, Sweden.
Ranges, 1987) and to induce tolerance to specific antigens (Benjamin et al., 1986; Gutstein et al., 1986; Waldman et al., 1987), to delay allograft rejection (Cobbold et al., 1987) and to retard experimental autoimmune diseases (Holmdahl et al., 1985; Ranges et al., 1985; Waldor et al., 1985; Wofsy, 1986). However, a major shortcoming of using various monoclonal anti-T cell antibodies for the investigation of in vivo immune processes by cell depletion is that some antibodies do not effectively eliminate their targets in vivo. Whether a monoclonal antibody will eliminate their target or not has been suggested to depend both on the structure of the Fc part of the antibodies (Ledbetter et al., 1979; Cobbold et al., 1984; Kaminski et al., 1986) and on the properties of the
0022-1759/88/$03.50 © 1988 Elsevier Science Pubfishers B.V. (Biomedical Division)
220 surface marker recognized by the antibody (Howard et al., 1982). One of the most commonly used antibodies for depletion studies in the mouse is the GK1.5 antibody which is specific for the CD4 receptor on murine T cells (Dialynas et al., 1983). Treatment with this rat IgG2b antibody effectively eliminates most CD4 + cells in vivo (Ranges et al., 1985; Waldor et al., 1985; Wofsy et al., 1985). Another rat monoclonal antibody directed against a spatially related determinant on the CD4 receptor is H129.19 (Pierres et al., 1984) which is of the IgG2a isotype. The results in this study show that this antibody binds to, but does not eliminate, the CD4 + target cells in vivo. Using these antibodies we report the development of a new approach for in vivo depletion of T cells which is based on the additional treatment with a homologous anti-rat antibody, MAR18.5 (Lanier et al., 1982).
Materials and methods Animals
Female D B A / 1 mice were bred and kept at the Biomedical Center in Uppsala. All mice were 8-12 weeks old and had an approximate weight of 20 g. All mice were age-matched at the beginning of the experiment. Cell culture
Hybridomas were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with glutamine 10 mM, penicillin 100 U / m l , streptomycin 100 # g / m l , 2-mercaptoethanol 5 x 10 -5 M and fetal calf serum 6-10% (KC Biological, Lennexa, KS). Some monoclonal antibodies were produced in serum-free medium supplemented by insulin (10 t~g/ml), transferrin (10/zg/ml) (Sigma, St. Louis, MO), ethanolamine (20 /~M) and nonessential amino acids (1%) (Flow Laboratories, U.K.) before being subjected to chromatography purification. Monoclonal antibodies
H y b r i d o m a supernatants were stored at - 2 0 o C. The antibodies were purified either by affinity chromatography on Protein A-Sepharose (Pharmacia, Uppsala, Sweden) (MAR18.5) or with
TABLE I CHARACTERISTICS OF MONOCLONAL ANTIBODIES USED FOR IN VIVO TREATMENT AND FOR IN VITRO ANALYSIS Hybridoma H129.19 MAR18.5 GK1.5 53.6.7 3A7 53.7.3 a
Isotype Rat IgG2a MouseIgG2a Rat IgG2b Rat IgG2a Rat IgG a Rat IgG2a
Target antigen CD4 (L3T4) Rat x chain CD4 (L3T4) CD8 (Ly2) Thyl CD5 (Lyl)
Reference Pierres(1984) Lanier(1982) Dialynas(1983) Ledbetter(1979) L~gdberg (1985) Ledbetter(1979)
The IgG subclass is not known.
cation exchange chromatography and gel filtration (Carlsson et al., 1985) (H129.19, 53.6.7 and GK1.5). In some experiments antibodies were used which had been concentrated by ammonium sulfate precipitation. The specific antibody concentrations were determined by ELISA using the corresponding purified monoclonal antibody as a standard (Holmdahl et al., 1985). All antibody preparations used for in vivo treatments were dialyzed against PBS and sterile filtered. No difference in in vivo depletion effects could be detected between purified antibodies and antibodies only subjected to enrichment and concentration by ammonium sulfate precipitation. Biotinylation of purified antibodies was performed as previously described (Holmdahl et al., 1986). The antibodies denoted 53.6.7 (anti-CD8), 53.7.3. (anti-CD5) and 3A7 (anti-Thyl) were used in the form of hybridoma supernatants. The various monoclonal antibodies that were used for in vivo treatment and for FACS analysis are listed in Table I. In vivo treatment
The mice were thymectomized at the age of 8-12 weeks, (day - 1 4 ) . 2 weeks later, on day - 1 and day 0, the anti-mouse T cell monoclonal antibodies (H129.19, GK1.5 and 53.6.7, respectively) were injected intraperitoneally. In the cases of combined treatment, the MAR18.5 antibody was injected intraperitoneally 1 h after injection of the primary antibodies (H129.19 and 53.6.7 respectively). Immunization
After the monoclonal antibody treatment on day 0, the mice were immunized by intradermal
221 injections of Freund's complete adjuvant, emulsified in phosphate-buffered saline (PBS), in the base of the tail and in both hind foot pads. The immunization was performed in order to enlarge the otherwise small lymph nodes to facilitate further phenotypic analyses of the cells within these lymph nodes. This treatment was accompanied by a relative decrease in the numbers of T ceils in the enlarged lymph nodes which explains the somewhat lower number of lymph node cells staining for the different T cell antibodies in this study as compared to certain other studies.
Fluorescence analysis Popliteal and inguinal lymph nodes were removed on day 4 - 5 and separate single cell suspensions were prepared from each mouse. The staining procedure of the lymph node cells was then performed in 96 well microtitration plates (Nunc, Copenhagen, Denmark). In the case of single staining, a two-step procedure was performed. 50 /~1 of a cell suspension containing approximately I x 106 cells/ml were incubated for 10 min at room temperature (or 30 min at 4 ° C), with 50/~1 of rat monoclonal antibody specific for the mouse T cell surface markers CD4, CD8 or CD5. As a control for non-specific binding, normal rat serum Ig ( N R I G ) was used. After incubation the cells were washed twice in PBS supplemented with 0.5% bovine serum albumin (Sigma, St. Louis, MO) to avoid plastic adherence and with 0.02% sodium azide to prevent capping of the surface
markers (Howard, 1982) and centrifuged 2 - 3 min between the different steps. Cell-bound rat antibody was then visualized by an affinity-purified F I T C (fluorescein isothiocyanate)-conjugated goat anti-rat Ig monoclonal antibody, which had been absorbed with mouse serum (Kirkegaard and Perry Laboratories, Gaithersburg, MD). In the case of double staining a purified biotinylated rat anti-mouse T cell monoclonal antibody was subsequently added to the cells. Binding of biotinylated antibodies was then visualized by a phycoerythrin-conjugated streptavidin (Becton Dickinson Immunocytometry Systems, Mountain View, CA). In order to avoid binding of the biotinylated rat antibody to free sites on the FITCconjugated goat anti-rat antibody, the single stained cells were first saturated with excess of N R I G . The cells were then analyzed by fluorescence-activated flow cytometry in a FACStar (Becton Dickinson). Lymphocytes were selected by forward and right angle light scattering and the viability was checked by exclusion of propidium iodide (Festin et al., 1987). In each sample 4000-8000 cells were counted. Results and discussion
Treatment with a combination of H 1 2 9 . 1 9 + MAR18.5 but not with H129.19 alone efiminates CD4 + cells from peripheral lymph nodes. In an initial experiment thymectomized mice were treated in vivo with GK1.5, with H129.19 or
TABLE II CYTOFLUOROMETRIC ANALYSIS OF CD4÷ LYMPH NODE CELLS AFTER TREATMENT WITH CD4÷ SPECIFIC MONOCLONALANTIBODIES Monoclonal antibody treatment (dose)
% CD4 + lymph node cells at various times after treatment 5 days a 8 weeks b 14 weeks b
% Thyl+, CD8lymph node cells 14 weeks c
Untreated control GK1.5 (2 x 100/~g) H129.19 (2 × 100/~g) H129.19 (2 x 100/Lg)+ MAR18.5 (2 × 100 t~g)
21 3 19 4
n.d. 12 23 9
28 6 n.d. 6
n.d. d 9 17 7
a Each value represents one mouse. Results were confirmed in three additional non-immunized mice. b Each value represents one mouse. c The double stainings at 14 weeks were performed on the same lymph node cells as the single stainings. d Not done.
222 with a combination of H129.19 and MAR18.5 antibodies. 5 days later lymph node cells from the mice were analyzed by cytofluorometry for effects on the CD4 ÷ cell subset (Table II). Both GK1.5 and combined H129.19 + MAR18.5 treatment substantially reduced the numbers of cells stained for CD4. The lymph node cells from the mice treated with H129.19 alone were, however, not different from untreated controls w h e n staining for CD4. This pattern was also seen when the analyses were carried out 8 and 14 weeks after antibody injection which indicated that the target cells had been eliminated rather than the CD4 receptor having been modulated. Thus if the antibodies had only modulated the CD4 receptors, their reappearance could be expected to occur when the added antibodies had been cleared from the circulation (MacDonald et al., 1986; Pedersen et al., 1987). To further confirm that the CD4 ÷ cells were eliminated we performed double staining with 53.6.7 (anti-CD8) and 3A7 (anti-Thyl) antibodies of the remaining cells 14 weeks after treatment (Fig. 1). Double staining for the p a n - T
CD8
cell marker Thyl and for CD8 on lymph node cells from untreated mice showed three distinct populations: (1) Thyl-, C D 8 - non-T cells; (2) CD8 +, Thyl ÷ T cells; (3) Thyl ÷, C D 8 - T cells which are mainly CD4 ÷ cells (Waldor et al., 1985). If the mice were anti-CD4 treated by either GK1.5 or H129.19 + MAR18.5, a reduction of Thyl ÷, C D 8 - lymph node cells was seen, corresponding to the reduction of CD4 ÷ cells detected by single staining by H129.19, indicating that the CD4 ÷ T cells were indeed eliminated (Table II). The elimination of C D 4 ÷ cells after GK1.5 or H129.19 + MAR18.5 was, however, not complete neither at 5 days nor at a longer time after injection. As indicated in Fig. 2 it was not possible to eliminate all cells with increasing amounts of antibody since there was no major difference between a total dose of 2 0 / , g compared with a total dose of 2 0 0 / , g H129.19. The reason for the inability to deplete the remaining 2-5% is unknown. Possible explanations are the existence of inaccessible CD4 + cells in the peripheral tissue, refractory CD4 ÷ non-T cells (Jefferies et al., 1985) or cells
..
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"
"
"
Thyl
Fig. 1. Flow cytometric analysis of double stained lymph node cells 4 days after in vivo anti-CD4 monoclonal antibody treatment. The cells were stained by an anti-Thyl pan-T cell marker monoclonal antibody, 3A7 (visualized by FITC-conjugated goat anti-rat Ig) and with biotinylated anti-CD8 monoclonal antibody, 53.6.7 (visualized by phycoerythrin-conjugated streptavidin), a shows lymph node cells from an untreated thymectomizedcontrol mouse and b shows lymph node cells from a thymectomizedmouse treated with H129.19 (200/*g) + MAR18.5 (200 ~tg).
223
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Fig. 2. Results from flow cytometry analyses of single stained l y m p h node cells obtained at 4 days after in vivo monoclonal antibody treatment in thymectomized mice. In a is seen a chequerboard titration of various doses of the monoclonal antibodies H129.19 and MAR18.5 administrated in vivo. The ordinate shows percent l y m p h node cells obtained 4 days after monoclonal antibody treatment and stained with H129.19 + FITC conjugated anti-rat Ig. The abscissa shows the administered dose of MAR18.5. Each point represents one mouse. The experiment shown is one of two experiments which yielded similar results. In b is seen staining on lymph node cells from an untreated mouse and from mice treated with either H129.19 or with H129.19 in combination with MAR18.5. The lymph node cells were stained with either H129.19 or normal rat Ig (NRIG) as primary antibodies and with FITC conjugated goat anti-rat Ig as secondary antibody. Each pair of N R I G or CD4 stained bars represents one mouse. The result shown is one of two experiments.
with a low density of CD4 receptors. Furthermore, the resident fraction of CD4 ÷ cells slightly increased with time, an expansion that was not dependent on the thymus since the mice had been
thymectomized. Several explanations for this expansion are possible and include the proliferation of remaining peripheral mature CD4 + cells or repopulation of the lymph nodes with immature
224 20 -la- 0 ,ug 53.6.7 •.e- 20 ~g 53.6.7 66 )Jg 53.6.7 -0- 200 ug 53.6.
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Fig. 3. Results from flow cytometryanalyses of single stained lymph node cells obtained 4 days after monoclonal antibody treatment of thymectomizedmice with various doses 53.6.7 and MAR18.5 antibodies. The ordinate shows percent lymph node cells stained with 53.6.7 followed by FITC-conjugatedgoat anti-rat Ig. The abscissa shows the administrated dose of MAR18.5. The indicated total doses of monoclonal antibodies represents the sum of two intraperitoneal injections given on 2 consecutive days. Each point represents one mouse. The experiment shownis one of two experiments which gave similar results.
CD4 + T lymphocytes, something that is known to occur in nude mice (Hunig, 1983).
An excess of MAR18.5 versus H129.19 is optimal for in oivo depletion To investigate which concentrations of H129.19 and MAR18.5 were optimal for in vivo depletion a chequerboard titration experiment was performed, the results of which are shown in Fig. 2. The antibodies H129.19 or MAR18.5, when given alone, did not have any detectable depletion effect. In the combined treatment of H129.19 + MAR18.5 the efficacy of depletion was critically dependent on the dose of MAR18.5. The optimal dose was the same or greater than that of H129.19. Essentially the same degree of depletion was obtained with all concentrations of H129.19 (20, 66 and 200/Lg) provided that MAR18.5 was given in an equal amount or in excess. However, a tendency towards an H129.19 dependent effect could be seen when the MAR18.5 was given in high doses (200 #g). The most likely explanation for the observed phenomena is that an excess of H129.19 versus MAR18.5 was ineffective since the administered MAR18.5 appears to bind to free H129.19, instead of to cell bound H129.19. Flow cytometric analysis of lymph node cells obtained from mice treated with H129.19 alone
showed positive staining even when no primary H129.19 antibodies were added in the staining procedure (Fig. 2b). The numbers of cells stained by the secondary antibody only in H129.19 treated mice were similar to the numbers of H129.19 stained cells in lymph nodes from untreated mice. These data suggest that when injected alone the H129.19 antibodies most likely bind to the cell surface neither modulating the CD4 receptors nor eliminating the CD4 ÷ cells.
Efimination of CD8 + target cells using the 53.6. 7 antibody is facilitated by the addition of MAR18.5 antibodies To investigate whether an enhanced depletion effect using a combination of primary antibody and MAR18.5 treatment, could be used for antibodies other than H129.19, we also analyzed the in vivo effects of treatment with the anti-CD8 specific, rat monoclonal IgG2a antibody, 53.6.7. Similar chequerboard titration experiments to those performed with H129.19 (anti-CD4) and MAR18.5 were carried out (Fig. 3) and the results, as analyzed by double staining on lymph node cells, are shown in Fig. 4 (in this case anti-CD5 was used as the pan-T cell marker). The results obtained were similar to those observed with H129.19. One difference, however, was that 53.6.7
225
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-~
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-
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CD4
.... ~
:~
PanT (CD5)
( b )
~,
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Fig. 4. Flow cytometry analysis of double stained lymph node cells obtained 4 days after in vivo treatment of thymectomizedmice. The cells were stained with an anti-CD5 pan-T cell marker monoclonal antibody, 53.7.3 (visualized by FITC-conjugated goat anti-rat Ig) together with a biotinylated anti-CD4 monoclonal antibody, H129.19 (visualizedby phycoerythrin-conjugated streptavidin). In a is seen the results of staining lymph node cells from an untreated control mouse and in b lymph node cells from a mouse treated with 53.6.7 (66 #g) + MAR18.5 (66/tg) antibodies.
given alone partly eliminated the target CD8 + cells and thus not only bound passively to the cell surface.
Conclusions Both the H129.19 and 53.6.7 antibodies used alone are functionally suboptimal for in vivo depletion of their respective target ceils. The antiC D 4 reactive H129.19 antibody did not initiate any killing of the target cells although the antib o d y did bind to the cell surface in vivo. The anti-CD4 reactive antibody, GK1.5, on the other hand, was effective when used alone in eliminating the CD4 target cells in vivo. It is possible that this difference was dependent on the isotype of the antibodies used since H129.19 is of the rat I g G 2 a and GK1.5 of the IgG2b subclass. It has been suggested that the IgG2b isotype is the most effective of the rat isotypes for in vivo depletion work (Cobbold et al., 1984). The depletion capacity in vivo is, however, not restricted to rat IgG2b isotypes since 53.6.7 which is an I g G 2 a protein, partially eliminates the CD8 target cells. Never-
theless, the present experiment shows that the capacity to deplete target cells in vivo can be facilitated by additional treatment with a mouse anti-rat x monoclonal antibody (MAR18.5). This mouse antibody is of the I g G 2 a isotype, which is the most effective of the mouse immunoglobulin isotypes in eliminating target cells in vivo (Kaminski et al., 1986). It is also possible that the addition of a secondary antibody induces cross-linking of the cell surface molecules which further strengthens the interaction with Fc-receptors on cytotoxic macrophages or enhances complement activation. This method of using autologous secondary antibodies m a y be of value in a number of different situations where monoclonal antibodies are required to eliminate their target cells.
Acknowledgements The technical assistance of Roger Festin is greatly appreciated. The work was supported by the Swedish Medical Research Council, King
226
Gustaf V 80-year foundation and the Swedish Technical Development Agency.
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