thymoma and severe hypogammaglobulinemia was stud- ied in special detail. In vitro the patient's cells were able to develop surface Ig in media supplemented ...
Proc. Nat. Acad. Scd. USA Vol. 71, No. 2, pp. 531-535, February 1974
Immunoglobulin Synthesis In Vitro by Lymphocytes from Patients with Immune Deficiency: Requirement for a Special Serum Factor (B-cell markers/B-cell differentiation/serum factor/T-cell influence/mitogen action)
P. WERNET, F. P. SIEGAL, H. DICKLER, S. FU, AND H. G. KUNKEL The Rockefeller University, New York, New York 10021
Contributed by Henry G. Kunkel, September 20, 1973 Certain patients with immune deficiency ABSTRACT were encountered whose peripheral blood lymphocytes included no immunoglobulin-bearing cells. However, other markers of B type lymphocytes were observed; lymphocytes isolated free of macrophages showed the presence of receptors for the Fc fragment of IgG and for the third component of complement. One patient with a syndrome of thymoma and severe hypogammaglobulinemia was studied in special detail. In vitro the patient's cells were able to develop surface Ig in media supplemented with fetal-calf serum or normal human serum; in media supplemented with autologous serum, the cells developed no surface Ig. During culturing antigenic determinants of immunoglobulin became detectable in the medium, and both medium and cell-surface immunoglobulin underwent a shift from specific IgM determinants early in the culture period to IgG and IgA determinants later. Normal lymphocytes and thymocytes activated by concanavalin A repaired the deficiency in the patient's serum. These data support the concept that a factor possibly derived from T cells is missing from this patient's serum and that this factor is required for the maturation of the B cell for im-
munoglobulin synthesis. A patient with X-linked agammaglobulinemia had a population of circulating lymphocytes with some surface characteristics that appeared similar to those of the B cells from the patient with thymoma. In contrast, however, no Ig synthesis by this patient's cultured cells could be demonstrated. The possible nature of the lymphocytes reacting with aggregated IgG in this case is discussed.
During the course of studies on the lymphocytes of a variety of patients with immunodeficiency, two special patients were investigated in particular detail because of an absence of immunoglobulin (Ig)-bearing peripheral blood lymphocytes (PBL), despite the presence of cells that carried receptors for aggregated human IgG; the latter, as well as surface Ig, have been shown to be B cell markers (1). The possibility arose that a population of abnormal nonimmunoglobulin-bearing B lymphocytes was present or alternatively that these cells represented a selective and excessive proliferation of the normal subpopulation of lymphocytes (K cells) which also possess an Fc receptor and are involved in antibody-induced lymphocyte-mediated killing of target cells (2, 3). Since one patient had a thymoma associated with the immune deficiency, it appeared of interest to ascertain whether a thymus defect was in some way involved in the occurrence of the unusual B cells Abbreviations: PBL, bone marrow-derived fetal calf serum; Fc, phytohemagglutinin; canavalin A.
peripheral blood lymphocytes; B cells, cells; T cells, thymus-derived cells; FCS, a fragment of immunoglobulin G; PHA, LPS, lipopolysaccharide; con A, con-
531
that were found. Therefore the patient's cells were put into culture, removed from their usual environment. When the cells were cultured in certain serum-containing media, but not when cultured in others, surface Ig did indeed develop, and antigenic determinants of immunoglobulin became detectable in the culture supernatants. It appeared that development of cell surface Ig depended upon a factor present in fetal-calf serum (FCS) and normal human serum but not in the serum of the patient. In contrast the second patient, with infantile X-linked agammaglobulinemia, failed to produce Igs in titro under similar conditions. MATERIALS AND METHODS
Patients. From a group of 14 patients with various types of immunodeficiency states, those showing no or minimal surface Ig but up to 15% Fc receptor staining of their PBL were selected. Of special interest was an adult, 63-year-old Caucasian female (Gol) known to have been hypogammaglobulinemic for 4 years. A thymoma was retrospectively identified in the anterior mediastinum on x-rays for 6 years prior to its excision. The tumor, consisting primarily of spindle cells, was benign. There was no hematologic or immunologic remission following thymectomy. During the 18-month period of study, the patient developed pure red cell aplasia; ultimately, complete bone marrow aplasia ensued. Cell-mediated immunity was apparently normal. Intradermal skin tests for mumps and intermediate strength PPD (purified protein derivative of tubercle bacteria) were positive. The lymphocytes responded to BCG (a vaccine prepared from the tubercle bacillus of Calmette and Guerin) in the direct lymphocyte migration inhibition system (4). There was an active incorporation of tritiated thymidine in response to PPD, phytohemagglutinin (PHA), concanavalin A (Con A), and allogeneic lymphocytes. A second patient of special interest was a 24-year-old man (Mas) with X-linked agammaglobulinemia and severe recurrent pulmonary infections. Leukocyte Isolation. Mononuclear cells were isolated under sterile conditions with Ficoll-Hypaque gradients (5) and washed three times in Hanks' balanced salts (GIBCO) prior to culturing. For immunofluorescence, the cells were prepared as previously described (6). To exclude phagocytic cells, in some experiments we allowed the cells to ingest latex particles prior to staining (7). Special care was exercised to insure that macrophage-type cells would not interfere with the evaluation of lymphocyte markers. Therefore in many experiments the PBL were isolated in a three-step procedure by (a) Ficoll-
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Wernet et al.
Hypaque flotation and (b) further passage through nylon wool columns and (c) finally through Sephadex G-10 columns. Where cell cultures (see below) were performed, steps b and c were carried out only at the end of the incubation periods. In combining morphological and functional criteria it was possible to insure almost complete elimination of the macrophages. Especially the incubation of the lymphocytes with Sephadex G-10 beads proved to be most efficient. Final lymphocyte preparations contained 99% lymphocytes that were 98-99% viable when freshly isolated (8).
Cell Culture Technique. All cell cultures were carried out using RPMI 1640 (GIBCO) or a mixture of RPMI 1640 and MEM (Dutton) supplemented with 20% serum from various sources. An atmosphere containing 5% C02, 9% 02, and 86% N2 was utilized. Medium was exchanged every third day by centrifugation at 130 X g and removal of 2/3 of the supernatant, which was replaced by an equal volume of fresh medium. Cultures were carried as long as the morphology and viability were acceptable, then terminated and the cells stained. Supernatants were snap frozen in isopentane-dry ice mixtures for later study for Ig determinants. Viabilities determined by trypan blue exclusion just prior to staining were 60-80% in all cultures submitted to staining. Preparation of mixed lymphocyte cultures (MLC) and mitogen stimulation were performed as described elsewhere (8). Hemagglutination Inhibition. Detection of Ig determinants in the culture supernatants was carried out as previously described (9). Preparation of Sera for the Tissue Culture Media. Serum from the patient as well as AB serum from six healthy individuals was heat inactivated at 56° for 30 min, Millipore filtered, snap frozen, and stored under sterile conditions at -20° until used. Fetal-calf serum (Rehuis Pharmacol. Co., lot no. 73208) was used as received from the processor, after heat inactivation. Portions of FCS were passed once or twice through sterile Seitz filters (Republic Filter Corp., Millvale, Connecticut, grades EK and S3, size 3) prior to use. For some cultures, a mixture of equal volumes of the patient's serum and FCS (not Seitz filtered) was utilized to supplement the medium. Immunofluorescent Studies. Antisera conjugated to tetramethylrhodamine isothiocyanate were prepared as previously described (6). A polyvalent screening antiserum capable of detecting mu, gamma, kappa, and lambda antigenic determinants was used in most of the experiments. Antisera monospecific for mu, gamma, and alpha were prepared by absorption of cruder preparations prior to their use. Staining was carried out at 4°. In one experiment, cells cultured 8 days were stained first with polyvalent anti-Ig, washed, then incubated with unlabeled 7S IgG, washed, and stained with fluoresceinated aggregated IgG, to determine the relationship of the populations of stained cells. The microscopy was carried out as previously described (6). Aggregated human IgG and fluorescein-conjugated aggregated human IgG were prepared as previously described (1). Aggregates of specific size (200300 S) were needed for optimal visualization and were obtained by preparative ultracentrifugation. Tests for lymphocyte membrane receptors for the third complement component were performed after Bianco et al. (10). Sheep red blood cell rosette tests were performed as described elsewhere (11). Mitogens-
Proc. Nat. Acad. Sci. USA 71 (1974)
phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) were obtained commercially. Lipopolysccharide (LPS) and lipid A from Salmonella R 595 was a gift from Drs. C. Galanos and 0. Luderitz. ThymocytesFresh human thymus was obtained from young patients undergoing cardiac surgery. The tissue was minced, passed through wire mesh and the cells were then processed as for PBL. Cell Surface Studies by Enzymatic Radioiodination. Uncultured and cultured lymphocytes were isolated by the threestep procedure. For the enzymatic surface radioiodination (SRI) a modification of the method described by Baur et al. (12) using lactoperoxidase (Sigma) to radioiodinate the lymphocyte membranes was applied. The radioiodinated lymphocytes were solubilized with the nonionic detergent NP-40 (Shell Oil Co.) and centrifuged for 25 min at 1000 X g. In a sandwich technique, aliquots of the supernatant were incubated, first with a portion of 30 jul of different rabbit anti-human Ig sera, and subsequently with goat anti-rabbit Ig. The specific precipitate formed was solubilized and subjected to electrophoresis on a 5% polyacrylamide gel containing 0.1% sodium dodecyl sulfate. The radioactive bands obtained represent immunoglobulin targets present on the lymphocyte surface. Radiolabelled marker proteins were: IgG (150,000 daltons), y-chains (50,000 daltons) and light (L) chains (24,000 daltons). RESULTS B-Cell Marker Discrepancies in Immunodeficiency. Studies of B and T cells have proven to be of considerable utility for the understanding of the great variety of immune deficiency states (11). Supplementation of surface Ig studies by other methods for B-cell analysis has been particularly valuable because marked discrepancies have been encountered that are not observed in normal PBL. The determination of the Fc receptor by uptake of aggregated IgG shows this most strikingly. Table 1 illustrates the results of such studies in four selected patients with immune deficiency as compared with normal individuals. In two of the patients the Fc receptor is clearly present. This suggested the presence of a significant number of B cells by this criterion since macrophages had been removed. In case 3 (Gol) no Ig-bearing cells were observed during initial studies. In case 4, a patient with X-linked agammaglobulinemia, some Ig-bearing cells were observed in the freshly drawn blood, but the Ig observed on the cell surfaces proved to be only IgG and disappeared after overnight culture. Evidence was obtained that this related to the presence of the Fc receptor, which picked up Ig, at least in part, from injected IgG. Lymphocyte Markers in the Patient Gol. Over a total period of 20 months, only 3/5942 Ig-positive lymphocytes were found, a frequency of 0.05%. (Normal is 21.5 ± 7.8%.) Over the same time period aggregate-binding lymphocytes, measured by both direct and indirect methods were constantly present, at 7.83 4 3.4%. (Normal is 21.8 + 8.2%.) Overall, there was a constant and marked discrepancy between the two B-cell markers, which normally parallel one another. On two occasions late in the patient's illness, the sheep erythrocyte rosette test was performed and the aggregate receptor was studied in aliquots of the same population of cells. There was complete accounting for the peripheral blood lymphocytes
Proc. Nat. Acad. Sci. USA 71
(1974)
T-Cell-Dependent Antibody Synthesis *
20
Ig staining
/L y -I- -+-
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o Aggregate staining
533
K
-+-
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-
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20
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9
0
0
2
4
6
8
10
12
14
16
18
10
30
20
40
50
60
Days of culture in FCS
FIG. 1. Development of lymphocyte surface Ig during culture of peripheral blood cells of patient Gol. The Fc receptor remains at a relatively constant level.
using the B and T markers on these occasions; normally, aggregate-reactive cells plus rosette-forming cells account for close to 100% of the peripheral blood lymphocytes (11). In two experiments 5 and 7% of the fresh lymphocytes also showed the receptor for the complement component C3, another B-cell marker. On one occasion, however, during an acute bacterial infection an 8% overlap of Fc receptor positive and SRBCrosette-forming lymphocytes was observed. Lymphocyte Markers after Culture. In media supplemented with FCS or AB serum for various periods of time, immunoglobulin determinants became detectable on some of the lymphocytes. These data are summarized in Fig. 1. Each point represents one culture experiment. There was a gradual increase with time in the percentage of lymphoid cells that bore immunoglobulin. Concurrently, there was no change in the percentage of cells binding direct aggregates. In a double-labeling experiment, on day 8 of culture in medium containing fetal-calf serum, cells were stained for both surface Ig and the aggregate receptor. An intermediate incubation intended to block the uptake of aggregates by unsaturated antiserum in the first layer was included. This procedure showed that the same population of cells expressed both surface Ig and aggregate binding. A few aggregatepositive cells that did not express surface Ig were also observed. Trypsinization and short term reculture confirmed the actual Ig production. To confirm these observations, two additional methods of Ig analyses were utilized. Hemagglutination inhibition was employed to detect the presence of immunoglobulin determinants in the culture media, after various periods in culture in the presence of fetal-calf serum. Medium at the start of the cultures contained no detectable human immunoglobulins. Early in the cultures, on about day 6, some IgM was detectable, as evidenced by the presence of both mu and light chain determinants; IgG was not present. Late in the same culture, on day 18, IgG and IgA were present, but IgM was no longer detectable. Thus the impression was gained that possibly a shift from mu to gamma production had occurred in the cultured cells. Immunofluorescent staining with anti-immunoglobulins specific for heavy chains gave further support to this possibility.
+-
-
+
ro0 E aL
10
20
30
40
50
60
Tube number
FIG. 2. (A) Sodium dodecyl sulfate-polyacrylamide-gel electrophoresis pattern for labeled lymphocyte surface Igs of patient Gol, after the cells had been in culture for 6 days. The predominance of pa chains over 'y chains is noted. (B) Sodium dodecyl sulfate-polyacrylamide-gel pattern of lymphocyte membranes of the same patient after 15 days of cell culture. The main component at this time is the Sy chain. NRS indicates that normal rabbit serum was used instead of anti-human Ig serum (see Methods) as a control.
Enzymatic radioiodination of the surfaces of cultured and uncultured lymphocytes was carried out with the enzyme lactoperoxidase. Results obtained by this technique are seen on Fig. 2A as the pattern of radioactivity from the lymphocyte surfaces obtained after 6 days of culture. The horizontal axis represents the sequential slices of the gel. The vertical axis represents the radioactivity present in each gel slice. It can be seen that the most radioactivity is located in the same slices where the mu chain marker appeared on a parallel gel. This pattern established the presence of IgM as the predominating species on the surface of the patient's lymphocytes after 6 days in culture. A small amount of IgG was also detectable. Fig. 2B shows the pattern of surface radioiodination after 15 days of culture. It is apparent that the pattern has changed considerably; now clearly IgG predominates. A small shoulder with the mobility of mu chain is still present, and the light-
534
Proc. Nat. Acad. Sci. USA 71 (1974)
Immunology: Wernet et al.
TABLE 1. Comparison of fluorescence staining for surface immunoglobulins and Fc receptors on PBL of normal individuals and patients with immune deficiency
16 I
14
12 0
Subject
0
*
10
1. 2. 3. Immune-deficient 1. 2. 3.
Normal
oB
*
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0
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2 0* r pl -I
*0
S
5 10 15 20 Autologous
5 10 15 20 Fetal colf
serum
serum
5 10 15 20 Pooled humon AB serum
5 10 15 20 Seitz filtered fetal calf serum
FIG. 3. Cell surface Ig, measured by immunofluorescence, as a function of medium supplements. The horizontal axis in each column is a time scale from 5 to 20 days of culture.
chain band persists. The pattern before culture is not shown. This gave -very low but definite evidence of surface IgM not detected by immunofluorescence. In order to investigate the nature of the deficiency in this patient, we cultured leukocytes in a variety of other media. These results are summarized on Fig. 3. The second column illustrates the results of cultures in medium supplemented with fetal-calf serum, as discussed before. Again, each point represents a single culture experiment. In pooled normal AB serum, a more limited number of experiments confirmed that the phenomenon was not restricted to cultures in fetal-calf serum alone. In sharp contrast, when cells were cultured in the patient's own serum there were practically no cells that exhibited surface Ig. The fourth column shows that the ability of normal fetal-calf serum to support the development of surface Ig was either markedly reduced or completely abolished by passage of the serum through a Seitz asbestos filter. The latter results suggested that FCS provides a nonspecific factor that is important for the differentiation of B cells and that was absent in the described patient. In tissue culture flasks with two compartments, separated by a cell-impermeable membrane, it was possible to show that Con A-activated lymphocytes and thymocytes from normal individuals supplied the necessary factor that enabled the patient's cells cultured in her own serum to form Ig. Positive immunofluorescence of up to 4% of the patient's lymphocytes after 5 days of such cultures demonstrated that activated T cells efficiently. provided the required factor. Since these results suggested a deficiency of a T lymphocyte function, various assays were performed to determine the in vitro response of the patient's PBL to T- or B-cell stimuli. Patient Gol did not show an apparent T-cell defect in the "classical" in vitro parameters, mixed lymphocyte cultures and Con A stimulation, although the response to PHA appeared somewhat weak. However, the response to pokeweed mitogen was clearly depressed. A surprising finding was a strong response of Gol lymphocytes to LPS from Salmonella R 595, a response which is ordinarily not observed with PBL lymphocytes. Some of these cells also expressed immunoglobulin on their surface after 4 days of LPS stimulation. Similar results were obtained also with the isolated Lipid A component, showing that this structure is the biologically active
Surface Ig % of PBL
Fc receptor*
23 14
22 14 27 2 4
25 1 3 0
Ot
% of PBL
12
15t
* As measured by binding of fluoresceinated and aggregated IgG. t Freshly isolated lymphocytes (16%) were positive for IgG; Overnight culture brought this figure down to zero, whereas the Fc-receptor-positive population stayed unchanged at
15%fO.
part of the LPS in human lymphocytes, as it has recently been shown to be in the mouse (13). The PBL of patient Gol are triggered by LPS in the same fashion as are otherwise normal human spleen cells (unpublished observation) or tonsil-
derived lymphocytes (14). The other deficiency patient, (number four in Table 1), the 24-year-old man (Mas) with sex-linked agammaglobulinemia, showed some similarities to the patient just described as far as apparent normal-cell-mediated immunity and the sharp discrepancy of B-cell markers are concerned. He had no Igpositive staining lymphocytes after short term culture but in some determinations as much as 21% of his peripheral blood lymphocytes were Fc-receptor-positive. C-receptor-positive lymphocytes were also present. In contrast to the first patient, no Ig synthesis by this patient's cultured cells could be demonstrated, thus pointing to a functionally different composition of his lymphocyte population. DISCUSSION
In the present studies the peripheral blood lymphocytes of special patients with immunodeficiency, although lacking in surface immunoglobulins, nevertheless had certain characteristics of B cells, in that they carried the receptor of aggregated IgG (Fc receptor), and the C3-receptor. The possibility arose that abnormal or incomplete B cells were present. In culture, the cells from one patient (Gol) were able to synthesize Ig and incorporate it into their membranes, indicating that the defect was not intrinsic to the cells, but related in some way to their usual environment. The expression of surface Ig was not accompanied by a generalized de-differentiation of the cells, which maintained the appearance of small lymphocytes and continued to express the receptor for aggregated IgG. Of interest is the sequential development of IgM, than IgG and IgA during the period of culture. A possible explanation for this transition is that once the cells expressed a receptor for antigen (the surface Ig), they were able to respond to antigens in the culture medium and undergo a shift from cells early in the B-cell line to more mature B cells. The development of Ig by this patient's cells was dependent upon a factor present in normal FCS or pooled normal human
AB serum which was not present in the patient's serum. In view of the capacity of normal serum or FCS to support the development of cell surface Ig even in the presence of autolo-
Proc. Nat. Acad. Sci. USA 71
the presence of an inhibitory factor in the patient unlikely (15). Fetal-calf serum known to support the expression of surface Ig was rendered incapable of doing so when passed twice through Seitz filters. Indeed, Byrd (16) recently reported a factor required for the development of in vitro immune response of thymectomized mice to sheep erythrocytes. This factor, present in some batches of FCS, was completely removed by Seitz filtration, though, as with the present factor, it could pass through Millipore filters. Various workers have presented evidence in experimental animals that humoral factors from thymocytes (16-21) or T cells may nonspecifically stimulate lymphoid populations deprived of T cells to function as if T cells were present. These studies differ from the present work in that normal B cells were present at the start of cultures; such previously described factors are assayed in systems such as that described by Mishell and Dutton (22). The present factor may act at an exceedingly early stage in the development of the B cell. The missing factor in Gol serum could be replaced by supernatants from Con A-activated T cells and thymocytes. The nature of the factor remains unclear but it appears to represent a B-cell maturation stimulator that is important for induction and proliferation of Ig synthesis. The factor possibly provides also the drive for the shift from IgM to IgG production and could well be identical to the T-cell product described by Schimpl and Wecker (18). It does not appear to be the same as colonystimulating factor or the specific fetal-calf serum factor described for the Mishell and Dutton System. Screening of individual normal as well as hypogammaglobulinemic sera from a number of sources by means of an in vitro test system will have to be carried out before the true significance of "undetectable" amounts of the factor can be assessed. It may be that some individual normal sera will similarly fail to support such a system. It is already known that different lots of FCS vary markedly in their ability to support in vitro immune responses. If such is the case, the defect in this present patient (Gol) may be unrelated to the thymoma and the cells of this patient merely served fortuitously as an assay system for the factor. The latter may be identical to the factor provided by nonspecifically activated T lymphocytes, thus representing a T-cell-dependent "second" signal which can modify and amplify an antigen-dependent "first" signal at the level of the B cell membrane. Cells similar to those seen in the thymoma patient were also observed in a patient with infantile X-linked agammaglobulinemia. The cells of this patient carried both the aggregate receptor and IgG. No IgM- or IgA-bearing lymphocytes could be detected in the patient's circulation. Evidence from trypsinization and short-term culture experiments indicated that the surface IgG was not intrinsic to these cells. Such short term culture experiments demonstrated the general importance of the distinction of "true" B cells carrying genuinely synthesized surface Ig from those that have picked up IgG through an Fc receptor. Longer culture studies similar to gous serum, seems
T-Cell-Dependent Antibody Synthesis
(1974)
535
those described above, however, failed to demonstrate any Ig production by this patient's lymphocytes in vitro. Thus no direct evidence for the presence of B cells in this case was obtained. The Fc receptor-bearing cells were nonphagocytic small lymphocytes and it appeared possible that they may represent the recently described "K" cells (2, 3) which react avidly with aggregated IgG and even unaggregated material. Froland et al. (23) have obtained evidence for such cells in human lymphocyte populations, utilyzing human erythrocytes coated with human or rabbit IgG. We are grateful to Drs. J. Zabriskie for performing the migration inhibition test and to P. Cheng for testing several sera in the Mishell-Dutton system. We are especially indebted to Drs. C. Galanos and 0. Liideritz, Max-Planck-Institut fur Immunbiologie, Freiburg, Germany, for their generous gift of LPS and Lipid A. P. W. was supported by Grant We 505/3 from the Deutsche Forschungsgemeinschaft. This investigation has been supported by U.S. Public Health Service Grants nos. AM 34761, RR-102, and Al 10811. 1. Dickler, H. & Kunkel, H. G. (1972) J. Exp. Med. 136, 191196. 2. MacLennan, I. C. M. (1972) Transplant. Rev. 13, 67. 3. Perlmann, P., Perlmann, H. & Wigzell, H. (1972) Transplant. Rev. 13, 91. 4. Zabriskie, J. B., Lewshenia, B., Wehle, B., M6ller, G. & Falk, R. E. (1970) Science 168, 1105-1108. 5. Boyum, A. (1967) Scand. J. Clin. Lab. Invest. 21: suppl 97, 77-89. 6. Siegal, F. P., Pernis, B. & Kunkel, H. G. (1971) Eur. J. Immunol. 1, 482-486. 7. Cline, M. J. & Lehrer, R. I. (1968) Blood 32, 423-435. 8. Wernet, P. & Kunkel, H. G. (i973) J. Exp. Med. 138,10211026. 9. Natvig, J. B. & Kunkel, H. G. (1971) Nature 215, 68-69. 10. Bianco, C., Patrick, R. & Nussenzweig, V. (1970) J. Exp. Med. 132, 702-720. 11. Bentwich, Z. & Kunkel, H. G. (1973) Transplant. Rev., in press. 12. Baur, S., Vitetta, E. S., Sherr, C. J., Schenkein, I. & Uhr, J. W. (1971) J. Immunol. 106, 1133-1142. 13. Andersson, J., Melchers, F., Galanos, C. & Luderitz, 0. (1973) J. Exp. Med. 137, 943-953. 14. Hoffmann, M., Schmidt, W. & Oettgen, H. F. (1973) Nature
243, 408-410. 15. Bullock, W. W. & M6ller, E. (1972) Eur. J. Immunol. 2, 514-517. 16. Byrd, W. (1971) Nature New Biol. 231, 280-282. 17. Sj6berg, O., Andersson, J. & Moller, G. (1972) J. Immunol. 109, 1379-1385. 18. Schimpl, A. & Wecker, E. (1973) J. Exp. Med. 137, 547-
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19.
20. 21. 22. 23.
Gorczynski, R. M., Miller, R. G. & Phillips, R. A. (1972) J. Immunol. 108, 547-551. Watson, J. & Thoman, M. (1972) Proc. Nat. Acad. Sci. USA 69, 594-598. Britton, S. (1972) Scand. J. Immunol. 1, 89-98. Mishell, R. I. & Dutton, R. W. (1967) J. Exp. Med. 126, 423-442. Frbland, S. S., Natvig, J. B. & Wisloff, F. (1973) Scand. J. Immunol. 2, 83 (Abstract.)
