Release of LT molecules with restricted physical heterogeneity by a continuous human lymphoid cell line in vitro.

Lymphotoxins released by lectin activated human lymphoid cells in vitro are complex and can be resolved by molecular sieving techniques into multiple classes of activity, termed complex, α,β and γ. The classes can be further resolved into sub-classes on the basis of their charge by ion-exchange chromatography and electrophoresis. We found the lytic activity spontaneously released by the continuous human B lymphoid cell line, PGLC-33H, to be of limited physical heterogeneity, and essentially identical to the α mol. wt class of human LT† molecules. This was determined by functional, physcial-chemical and immunologic means. We found that most of the α-LT charge sub-classes present in supernatants from mitogen stimulated normal lymphocyte cultures were also detected in these supernatants. These studies suggest: (a) this cell line provides a source of human LT for study with restricted heterogeneity; (b) different lymphoid cells (T and B) may be capable of releasing different LT molecules; and (c) use of selected human lymphoid cell lines may be useful to examine the biokinetics of LT synthesis and release.


INTRODUCTION
In-vitro stimulation of lymphocytes from experimental animals and man by specific (antigens) or non-specific (mitogens) means results in the release into the culture medium of a complex family of soluble molecules (Lawrence & Landy, 1969;Granger, 1972, Ruddle, 1972. It has been suggested that these materials, termed lymphokines (LK), may be important effecters in causing the various manifestations of cell-mediated immunity (CMI) observed in vitro and in vivo. One family of LK, termed lymphotoxins (LT), has been shown to be cytotoxic or growth inhibitory to cells in vitro, depending on the concentration of the cytoxin and the particular indicator target cell employed (Williams & Granger, 1973;Jeffes & Granger, 1976;Walker & Lucas, 1972;Namba & Waksman, 1975). The actual role of LT in CM1 reaction is not yet clear. However, it is becoming more plausible, for several studies, employing antisera which will neutralize their activity in vitro, indicate they are lytic effecters in certain types of lymphocyte induced target cell destructive reactions in vitro (Gately et al., 1976;Hiserodt & Granger, 1977).
The human LT family of effecters is complex and can be separated into various classes based on differences in their mol. wt (Walker et al.. 1976;Granger et al., 1978) and several classes can be further resolved into sub-classes on the basis of differences in their charge (Lee and Lucas, 1976;Hiscrodt et al., 1976~;Granger et al., 1978). The first class, complex, is greater than 150,000 daltons, the second, X, is 70-90,000 daltons. Both classes are relatively stable to heating, storage and manipulation (Walker et al., 19766;Granger ef al., 1978). The 61 class contains 3-6 sub-classes, which can be resolved by chromatography on diethyamino ethyl cellulose and phosphocellulose (Lee & Lucas, 1976;Hiserodt et al., 19766, Granger et al., 1978). The third class, p, is composed of at least two sub-classes; one relatively stable, the other highly unstable (Hiserodt & Granger, 1976~). Finally, a third, short-lived small mol. wt class, termed y, of 10-l 5,000 daltons, which is highly unstable and little studied, has also been described (Hiserodt et al., 1976h).
In order to obtain large quantities of human LK for purification from both a uniform source and identifiable cell type, a number of investigators have examined supernatants from continuous human lymphoid cell lines. These studies revealed that many human lymphoid cell lines are spontaneously releasing LK-like materials (Granger et al., 1976;Papageorgiou et al., 1972;Amino et al., 1974). Several studies reported LT activity in the supernatants from different cell lines possessed physical-chemical properties similar to those described for the a-LT class (Granger er al., 1976;Amino ef al., 1974). However, the activity detected in these supernatants was only partially characterized.
We have shown in vitro that lectin activated human T and B cells can release LT, and moreover, T cells can express LT on their surface plasma membranes (Hiserodt et al., 1977). It has been shown that the human continuous B cell line PGLC- Human tonsil and adenoid lymphocytes were prepared as a single cell suspension as described previously (Kramer & Cranger, 197.5). The lymphocytes in these suspensions were adjusted to 5 x IO0 ciable cells ml in MEM containing lo",, INCS. non-essential amino acids and 1 miCl pyruvate.  n Tube cultures of target monolayer L cells were exposed to serial dilutions of LT-containing medium. After 1 hr, this media was discarded, and the monolayer washed and replaced with fresh media. Cultures were assayed for the units of detectable LT activity after 20 hr, as described in the text.

Phytohemaggintinin
'Activity is expressed as units of LT/ml supernatant. determined as described in the text. members of the fl or y classes. After overnight incubation at 4-C, rabbit antisera LT complex was precipitated by addition of either goat anti-rabbit IgG or sheep anti-rabbit serum. After an additional 12 hr of incubation at 4°C the precipitate was removed by centrifugation, and the supernatant was diluted with fresh media containing 3% NCS. Duplicate samples of each dilution were tested on MC treated L-929 cells.
Serial dilutions of PGLC or control supernatants (concentrated IO-fold) were made in MEM + 3% FCS, (MEMS) and added to three parallel sets of duplicate tubes containing mitomycin C treated L cells. Two sets of tubes were then directly incubated at either 37°C or at 34°C without further manipulation. The remaining set of tubes were incubated for 1 hr at 37°C and the free LT removed by washing the monolayers 5 times with a total of 15 ml of PBS. After the adddition of fresh MEMS, these tubes were incubated for 20 hr at 37°C the total adherent cell number in all cultures was determined. The number of LT units detected in each set of tubes is shown in Table  1. First, a small but significant amount of PGLC-LT did bind to the L-929 target cells after a 1 hr incubation at 37°C. Second, the activity of PGLC-LT was dramatically reduced by incubating the cultures at 34°C when compared with the cultures incubated at 37°C.

Assessment of binding, growth inhibition and effect of temperature on lysis of target L cells in vitro by PGLC-33H supernatants
The mechanism of in vitro target L-cell destruction induced by LT generated from mitogen activated human lymphocytes at least two parameters. First, LT may rapidly bind to the target cell (Hessinger et al., 1973) and second, the cytolytic step of the reaction appears to be temperature dependent.
Kramer and Granger (1976) reported a 97% reduction in LT It has been previously suggested that LT released by activated human lymphocytes at low, sub-toxic levels can cause growth inhibition of cells in vitro (Jeffes & Granger, 1976;Walker & Lucas, 1972;Namba & Waksman, 1975). In order to continue a functional comparison of the LT released from mitogen activated lymphoid cells with the LT-like material secreted by PGLC-33H cells, we tested the ability of the latter cytoxin(s) to growth inhibit HeLa target cells, as previously described (Jeffes et al., 1976). PGLC or control supernatants were concentrated lo-fold, serially diluted, and cultured on growing HeLa cell monolayers for 48 hr at 37°C. In addition, the same concentrates were tested on MC treated L-929 cells to determine the number of units of cytolytic activity in   and sub-classes. Particularly important was to examine the possibility that a continuous lymphoid cell line may only secrete a single LT class or sub-class in a fashion analogous to the production of a single ig product seen with both human and murine myeloma cells.

Heat stability of' PGLC-LT
We first performed a heat inactivation study; the results of which can be seen in Table 2. Supernatants were heated and dilutions tested for LT activity, as described in Materials and Methods. As previous investigators have shown for supernatants from both continuous culture lymphoid cells (Amino rt al., 1974) and mitogen activated human lymphocytes (Walker et ai., 1976;Peter et&., 1973;Kolb & Granger, 1968), the lytic activity was destroyed at 85°C and stable at 37°C and 56'C, following 15 mm exposure at each given temperature.

Gel,filtration
In order to determine the number of LT classes, both the supernatants from PGLC-33H and PHA-P, activated human tonsil lymphocytes were fractionated by gel filtration. We did not store these supernatants, but they were fractionated immediately, in order to insure detection of the unstable p-class LT activity, if present. Supernatants were harvested, concentrated IO-17-fold, and then fractionated over Sephadex G-150. The data shown in Figs. 2(A) and(B) demonstrates that r class LT was the major LT class present in the PGLC supernatants.
fn addition, the activity of PGLC-LT eluting from the Sephadex G-150 column was further tested on growing L-929 cells. Although the data are not shown. the ability of PGLC LT eluting from the column to growth inhibit L-929 coincided with the cytotoxic activity observed on MC treated L-929 cells [ Fig.  Z(B)]. In contrast, if dividing HeLa cells were utilized, inhibitory activity eluted from the sephadex column over a more broad range of apparent mol. wt (from 40 to 90,000 daltons). Next, PGLC-LT fractions and whole supernatants were tested for activity after three months of storage at 4'C, in order to monitor the stability of this material. The levels of activity and elution profiles of cytotoxic activity coincided with those observed upon initial fractionation.
Hence. it appears that PGLC released cytoxin(s) are composed of a stable, single molecular weight class similar to tl-LT from mitogen stimulated human cells, which exhibits both cytotoxic and growth inhibitory activities on L-929 cells.

Ion exchange
Recently, Lee and Lucas (1976) demonstrated that the human r-LT class contains various sub-classes which can be separated on the basis of differences in their charge. To examine if the PGLC released cell toxin(s) contained sub-classes, we separated PGLC supernatants on phosphocellulose and DEAEcellulose columns.
Initially, PGLC supernatants were concentrated IOfold, dialyzed against PBS, and subjected to DNASE treatment (80 units pancreatic DNase A/ml) for 2 hr at room temperature in the presence of 10 mM MgCl,, 10 mM CaCl,. After dialysis in 10 mM potassium phosphate, 0.1 mM EDTA (pH 6.6), the PGLC-LT was chromatographed over phosphocellulose. As shown in Fig. 3, nearly all of the cytoxic material was retained by this resin under theseconditions.
In several experiments, we found a small amount of activity did not bind to the column, and eluted with the first protein peak. The majority of lytic activity began to elute from the column after a concentration of 0.15 M NaCl, and a second peak was included with the 1 M NaCl wash.
The entire LT-containing eluate from the PC column was then concentrated to 5-10 ml by ultrafiltration and dialyzed against 50 mM Tris, 0.1 mM EDTA (pH 9.0) and applied to a DEAE-cellulose column. Figure 4 depicts the elution profile of the lytic activity. Two fractions of LT-like material were eluted. The first failed to bind to the DEAE-cellulose under these conditions, while the second broad profile was only slightly bound and eluted shortly after the salt gradient was initiated. If the equilibrating buffer was decreased to 10 mM Tris, no difference was observed in the elution profile. This suggested then that there were two separate activities, and that they were not attributed to a single molecular species of LT whose ability to bind to the column was dependent on the ionic strength of the Tris buffer. In a number of experiments, a third LT activity was observed to elute as a shoulder on the high salt side of the second LT activity. These components of LT which emerge from  the DEAE-cellulose column with increasing salt were consistent with those activities previously defined as xi-LT, a,-LT and z,-LT. respectively, found as subclasses of r-LT secreted by mitogen activated human lymphocytes in vitro (Granger et cd., 1978).

Rihonurlease i RNaw) activity
Recently, Lee and Lucas (1976) have reported an RNase activity which copurifies along with the various X-LT components.
Because of the important impli~tions of these findings to the mechanism of action of LT on target cells, we undertook a study to determine if there was RNase activity associated with various r sub-classes of PGLC-LT and LT from PHA activated human lymphocytes.
DEAE fractions of PGLC-LT were generated, concentrated, and samples adjusted to contain high levels of LT activity (ZO~iOOO units/ml). In addition, supernatants from PHA stimulated lymphocytes were separated over Sephadex G-150, and the a-LT class further separated into sub-classes on DEAE. Each set of r-LT sub-class Vr;as incubated with i4C-or 3Hlabeled yeast or fruit fly RNA as described in the Methods section. Table  3 depicts a representative experiment employing "C-labeled yeast RNA, It is clear from this study that no RNase activity was detected in separated r-LT sub-classes obtained from either PGLC or mitogen stimulated human lymphocytes. However, positive controls incubated with low levels of pancreatic RNase A showed significant degradation of RNA.

.4ntigenic properties of PGLC-LT
Previous studies indicated that antisera from rabbits injected with fractions contain all the X-LT sub-classes released by mitogen activated human lymphocytes will neutralize the lytic activity of these molecules in vifro (Lewis et ul., 1977;~amamoto er al., 1978). We used preselected antisera (Rabbit A9) which completely neutralizes all Z-LT class activity from mitogen activated human lymphoid cells, but does not CTOSSreact with any of the /Y-LT classes (Lewis et al., 1977). In preliminary studies, we found that antisera to r-LT. but not antisera to P-LT, could neutralize PGLC-LT activity. To further investigate this finding, we decided to physically remove the PGLC-LT-antibody complex, utilizing an indirect antibody precipitation method. One milliliter of PGLC-LT was incubated with 0.1 ml of rabbit anti-human LT or normal rabbit serum overnight at 4°C. This complex was then precipitated with either sheep anti-rabbit serum or goat anti-rabbit IgG after incubation overnight at 4"C. The precipitate was removed by centrifugation, and the supernatants diluted and tested for LT activity. Table 4 indicates that the indirect antibody precipitation removed greater than 90% of the PGLC activity (reduction from 700 units to < 5 units). Hence, all the PGLC-LT activity could be removed by antisera specific for r-LT antigenic determinants.

Continuous
human lymphoid cell lines with characteristics of T and B cells have been previously shown to be capable of releasing lymphokines in r&o "SAL from PGLC was incubated with anti-r-LT sera and then treated with sheep or goat anti-rabbit serum. The immune complex was removed by sedimentation, and the supernatant tested for LT activity. as described in Materials and Methods.
"Sheep anti-rabbit serum used to precipitate the PGLC-LT + anti-x-LT complex.
"Goat anti-rabbit IgG used to precipitate the PGLC-LT + anti-r-LT complex.
( Papageorgiou et al., 1972;Lewis et al., 1977;Yoshida et al., 1976). While the studies were not extensive, physical characteristics of human MIF (Tubergen et al., 1972;Yoshida et al., 1976), chemotactic factor (Yoshida et al., 1976), lymphotoxins (Granger et al., 1970Papageorgiou et al., 1972;Amino et al., 1974) and interferon (Ware & Granger, in preparation), secreted by continuous lymphoid cell lines, appeared to be generally similar to their counterparts released by mitogen or antigen activated human lymphocytes. Glade and colleagues (Papageorgiou et al., 1972) have repeatedly shown that the human lymphoid cell line, PGLC-33H releases MIF and we demonstrated it releases LT (&anger et al., 1970). It is clear that LT activity released in vitro by mitogen stimulated human lymphoid cells represent a complex 'family of cytotoxins composed of multiple classes and sub-classes of molecules'. The various LT classes are defined by their mol. wt; complex > 150,000 daltons (d), alpha, 70-90,000 d;beta, 30-50,000 d;and gamma, 15-20,000 d (Walker et al., 1976;Hiserodt et al., 19766;Granger et al., 1978). Also, each LT class contains several sub-classes which can be resolved by ion exchange chromatography and electrophoresis (Walker et al., 1976;Hiserodt et al., 1976~). We undertook our investigation of the cell-toxins released by the continuous B cell lymphoid culture, PGLC-33H, to compare and characterize the heterogeneity of these molecules to those found in supernatant from mitogen activated normal lymphoid cells (SAL).
Initial functional and stability studies suggested PGLC-33H toxins were similar to the stable a-LT class molecules.
Cytolytic activity of both types of supernatants was inhibited by 34°C and there was a certain amount of binding to the target L cells. Also, PGLC-LT was stable at 4°C for long periods and at 56°C which is a distinct characteristic of the r-LT class (Walker et al., 1976;Hiserodt ef al., 1976~;Lee & Lucas 1976). Growth inhibition measured on L-929 or Hela cells by PGLC supernatants were indistinguishable from SAL, as described previously (Williams & Granger, 1973;Jeffes & Granger, 1976). The growth inhibition exhibited by PGLC supernatants was dependent on concentration and the particular cell line employed in the assay. However, in contrast to L cells, growth inhibition of HeLa cells by PGLC-LT fractionated over Sephadex G-150 indicated that the effect was not restricted to molecules possessing LT activity, but also possessed by material(s) in the 30-50,000 d mol. wt range. Additional studies have suggested that interferon is probably the active material in these fractions, and this is a species-specific effect (Ware & Granger. in preparation).
The physical characteristics of PGLC-LT appear to be very similar to those seen for the r-LT class released by mitogen activated human lymphocytes. Almost all of the LT activity eluted from Sephadex G-l 50 is in the 70-90,000 d fraction, characteristic of the r-LT class. Additional separation on ion exchange columns revealed three separable sub-classes of this activity. The majority of PGLC-LT was observed to bind to phosphocellulose during chromatography at pH 6.6 in a linear gradient of NaCl from 0 to 0.3 M. However, on several occasions, a small amount of material not binding to the column was observed.
Extensive experiments revealed that this was not due to overloading, because this material was not retained by the resin upon rechromatography under the same conditions. Lee and Lucas (1976) have reported, and we have confirmed. that there is a major LT peak which does not bind to PC columns under these conditions in the r-LT class from mitogen stimulated cells (Granger et al., 1978). It appears that PGLC-33H cells release only small amounts of this sub-class of r-LT activity. The PGLC-LT which was retained by phosphocellulose, and subsequently chromatographed over DEAE-cellulose, exhibited three characteristic sub-class activities observed for r-LT. These three sub-class activities have been defined by their elution order from DEAE-cellulose at pH 8.0 in a linear NaCl gradient of O-O.3 M. The first, xl, does not bind, the second, x2, binds weakly and the third, x3, binds strongly. Each sub-class, when subjected to separation on PAGE, exhibited somewhat broad but distinctive migration profiles. The PAGE profiles of various r-like LT from PGLC-LT were very similar to those observed for s( sub-classes released by lectin stimulated normal human lymphocytes (Granger et al., 1978). These data would indicate that the PGLC cells are able to release most of the r-LT class of molecules observed in cultures of mitogen activated lymphocytes.
The basis of the heterogeneity of the x-LT class of LT activity is not yet clear. Immunologic and physical-chemical studies of these molecules suggest thay arc similar, and indeed may be isomer% forms of one another. Other investigators have studied LT-like material(s) from other lymphoid cell lines. Their results suggest that the activity was probably due to member(s) of the 7&YO,OOO mol. wt r-LT class. but these studies did not describe evidence of sub-class activity. Our finding multiple r-LT sub-classes is due to several reasons: (a) PGLC33H sccrctcs high enough levels of LT (3&lOO units:ml) to permit moreextensive physical studies; (b) we employed a sensitive assay system able to detect low levels of activity: and (c) wc employed more extensive separation methods.
We found the r-LT class of cytotoxins released by lectin activated human lymphocytes or PGLC-33H cells does not appear to possess RNase activity. This is in contrast to the findings of Lee and Lucas. who reported that all fractions containing r-LT sub-classes possess RNase activity (Lee & Lucas. 1976). None of the various X-LT containing fractions in these studies. obtained from either PGLC'-33H or normal lectin stimulated lymphocytes. possessed RNase activity that we could detect. However. positive controls indicated our assay procedure was able to detect less than 50 ng of RNase activity. We have no direct explanation for these conflicting results. The total abrogation ofPGLC-LT activity on target cells in t,itro by addition of rabbit anti-x class antisera further supports the hypothesis that the LT secreted by this cell line is essentially the same as r-LT released by activated normal human lymphocytes.
This was similar to the findings of Ammo c't ml. (1974). in that antisera dlrected toward the LT secreted by a continuous lymphoid cell line could neutralize LT produced from mitogen or antigen activated human lymphocytes.
It is important to consider why supernatants from PGLC-33H only contain r-LT class of molecules. There are several possibilities; those based on the concept that the various classes are unrelated. and the other that they are related forms of one another. Under the former situation.
this cell line could represent a variant at the level of LT release, a selective synthesis of only the r classes of LT. or the other soluble phase LT classes are selectively degraded. The latter possibility does not appear to be the case, for mixing experiments revealed that PGLC supernatants do not selectively degrade the complex. b or LT forms. The alternative is that z-and [I-LT classes are related. and the PGLC-33H cell line lacks the ability to convert the z to the /I-LT form. Physical and immunologic studies indicate this latter possibility may actually be the case (Yamamoto C/ ~1.. 197X). Additional studies have revealed different populations or subpopulations of human lymphocytes, ie. T or B, are capable of releasing different soluble phase LT classes when activated wjith lcctln ,,I t'i/ro (Harris ('/ rri.. 111 preparation).
It appear\ that human I'ccll\ can rclc;txc all classes. but B cells only release x class LT molecules. The present studies suggest that the examination of other human lymphoid ccl1 lines may provide insight into revealing the genetic regulation and control of lymphokines in general.