Immunology 197938 827

Changes occurring on the surface of mouse T cells during concanavalin A-induced lymphoblastic transformation

F. KIERSZENBAUM & DELIA B. BUDZKO Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan, U.S.A.

Acceptedfor publication 19 July 1979

of surface changes during mitogen-induced blastogenesis and suggested a role for sialic acid residues in interference with C-dependent cytotoxicity.

Summary. Thy- I antigen and GPCA-the mouse T-cell surface component responsible for activation of guinea-pig complement (C)-were readily demonstrated on unstimulated but not on Con A-stimulated thymus or spleen cells when (guinea-pig) C-dependent cytotoxic reactions were used. Mitogenic stimulation was a necessary condition for these changes to occur since mere incubation of thymocytes in the absence of Con A failed to alter the sensitivity of these cells to treatment with either anti-Thy-1.2 antibodies plus C (devoid of non-specific cytotoxicity) or untreated guinea-pig serum (GPS) alone. Both Thy-1.2 and GPCA were, however, readily detectable on the Con A blasts when tested for by indirect immunofluorescence and immune adherence, respectively, i.e. by using tests which do not involve C-effected lysis. That Thy- I was indeed expressed on the T-cell blasts was further indicated by their capacity to absorb anti-Thy-1.2 activity from specific antiserum. Con A, whether bound to the cells or added in excess to the reaction mixture, did not interfere with C-mediated lysis and was ruled out as a possible C inactivator. Pre-treatment of the lymphoblasts with neuraminidase rendered these cells sensitive to lysis by either anti-Thy-1.2 plus C or GPS. These results, highlighting altered reactivity of mouse T lymphoblasts with guinea-pig C, indicated the occurrence

INTRODUCTION

Characterization of the lymphocyte membrane and surface markers has been instrumental in furthering our understanding of important immunological processes such as lymphocyte differentiation, interplay among functionally distinct cell subpopulations and regulation of the immune response by soluble cell products (Wedner & Parker, 1976; Oppenheim & Rosenstreich, 1976; Williams, 1978; Williams & Standring, 1977; Cantor & Boyse, 1977; Waksman & Namba, 1976). Little is known, however, about the dynamic changes occurring at the cell surface level that may affect cell physiology and expression of these markers during lymphoblastic transformation, a key process in manifestation of immune responses and reactions. In this work, we have centred our attention on the alterations in expression of two murine T-cell markers occurring during mitogen-induced lymphoproliferation and the possible reason why these alterations may occur. Two cell surface components were chosen: Thy- I, probably the best defined T-cell marker to date, and the guinea-pig complement activator (GPCA) responsible for the susceptibility of mouse T lymphocytes to the non-specific but selective cytotoxic effects of normal guinea-pig serum (GPS) (Budzko,

Correspondence: Dr F. Kierszenbaum, Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824, U.S.A. 0019-2805/79/1200-0827$02.00 () 1979 Blackwell Scientific Publications

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F. Kierszenbaum & Delia B. Budzko

Kierszenbaum & Waksman, 1977, Kierszenbaum & Budzko, 1979). It will be shown in this paper that concanavalin A (Con A)-induced blastic transformation brings about surface alterations affecting the detectability-but not the presence-of both Thy-1 and GPCA in complement(C)-dependent cytotoxicity assays, and that surface sialic acid residues are likely to be relevant to occurrence of these phenomena.

MATERIALS AND METHODS

Cells Thymocytes and spleen cells used in this study were obtained from inbred CBA/J mice purchased from Jackson Laboratories (Bar Harbor, Maine). Cells were dispersed in cold RPMI-1640 medium (Grand Island Biological Company, Grand Island, New York) using a Ten Broeck tissue grinder (two strokes only). Cellular clumps and debris were removed by filtration through nylon gauze and cells were washed twice with the same medium. Suspensions used in cytotoxicity tests were adjusted to contain 1 x 107 trypan-blue-excluding nucleated cells/ml. Initial viability of cell preparations was always greater than 95%. Sera and anti-Thy-i.2 ascites fluid Blood was obtained from Hartley guinea-pigs (Isaacs Breeding Laboratory, Litchfield, Illinois) and CBA/J mice by cardiac puncture. Sera were separated after clotting at room temperature for I h. GPS was used immediately after separation. Normal mouse serum used in reference cytotoxicity assays was previously heat-inactivated (HI-NMS) at 56° for 1 h. AntiThy- 1.2 antibodies were used in the form of heat-inactivated ascites fluid from AKR/J mice (Jackson Laboratories) immunized with CBA/J mouse thymocytes (HI-anti-Thy-1.2AF) (Reif & Allen, 1966; Budzko et al., 1977). The cytotoxic titre of this preparation was 1/2560 when tested on normal CBA/J thymus cells in conjunction with a suitable source of C (see below). Foetal calf serum was purchased from Microbiological Associates (Walkersville, Maryland) and inactivated by heating at 560 for 1 h (HI-FCS).

Specific anti- T cell cytotoxicity assay The quantitative test used to determine T cells in cell suspensions by using HI-anti-Thy-1.2AF plus C has been previously described in detail (Budzko et al., 1977). In the present assays, GPS diluted 1/20 in

RPMI- 1640 medium was used as a source of C activity and was free of nonspecific cytotoxicity (Kierszenbaum & Budzko, 1978).

Non-specific GPS cytotoxicity test The cytotoxic effect of GPS on lymphoid cells was determined as described in an earlier publication (Kierszenbaum & Budzko, 1977). Briefly, 0 2 ml of fresh GPS was incubated at 370 for I h with 0 1 ml of cell suspension containing I x 106 viable cells in the presence of divalent cations added for optimal C activity. After cooling the reaction mixture in an ice bath, viable cells were counted microscopically using a Neubauer haemocytometer. Results were expressed as percentage cytotoxicity with reference to the number of viable cells present in control reaction mixtures in which HI-NMS had been substituted for GPS (0% cytotoxicity). The lack of cytotoxicity of HI-NMS on mouse lymphocytes was established in previous work (Budzko et al., 1977; Kierszenbaum & Budzko, 1977). Preparation of mouse lymphoblastic cells Thymus or spleen cell suspensions in RPMI-1640 containing 5 x 106 viable, nucleated cells/ml and 1 yg/ml Con A (Sigma Chemical Company, St Louis, Missouri) were incubated in sterile tissue culture flasks (Falcon, Oxnard, California) at 370 for 48 h in a 5% C02-in-air atmosphere saturated with water vapour. Cultures were performed either in the presence or absence of 2% HI-FCS (Microbiological Associates, Walkersville, Maryland). After incubation, the cells were washed three times with RPMI-1640 medium and adjusted to I x 107 viable cells/ml in the same medium. Thymus cell preparations to be used in some control experiments were incubated with Con A for only 30 min under the same conditions. After washing, these cells were used in cytotoxicity assays to establish if the presence of Con A on the cell surface could interfere with guinea-pig C activation. In additional control experiments untreated cells were used and Con A was directly added to the reaction mixtures. Cells were also incubated in the absence of Con A for use in further control cytotoxicity assays. Proportions of blastic cells present in cell suspensions were determined by microscopic examination of cell preparations fixed in methanol and stained with Giemsa.

Indirect immunofluorescence Cells suspended in Eagle's minimal essential medium (Flow Laboratories, Rockville, Maryland) buffered

Surface changes on mouse T lymphoblasts with HEPES were incubated in an ice bath for 30 min with a 1/20 dilution of either HI-anti-Thy-1 2AF or heat-inactivated ascites fluid from normal, nonimmunized mice (HI-NAF). After being washed five times with the same medium, the cells were further incubated in an ice bath with a solution of fluoresceinlabelled goat anti-mouse Ig (Antibodies Incorporated, Davis, California) for 30 min. The cells were then washed five more times and examined under a Zeiss fluorescence microscope. Immune adherence Cells, 2 x 105, suspended in 50 p1 of RPMI-1640 medium were mixed with an equal volume of fresh GPS and incubated at 370 for 30 min. After washing three times, the cells were resuspended in 50 MI veronal buffer pH 7-5 and 50 p1 of O-positive red cell suspension (2 x 108 red cells/ml) was added. The mixture was incubated at 370 for 1 h. After this period, proportions of rosette-forming cells (RFC) were determined microscopically among the live and dead cells. Viability was defined by trypan blue exclusion. HI-GPS or serum-free medium was substituted for GPS in control reactions. Disodium ethylenediaminotetraacetate (EDTA) or sodium ethyleneglycoltetraacetate (EGTA), when added to the reaction mixtures, were present at a concentration of 10 mm. Excess magnesium ions (1 mM) were added when EGTA was used.

Absorptions HI-anti-Thy-12 AF diluted 1/10 in RPMI-1640 medium was incubated at room temperature for 1 h with either normal thymocytes of Con A-induced thymoblasts at a concentration of 1 5 x 108 cells/ml. Cells were then removed by centrifugation and the residual cytotoxic activity of the absorbed material was titrated using normal thymocytes as target cells. A control titration was performed with an aliquot ofthe HI-antiThy-1.2 AF incubated in the absence of cells. Additional controls were included to independently test the source of C, GPS 1/20. Neuraminidase treatment Suspensions containing 1 x 107 cells and 50 units of Vibrio cholerae neuraminidase (VCN, Calbiochem, San Diego, California) per ml were incubated at 370 for 45 min. After washing three times with cold RPMI-1640 the cells were counted and adjusted to 1 X 107 viable cells/ml for use in cytotoxicity assays. Control treatments were performed with aliquots of

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VCN previously inactivated by heating at 70° for 45 min (HI-VCN).

RESULTS Insensitivity of T lymphoblasts to guinea-pig C-dependent lysis Cytotoxicity assays using CBA/J mouse thymocytes as target cells readily confirmed the high degree of susceptibility of these cells to lysis by either GPS or treatment with allogeneic anti-Thy- 1.2 antibodies plus C (Table 1). By contrast, preparations containing over 80% lymphoblasts induced by stimulation with Con A were markedly resistant to killing by either treatment. Incubation of mouse thymocytes with Con A for only 30 min was insufficient to render cells insensitive to killing by either GPS or anti-Thy- 1.2 plus C. The small proportion of lymphoblasts present in this preparation was comparable to that found in suspensions of unstimulated thymocytes. Con A stimulation, however, was a requirement for the development of cell resistance to lysis since mere incubation in the absence of the mitogen failed to alter the high degree of sensitivity of thymocytes to either cytotoxic treatment. Con A-induced blasts originating from mouse spleen cells were also resistant to the non-specific, though selective (Budzko et al., 1977), cytotoxic effect of GPS and they were insensitive to specific lysis by anti-Thy- 1.2 and C (Table 2). The presence or absence of HI-FCS in the culture medium did not influence the results, indicating that the apparent lack of expression of Thy- 1 and GPCA was not a consequence of the absence of essential serum components in the medium. Additional control assays were included in this experiment which involved (a) the use as target cells of thymocytes previously incubated with Con A for 30 min and (b) testing the susceptibility of untreated thymocytes to lysis by either anti-Thy-1.2 plus, C or GPS in the presence of excess Con A. The amount of Con A added to the reaction mixture represented a twenty-eight-fold increase in concentration over that initially present in stimulated cell cultures. These conditions failed to interfere with lysis of the highly susceptible thymocytes (which provide a more sensitive

cytotoxicity assay). Detection of Thy-1.2 and GPCA on mouse T lymphoblasts In view of past reports by other investigators that

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F. Kierszenbaunrn& Delia B. Budzko

Table 1. Lack of detection of the Thy-1.2 and GPCA markers on Con A-induced mouse thymoblasts in C-dependent cytotoxicity tests

% Cytotoxicity Untreated Thymocytes incubated Thymocytes incubated thymocytes with Con ASor 48 h* with Con A for 30 min

Material tested GPS

HI-anti-Thy-1.2AF+GPS 1/20 HI-anti-1.2AF GPS 1/20 HI-NMS

7*T

85 2 93 4 0

9

0

°

Thymocytes incubated in the absence of Con A for 48 h*

92-6 95 0 0

85 7 90 4 21

0

0

1 5 (0-3)

0-2 (0-1)

0>

%/ Blasts 2 3 (2-3)t

85-0 (&L-92)

* Serum-free cultures. t Numbers in parentheses represent extreme range values.

Table 2. Resistance of mouse spleen T cell blasts induced by Con A to lysis by GPS or anti-Thy- 1.2 plus C

% Cytotoxicity Spleen cells Spleen cells incubated-with incubated with Untreated Con A for 48. h Con A for 48 h Untreated Thymocytes incubated spleen cells (serum-free) (HI-FCS present) thymocytes with Con A for 30 min

Material tested GPS

HI-anti-Thy-1.2AF+GPS 1/20 HI-anti-Thy-1.2AF GPS 1/20 GPS+28 pg/ml Con A HI-anti-Thy-1 .2AF + GPS 1/20 + 28 ,g/ml Con A HI-NMS

53'

412 39-7 0 0 ND*

5-3i 0 0 ND

32 7-2 0 0 ND

932 99 1 10 0 90.0

926 95 0 0 0 ND

ND 0

ND 0

ND}

98-3 0

ND 0

ND

ND

0 '

4*1 (3 3-5 4)t

88-1 (83 4-92 7y)

Blasts

938 (89-7-96 8)

* ND, not determined. t Numbers in parentheses represent extreme range values.

Surface changes on mouse T lymphoblasts

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Table 3. Detection of Thy-1.2 on mouse Con A thymoblasts by indirect immunofluorescence

Fluorescent staining* after pre-incubation with

Cells

HI-anti-Thy-1.2AF

NI-NAF

Unstimulated thymocytes Con A-thymoblastst

Positive (97%) Positive (98%)$

Negative (< 1%) Negative (< 2%)

* Fluorescein-labelled goat anti-mouse Ig was used (see Materials and Methods). t Eighty-seven per cent of the cells present in this suspension were lymphoblasts. $ Numbers in parentheses represent percentages of fluorescent cells in each of the suspensions.

Thy- I antigen can be detected on murine lymphoblasts by immunofluorescence and indirect autoradiography (Vischer, 1972; Jones, 1972; Boyd, Rolland & Cauchi, 1974) and the fact that we had used C-dependent cytotoxicity tests, detection of Thy-1.2 and GPCA on lymphoblastic cells was further attempted by using alternative methods. Table 3 exhibits the results of a representative experiment showing not only that Thy-1.2 was expressed on the surface of Con A thymoblasts but also that this antigen was readily accessible to specific antibodies for binding. Thus, virtually all of the cells (98%) present in the suspension, containing 87% cells in blastic stages, were positively stained by fluorescein-labelled goat anti-mouse Ig after pre-incubation with HI-anti-Thy-1 .2AF fol-

lowed by washing. Control immunofluorescence tests using cells which had been pre-incubated with HINAF gave negative results. Similar results were obtained with unstimulated thymocytes, except that the proportion of blasts in this preparation was

1%. Absorption of diluted HI-anti-Thy-i .2AF with thymoblasts resulted in a considerable loss of specific lytic activity of this reagent when tested in the presence of GPS diluted 1/20 as a source of C (Table 4). A comparison of titrations of aliquots of HI-anti-Thy- I .2AF absorbed with either normal thymocytes or Con A thymoblasts failed to reveal any significant differences. Conditions resulting in partial absorption of anti-Thy-1.2 were selected for this type of experiment

Table 4. Absorption of anti-Thy- 1.2 antibodies by normal and Con A-induced blastic thymocytes

HI-anti-Thy- I.2AFtabsorbed with Dilution* of HI-anti-Thy-i .2AF or other material tested Nothing Thymoblasts Thymocytes 1/10 1/20

1/40 1/80 1/160 1/320

1/640 GPS 1/20

HI-NMS

100+ 100 93 8 984 96 9 90 8 93-8 0 0

17 6 108 31 0 08 0 0 0 0

20-0 115 46 15 0 0 0 0 0

* Dilutions were made in RPMI- 1640 medium.

t Diluted 1/10 before absorption.

t GPS 1/20 was used as a source of C. Results are expressed as % cytotoxicity.

F. Kierszenbaum & Delia B. Budzko

832

to be able to compare the relative absorptive capacities of thymocytes and thymoblasts. Reactivity of Con A thymoblasts with guinea-pig C was evidenced by the capacity of these cells to form immune adherence rosettes following incubation with GPS (Table 5). GPCA, which was identified on the

Restoration of Con A blast sensitivity to guinea-pig C-dependent lysis by treatment with VCN

Table 5. Guinea-pig C reactivity of mouse Con A thymoblasts (detection of GPCA)

°OORFC* among Treatment of cells

Live cells Dead cells

GPS GPS+ l0mM EGTA+ I mM Mg2+ GPS+ IO mM EDTA HI-GPS RPMI-1640

340 25 9 0 0

507 31.0 0 0 0

0

No. live (or dead) RFC *

RFC=

x

100

Total No. live (or dead) cells

surface of live as well as dead lymphoblasts, was also detectable after reacting the cells with GPS in the presence of EGTA to chelate calcium ions, i.e. to inhibit C activation via the classical pathway. Addition of EDTA, which chelates both calcium and magnesium ions, inhibited the reaction. No rosette formation occurred when HI-GPS or RPMI-1640 medium was used instead of GPS.

Lysis of murine thymocytes by GPS has been shown to require activation of C via the alternative pathway but not antibody (Budzko et al., 1977; Kierszenbaum & Budzko, 1977). Furthermore, activation ofthe alternative C pathway in human serum by sheep erythrocytes has been shown to require removal of a substantial amount of sialic acid residues from the cell surface (Fearon, 1978). Therefore, we decided to test the effects of VCN treatment, which specifically cleaves sialic acid residues, on the reactivity of mouse lymphoblasts with guinea-pig C. Concomitantly, we tested the susceptibility of these cells to specific killing by anti-Thy-1.2 antibodies and C. Thymoblasts treated with VCN, but not those treated with HI-VCN, were readily killed by GPS as well as by anti-Thy-1.2 plus C (Table 6). Neither HI-antiThy-1.2AF nor GPS diluted 1/20 were capable, separately, of causing lysis of the VCN-treated blasts. Results of control assays using thymocytes not incubated with Con A revealed the presence of the corresponding activities in the cytotoxic reagents employed. As in previous experiments, the thymoblasts were insensitive to lysis by GPS or anti-Thy-1.2 and C.

DISCUSSION These results indicate the occurrence of significant changes in surface characteristics of mouse T lympho-

Table 6. Sensitivity of VCN-treated Con A thymoblasts to lysis by GPS or anti-Thy- 1.2 antibodies plus C

% Cytotoxicity Thymoblasts* subjected to Normal Material tested GPS

HI-anti-Thy-1.2AF+GPS 1/20 HI-anti-Thy- 1.2AF GPS 1/20 HI-NMS

thymocytes No treatment VCN HI-VCN 92-3 93 0 0 0 0

0 4-0 0 0 0

93-8 95-0 0 0 0

0 43 0

0 0

* Mouse thymus cells incubated with Con A (1 pg/ml) at 37° for 48 h in serum-free RPM I- 1640 medium and washed three times with the same medium. The suspension contained 91% + 5% blasts.

Surface changes on mouse T lymphoblasts cytes while undergoing Con A-induced blastic transformation. A notable consequence of these changes was the lack of detectability of Thy-I and GPCA by means of C-dependent cytotoxic reactions despite the actual presence of these markers on the cell membrane. Identification of both Thy-I and GPCA on the lymphoblasts by using the same types of reactions was possible, however, following treatment of these cells with neuraminidase, thus implicating surface sialic acid residues in the production of the observed alterations. Several findings indicated that lack of detectability of Thy-1.2 and GPCA in terms of cell insensitivity to killing by anti-Thy-1.2 plus C and GPS, respectively, was dependent upon effective lymphocyte transformation. Thus, lymphocytes cultured in the absence of Con A were readily lysed by these treatments. Furthermore, incubation with Con A for only 30 min, a period of time which was insufficient for the stimulated lymphocytes to attain blastic status, failed to induce any noticeable changes in detectability of Thy- 1.2 and GPCA. Further, Con A added to reaction 'mixtures at concentrations twenty-eight times higher than used to induce blastic transformation failed to modify the susceptibility of the highly sensitive murine thymocytes to undergo lysis by either GPS or anti-Thy- I plus C. After washing the cultured cells, a certain amount of dead cells and some cellular debris were still present in the blast suspensions used in cytotoxicity assays. Attempts were made to isolate the living blasts from the other components of the mixture but quantitative recovery could not be achieved. To circumvent having to deal in our tests with selected blast subpopulations which might have not been representative of the whole pool, cytotoxicity assays were performed with relatively 'dirty' cell suspensions. In view of this circumstance, the possibility was considered that C consumption or competitive binding of anti-Thy- 1.2 antibodies by markers present on dead cells could have resulted in an apparent blast cell insensitivity to lysis. This possibility was precluded by two findings. First, thymocytes cultured in the absence of Con A were readily killed by GPS or anti-Thy- I plus C despite the presence of dead cells and cellular debris in the suspension. Second, VCN-treated blast suspensions, which also contained the contaminants, underwent lysis when incubated with these reagents. It seems highly unlikely that VCN may have affected only surface components present in dead cells, sparing those expressed on intact blasts. B cells are insensitive to GPS cytotoxicity (Budzko

833

et al., 1977) and certainly not affected by anti-Thy-I plus C. On this basis, B lymphoblasts would not be expected to be sensitive to these reagents. Possible recruitment of B lymphocytes present in spleen cell suspensions by some of the Con A-activated T lymphocytes could conceivably complicate interpretation of our results. Two facts tend to minimize concern over this matter however. First, results obtained by using thymus cell suspensions, whose B-cell content is negligible, were essentially the same as with splenocytes. Second, 95% of the cultured spleen cells survived treatment with either GPS or anti-Thy-I plus C. It is difficult to envisage that such a high proportion of B-cell blasts would be present in cultures of mouse lymphocytes triggered with a specific T-cell mitogen such as Con A. Detection of Thy- 1.2 by indirect immunofluorescence and absorption of anti-Thy- 1.2 leaves no doubt that Thy-1.2 was present on the surface of the Con A blasts. These results are in agreement with those of other investigators who reported the presence of this marker on murine T lymphoblasts by either immunofluorescence or autoradiography (Vischer, 1972; Jones, 1972; Boyd et al., 1974). Since both positive immunofluorescence and absorption reveal actual binding of anti-Thy-1.2 to surface Thy-1.2, one must infer that lack of cytotoxicity is a consequence of interference with C-effected lysis. A similar conclusion was reached with respect of GPCA, whose presence was clearly demonstrated on the Con A blasts by immune adherence. Since the latter reaction represents C activation, it would appear that lack of production of cell killing by GPS reflects interference with C after its initial triggering. Immune adherence was also observed after the blasts were incubated with GPS in the presence of EGTA, indicating involvement of the alternative but not the classic C pathway. In this context, it is noteworthy that GPS killing of murine T cells proceeds exclusively via the alternative C pathway (Budzko et al., 1977; Kierszenbaum & Budzko, 1977, 1978). Therefore, detention of the lytic process occurs after activation of C3. The mechanism and precise surface alterations affecting detection of Thy- 1.2 and GPCA in C-dependent cytotoxicity tests are not revealed by the present results. However, restoration of sensitivity of the lymphoblasts to either anti-Thy- 1.2 plus C or GPS by treatment with neuraminidase indicates that surface sialic acid residues play a relevant role in preventing C-effected killing. This observation is reminiscent of the phenomenon described by Fearon (1978) in which

F. Kierszenbaum & Delia B. Budzko

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removal of sialic acid rendered sheep red blood cells sensitive to non-specific lysis by normal human serum. It should be noted, however, that the present findings, which apply to guinea-pig C, should not be extrapolated to other sources of C. Although it is conceivable that blastic transformation as induced in this work may lead to a reduction in cell concentration of Thy-I or GPCA with respect to unstimulated lymphocytes, this possibility was not supported by the results of our experiments showing that normal thymocytes and blasts had comparable absorptive capacities under conditions which did not allow for complete removal of antibody activity. The results, highlighting changes in reactivity of mouse T lymphoblasts with guinea-pig C, add to the description of surface alterations occurring during lymphocyte transformation (Kerbel, 1977; Robinson, Anderton & Roitt, 1977; Speckart, Boldt & Ryerson, 1978). Furthermore, Paraskevas & Lee (1976) reported a striking contrast between unprimed and antigen-primed T cells in terms of abrogation of helper cell function after treatment with anti-Thy-l and C; only the latter were unaffected. Therefore, it is conceivable that surface alterations manifested by lymphoblastic T cells such as described here may not occur exclusively on Con A-induced blasts. If this turns out to be the case, selective lysis of T lymphocytes other than those engaged in lymphoblastic transformation may afford a new means for separating specific antigen-reactive T lymphocyte clones. Such a possibility is subject of current studies in our laboratory.

ACKNOWLEDGMENTS The work was supported in part by grants from the National Institutes of Health (CA-25042 and AI-14848) and the Michigan Heart Association. Published as Michigan Agricultural Experiment Station Journal Article 8721. REFERENCES BOYD R.L., ROLLAND J.M. & CAUCHI M.N. (1974) Membrane antigenic changes associated with PHA transformation of mouse spleen cells in vitro. Immunol. Commun.

3,337. BUDZKO D.B., KIERSZENBAUM F. & WAKSMAN B.H. (1977) Cytotoxic effects of normal sera on lymphoid cells. 111. Selective killing of mouse T cells by normal guinea pig serum. J. Immunol. 119, 381.

CANTOR H. & BOYSE E.A. (1977) Regulation of cellular and humoral immune responses by T-cell subclasses. Cold Spring Harbor Symp. Quant. Biol. 41, 23. FEARON D.T. (1978) Regulation by membrane sialic acid of /31H-dependent decay-dissociation of amplification C3 convertase of the alternative complement pathway. Proc. natn. Acad. Sci. (U.S.A.), 75, 1971. JONES G. (1972) Lymphocyte activation. I. Expression of theta, H-2 and immunoglobulin determinants on lymphocytes stimulated by phytohaemagglutinin, pokeweed mitogen, concanavalin A or histocompatability antigen. Clin. exp. Immunol. 12, 391. KERBEL R.S. (1977) Surface changes accompanying lymphocyte activation. Scand. J. Immunol. 6, 1029. KIERSZENBAUM F. & BUDZKO D.B. (1977) Cytotoxiceffectsof normal sera on lymphoid cells. I. Antibody-independent killing of heterologous thymocytes by guinea pig, rabbit, and human sera: role of the alternative pathway of complement activation. Cell. Immunol. 29, 137. KIERSZENBAUM F. & BUDZKO D.B. (1978) Identification and characterization of a new subset of rat T lymphocytes. J. Immunol. 121, 166. KIERSZENBAUM F. & BUDZKO D.B. (1979) The guinea pig complement activating surface marker (GPCA): functional evidence for its characteristic presence on murine T lymphocytes. In: The Molecular Basis of Immune Cell Function (Ed. by J. G. Kaplan). Elsevier/North-Holland Biomedical Press B.V. Amsterdam. (In press.) OPPENHEIM J.J. & ROSENSTREICH D.L. (1976) Signals regulating in vitro activation of lymphocytes. Progr. Allergy, 20, 65. PARASKEVAS F. & LEE S.T. (1976) Helper cell function of primed T cells. II. T-T cell synergism between Ig+ and Ig- subpopulations of primed thymocytes: a mechanism for amplification of helper cells function. Europ. J. Immunol. 6, 862. REIF A.E. & ALLEN J.M. (1966) Mouse thymus iso-antigens. Nature (Lond.), 209,521. ROBINSON P.J., ANDERTON B.H. & RoITT I.M. (1976) Surface membrane changes in mitogen-transformed lymphocytes. Exp. Cell Res. 102, 43 1. SPECKART S.F., BOLDT D.H. & RYERSON K.L. (1978) Cell surface changes in transformed human lymphocytes. I. Con A and E-PHA induced unique changes in surface topography. Exp. Cell Res. 111,385. VISCHER T.L. (1972) Immunoglobulin-like surface molecules and theta antigen during the specific and non-specific stimulation of mouse spleen cells in vitro. Clin. exp. Immunol. 11, 523. WAKSMAN B.H. & NAMBA Y. (1976) On soluble mediators of immunologic regulation. Cell Immunol. 21, 161. WEDNER H.J. & PARKER C.W. (1976) Lymphocyte activation. Progr. Allergy, 20, 195. WILLIAMS A.F. (1978) Differentiation antigens of the lymphocyte cell surface. Contemp. Top. mol. Immunol. 66, 116. WILLIAMS A.F. & STANDRING R. (1977) Glycoproteins and differentiation antigens of the cell surface of rodent lymphocytes. Progr. Immunol. 3, 41.

Changes occurring on the surface of mouse T cells during concanavalin A-induced lymphoblastic transformation.

Immunology 197938 827 Changes occurring on the surface of mouse T cells during concanavalin A-induced lymphoblastic transformation F. KIERSZENBAUM &...
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