Growth of an interleukin 2/interleukin 4-dependent T cell line induced by granulocyte-macrophage colony-stimulating factor (GM-CSF)

Lymphokine activities in conditioned medium from activated helper T cell lines are most commonly defined by the proliferation of "specific" lymphokine-dependent cell lines. Various sublines of IL 2-dependent (and ostensibly specific) HT-2 and CTLL cells have now been shown to proliferate in response to BSF-1/IL 4 as well. After activation with antigen or mitogen, D10.G4.1, an antigen-specific cloned T helper cell that has recently been shown to produce IL 4 but not IL 2, secretes two distinct cytokines that induce the growth of HT-2 cells. These "T cell growth factors" (TCGF) can be separated by reversed phase high-performance liquid chromatography (RP-HPLC). The TCGF activity of one of these factors can be blocked by 11B11, an antibody specific for IL 4. The second TCGF activity is not affected by 11B11 or by antibodies specific for IL 2. This TCGF activity can be neutralized by a goat polyclonal antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF), and has a RP-HPLC elution profile identical to that of recombinant GM-CSF. Recombinant GM-CSF induces both proliferation and long-term growth of HT-2 but not CTLL cells, and this activity can be neutralized by the same antibody to GM-CSF. GM-CSF is best known as a factor that induces the maturation and growth of granulocytes and macrophages from bone marrow-derived hematopoietic precursor cells. The ability of GM-CSF to induce the growth of certain T cell lines indicates that this molecule may play a role in T cell-mediated immune responses, either as an autocrine growth factor or a paracrine stimulus from both lymphoid and nonlymphoid tissues that produce this cytokine.     

Immunoregulation by mouse T-cell clones. I. Suppression and amplification of cytotoxic responses by cloned H-Y-specific cytolytic T lymphocytes

H-Y-specific and H-2Db-restricted, Lyt-1-2+ T-cell clones ( CTLL ) with graded specific cytotoxic activities on male C57BL/6 (B6) target cells ( 1E3 , ; 2C5 , ++; 2A5 , +, 3E6 , +/-) were tested for their capacity to inhibit the generation of H-Y-specific cytotoxic T lymphocytes (CTL) in vitro. Addition of irradiated lymphocytes of CTLL 1E3 and CTLL 3E6 but not those of CTLL 2A5 or CTLL 2C5 abolished the generation of CTL from in vivo primed H-Y-specific precursor cells (CTLP) when added to fresh mixed-lymphocyte cultures (MLC). Exogenous sources of T-cell growth factors (TCGF) did not overcome suppression. Rather the presence of TCGF resulted in a further enhancement of suppressive activities in CTLL 1E3 and 3E6 and the induction of similar activities in cells from CTLL 2A5 and 2C5 , which by themselves were not inhibitory. Moreover when added to similar MLC on Day 1 instead of Day 0, only irradiated cells of CTLL 3E6 but not those of the other three CTLL were suppressive. Induction of suppressive activities in H-Y-specific CTLL was independent of the appropriate male stimulator cells since it was also observed in MLC induced by irrelevant antigens (H-2, trinitrophenol). Furthermore at low cell numbers, irradiated lymphocytes from any of the CTLL consistently enhanced CTL activities generated from H-Y-specific CTLP. This augmenting activity, which was not TCGF, could be transferred by soluble mediators present in antigen-sensitized CTLL cultures. Thus, these data indicate (i) that cytotoxic effector cells can function as suppressor cells in the generation of CTL, (ii) that the cytotoxic activity of cloned CTL does not correlate with their capacity to suppress CTL responses, (iii) that the inhibition of CTL responses by CTLL is not due to simple consumption of T-cell growth factors produced in MLC, and (iv) that different CTL clones may interfere with the generation of CTL at different stages of their maturation. Moreover, the experiments suggest an antigen-independent enhancement of suppression by the interaction of CTL with lymphokines. Together with the augmenting activity evoked by cloned CTL the data provide strong evidence for the expression of multiple immunological functions by one particular subset of T cells and suggest that cytotoxic effector cells can differentially regulate the maturation and/or clonal expression of their precursor cells.     

The effect of T cell growth factor on the generation of cytolytic T cells.

The development of cytotoxic effector cells through primary allogeneic mixed tumor-lymphocyte culture (MTLC) was found to be accompanied by the production of T cell growth factor (TCGF). Addition of supplemental TCGF to MTLC resulted in the generation of significantly greater quantities of effector cells, and these effector cells displayed augmented cytotoxic activity. The TCGF-induced effect could not by duplicated by the addition of fresh medium or a mitogenic concentration of concananvalin A. Although TCGF augmented the proliferation of antigen-nonreactive cells, antigen-reactive cells appeared to be preferentially stimulated by TCGF. Finally, it was shown that depletion of TCGF from MTLC resulted in an impairment of proliferation and differentiation of cytotoxic effector cells. These findings demonstrate that soluble factors are involved in the regulation of in vitro cell-mediated immune responses in an analogous manner to similar factors that have been shown to regulate humoral immune responses. Therefore, the forces affecting TCGF production may modulate the amplitude of a T cell-mediated cytolytic response.     

Dexamethasone-mediated inhibition of human T cell growth factor and gamma-interferon messenger RNA

Glucocorticoids suppress the proliferation of human T lymphocytes. Activated T lymphocytes require T cell growth factor (TCGF) for proliferation. TCGF is produced by a subset of T lymphocytes, and this production is regulated at the TCGF mRNA level. Dexamethasone, a synthetic glucocorticoid, strongly inhibits the synthesis of TCGF mRNA in human normal peripheral blood lymphocytes stimulated in culture with phytohemagglutinin. It also inhibits the accumulation of gamma-interferon mRNA in these cells. This dual effect may in part explain some of the immunosuppressive and anti-inflammatory effects of glucocorticoids.     

Cell lines derived from human autorosette-forming cells with suppressive activity

Concanavalin-A-stimulated human T lymphocytes from healthy donors and from patients suffering from diverse immune disorders were fractionated into rosette-forming (R) and nonrosette-forming (NR) cells. The separation method is based upon the ability of the lymphocytes to bind autologous erythrocytes and form autorosettes. Long-term cultures of the R and NR subpopulations were established. The activity of the culture supernatants on the T cell proliferation of normal human phytohemagglutinin (PHA)-induced lymphocytes and of a murine, interleukin-2 (IL-2)-dependent cytotoxic T cell line (CTLL) was investigated. Only the R cell line-derived supernatants from almost all patients tested evinced potent suppressor activity, those from healthy donors less so. The suppressive function was demonstrated not to be due to a cytotoxic effect since preincubation of the PHA-induced lymphocytes and CTLL cells with the factor did not diminish their proliferative capacity. Our study indicates the existence of a competitive relationship between the suppressor factor and IL-2. We found that inhibition of the proliferation decreased with the addition of increasing quantities of exogenous IL-2. We also observed that preincubating the CTLL cells with IL-2 prior to exposing them to the suppressive factor precludes inhibition of their proliferation. Phenotypic analysis of the suppressor cell line revealed that they were comprised of a T cell population which included OKT4+ and OKT8+ cells and that 99% of the cells formed autorosettes. Preliminary purification of the suppressive factor was performed by ultrafiltration and maximal suppression was exhibited by the fraction of less than 10,000 daltons. The development of suppressor cell lines from the unique population of autologous rosette-forming cells may be very helpful in studying the immunoregulatory properties of these cells and their suppressor activity.     

Biological and biochemical characterization of bovine interleukin 2. Studies with cloned bovine T cells

Bovine peripheral blood lymphocytes stimulated with the T cell mitogen concanavalin A (Con A) secrete a lymphokine with biological properties similar to T cell growth factor (TCGF) or interleukin 2 (IL 2) from other species. The material supports proliferation of Con A-derived T cell blasts, limiting dilution cloning of T cell blasts, and continuous growth of T cell clones for over 6 mo in vitro. A quantitative microassay with the use of TCGF-dependent, Con A-unresponsive cloned T cells was used to determine the biological activity during purification of IL 2. A single peak of activity with an apparent m.w. of 25,000 to 28,000 was recovered after gel filtration. This material eluted from DEAE-Sephacryl between 135 and 165 mM NaCl. After isoelectric focusing, high pressure liquid chromatography, and gel electrophoresis under reducing conditions, peak IL 2 activity was associated with proteins having m.w. of 20,000 and 23,000.     

Preparing T cell growth factor from rat splenocytes

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization (3). Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro (3). Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later and excess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20 degrees C.     

Detection of HSV-1-induced lymphokine production in human cells by a direct indicator cell addition assay

These studies present an efficient and sensitive method for detection of T cell growth factor (TCGF) activity in human lymphocyte cultures and illustrate that T cell growth factors are associated with T lymphocyte-mediated anti-HSV-1 responses. Secretion of TCGF is induced after stimulation of human peripheral blood mononuclear cells ( PBMNC ) with herpes simplex virus type 1 (HSV-1). Lymphokine activity is detected in a simple, sensitive method by studying [3H]thymidine incorporation after the addition of murine CTLL -20 cells to cultures of gamma-irradiated (4000 R), virus-stimulated PBMNC . By using this assay, we find that PBMNC from seropositive but not seronegative individuals produce detectable TCGF activity in a dose-dependent manner after incubation with HSV-1. Maximum activity is detected between 24 to 48 hr of incubation and correlates with in vitro proliferation of nonirradiated PBMNC in response to the virus. In addition, gamma-irradiated (1000 to 3000 R) PBMNC , which are frequently used as a source of antigen-presenting cells (APC), can secrete TCGF after contact with HSV-1. Lymphokine production by the APC-containing population is eliminated by gamma-irradiation (5000 R); such APC can still present UV-inactivated HSV-1 to HSV-1-responsive lymphoblasts, indicating that lymphokine production by T cells residing in the APC population is not essential for antigen presentation.     

Three different signals are required for the induction of cytolytic T lymphocytes from resting precursors

The requirement for the signals in induction of cytolytic T lymphocytes (CTL) has been investigated. C57BL/6 X CBA/T6 F1 spleen cells stimulated with the lectin leukoagglutinin (L-A) failed to show CTL activity in a PHA-facilitated assay, although L-A-activated splenic T cells were able to respond to T cell growth factor (TCGF). Concanavalin A (Con A) on the other hand was able to induce cytolytic activity from CTL-P, as well as to render splenic T cells responsive to TCGF. Furthermore, L-A-activated splenic T cells could generate cytolytic activity upon subsequent culture in secondary mixed leukocyte culture supernatant (2 degrees MLC SN). In contrast, EL-4-derived SN (EL-4 SN) was unable to induce cytolytic activity from L-A-activated spleen cells. In addition, proliferation of L-A-activated spleen cells cultured in EL-4 SN was similar to those cultured in 2 degrees MLC SN. Nonactivated spleen cells were totally unresponsive to both SN in proliferation and generation of CTL. Analysis of T cell clones for the production of a factor necessary for induction of cytolytic activity revealed that both cytolytic and noncytolytic T cell clones were able to produce a factor(s) for the generation of cytolytic activity from L-A-activated T cells. On the other hand, SN from certain antigen-stimulated T cell clones produced factors capable of inducing cytocytic activity by L-A-activated T cells only in the presence of EL-4 SN. Neither EL-4 SN nor cloned T cell SN alone had such a capacity. The nature of the necessary lymphokines in the SN from the clone cells or from the EL-4 is unknown. In the case of the EL-4 SN, it is not known whether the presence of TCGF plays a role or whether that role is perhaps more differentiative than proliferative. This study provides evidence that the induction of CTL from CTL-P can be dissociated into activation, which is required to render T cells responsive to second signals, and differentiation, which is mediated by two different factors.     

Effect of cyclosporin A on human lymphocyte responses in vitro. III. CsA inhibits the production of T lymphocyte growth factors in secondary mixed lymphocyte responses but does not inhibit the response of primed lymphocytes to TCGF

The mechanism whereby Cyclosporin A (CsA) inhibits secondary mixed lymphocyte responses was assessed. CsA added to secondary MLR cultures inhibited proliferation and induction of cytolytic lymphocyte activity. This inhibition was found to be associated with the inhibition of T lymphocyte stimulating growth factor(s) (TCGF) production in the supernatants of secondary MLR cultures. As little as 1.0 micrograms/ml of CsA added to secondary MLR cultures resulted in no measurable TCGF activity. In contrast, moderate doses of CsA (1.0, 2.5 micrograms/ml), which completely inhibited the secondary MLR response to alloantigen, did not inhibit the proliferative and CML response of alloantigen-primed lymphocytes to these stimulating growth factors. Even at high doses of CsA (20 micrograms/ml), substantial levels of proliferation (50% of control response) and CML induction (60% of control response) were observed when the primed cells were exposed to secondary MLR supernatants containing TCGF activity. It was concluded that inhibition of secondary mixed lymphocyte responses by CsA may be due in part to the inhibition of TCGF production rather than the inhibition of the effect of TCGF on mature cytotoxic T lymphocytes.     

Contra-IL 2; a suppressor lymphokine that inhibits IL 2 activity

Suppressive activity of culture supernatant of AS-9 (AS-9 CS), a T cell hybridoma line that was derived from fusion of BW5147 thymoma and splenic T cells of anti-lymphocyte serum-treated C3H mice, was analyzed. AS-9 CS inhibited allogeneic cytotoxic T lymphocyte (CTL) generation as well as T cell proliferation to alloantigens and mitogens, but failed to inhibit B cell response to lipopolysaccharide or growth of tumor and fibroblast cells. Although addition of AS-9 CS to the allogeneic sensitization culture as late as on day 2 of incubation resulted in maximal inhibiton of CTL generation, removal of AS-9 CS on day 3 of incubation abolished its inhibitory effect. Addition of purified IL 2 together with AS-9 CS to the allogeneic sensitization cultures only partially abrogated the suppression. Experiments with IL 2-dependent cytotoxic T cell line (CTLL) showed that AS-9 CS suppressed the IL 2-induced proliferation of CTLL. Preincubation of AS-9 CS with CTLL removed its inhibitory effect on CTL generation. These results indicate that AS-9 CS interferes with the mechanism of T cell activation by IL 2. On this basis, AS-9 CS was named contra-IL 2.     

Biological Activities of Guinea Pig Mitogenic Factor from Immune Lymphocytes Stimulated with Antigen

Mitogenic factor was produced by sensitized guinea pig lymph node cells stimulated with a specific antigen. Both T lymphocytes and macrophages were required for the production of this factor. The culture supernatant of lymphocytes containing the mitogenic factor exhibited a strong helping effect on the proliferative response of T lymphocytes to phytohemagglutinin (PHA). Mitogenic factor and the factor with the helping activity coeluted in the molecular weight range of 25,000-35,000 daltons in gel filtration. Furthermore the fraction containing mitogenic factor was found to support the proliferation of lymphoblasts induced by PHA or antigen, suggesting that the mitogenic factor may be the guinea pig equivalent of T cell growth factor (TCGF) reported in the mouse, rat, and human. On the other hand, the T cell-activating monokine of the guinea pig, possessing the helping activity for the proliferative response of T lymphocytes to PHA, never exhibited TCGF-like activity.     

Antigen-dependent proliferation of cloned continuous lines of H-2-restricted influenza virus-specific cytotoxic T lymphocytes

The antigenic requirements for in vitro proliferation of several cloned continuous lines of H-2-restricted influenza virus-specific cytotoxic T lymphocytes (CTL) has been examined. The cloned CTL lines were established from individual splenic CTL precursors obtained from A/JAPAN/305/57 (H2N2)-immune F1 (C57BL/6 X BALB/c) donors. The lines were isolated (by limiting dilution in liquid culture) and expanded in the presence of A/JAPAN/305/57-infected irradiated syngeneic (F1) spleen cells and T cell growth factor (TCGF) of rat spleen origin. Optimal proliferation (and long-term in vitro cultivation) of these H-2-restricted CTL lines required both specific antigenic stimulation in the form of virus-infected syngeneic spleen cells and an exogenous source of TCGF. In addition, the antigenic requirements for proliferation of these lines directly reflected the pattern of H-2-restricted influenza virus-specific recognition at the level of target cell recognition and lysis.     

Regulation of the rate of cell cycle progression in quiescent cytolytic T cells by T cell growth factor: analysis by flow microfluorometry

We have previously shown that greater than 90% of B6.1 cells, a murine cytolytic T lymphocyte (CTL) cloned line which is solely dependent on T cell growth factor (TCGF) for continuous growth in vitro, accumulates in the G1 phase of the cell cycle after transfer into culture medium containing no TCGF. Moreover, when such quiescent cells are exposed again to TCGF, greater than 85% reenter the S phase and subsequently divide in a relatively synchronous fashion. In this study, the regulation of the rate of cell cycle progression of quiescent B6.1 cells after exposure to TCGF was analyzed using two complementary DNA staining techniques, namely, the propodium iodide method (to enumerate cells entering the S phase) and the Hoechst 33342-bromodeoxyuridine substitution technique (to enumerate cells which have gone through mitosis). After TCGF addition, quiescent B6.1 cells resumed DNA synthesis and divided after a lag phase of 10 and 20 h, respectively. The duration of the lag phase was found to be dependent on the length of time during which quiescent B6.1 cells had been deprived of TCGF, but was independent of the concentration of TCGF used for restimulation. In contrast, the proportion of cells responding to TCGF as well as the rate of their first passage through mitosis was dependent on TCGF concentration. The presence of TCGF for at least 6 h was required for a maximal response. Moreover, direct evidence was obtained that TCGF by itself was able to stimulate proliferation of quiescent B6.1 cells in the absence of other growth factors and serum constituents other than bovine serum albumin, transferrin, and lipids.     

Functional characteristics of cytotoxic T lymphocyte polyoma virus-transformed fibroblast hybrids

By two different fusions between a cytotoxic T-lymphocyte (CTL) clone and a polyoma virus (Py)-transformed fibroblast line, 40 hybrid clones have been generated. It has been demonstrated that they were all TCGF independent for multiplication. Moreover, some of these hybrids were functional for cytolytic expression, whether or not TCGF was present either at the time of fusion or in the selective media. Two clones generated from the same fusion were markedly cytolytic and were able to remove TCGF from their culture medium, suggesting that they possessed TCGF receptors. These clones also secreted discrete amounts of a TCGF-like factor. The effect of TCGF on hybrid cell proliferation is discussed.     

Biochemical characterization of lymphocyte regulatory molecules. II. Purification of a class of rat and human lymphokines

Rat spleen cells activated in vitro by concanavalin A produce lymphokine molecules that possess biologic activity in a number of murine lymphocyte response assays. A single class of lymphokine most adequately described as T cell growth factor (TCGF, Interleukin-2) with a m.w. of 15,000 as estimated from gel filtration studies and with an isoelectric range of 5.4 to 5.6 stimulates i) the growth of established T cell lines in culture, ii) the proliferation of thymocytes in the presence of Con A under culture conditions where Con A alone is non-mitogenic, iii) the induction of antibody responses to heterologous erythrocyte antigens in athymic (nude) mouse spleen cell cultures, and iv) the generation of cytotoxic T lymphocytes (CTL) in thymocyte cultures and in nude mouse spleen cell cultures. We suggest that in each of the assay systems tested, this class of rat lymphokine acts directly on activated T cells. Nonactivated T cells must be stimulated by either mitogen or antigen before becoming responsive to lymphokine, but do not require antigen or mitogen for continued lymphokine-dependent proliferation. Similarly, human peripheral blood mononuclear cells activated by phytohemagglutinin (PHA) produce a class of lymphokines of identical size with an isoelectric point of 6.0 to 6.5 that possess the same biologic properties as measured in murine lymphocyte response systems.     

Helper factor production in murine secondary syngeneic mixed leukocyte reactions

Culture supernatants of murine thymocytes or spleen cells responding in a secondary syngeneic mixed leukocyte reaction (SMLR) were studied for their biologic effects on cell-mediated immune responses in vitro. Such supernatants contained helper factor(s) that facilitated the development of alloantigen-specific cytotoxic T lymphocyte (CTL) responses from thymocyte precursors. Thymocytes, but not spleen cells, required activation by allogeneic effect factor (AEF) in primary culture in order to proliferate and produce biologically active mediator(s) during a secondary SMLR. The same culture supernatants possessed, in some instances, weak T cell growth factor (TCGF; IL 2) activity. However, TCGF activity could be dissociated from helper factor(s) active in the CTL induction assay because some culture supernatants that had potent helper activity were devoid of TCGF activity. This lack of TCGF activity was not due to a lower degree of sensitivity of the TCGF assay or to the presence of a selective TCGF inhibitor in the SMLR-derived supernatants, indicating that the helper factor(s) studied is distinct from TCGF. Production of immunoregulatory lymphokines during the SMLR may serve as a physiologically relevant model for studying the role of T cell-derived lymphokines in immunoregulation.     

A permanent human cytotoxic T-cell line with high killing capacity against a lymphoblastoid B-cell line shows preference for HLA A,B target antigens and lacks spontaneous cytotoxic activity

The optimal conditions for the generation of highly cytotoxic human T lymphocytes (CTL) in vitro against a lymphoblastoid B-cell line (JY) in primary and secondary mixed lymphocyte cultures (MLC) were investigated. Variation of the stimulator:responder (S:R) cell ratio influenced both the specific cytotoxicity and the spontaneous cytotoxicity as well as the recovery of the responder cells. T lymphocytes of donor JR (HLA A23,29; B7,7; DRw5) were stimulated with JY (HLA A2,2; B7,7; DRw4,6) in primary and secondary MLC at S:R ratios of 1:50 and 1:10, respectively, since stimulations at these S:R ratios resulted in the highest specific cytotoxicity against JY, the lowest spontaneous cytotoxicity against K562 and Daudi, and in a good recovery of the responder cells. From these T-lymphocyte cultures an exponentially growing CTL line (JR-2) was obtained by weekly stimulations with irradiated JY cells at a S:R ratio of 1:1. After 2 months of culturing the growth rate of the JR-2 cells declined, but could be restored by the addition of conditioned medium, containing T-cell growth factor (TCGF). Irradiated JY cells or TCGF alone were insufficient to maintain proliferation. JR-2 cells were strongly cytotoxic for JY (50% lysis was obtained at an effector:target ratio of 1:2) but the cytotoxic activity against a classical target cell for spontaneous cytotoxicity (K562) was negligible. The cytotoxic activity of JR-2 cells against JY could be inhibited by a monoclonal antiserum W6/32, which recognizes all HLA A, B, and C specificities, and by a monoclonal antiserum directed against β2 microglobulin, whereas monoclonal anti-Ia antisera showed no inhibition. JR-2 cells lysed fresh HLA A2-homozygous lymphocytes more efficiently than HLA A2-heterozygous lymphocytes, whereas the latter were better killed than HLA A2-negative lymphocytes.     

Human T-cell cultures with selective autotumor reactivity

Summary T-cell cultures derived from the blood of 14 patients with solid tumors were propagated with T-cell growth factor (TCGF). The cultures were initiated from lymphocytes exposed to autologous tumor-biopsy cells. TCGF was added either immediately or 3–10 days later. In the former culture type the cell yield on day 7 was considerably higher. The cytotoxic potential of the cultured cells was assayed on two occasions, between days 7 and 10 and between weeks 5 and 8. Cells of all but two cultures had the potential to lyse autologous tumor-biopsy cells.On the population level, cytotoxicity was specific for autologous tumor in those cultures that were driven to growth with TCGF after the 3rd day. These lymphocytes did not lyse allogeneic tumor-biopsy cells. In contrast, all five cultures initiated in the presence of TCGF exhibited a broader cytotoxic potential, i.e., in addition to the stimulator autologous-tumor cells, they also lysed other targets. Another difference between the two culture types was their behavior toward K562. Tested on the 7th day they all lysed K562; however, this function declined in strength or disappeared later in the cultures exposed to TCGF after the 3rd day.Reexposure of the lymphocytes to autologous tumor-biopsy cells after 2 weeks of culture period, but not on the 7th day, induced DNA synthesis. This secondary response was specific inasmuch as allogeneic tumor cells had little or no effect.One of the autotumor restimulated cultures was tested for cytotoxic potential. It increased against the autologous but not against other tumors or K562 cells.