T-cell responses induced by the parenteral injection of antigen-modified syngeneic cells. III. Dissociation of primed cytolytic T-cell and efferent suppressor-T-cell activity following intravenous injection of trinitrophenol-modified syngeneic spleen cells

The parenteral injection of ligand-coupled syngeneic spleen cells has profound effects on immune responsiveness. In this regard, it was examined whether the primed in vitro trinitrophenol (TNP)-specific cytotoxic T-lymphocyte (CTL) responses observed in splenic T-cell populations from mice injected intravenously (iv) with syngeneic TNP-modified spleen cells (TNP-SC) are related to the efferent-acting suppressor-T-cell (Ts) activity observed in splenocytes from iv primed mice. Treatment of mice with cyclophosphamide, adult thymectomy, or monoclonal anti-I-J antiserum prior to the iv injection of TNP-SC was found to eliminate the ability of splenic Ts from these mice to suppress the passive transfer of delayed-type hypersensitivity (DTH) mediated by trinitrochlorobenzene-immune T cells. In contrast, spleen cells from these pretreated mice showed no impairment in the development of augmented TNP-specific CTL responses upon in vitro restimulation with TNP-SC. Separation of the two activities was also achieved in a kinetic analysis. It is concluded that specific enhancement of CTL responsiveness induced by the iv injection of TNP-SC is related to the expansion of a population prelytic Lyt 2+ CTL effector cells which does not appear to contain efferent-acting Lyt 2+ Ts active in suppressing DTH expression.     

The cytolytic T lymphocyte response to trinitrophenyl-modified syngeneic cells. I. Evidence for antigen-specific helper T cells.

Mice were primed subcutaneously with trinitrophenyl (TNP)-modified syngeneic spleen cells. Seven days later, spleen cells from these in vivo primed mice, or spleen cells from naive mice, were co-cultured with TNP-modified syngeneic cells. Spleen cells from the in vivo primed mice demonstrated augmented cytolytic T lymphocyte (CTL) activity. The spleens of these in vivo primed mice contained a population of radioresistant, antigen-specific, helper T cells. Specifically, spleen cells from these mice, after x-irradiation, were able to augment the in vitro CTL response of normal spleen cells to TNP-modified syngeneic cells.     

Regulation of the hapten-specific T cell response. I. Preferential induction of hyporesponsiveness to the D-end of the major histocompatibility complex in the hapten-specific cytotoxic T cell response

In this paper we have examined the phenomenon of hapten-specific tolerance in the cytolytic T lymphocyte (CTL), using the trinitrophenyl (TNP) and azobenzenearsonate haptens. We found that the H-2 K and H-2 D-end restricted CTL in H-2a mice are differentiable in the ease with which they are tolerized to the TNP hapten. With TNP modified syngeneic spleen cells (TNP-SC), or low amounts of trinitrobenzylsulfonic acid as tolerogen, preferential hyporesponsiveness of D-end restricted CTL can be observed. Larger doses of hapten, e.g. a higher amount of trinitrobenzylsulfonic acid, will tolerize both K- and D-end restricted TNP-specific CTL in H-2a mice. The phenomenon of preferential D-end restricted CTL hyporesponsiveness is not observed in H-2d, H-2k, or H-2b mice, nor is it observed in H-2a mice with respect to the azobenzenearsonate hapten. We have also shown that the clones of CTL responsible for lysis of TNP-modified allogeneic targets (cross-reactive lysis) in H-2a mice probably overlap with the D-end restricted TNP-specific CTL since D-end restricted hyporesponsiveness induced by intravenous injection of TNP spleen cells also results in the elimination of cross-reactive lysis of TNP-modified allogeneic targets. The possible mechanisms of preferential D-end hyporesponsiveness to the TNP hapten in the H-2a mice as well as its significance and relationship to previous work in this area are discussed in this paper.     

Activation of idiotype-specific suppressor T cells by injection of antibody carrying a recurrent idiotype

Intravenous injection of spleen cells (SC) coated with an antitrinitrophenyl (anti-TNP) IgM monoclonal antibody, Sp6 (Sp6-SC), which carries a recurrent idiotype, resulted in activation of a Lyt-2-positive population which did adhere to Sp6-coated plates. No effect of Sp6-SC injection could be observed in vivo on an anti-TNP B-cell response when mice were primed with an immunogenic dose of TNP-horse red blood cells (HRBC), but an anti-TNP response was observed when Sp6-SC-injected mice were primed with a subimmunogenic dose of TNP-HRBC. Furthermore, after intravenous (iv) injection of Sp6-SC, it was no longer possible to suppress a primary anti-TNP response by iv injection of TNP-haptenized thymocytes. In vitro analysis showed that the Sp6-induced suppressor T cell (Ts) population had no measurable influence on TNP-specific naive B cells, nor did it suppress TNP-specific helper T cells (THTNP), but it did lead to counterregulation of TNP-specific suppressor T cells (TsTNP). Hence, iv injection of antibody carrying a recurrent idiotype resulted in activation of a Ts population which functioned as inhibitor of suppression, thus displaying a helper effect.     

Comparison between the in vitro activities of in vivo-induced hapten-specific suppressor cells and supernatants of a hapten-specific suppressor hybridoma

A trinitrophenyl (TNP)-specific suppressor hybridoma was obtained by fusing hapten-binding spleen cells (SC) of BALB/c mice 1 week after intravenous (iv) injection of TNP-modified syngeneic lymphocytes with the AKR lymphoma BW5147. The suppressive activity of supernatants from one clone (TNP-44) was compared with that of in vivo-induced TNP-specific suppressor cells. Both the TNP-specific suppressor cells (TsTNP) and the TNP-44 were hapten binding and hapten specific. They suppressed the functional activity of TNP-haptenized T as well as B cells. TNP-44 supernatant also inhibited the proliferation of TNP-modified cells. Using native target cells, both TNP-44 supernatant and the in vivo-induced suppressor cells suppressed the anti-TNP B-cell response to TNP-bound T-dependent soluble or cellular antigens, but not to TNP-lipopolysaccharide (LPS). Furthermore, the function of TNP-specific helper T cells (THTNP) was impaired in the presence of TSTNP or supernatant from TNP-44. From these observations it was concluded that both the TSTNP and a TNP-specific factor derived from a suppressor hybridoma function via an antigen bridge at the TH or at the TH-dependent B-cell subset.     

Non-H-2-linked genetic regulation of cytotoxic responses to hapten-modified syngeneic cells. I. Non-H-2-linked Ir gene defect expressed on T cells is not predetermined at the stage of bone marrow cells

Spleen cells from C3H/He or BALB.K mice immunized to the newly synthesized amino-reactive hapten 5-sulfo-1-naphthoxy acetic acid N-hydroxysuccinimide ester (AED-NH2) were stimulated in vitro with AED-NH2-modified syngeneic cells. After 5 days of culture, effector cells were assayed for their cytotoxic activity against AED-NH2-modified target blast cells. C3H/He and BALB.K mice exhibited the respective high and low anti-AED-NH2 cytotoxic T lymphocyte (CTL) responses. This contrasted with the observation that both of these H-2k strains generated potent CTL responses against aminoreactive haptens, e.g., trinitrophenyl (TNP). Because C3H.SW and BALB.B strains, which are the H-2b counterpart of the above two strains, also represented the respective high and low responders to AED-NH2 hapten, this hapten model enabled us to investigate cellular mechanisms underlying the above non-H-2-associated genetic regulation of CTL responses (C3H vs BALB non-H-2 backgrounds). The results demonstrated that there was no detectable difference between C3H/He and BALB.K strains in the lysability of target cells and the ability of stimulating cells to activate primed spleen cells. Anti-AED-NH2 CTL responses were only marginal when antigen-presenting cells (APC) were eliminated from the primed spleen cells of high responder C3H/He or (C3H/He X BALB.K)F1 mice. The addition of APC to cultures free of APC regained an appreciable CTL response in C3H/He or (C3H/He X BALB.K)F1 mice, irrespective of whether APC were derived from high (C3H/He) or low (BALB.K) responders. We have also demonstrated that allogeneic radiation bone marrow chimera (BALB.K----C3H/He) exhibited a CTL response comparable to that induced by C3H/He mice, whereas the reverse direction of allogeneic chimera (C3H/He----BALB.K) induced a marginal CTL response. These results indicate that this non-H-2-associated Ir gene defect is expressed on T cells (CTL precursors and/or helper T cells) rather than APC, and that this T cell defect is not predetermined at the level of bone marrow cells. The results are discussed in the light of the genetic and cellular mechanisms underlying non-H-2-linked Ir gene control.     

Time course analysis of regulation of cytotoxic T cells

The frequency of cytotoxic T-cell precursors (T(K] was determined in spleen cells (SC) of naive mice and after subcutaneous (sc) or intravenous (iv) priming with trinitrophenyl (TNP)-haptenized syngeneic thymocytes by limiting dilution (LD) analysis in cultures containing exogenous interleukin 2 (IL-2). The frequency of TNP-specific T(K) (T(K)TNP) was found to be 1/4500 +/- 1097 in SC of naive mice. After treatment, the frequency of T(K)TNP increased up to threefold. An evaluation of regulatory elements (helper and suppressor T cells) (TH, TS) was possible by comparing SC from naive and primed animals after prolonged in vitro culture periods in the absence of exogenous IL-2. The experiments indicated that after 7 days of culture, activation of T(K) was limited by the supply of help. After 2-3 weeks of culture, sufficient help was available, especially in SC populations of primed mice, i.e., priming resulted in activation of the helper compartment with gradual differences depending on the route of priming (sc priming was more efficient than iv priming). But, after prolonged culture periods, cytotoxic activity was counterregulated by TS in naive and primed animals. While sc priming was a minor influence on the suppressor compartment, iv priming led to activation and numerical increase of TS. Finally, the activation status of effector and regulatory cells was tested at various times after antigenic stimulation. Four to five weeks after in vivo priming the system was found to move back into a status similar to that of naive mice, except that a small population of "nonsuppressible" T(K) appeared.     

Helper cells in the autologous mixed lymphocyte reaction. I. Generation of cytotoxic T cells recognizing modified self H-2

The autologous mixed lymphocyte reaction (AMLR) in mice measures the proliferative response of T cells to determinants on syngeneic non-T spleen cells. Normally, cytotoxic T lymphocytes (CTL) are not generated in this reaction. However, the addition of trinitrophenyl-modified mitomycin C-treated syngeneic T cells (TNP-Tm) to the AMLR results in the generation of TNP-specific CTL but does not alter the proliferative response. Significant cytotoxic activity is not detectable against TNP in association with Ia unless TNP is present on cells bearing those determinants. Thus, if unselected spleen cells are TNP-modified and used as stimulators in the AMLR, the proliferative response is enhanced and CTL are generated that recognize TNP in association with K, D, and I region-encoded determinants. The CTL generated in the AMLR are H-2 restricted and dependent on the presence of adherent cells in the sensitization cultures. The experiments presented here suggest that the AMLR can provide the help necessary for generating cytotoxic T cells in vitro.     

Predominant T cell receptor gene elements in TNP-specific cytotoxic T cells.

H-2b class I-restricted, TNP-specific CTL clones were obtained by limiting dilution cloning of either short term polyclonal CTL lines or spleen cells of TNP-immunized mice directly ex vivo. Sequence analyses of mRNA coding for TCR alpha- and beta-chains of 11 clones derived from CTL lines from individual C57BL/6 mice revealed that all of them expressed unique but clearly nonrandom receptor structures. Five alpha-chains (45%) employed V alpha 10 gene elements, and four of those (36%) were associated with J beta 2.6-expressing beta-chains. The alpha-chains from these four TCR, moreover, contained an acidic amino acid in position 93 of their N or J region-determined sequences. Clones isolated directly from spleen cells carried these types of receptors at lower frequency, 27% V alpha 10 and 19% J beta 2.6, indicating that bulk in vitro cultivation on Ag leads to selection for these particular receptors. However, even in TNP-specific CTL cloned directly ex vivo, V alpha 10 usage was increased about fivefold over that in Ag-independently activated T cells in H-2b mice (4 to 5%). The selection for V alpha 10/J beta 2.6-expressing cells was obtained repeatedly in other TNP-specific CTL lines from C57BL/6 mice but not in FITC-specific CTL from the same strain or in TNP-specific CTL lines from B10.BR (H-2k) or B10.D2 (H-2d) mice. We conclude from this (a) that the selection for V alpha 10/J beta 2.6+ T cells is driven by the complementarity of these receptors to a combination of TNP and MHC epitopes and (b) that predominant receptor structures reflect the existence of a surprisingly limited number of "T cell-relevant" hapten determinants on the surface of covalently TNP-modified cells.     

Helper T cells against tumor-associated antigens (TAA): preferential induction of helper T cell activities involved in anti-TAA cytotoxic T lymphocyte and antibody responses

This study establishes assay systems for helper T cell activities assisting cytotoxic T lymphocyte (CTL) and antibody responses to tumor-associated antigens (TAA) and demonstrates the existence of TAA that induce preferentially anti-TAA CTL helper and B cell helper T cell activities in two syngeneic tumor models. C3H/HeN mice were immunized to the syngeneic X5563 plasmacytoma or MH134 hepatoma. Spleen cells from these mice were tested for anti-TAA helper T cell activity capable of augmenting anti-trinitrophenyl(TNP) CTL and anti-TNP antibody responses from anti-TNP CTL and B cell precursors (responding cells) by stimulation with TNP-modified X5563 or MH134 tumor cells. The results demonstrate that cultures of responding cells plus 85OR X-irradiated tumor-immunized spleen cells (helper cells) failed to enhance anti-TNP CTL or antibody responses when in vitro stimulation was provided by either unmodified tumor cells or TNP-modified syngeneic spleen cells (TNP-self). In contrast, these cultures resulted in appreciable augmentation of anti-TNP CTL or antibody response when stimulated by TNP-modified tumor cells. Such anti-TAA helper activities were revealed to be Lyt-1+2- T cell mediated and TAA specific. Most interestingly, immunization with X5563 tumor cells resulted in anti-TAA helper T cell activity involved in CTL, but not in antibody responses. Conversely, TAA of MH134 tumor cells induced selective generation of anti-TAA helper T cell activity responsible for antibody response. These results indicate that there exists the qualitative TAA-heterogeneity as evidenced by the preferential induction of anti-TAA CTL- and B cell-helper T cell activities. The results are discussed in the light of cellular mechanisms underlying the preferential anti-TAA immune responses, and the interrelationship between various types of cell functions including CTL- and B cell-help.     

Production and characterization of T-cell clones specific for trinitrophenyl (TNP)-modified syngeneic spleen cells (TNP-self)

Using serial antigenic challenge as the method of selection and stimulation, continuous lines of cytotoxic T-lymphocytes (CTL) directed against TNBS-modified syngeneic spleen cells (TNP-self) have been generated. Spleen cells from C3H/HeJ (H-2k) mice were primed in vitro with autologous spleen cells modified with TNBS, and subsequently cloned by limiting dilution and in soft agar in the presence of IL2. These CTL clones grew continuously in medium supplemented with IL2 and in the presence of antigen. They are antigen specific and H-2 restricted in their target cell recognition. They all express Thyl and Lyt2 surface markers. None of the clones exhibit natural killer (NK) cell activity. All CTL clones tested so far are restricted in their target cell recognition to H-2Kk-TNP and none were found to be restricted to H-2Dk-TNP. These findings demonstrate at the clonal level the H-2K/D restriction of TNP-self specific CTL. These clones provide tools that may facilitate an understanding of the development and regulation of antigen specific CTL. They may also serve as models useful towards an understanding of the mechanism of lysis by CTL.     

Defining cytolytic T lymphocyte recognition of chemically modified self. I. Response to trinitrophenyl-H-2Kk

We used purified class I antigen incorporated into liposomes to examine the response of secondary cytolytic T lymphocytes (CTL) to chemically modified self. By generating the secondary response in the presence of T cell helper factor, the level of CTL response was limited by CTL recognition of added antigen rather than by helper cell generation of lymphokines. We found a strong secondary response against chemically modified self when spleen cells from trinitrophenyl (TNP)-primed C3H/HeJ mice were stimulated with a) TNP-modified liposomes containing H-2Kk, b) liposomes containing H-2Kk purified from TNP-modified RDM-4 (H-2k) cells, or c) liposomes containing the limited trypsin proteolysis product of H-2Kk that had been directly modified with TNP. In contrast, we were not able to generate a significant CTL response with unmodified H-2Kk incorporated into vesicles along with TNP-modified membrane components lacking H-2Kk. These results suggest that TNP-modified H-2Kk is a major antigenic site recognized by CTL from C3H/HeJ mice after priming against TNP-modified self.     

Augmentation of killer activity of murine spleen cells against allogeneic and syngeneic tumor cells by inoculation of alloantigen in vivo

After C57BL/6 (B6) mice were inoculated with BALB/c spleen cells via tail vein, kinetics of cytotoxic activities in the B6 mice against sensitizing alloantigens (H-2d) and against syngeneic antigens were investigated using, as target cells, P815 mastocytoma cells (H-2d) and B16 melanoma cells (H-2b). Cytotoxic activity against P815 in the B6 spleen cells reached a peak 3 days after alloantigen inoculation, decreased drastically on day 5 and rose again thereafter. The profile of anti-B16 cytotoxic activity was similar to that of anti-P815 activity. The cytotoxic activity against P815 was inhibited partially by cold B16, but that against B16 was not inhibited by cold P815. Surface phenotype of cytotoxic cells against P815 was Lyt2+, Thy1+, Asialo GM1+ and that of cytotoxic cells against B16 was Lyt2-, Thy1+/-, and Asialo GM1+. The results indicate that inoculation of B6 mice with allogeneic BALB/c spleen cells induce two types of cytotoxic cells; one is similar to lymphokine-activated killer (LAK) cells and the other is activated natural killer cells.     

Significant frequency of cytotoxic T lymphocyte precursor cells specific for TNP-modified allogeneic cells in normal lymphocytes

It was tested whether the cytotoxic T-lymphocyte precursor (CLP) repertoire in normal mice is biased toward recognizing foreign antigen in association with self H-2 as opposed to allogeneic H-2. The frequencies of CLPs in normal mice (H-2b,k,d) specific for TNP-modified syngeneic and TNP-modified allogeneic cells have been compared by limiting dilution analysis. Normal spleen cells were cultured at a limiting dilution with TNP-modified (TNP-self) or TNP-modified allogeneic (TNP-allo) stimulator cells. Cultures were split into four aliquots and assayed against TNP-self, TNP-allo, unmodified syngeneic, and unmodified allogeneic Concanavalin A blast targets and classified for cytotoxic activity directed against TNP-self, TNP-allo, and allo H-2 determinants. In disagreement with our expectations from the literature, the frequencies of CLPs in H-2b and H-2d responder cells recognizing TNP-modified H-2k were higher than the frequencies of CLPs recognizing TNP-self. There was no clear preference for TNP-self in the case of H-2b responder and H-2d allogeneic cells, nor vice versa. Only in the case of H-2k responder cells was there a distinct preference for TNP-self. The significance of a considerable number of TNP-specific, allo H-2-restricted CLPs in normal lymphocytes is discussed.     

Suppressor T cells, distinct from "veto cells," are induced by alloantigen priming and mediate transferable suppression of cytotoxic T lymphocyte responses in vivo

Primary and secondary cytotoxic T lymphocyte responses to minor alloantigens can be suppressed by priming host mice with a high dose (10(8) cells) of alloantigenic donor spleen cells (SC). Such suppression is antigen specific and transferable into secondary hosts with T cells. One interpretation of this is that antigen-specific host suppressor T cells (Ts) are activated. Alternatively, donor Lyt-2+ T cells, introduced in the priming inoculum, may inactivate host CTL precursors (CTLp) that recognize the priming (donor) alloantigens. Donor cells that act in this way are termed veto T cells. The experiments described here exclude veto T cell participation in transferable alloantigen-specific suppression, and demonstrate the operation of an alloantigen-specific host-derived T suppressor (Ts) cell. The origin of the Ts has been studied directly by using Thy-1-disparate BALB/c mice. The cell responsible for the transfer of suppression of a secondary CTL response to B10 minors was of the host Thy-1 allotype, and so originated in the host spleen and was not introduced in the priming inoculum. Secondly, antigen-specific Ts generated in CBA female mice against B10 minors could act on CTL responses to an unequivocally non-cross-reactive-third party antigen (H-Y), provided the two antigens were expressed on the same cell membrane. Such third-party suppression is incompatible with the operation of veto T cells. Depletion of Thy-1.2+ or Lyt-2+ cells from the suppression-inducing donor SC inoculum did not abrogate suppression induction in BALB/c mice; instead, suppression was enhanced. The demonstration of veto cell activity in similarly primed mice by other groups of investigators indicates that both types of suppression may operate. However, our results show that only antigen-specific Ts can mediate the transferable suppression of CTL responses to alloantigens.     

Studies on T helper cells and the specificity of their soluble products for induction of cytotoxic T cells directed against trinitrophenyl-modified self

The role of T helper cells (Th) and their soluble products in the generation of a cytotoxic T-cell (CTL) response of thymocytes to trinitrophenyl (TNP)-modified syngeneic cells was investigated. The Th have a Thy 1⁺ Lyt 1⁺2^? surface phenotype, and produce at least two soluble helper factors. Production of factors requires stimulation of primed Th by specific antigen (self-TNP), and depends on a Thy 1⁺ Lyt 1⁺2^? cell. Factors present in supernatants after 5 hr of stimulation act preferentially on antiallogeneic precursor CTL (pCTL); factors present at 24 hr act preferentially on self-TNP-specific pCTL with a variable activity for alloantigen-specific pCTL. These results are interpreted as suggesting a possible role for helper factors having selective action in generation of CTL responses.     

Calorie restriction modifies the delayed-type hypersensitivity response to the hapten trinitrobenzenesulfonic acid and to hapten-modified syngeneic spleen cells

We have studied the influence of different degrees of calorie restriction on the induction and the regulation of the delayed type hypersensitivity (DTH) response to trinitrobenzenesulfonic acid (TNBS) and TNBS-modified spleen cells (TNBS-SC), injected by the sc or the iv route. Immediately after weaning, BALB/c mice were placed on restricted diets for either 2 or 4 weeks and then the DTH response was induced. The results showed that a 37.5% restriction in the food supply significantly depressed the level of the DTH response induced by the sc injection of TNBS-SC. In contrast, a 25% restriction in the food supply was insufficient to depress the response. Calorie restriction did not modify the inhibitory influence of an iv injection of TNBS-SC on the DTH response. However, iv presensitization with free hapten or the simultaneous injection of TNBS-SC by the iv and the sc routes did not significantly depress the DTH response in calorie-restricted mice, indicating a defect in the inhibitory regulation of the DTH response in these dietary groups.     

The use of cytotoxic T cells in the regulation of tumour growth in syngeneic mice

Summary Normal C57BL/6 (B6) spleen cells were cultured with syngeneic EL4 tumour cells, expanded in IL2-containing medium, and tested for anti-tumour activity in vitro and in vivo. The activated cells were highly cytotoxic for EL4 and to a lesser degree killed syngeneic B6 blasts and allogeneic (D2) P815 tumour cells. B6 or BDF1 mice that received these cultured cells by IP injection cleared ¹²⁵IUdR-labelled EL4 cells faster than untreated mice. However, this enhanced clearance was evident only 7–12 days after injection. Since the injected cells had a short half-life (<10% remaining after 48 h) the effect of these cells in vivo was most probably due to the activation of the host's immune system. Mice that received cultured cells survived significantly longer than untreated mice following a lethal dose of EL4 cells. Cultured cells were much more effective in prolonging survival when used in conjunction with cyclophosphamide (CY). In animals receiving either cultured cells with or without CY or CY alone tumour clearance was markedly enhanced 7–12 days after injection.When challenged with a small dose of EL4 tumour cells (1×10⁴ SC per mouse) three of ten B6 mice treated with B6 anti-EL4 cultured cells were able to survive indefinitely. The frequency of CTL precursors to EL4 from the spleen cells of these surviving animals was about five-fold higher than that of normal spleen cells. Furthermore, CTL derived from primed spleen cells were more specific for EL4 than those derived from normal spleen cells.Abbreviations B6 C57BL/6J - BDF1 (C57BL/6J×DBA/2J) F1 - ConA SN concanavalin A supernatant - CTL cytotoxic T lymphocytes - CTL-P cytotoxic T-lymphocyte precursors - CY cyclophosphamide - E/T effector-to-target ratio - IL2 interleukin 2 - IP intraperitoneal - IUdR iododeoxyuridine - IV intravenous - LPS lipopolysaccharide - MST mean survival time     

Regulatory mechanism of delayed-type hypersensitivity in mice. I. Properties of memory cpells and suppressor cells for delayed-type hypersensitivity against ovalbumin.

Delayed-type hypersensitivity (DTH) response in mice induced by sc injection of alum-absorbed ovalbumin (OA) was accelerated and enhanced by priming sc with a low dose of urea-denatured ovalbumin (UD-OA), 2 or more days earlier, whereas it was suppressed by priming sc with a high dose of UD-OA, 0 or more days earlier. The ability in primed mice to accelerate or suppress the DTH response could be transferred antigen specifically into cyclophosphamide (CY)-pretreated recipients or normal recipients by spleen cells from primed mice, but not by the T-cell-depleted spleen cells. Furthermore, the ability of spleen cells to transfer the acceleration or the suppression appeared transiently around 7 or 4 days after priming, although the acceleration or the suppression in donor mice persisted for a much longer time. Pretreatment with CY abolished the suppression of DTH response in high dose-primed mice and resulted in the acceleration of DTH response. These results suggest that the activity of DTH-related memory T cells which accelerate and enhance the response can be inhibited by suppressor T cells for the DTH response.     

Augmentation of the generation of cytotoxic T lymphocytes against syngeneic tumor cells by recombinant human tumor necrosis factor

In order to clarify the effect of recombinant human tumor necrosis factor (rHu-TNF) on the antitumor T cell immune response, we examined the effect of rHu-TNF on the generation of cytotoxic T cells (CTL) against syngeneic tumor cells. Spleen cells from X5563 plasmacytoma-transplanted mice were stimulated in vitro with mitomycin C-treated X5563 cells in the presence or absence of rHu-TNF. The generation of CTL was augmented in a dose-dependent manner by the addition of rHu-TNF. The augmenting effect of rHu-TNF was more marked when indomethacin was added to the culture. The augmenting effect was observed only when rHu-TNF was added at the early stage of the generation of CTL. The cell surface phenotype of CTL generated was L3T4- and Lyt2+. The augmentation was shown not only by the chromium-51 release assay but also by the Winn assay. As to the specificity, the augmentation of CTL generation was observed by the addition of rHu-TNF when responder-primed spleen cells were stimulated with the tumor cells in vitro. On the other hand, augmentation was not observed when responder spleen cells were not stimulated with the tumor cells in vitro, or when responder spleen cells were obtained from normal mice. The CTL generated was not cytotoxic against other tumor cells of the same haplotype. Thus, rHu-TNF augmented the generation of CTL against syngeneic tumor cells in an antigen-specific manner. The in vivo effect of rHu-TNF was examined by administering rHu-TNF into X5563-bearing mice. The spleen cells of rHu-TNF-injected mice generated a much higher CTL activity against X5563 cells in vitro than did the spleen cells of uninjected mice. From these results, a possibility can be considered that in some cases, rHu-TNF may exert its antitumor activity by stimulating the immune system.