Suppression of alloimmune cytotoxic T lymphocyte (CTL) generation by depletion of NK cells and restoration by interferon and/or interleukin 2

By using rabbit antiserum to a glycolipid, ganglio-n-tetraosylceramide (ASGM1), the accessory effect of natural killer (NK) cells on the generation of alloimmune CTL in mice was investigated. When normal C3H/He mice were immunized with C57BL/6 or BALB/c spleen cells, they generated alloimmune CTL with a surface marker phenotype of Thy-1+ Lyt-1-2+ ASGM1-, preceded by early augmentation of cytotoxic activity of NK cells with a Thy-1-Lyt-1-2-ASGM1+ phenotype. Administration of anti-ASGM1 (10 microliters) in mice resulted in a complete depletion of NK activity and ASGM1+ cells in the spleen even 1 day after injection, but no changes in the proportions of T (Thy-1+) cells and their Lyt-1 and Lyt-2 subsets as revealed by an immunofluorescence analyzer (FACS) and phagocytic cells. When these anti-ASGM1-treated mice were immunized with allogeneic cells, they showed neither augmented NK activity nor generation of alloimmune CTL, and spleen cells isolated from these anti-ASGM1-treated mice produced no CTL response to alloimmunization in vitro. Normal spleen cells treated with the antiserum and complement in vitro also showed a complete NK depletion without any deterioration of T cells and their Lyt-1 and Lyt-2 subsets, and when stimulated with allogeneic cells they generated no CTL. Spleen NK (ASGM1+) cells were purified by Percoll-gradient centrifugations followed by complement-dependent killing of T cells with the use of anti-Thy-1 monoclonal antibody, and were further purified by panning methods with anti-ASGM1, giving a preparation consisting of greater than 90% ASGM1+, Ly-5+ cells, and less than 0.5% of Thy-1+, Lyt-1+, and Lyt-2+ cells. These purified ASGM1+ Thy-1- cells alone generated no alloimmune CTL in response to alloantigens, suggesting that ASGM1+ NK cells contained no precursors of alloimmune CTL. When added into NK-depleted spleen cells, they restored the normal alloimmune CTL response of the spleen cells, indicating that ASGM1+ fractions contained cells to provide an accessory function for CTL generation. Lyt-1+ cells purified by panning methods did not restore the CTL response of NK-depleted spleen cells. These results indicate that ASGM1+ NK cells, but not Lyt-1+ helper T cells contaminating ASGM1+ fractions at undetectable levels, are responsible for the accessory function. When these purified ASGM1+ Thy-1- cells were stimulated with allogeneic cells, they produced IL 2 and IFN.(ABSTRACT TRUNCATED AT 400 WORDS)     

Depletion of macrophages expressing I-J antigen results in efficient generation of alloreactive cytotoxic T lymphocytes

The role of suppressor macrophages (S-M phi) produced during generation of cytotoxic T lymphocytes (CTL) stimulated with allogeneic lymphocytes was investigated. Splenic CTL from C3H/He mice (H-2k) were generated by in vivo immunization and subsequent in vitro stimulation by splenic lymphocytes from C57B1/6 mice (H-2b) in mixed lymphocyte reaction (MLR). In addition to in vitro standard 51Cr release assay, the CTL activity was mainly measured in vivo using the Winn assay against EL-4 thymoma cells in B6C3F1 mice (H-2b/k). In mice injected with CTL plus EL-4 cells survival rate was 20% compared with no survival of mice treated with normal spleen cells plus EL-4 cells. The antitumor activity of the CTL was significantly increased when immunized mice were treated with a 5 mg/kg ip dose of indomethacin at the time of immunization (80% survival). Macrophages were depleted from spleen cells of immunized mice by plastic adherence or carbonyl-iron treatment, replaced with an equivalent number of M phi from normal mice, and then introduced into a 5-day MLR. When the antitumor activity of the cells isolated from this MLR was measured in the Winn assay, 90-100% survival in EL-4-bearing mice was observed. In contrast, none of the mice inoculated with EL-4 alone and 20% of the mice that received CTL obtained after alloimmunization followed by MLR in addition to EL-4 survived. These results of CTL activity were confirmed by in vitro cytotoxicity tests. When the M phi isolated from spleens of immunized mice were analyzed for I-Jk antigen expression, a 2.5-fold increase was detected, compared with splenic M phi obtained from normal C3H/He mice. In contrast, Ia and I-Ak antigen expression was equivalent in M phi isolated from normal or immunized C3H/He mice. When immune spleen cells were treated with anti-I-Jk antiserum followed by complement and then, subjected to the MLR, the antitumor activity of CTL was significantly enhanced (80% survival). However, treatment of these cells with anti-I-Ak antiserum and complement did not alter CTL activity. These data suggest that the increase of S-M phi expressing I-Jk+ antigen to be induced during alloimmunization results in suppression of allospecific CTL-generation in MLR.     

Veto cell H-2 antigens: veto cell activity is restricted by determinants encoded by K, D, and I MHC regions

Responder cells from primary syngeneic and allogeneic one-way mixed-lymphocyte cultures (MLC) specifically inhibit the development of cytotoxic T lymphocytes (CTL) directed against the major histocompatibility complex (MHC) antigens of the MLC responder cells. This special kind of suppressor activity is known as veto suppression. Ia+ cells with veto activity obtained from H-2 recombinant mouse strains were shown to downregulate alloantigen (class II)-specific helper activity for class I-specific CTL development in a primary MLC provided that the veto cells expressed the same I-E alpha subregion as the MLC stimulator cells. The veto-induced suppression of allo-help was prevented by the addition of supernatant from concanavalin A-stimulated spleen cells (Con A-SN) and was inhibited considerably by very high amounts of recombinant interleukin-2 (IL-2). In the presence of Con A-SN, CTL precursors recognizing either the K end or the D end of the veto cell MHC were found to be inactivated. Thus, our results indicate that MLC responder cells include active veto cells expressing Ia region-encoded restriction elements for allospecific T helper cells, as well as K- or D-encoded restriction elements for allospecific T cytotoxic cells.     

Species-restricted recognition of transfected HLA-A2 and HLA-B7 by human CTL clones

Human cytolytic T lymphocytes (CTL) clones and HLA-A2- and HLA-B7-transfected human, monkey, and mouse cell lines were used to investigate the basis for species-restricted antigen recognition. Most allospecific CTL clones obtained after stimulation with the human JY cell line (source of HLA-A2 and HLA-B7 genomic clones) recognized HLA antigens expressed in human and monkey cell lines but did not recognize HLA expressed in murine cells. By initially stimulating the responder cells with HLA-transfected mouse cells, two CTL clones were obtained that recognized HLA expressed in murine cells. Functional inhibition of these CTL clones with anti-class I monoclonal antibodies (MAb) indicated that clones reactive with HLA+ murine cells were of higher avidity than clones that did not recognize HLA+ murine target cells. MAb inhibition of accessory molecule interactions demonstrated that the LFA-1 and T8 surface molecules were involved in CTL-target cell interactions in all three species. In contrast, the LFA-2/CD2 molecule, previously shown to participate in a distinct activation pathway, was involved in the cytolysis of transfected human and monkey target cells, but not in the lysis of HLA+ murine cells. Thus transfection of HLA genes into different recipient species cell lines provides us with the ability to additionally delineate the functional requirements for allospecific CTL recognition and lysis.     

Dissociation of H-2 recognition by antibody and cytotoxic T cells of a cloned murine leukemia cell line

The differential expression of H-2 specificities recognized by antibody and by cytotoxic T lymphocytes (CTL) has been studied using a clone (FY7) of the C57BL/6 leukemia cell line FBL-3 (H-2b/H-2b). Unlike C57BL/10 spleen cells, EL-4 lymphoma cells and Y57-2C leukemia cells (all H-2b/H-2b), FY7 failed to induce the primary in vitro generation of anti-H-2b CTL by (B10.A x A)F1 (H-2a/H-2a) or B10.D2 x BALB/c)F1 (H-2d/H-2d) responder spleen cells. In addition, FY7 was not lysed by, and did not competitively inhibit anti-H-2b CTL. Quantitative absorption tests with H-2Kb and H-2Db antisera revealed that FY7 expressed these antigens in quantitatively similar amounts to EL-4. The H-2Kb product of FY7 appeared to be identical with that of C57BL/10 spleen cells both in apparent molecular weight and isoelectric point. Yet FY7 failed to inhibit anti-H-2Kb CTL competitively in a cold target inhibition assay. Possible mechanisms are discussed for the lack of T-lymphocyte recognition of the H-2Kb-gene product expressed by FY7.     

Development of CD8+ effector T cells is differentially regulated by IL-18 and IL-12

We investigated the effects of IL-18 on the development of CD8+ effector T cells in DBA/2 anti-BDF1 whole spleen cell MLC and compared the results with those of IL-12. Addition of IL-18 to the MLC resulted in a twofold increase in CD8/CD4 ratios compared with the control cultures when cells were expanded in IL-2-containing medium following MLC. Purified CD8+ T cells recovered from the IL-18-stimulated MLC produced 20- to 30-fold more IFN-gamma after secondary stimulation with C57BL/6 spleen cells or anti-CD3 mAb, and exhibited strong allospecific CTL activity. Neither IL-18 nor IL-18-supplemented culture supernatants from DBA/2 anti-BDF1 MLC induced type I CD8+ effector T cells when purified CD8+ T cells were used as responder cells in primary MLC. Furthermore, CD4+ T cell depletion from the responder cells abrogated the IL-18-induced increase in secondary IFN-gamma production by CD8+ T cells, suggesting that IL-18-induced type I effector CD8+ T cell development was CD4+ T cell dependent. In marked contrast, adding IL-12 to primary MLC decreased CD8/CD4 ratios by 50% and suppressed secondary IFN-gamma production and CTL activity by CD8+ T cells regardless of concentration, whereas Th1 development was promoted by IL-12. Moreover, both IL-12 and IL-18 efficiently induced type I CD8+ effector T cells in C57BL/6 anti-BDF1 MLC. These findings show that IL-18 plays an important role in the generation of type I CD8+ effector T cells, and further suggest that functional maturation of CD8+ T cells is differentially regulated by IL-18 and IL-12.     

Induction and characterization of minor histocompatibility antigens. Specific primary cytotoxic T lymphocyte responses in vitro

A definite cytotoxic activity was developed in a BALB/c (H-2d) anti-DBA/2 primary mixed leukocyte culture (MLC), which received interleukin 2 (IL-2) on day 3 of culture. This cytotoxic activity was minor histocompatibility antigens (MIHA)-specific at the stimulator level, and was not developed in a syngeneic (BALB/c anti-BALB/c) MLC. The addition of IL-2 on day 3 of culture was crucial; no or very weak cytotoxic activity was developed in MLC receiving IL-2 on day 0 or on both day 0 and day 3. Only appropriate MIHA-allogeneic tumor cells were lysed as the target of the cytotoxic activity. The cytotoxic activity seemed MIHA-specific also at the target level; it lysed tumor cells of DBA/2 mouse origin but not those of BALB/c (syngeneic) origin. Phenotypes of the cytotoxic effector cell were Thy-1+ Lyt-2+. We concluded from these results that MIHA-specific cytotoxic T lymphocytes (CTL) were generated in the MIHA-allogeneic primary MLC. In this newly developed system, we studied genetic and antigenic requirements for primary anti-MIHA CTL responses in vitro. We demonstrated; among spleen cells (SC) of seven B10 H-2-congenic strains only SC of B10.D2 strain whose major histocompatibility complex (MHC) (H-2d) was compatible with the responder MHC effectively stimulated responder BALB/c (H-2d) SC for an anti-MIHA (DBA-C57BL-common) CTL response. Similarly, only SC of two out of seven C x B recombinant inbred strains (C x B.H and C x B.D), which were compatible at the MHC with responder SC, activated responder BALB/c SC for the response. The possibility that cells responding to H-2 alloantigens suppressed the anti-MIHA response was ruled out. Additional experiments showed that compatibility at the H-2K-end or the H-2D-end of the MHC was sufficient for a definite anti-MIHA response. These provided formal evidence that primary anti-MIHA CTL responses in vitro were MHC-restricted at the stimulator level. We then showed that sonication-disrupted SC or Sephadex G-10 column-passed nonadherent SC failed to stimulate responder SC for a primary anti-MIHA CTL response, whereas G-10-passed nonadherent SC responded well to adherent stimulator cells. Further study demonstrated that Ia+ adherent cells were the most active cell type as stimulator. Finally, we confirmed that the primary anti-MIHA CTL responses to adherent stimulator cells was MHC-restricted.     

Recognition of MHC class I allodeterminants regulates the generation of MHC class II-specific CTL

We have analyzed the signals influencing the generation of major histocompatibility complex (MHC) class II allospecific cytolytic T lymphocytes (CTL) and have found that the development of these CTL is actively regulated in primary in vitro cultures by Lyt-2+ T cells triggered in response to MHC class I alloantigens. Class II allospecific CTL can be readily stimulated in primary cultures, but the presence of a simultaneous class I MHC stimulus in these cultures causes a marked reduction of class II-specific CTL activation. This reduction can be prevented by adding to culture a dose of monoclonal anti-Lyt-2 antibody (in the absence of complement) that does not block the generation of class I-specific CTL. The role of MHC class I alloantigens in the regulation of class II allospecific responses illustrates that T cells recognizing class I and class II MHC antigens in mixed leukocyte cultures interact in a complex and nonreciprocal manner to influence the final effector T cell repertoire elicited by this complex immunogenic challenge.     

Epidermal cells as accessory cells in the generation of allo-reactive and hapten-specific cytotoxic T lymphocyte (CTL) responses

The capacity of epidermal cells (EC) to stimulate T cell activation is a Langerhans cell (LC)-dependent phenomenon. In all in vitro assays probed, LC subserve antigen-presenting cell functions in that they display surface-bound foreign or altered-self structures and thereby activate T cell responses. In contrast, attempts to demonstrate accessory cell (ACC) function of LC-containing EC have yielded negative results, i.e., EC lacking foreign cell surface antigens were not able to restore cytotoxic T lymphocyte (CTL) responses in Ia+ adherent cell-depleted cultures. Reasoning that the ACC function of EC might be critically linked to cluster formation between LC and other cell types involved, we tested the ACC function of EC under experimental conditions that allow a close physical contact between the cell types involved (round-bottomed microtiter plates and brief centrifugation of culture plates). By using these modifications, the failure of highly purified B6 T cells to develop alloreactive CTL activity when stimulated with either highly purified, mitomycin C-treated C3H or B6CF1 T cells was restored by the addition of B6 EC. The CTL thus generated produced significant lysis of Con-A-stimulated C3H or BALB/c, but not B6, spleen cell targets. In a similar fashion, TNP- or FITC-specific CTL were generated when (in a syngeneic system) mitomycin C-treated TNP- or FITC-modified stimulator T cells and responder T cells were co-cultured in the presence, but not in the absence, of unmodified EC. The capacity of EC to restore CTL activity in a culture system depleted of Ia-bearing cells was not dependent upon their H-2 type, but was critically linked to the presence of Ia-bearing LC. We therefore conclude that LC-containing EC can subserve the ACC function in the generation of H-2-restricted CTL, provided that culture conditions are chosen that allow a close physical contact between the cell types involved.     

Asialo-GM1-positive T killer cells are generated in F1 mice injected with parental spleen cells

(C57BL/6 x DBA/2)F1 mice transplanted with parental C57BL/6 spleen cells become splenic chimeras, show donor antihost cytotoxic T cell activity, and lose their T cell-mediated, humoral, and natural immunity. Injection of anti-asialo-GM1 (ASGM1) into transplanted mice strongly suppresses splenic cytotoxic activity and causes a significant reduction of spleen cells expressing ASGM1, Thy-1, and Lyt-2. In vitro treatment of spleen cells from transplanted mice with antibody and complement shows that the cytotoxic effector cells are ASGM1+, Thy-1+, Lyt-2+, L3T4-, NK1.1-, and H-2d-, hence of donor origin. The cytotoxic effector cells are specific for H-2d targets and lack NK activity. In an attempt to explore whether in vivo elimination of the cytotoxic effector cells has any influence on splenic chimerism or humoral immunity, F1 mice injected with parental splenocytes were treated with anti-ASGM 1. Results show that this treatment eliminates a substantial proportion of cytotoxic effector cells but has no effect on splenic chimerism or restoration of humoral immunity. It therefore appears that cytotoxic effector cells are not primarily responsible for induction of chimerism or suppression of humoral immunity. In support of this injection of parental spleen cells with the nu/nu mutation induces killer cells in F1 mice but fails to induce splenic chimerism or immunosuppression. In contrast, injection of parental spleen cells with the bg/bg mutation generates both splenic chimerism and suppression of humoral immunity although their ability to generate cytotoxic effector cells in F1 hosts is seriously impaired and comparable to the cytotoxic potential of C57BL/6 nu/nu cells. It is concluded that the ASGM1 + cytotoxic T cells are not primarily responsible for splenic chimerism and suppression of humoral immunity and that the two effects are likely caused by parental cells with a different phenotype and function.     

IL-6/BSF-2 functions as a killer helper factor in the in vitro induction of cytotoxic T cells

rIL-6/B cell stimulatory factor 2 was found to augment CTL generation from mature as well as immature human T cells stimulated with UV-treated allogeneic cells. rIL-6 also acted on peanut agglutinin-positive murine thymocytes and Lyt-2-positive splenic T cells to give rise to CTL. rIL-6 alone could not induce CTL generation, the presence of IL-2 during the early phase of culture period was found to be essential for the IL-6 activity in the induction of CTL. The effect of rIL-6 was not mediated by the induction of IL-2 inasmuch as rIL-6 did not augment IL-2 production in MLC and anti-IL-2 antibody could not neutralize IL-6 activity. rIL-6 augmented CTL generation even when added 72 h after the initiation of cultures. The enhancing activity of rIL-6 could be neutralized with anti-IL-6 antibodies even when added 72 h after the initiation of cultures. The present data indicate that IL-6 acts in the late phase of CTL generation.     

Characterization of the Fas ligand/Fas-dependent apoptosis of antiretroviral, class I MHC tetramer-defined, CD8+ CTL by in vivo retrovirus-infected cells

C57BL/6 (B6; H-2(b)) mice mount strong AKR/Gross murine leukemia virus (MuLV)-specific CD8(+) CTL responses to the immunodominant K(b)-restricted epitope, KSPWFTTL, of endogenous AKR/Gross MuLV. In sharp contrast, spontaneous virus-expressing AKR.H-2(b) congenic mice are low/nonresponders for the generation of AKR/Gross MuLV-specific CTL. Furthermore, when viable AKR.H-2(b) spleen cells are cocultured with primed responder B6 antiviral precursor CTL, the AKR.H-2(b) cells function as "veto" cells that actively mediate the inhibition of antiviral CTL generation. AKR.H-2(b) veto cell inhibition is virus specific, MHC restricted, contact dependent, and mediated through veto cell Fas ligand/responder T cell Fas interactions. In this study, following specific priming and secondary in vitro restimulation, antiretroviral CD8(+) CTL were identified by a labeled K(b)/KSPWFTTL tetramer and flow cytometry, enabling direct visualization of AKR.H-2(b) veto cell-mediated depletion of these CTL. A 65-93% reduction in the number of B6 K(b)/KSPWFTTL tetramer(+) CTL correlated with a similar reduction in antiviral CTL cytotoxicity. Addition on sequential days to the antiviral CTL restimulation cultures of either 1) AKR.H-2(b) veto cells or 2) a blocking Fas-Ig fusion protein (to cultures also containing AKR.H-2(b) veto cells) to block inhibition demonstrated that AKR.H-2(b) veto cells begin to inhibit B6 precursor CTL/CTL expansion during days 2 and 3 of the 6-day culture. Shortly thereafter, a high percentage of B6 tetramer(+) CTL cocultured with AKR.H-2(b) veto cells was annexin V positive and Fas(high), indicating apoptosis as the mechanism of veto cell inhibition. Experiments using the irreversible inhibitor emetine demonstrated that AKR.H-2(b) cells had to be metabolically active and capable of protein synthesis to function as veto cells. Of the tetramer-positive CTL that survived veto cell-mediated apoptosis, there was no marked skewing from the preferential usage of Vbeta4, 8.1/8.2, and 11 TCR normally observed. These findings provide further insight into the complexity of host/virus interactions and suggest a fail-safe escape mechanism by virus-infected cells for epitopes residing in critical areas of viral proteins that cannot accommodate variations of amino acid sequence.     

Antiretroviral cytolytic T-lymphocyte nonresponsiveness: FasL/Fas-mediated inhibition of CD4(+) and CD8(+) antiviral T cells by viral antigen-positive veto cells

C57BL/6 (H-2(b)) mice generate type-specific cytolytic T-lymphocyte (CTL) responses to an immunodominant Kb-restricted epitope, KSPWFTTL located in the membrane-spanning domain of p15TM of AKR/Gross murine leukemia viruses (MuLV). AKR.H-2(b) congenic mice, although carrying the responder H-2(b) major histocompatibility complex (MHC) haplotype, are low responders or nonresponders for AKR/Gross MuLV-specific CTL, apparently due to the presence of inhibitory AKR. H-2(b) cells. Despite their expression of viral antigens and Kb, untreated viable AKR.H-2(b) spleen cells cause dramatic inhibition of the C57BL/6 (B6) antiviral CTL response to in vitro stimulation with AKR/Gross MuLV-induced tumor cells. This inhibition is specific (AKR.H-2(b) modulator spleen cells do not inhibit allogeneic MHC or minor histocompatibility antigen-specific CTL production), dependent on direct contact of AKR.H-2(b) cells in a dose-dependent manner with the responder cell population, and not due to soluble factors. Here, the mechanism of inhibition of the antiviral CTL response is shown to depend on Fas/Fas-ligand interactions, implying an apoptotic effect on B6 responder cells. Although B6.gld (FasL-) responders were as sensitive to inhibition by AKR.H-2(b) modulator cells as were B6 responders, B6.lpr (Fas-) responders were largely insensitive to inhibition, indicating that the responder cells needed to express Fas. A Fas-Ig fusion protein, when added to the in vitro CTL stimulation cultures, relieved the inhibition caused by the AKR.H-2(b) cells if the primed responders were from either B6 or B6.gld mice, indicating that the inhibitory AKR.H-2(b) cells express FasL. Because of the antigen specificity of the inhibition, these results collectively implicate a FasL/Fas interaction mechanism: viral antigen-positive AKR.H-2(b) cells expressing FasL inhibit antiviral T cells ("veto" them) when the AKR.H-2(b) cells are recognized. Consistent with this model, inhibition by AKR.H-2(b) modulator cells was MHC restricted, and resulted in approximately a 10- to 70-fold decrease in the in vitro expansion of pCTL/CTL. Both CD8(+) CTL and CD4(+) Th responder cells were susceptible to inhibition by FasL+ AKR.H-2(b) inhibitory cells as the basis for inhibition. The CTL response in the presence of inhibitory cells could be restored by several cytokines or agents that have been shown by others to interfere with activation-induced cell death (e.g. , interleukin-2 [IL-2], IL-15, transforming growth factor beta, lipopolysaccharide, 9-cis-retinoic acid) but not others (e.g., tumor necrosis factor alpha). These results raise the possibility that this type of inhibitory mechanism is generalized as a common strategy for retrovirus infected cells to evade immune T-cell recognition.     

Interleukin-7 mediates the generation and expansion of murine allosensitized and antitumor CTL.

Interleukin-7 (IL-7) is a 25-kDa cytokine that was initially described as a pre-B cell growth factor and more recently has been shown to cause T cell proliferation. We have investigated the in vitro effects of IL-7 on mature T cells to include the generation and further expansion of allospecific and antitumor CTL. B6 anti-DBA allospecific CTL were generated in the presence of IL-7, IL-2, the combination IL-7 plus IL-2, or no cytokine. IL-7 alone or when combined with IL-2 enhanced the generation of allospecific CTL. To evaluate the proliferative effects of IL-7, 4-day B6 anti-DBA cultures were cultured in IL-7, IL-2, or no cytokine. Cell proliferation and duration of growth of cells cultured in IL-7 were significantly greater than cells cultured in IL-2 or in the absence of cytokine. Allospecific cytolytic activity was maintained during proliferation in IL-7 to a maximum of 60 days. In contrast with the ability of IL-2 to generate LAK cells, murine splenocytes cultured at varying doses of IL-7 (1 to 10,000 ng/ml), resulted in minimal LAK cell activity. The effect of IL-7 in the generation of CTL with antitumor activity was also studied. Seven days after footpad injection of MCA 203 or 205 sarcoma, draining lymph nodes (DLN) were harvested and restimulated in vitro with MCA 203 or 205, respectively, and maintained in culture with either IL-7, IL-2, the combination of IL-7 plus IL-2, or no cytokine. After 10 days in culture, cells generated in IL-7 or IL-2 exhibited similar cytotoxicity against the syngeneic autologous MCA tumor. IL-7 generated cells, however, showed specificity when tested by 51Cr release which was not seen with IL-2-generated cells. Cells generated in IL-7 plus IL-2 were more cytolytic than cells cultured with either cytokine alone. To further define the mechanism of action of IL-7 in antitumor CTL cultures, a monoclonal antibody, S4B6.1, capable of blocking murine-specific IL-2 was employed. The partial inhibition by this mAb of the generation of antitumor CTL demonstrated that IL-7 acted, in part, by an IL-2-dependent mechanism. Finally, IL-7 cultures restimulated at Day 11 with autologous MCA 203 showed greater proliferation than IL-2 cultures and remained lytic at Day 21 of culture.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pretreatment with minor histocompatibility antigens prevents the development of functional CTL helper cell activity

We have been studying the regulation of allogeneic cytotoxic cells (CTL) in vivo. CBA/J (H-2k, mls d) responder mice are unable to develop CTL after an allogeneic footpad immunization if they are pretreated i.p. with spleen cells from either C3H/HeN (H-2k, mls c) or B10.BR (H-2k, mls b) mice. These mouse strain combinations are H-2 compatible but differ at the Mls and other minor histocompatibility loci. We reported that this state of CTL unresponsiveness is specific and that the allogeneic cells used for footpad immunization and the pretreatment strain must share both minor antigens and part of the MHC. In this paper, we describe some of the characteristic features of this CTL unresponsiveness. The CBA host plays an active role and appears to down-regulate its subsequent response against minor antigens after the initial pretreatment. This statement is based on the following: 1) The inhibition of in vivo CTL generation can be achieved by injecting F1 or irradiated C3H cells, i.e., under conditions where GVHD was not a factor; and 2) the state of unresponsiveness is abolished by host treatment with cyclophosphamide. In addition, we demonstrate that the lack of CTL development in pretreated responder animals is the result of impaired helper cell activity. Draining LNC from unresponsive mice can become functionally cytolytic if cultured in a Con A-activated spleen cell supernatant. However, normal CTL responses were not restored after adult thymectomy or splenectomy. Thus, the state of CTL inhibition that is induced by the minor antigen pretreatment is the result of a host-mediated regulatory circuit.     

Generation of cytotoxic T lymphocytes in vitro. VII. Suppressive effect of irradiated MLC cells on CTL response.

Irradiated cells obtained from MLC at the peak of the CTL response caused profound suppression of generation of CTL when added in small numbers at the initiation of primary MLC prepared with normal spleen cells. The inhibitory activity of the MLC cells was not affected by irradiation (1000 rads) but was abolished by treatment with anti-theta serum and complement. The suppression was immunologically specific. The response of A (H-2a) spleen cells toward C3H (H-2k) alloantigens was suppressed by irradiated MLC cells obtained from MLC prepared with A spleen cells and irradiated C3H-stimulating cells, whereas the response of A spleen cells toward DBA/2 (H-2d) alloantigens was affected relatively little. However, if irradiated C3H X DBA/2 F1 hybrid spleen cells were used to stimulate A spleen cells in MLC, addition of irradiated MLC cells having cytotoxic activity toward C3H antigens abolished the response to both C3H and DBA/2 antigens. The response to DBA/2 antigens was much less affected when a mixture of irradiated C3H and DBA/2 spleen cells was used as stimulating cells. Thus, the presence of MLC cells having cytotoxic activity toward one alloantigen abolished the response to another non-cross reacting antigen only when both antigens were present on the same F1 hybrid-stimulating cells. This suppression of generation of CTL by irradiated MLC cells apparently involves inactivation of alloantigen-bearing stimulating cells as a result of residual cytotoxic activity of the irradiated MLC cells. This mechanism may be active during the decline in CTL activity noted in the normal immune response in vivo and in vitro.     

Intrathymic differentiation of cytotoxic T lymphocyte (CTL) precursors. I. The CTL immunocompetence of peanut agglutinin-positive (cortical) and negative (medullary) Lyt 123 thymocytes

To analyze the developmental and functional interrelationship between cortical and medullary thymocytes, the peanut agglutinin-(PNA) binding capacity was used to separate thymocytes into PNA+ (cortical) and PNA- (medullary) thymocytes. Virtually, all positively selected PNA+ thymocytes (90% of the overall thymocyte population) expressed the Lyt 123 phenotype, whereas 90% of negatively selected PNA- thymocytes expressed Lyt 1 alloantigens, about 10% being Lyt 123 thymocytes. Provided, the requirement of Lyt 1 T helper cells was bypassed by Interleukin 2, a nonspecific mediator of T help, PNA+ Lyt 123 thymocytes mounted cytotoxic T cell responses comparable in magnitude to that of peripheral T cells. Their repertoire included antigenic disparities coded for by the complete MHC complex, H-2K, I-A, H-2D, mutational events at H-2K, as well as antigenic disparities expressed on TNP conjugated- and Sendai virus-infected syngeneic cells. PNA- Lyt 123 thymocytes represent a highly reactive pool of primary cytotoxic T lymphocyte (CTL) precursors for both alloreactive and H-2-restricted CTL responses. Since PNA- thymocytes include also Lyt 1 T helper cells, PNA- responder thymocytes are able to mount autonomously (CTL responses. Our data are first to provide direct evidence that Lyt 123 cells represent a common source of alloreactive and H-2-restricted CTL precursors in unprimed lymphocyte populations. Moreover, the apparent immunocompetence of cortical PNA+ thymocytes is now explained by their lack of T helper cells.     

CD4-CD8+ T lymphocytes mediate AKR/gross murine leukemia virus nonresponsiveness in moderately aged AKR.H-2b:Fv-1b mice.

Previously we reported that as AKR.H-2b:Fv-1b mice become older than 9 wk of age they begin to specifically lose the ability to generate anti-AKR/Gross murine leukemia virus (MuLV) CTL responses after immunization and in vitro restimulation with cells expressing AKR/Gross MuLV-encoded Ag. Interestingly, the frequency of virus-specific precursor cytotoxic T lymphocytes (CTL) observed in moderately-aged AKR.H-2b:Fv-1b mice was not substantially decreased from that found in their young responder counterparts. To further investigate the mechanism(s) responsible for the inability of moderately-aged AKR.H-2b:Fv-1b mice to mount AKR/Gross MuLV-specific CTL responses, adoptive transfer experiments were performed in the present study. Transferring splenocytes from moderately-aged AKR.H-2b:Fv-1b donors into young AKR.H-2b:Fv-1b recipients resulted in inhibition of AKR/Gross MuLV-specific CTL responsiveness. Anti-Thy-1.1 plus complement depletion of T cells from the donor cell population before adoptive transfer resulted in a near complete restoration of AKR/Gross MuLV responsiveness of young recipient AKR.H-2b:Fv-1b mice suggesting that the inhibition observed in moderately aged mice was mediated by T lymphocytes. Additional experiments using depletion of T subsets before cell transfer demonstrated that inhibition of AKR/Gross MuLV-specific CTL responsiveness was mediated by a CD4-CD8+ T lymphocyte.     

IL-2 and IFN-gamma are two necessary lymphokines in the development of cytolytic T cells

A filler cell-free limiting-dilution microculture system has been developed for the expansion and differentiation of a high proportion of single CD4-CD8+ T cells into cytolytic T cell (CTL) clones. The stimulus used was PMA together with the calcium ionophore ionomycin. The growth and differentiation factors were rIL-2, together with either a Con A-stimulated spleen cell supernatant (CAS) or rIFN-gamma. CTL activity was monitored by an autoradiographic 111In-release assay. With CAS and rIL-2 present, 50% of all potential precursors (CTL-p) produced cytolytic clones. Substitution of rIFN-gamma for CAS gave a similar efficiency with up to 42% of CTL-p producing cytolytic clones. rIL-2 alone allowed only a small proportion (6%) of CD4-CD8+ T cells to become cytolytic clones. Addition of rIL-2 and rIFN-gamma at various stages of the culture demonstrated that IL-2 was required throughout, but exogenous IFN-gamma was required only during the early stages. It is concluded that for at least 40% of all CTL-p, the lymphokines IL-2 and IFN-gamma are essential and act in synergy to induce proliferation and differentiation into CTL.     

Antiviral protection by CD8+ versus CD4+ T cells. CD8+ T cells correlating with cytotoxic activity in vitro are more efficient in antivaccinia virus protection than CD4-dependent IL.

T cell-mediated protection against a recombinant vaccinia virus was evaluated in mice with respect to the relative contributions of CTL vs that of T cell-dependent IL and of CD4+ cells. H-2b mice primed with the wildtype of vesicular stomatitis virus serotype Indiana (VSV-IND wt) mount an in vitro measurable cytotoxic response against the nucleoprotein (NP) of VSV-IND and are protected against a challenge infection with a vaccinia-VSV recombinant virus expressing the NP of VSV-IND (vacc-IND-NP). Their protective mechanism was highly susceptible to in vivo depletion of CD8+ T cells, but resistant to CD4+ depletion or treatment with anti-IFN-gamma and anti-TNF-alpha. Surprisingly, also VSV-CTL nonresponder H-2k mice were protected against a challenging infection with vacc-IND-NP when primed with VSV-IND wt. In contrast to the CTL responder H-2b mice, this protection was highly susceptible to CD4+ T cell depletion and to anti-IFN-gamma or anti-TNF-alpha treatment, but resistant to CD8+ T cell depletion. Antibodies were not responsible because they failed to transfer protection; in contrast CD4+ T cells conferred significant protection. VSV-CTL responder H-2b and nonresponder H-2k mice were protected almost equally well against a challenge dose of 10(3) pfu vacc-IND-NP inoculated intracerebrally. However, after intracerebral challenge with 5 x 10(6) pfu vacc-IND-NP, the CTL nonresponder mice died, whereas the CTL responder mice eliminated the virus by day 5. These results collectively show that CD4+ T cell-dependent IL may mediate antiviral protection, but their efficiency is relatively weak compared with CD8-mediated protection correlating with cytotoxic activity in vitro.