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.     

IL-4 (B cell-stimulatory factor 1) regulates multiple aspects of influenza virus-specific cell-mediated immunity

The effect of endogenous and exogenous IL-4 on the generation of influenza virus-specific cell-mediated immunity was examined. When added at the onset of the culture, IL-4 augmented both cluster of differentiation (CD)8+ lymphoproliferation and MHC-restricted, influenza virus-specific cytotoxicity. When added 5 or 6 days after initiation of cultures, IL-4 was highly effective at augmenting cytotoxicity, whereas no augmentation of proliferation was observed. This disassociation of the effect of IL-4 on lymphoproliferation and cytotoxicity indicated that IL-4 was providing a late signal in CTL generation. Studied at the level of CTL precursor maturation in microcultures, IL-4 was found not to increase cytotoxicity but to be required, in some cases, for the generation of cytotoxicity. Endogenous IL-4 production was observed and demonstrated to be important because neutralizing antiserum to IL-4 suppressed CTL development. In contrast to the effects of IL-4 when added later to the cultures, pulsing the lymphocytes with IL-4 before, or shortly after, exposure to antigen resulted in suppression of the CTL response. These results indicate that IL-4 has differentiative, proliferative, and suppressive effects on cell-mediated immune responses.     

Antigen specific and MHC nonrestricted cytotoxicity of T cell receptor alpha beta+ and gamma delta+ human T cell clones isolated in IL-4

IL-4 has been shown to act as a growth factor for human T cells. In addition, IL-4 can enhance CTL activity in MLC, but blocks IL-2 induced lymphokine activated killer cell activity in PBL. In our study, the cloning efficiencies, Ag-specific CTL activity and non-MHC-restricted cytotoxicity of CTL clones generated in IL-2 were compared to those generated in IL-4. In a first experiment, T cells were stimulated with the EBV-transformed B cell line JY and cloned 7 days later with feeder cells and either IL-2 or IL-4. In a second experiment, stimulation of the T cells was carried out in the presence of IL-2 plus anti-IL-4 antibodies or IL-4 plus anti-IL-2 antibodies in order to block the effects of IL-4 and IL-2, respectively, produced by the feeder cells. Although the cloning efficiencies in the second experiment were lower than those obtained in the first experiment, the cloning efficiencies obtained with IL-2 or IL-4 were similar in both experiments. The overall proportion of TCR alpha beta+ T cell clones cytotoxic for the stimulator cell JY established in IL-2 or IL-4 were comparable. A striking difference between the clones obtained in IL-2 or IL-4 was that a large proportion of the clones obtained in IL-4 expressed CD4 and CD8 simultaneously, whereas none of the clones isolated in IL-2 were double positive. Also gamma delta+ T cell clones could be established with IL-4 as a growth factor. TCR gamma delta+ T cell clones isolated in either IL-2 or IL-4 were CD4-CD8- or CD4-CD8+, but the proportion of CD4-CD8+ clones isolated in IL-4 was higher. Interestingly, one TCR gamma delta+ clone isolated in IL-2 was CD4+CD8-. Most of the TCR alpha beta+ and TCR gamma delta+ CTL-clones isolated in IL-2 lysed the NK cell sensitive target cell K562. In contrast, only a small proportion of the TCR alpha beta+ or TCR gamma delta+ CTL clones isolated in IL-4, lysed K562. One TCR gamma delta+ T cell clone (CD-124) isolated in IL-4 and subsequently incubated in IL-2 acquired lytic activity against K562.(ABSTRACT TRUNCATED AT 400 WORDS)     

Different signaling pathways induced by alpha-CD3 monoclonal antibody versus alloantigen on the basis of differential ornithine sensitivity.

Previously we reported that 10 mM ornithine (Orn) selectively inhibits the development of CD8+ CTL in MLC. Herein we show that induction by alpha-CD3 mAb of CD8+ killer cells which manifest antibody-redirected cytotoxicity (ARC) of FcR+ targets is not Orn sensitive. Orn resistance was independent of activation kinetics or alpha-CD3 mAb concentration. alpha-CD3 mAb added to the cytotoxicity assay did not reveal a cytolytic potential in CTL from an Orn-treated MLC when the target cells bore both the inducing alloantigen and FcR. Addition of alpha-CD3 mAb to MLC failed to overcome Orn inhibition of CTL and yet induced ARC activity in the same culture. Expression of mRNA for pore-forming proteins (PFP) and granzyme B was inhibited by Orn in CTL but not in ARC killer cells. Our results demonstrate differences in the T cell activation process stimulated by alloantigen or alpha-CD3 mAb.     

Effects of interferon-gamma on the activation of human T lymphocytes

The role of interferon (IFN)-gamma in the activation of human T cells was investigated. Addition of IFN-gamma to mixed-lymphocyte cultures (MLC) augmented both the proliferation and the development of T-cell-mediated cytotoxicity. IFN-gamma also augmented the early expression on CD8+ but not CD4+ lymphocytes of IL-2 receptor alpha chain (Tac antigen) and Class II major histocompatibility antigen (HLA-DR). This effect synergized with that caused by interleukin 2 and was not observed with IFN-alpha. The addition of neutralizing antibody against IFN-gamma to MLC suppressed the development of cytotoxicity and proliferation and the expression of activation antigens on CD8+ cells. In experiments in which highly purified CD8+ T cells were activated with cell-free stimuli, IFN-gamma slightly but significantly augmented proliferation, antibody to IFN-gamma suppressed proliferation, and excess IFN-gamma reversed this suppression. It is concluded that (i) IFN-gamma augmented activation of T cells in human MLC, (ii) IFN-gamma exerted effects directly on T cells, and (iii) IFN-gamma preferentially augmented CD8+ cell activation.     

IL-7 regulation of cytotoxic lymphocytes: pore-forming protein gene expression, interferon-gamma production, and cytotoxicity of human peripheral blood lymphocytes subsets.

The effects of IL-7 on the generation of cytolytic human peripheral blood lymphocytes (PBL) were investigated. Induction of T-cell pore-forming protein (PFP) mRNA and cytotoxic potential by IL-7 was both slow and minor compared with that observed in IL-2-cultured T cells. IL-7 and suboptimal doses of IL-2 (10 U/ml) were found to costimulate PFP mRNA expression and cytotoxic potential in T cells. Clearly, however, both IL-7 and IL-2/IL-7 induced the PFP gene expression and cytotoxic potential of CD8+ T cells and not CD4+ T cells. In addition, neither monoclonal antibodies (mAb) to the p55 or p75 IL-2-receptor subunits had any effect upon IL-7 induction of CD8+ T-cell cytotoxicity, indicating that IL-7 induction of cytotoxic CD8+ T cells was IL-2 independent. IL-7 induction of CD3- large granular lymphocyte (LGL) and PB gamma delta T-cell cytotoxicity was also delayed and reduced compared with that effected by IL-2. IL-7 (10 or 1000 U/ml, 72 hr) enhanced the NK and LAK cytotoxic of LGL and PB gamma delta T cells. By contrast IL-7 or IL-2 augmented the redirected cytotoxic potential of PB gamma delta T cells, but not that of LGL, and neither lymphokine had any effect on constitutive PFP mRNA expression in either lymphocyte subset. In addition, IL-7 induction of LGL IFN-gamma production was weak and delayed compared with that effected by IL-2 and neither IL-2 nor IL-7 stimulated IFN-gamma production in PB gamma delta T cells. Therefore, overall the effects of IL-2 and IL-7 on various cytotoxic human PBL were qualitatively similar, but quantitatively and kinetically different.     

Suppression of lymphocyte alloreactivity by early gestational human decidua. II. Characterization of the suppressor mechanisms

We have earlier shown that first trimester human decidual cells (typical decidual cells and decidual macrophages) suppress lymphocyte alloreactivity (MLR and CTL generation) in vitro in an MHC-unrestricted manner and that this suppression is mediated by PGE2. The present study explored the mechanisms underlying this suppression by noting the effects of decidual cells (+/- indomethacin or anti-PGE2 antibody) or chemically pure PGE2 on numerous T lymphocyte activation events following allogeneic stimulation in mixed lymphocyte culture (MLC) or polyclonal activation with Con A. The results revealed that this suppression was the net result of an action of PGE2 on at least two events during lymphocyte activation: (i) a down-regulation of IL-2 receptor development on lymphocytes, quantitated with a radioimmunoassay and radioautography; this was noted in MLC or Con A-stimulated lymphocyte cultures in the presence of decidual cells (reversible in the presence of indomethacin or anti-PGE2 antibody), or PGE2, but not PGF2 alpha; (ii) an inhibition of IL-2 production in the MLC, measured with a bioassay using an IL-2-dependent T cell line (CTLL-2) and a recombinant IL-2 standard. These effects blocked cell proliferation and eventual generation of killer cells in the MLC. Decidual cells or PGE2 did not interfere with IL-2-dependent proliferation of CTLL-2 cells, which require an interaction between IL-2 receptors on these cells and IL-2. Finally, neither agent interfered with the lytic function of CTL, once generated. These results indicate that the PGE2-mediated immunosuppressor function of early gestational human decidual cells is accomplished by an afferent blockade of the early events in T lymphocyte activation.     

Generation of MHC-nonrestricted and restricted oncolytic subsets from human bone marrow.

We analyzed the cytotoxicity and characterized the phenotype of oncolytic bone marrow (BM) lymphocyte subsets generated in vitro by interleukin-2 (IL-2) and stimulator cells (SC). Two irradiated B-lymphoblastoid cell lines (Daudi and EBV-transformed BSM) and fresh human acute myelogenous leukemia (AML) were used as SC. Stimulation with Daudi and IL-2 resulted in a substantial increase in cytotoxic activity (100- to 1000-fold) against a broad range of tumor targets, and total cellular expansion was higher compared to stimulation with IL-2 alone. The most prominent increase was observed in the CD16+ and CD56+/CD3- natural killer (NK) cell subset; however, a significant increase was also observed in CD56+/CD3+ T cells. Functional analysis of Daudi- and IL-2-generated subsets using fluorescence-activated cell sorting (FACS) revealed that most of the lytic activity was mediated by NK cells. Significant potentiation of oncolytic activity and cell growth was also seen in the cultures stimulated with BSM or fresh AML and IL-2. The highest oncolytic activity in the latter cultures was mediated primarily by CD8+, CD3+, and CD56- T cells, although NK cells also participated in cytotoxic activity. The T cell-mediated cytotoxicity was restricted by the major histocompatibility complex (MHC), since most cytotoxicity could be blocked by HLA I antibodies. Additionally, we observed that optimum stimulation of cytotoxicity required effector cell-stimulator cell contact. These data indicate that depending on the tumor used for stimulation, different lymphocyte subsets may be generated in IL-2 cultures. These different approaches may be useful in both specific and nonspecific immunotherapy.     

Human vascular endothelial cells stimulate memory but not naive CD8+ T cells to differentiate into CTL retaining an early activation phenotype

Endothelial cell (EC)-selective alloreactive CTL may mediate alloimmune vascular injury. In the present study, EC-selective CTL were generated in cocultures of purified human CD8+ T cells with allogeneic EC and were compared with conventional CTL against corresponding B lymphoblastoid cells (BLC). EC caused activation and expansion of memory but not naive CD8+ T cells, which differentiated into EC-selective CTL that retained high surface expression of CD69, CD25, and CD62L and displayed low intracellular perforin content. In contrast, BLC-stimulated CTL could be generated from naive or memory CD8+ T cells and showed a more mature phenotype (low CD69, CD25, and CD62L with higher levels of perforin). The expansion of alloreactive T cells by EC stimulation was 5- to 20-fold less effective than in corresponding BLC-stimulated cultures, accounting for a reduction in the assayable cytotoxicity of individual microcultures. In these IL-2-supplemented cocultures, no effect on CTL generation or phenotype was observed by mAb blocking of costimulation provided by LFA-3, ICAM-1, or CD40, by addition of comitogenic anti-CD28 mAb, or by preactivation of EC with CD40 ligand. Cyclosporine inhibited CTL expansion and cytotoxicity similarly in both EC- and BLC-stimulated cultures but did not affect the phenotype of those CTL that did emerge. This study extends the characterization of endothelium as an immunoregulatory cell type distinct from conventional APC and may explain why graft rejection within the arterial intima, an anatomic compartment in which EC may be the primary type of APC, is separable from rejection in the graft parenchyma.     

The anti-idiotypic antibody 1F7 selectively inhibits cytotoxic T cells activated in HIV-1 infection

Circulating CD8+ T lymphocyte numbers rise substantially following infection with HIV-1. This expanded CD8+ T cell population includes HIV-specific CTL and CTL that kill activated uninfected CD4+ lymphocytes. Experimental, epidemiological and clinical evidence supports the possibility that expansion of CD8+ CTL contributes to CD4+ T cell depletion and disease progression in human HIV infection. Therefore, modulation of CD8+ T cell numbers or of certain CD8+ CTL activated in HIV-infected individuals may be beneficial. It was found that 1F7, a mAb against an idiotype common to anti-HIV and anti-simian immunodeficiency virus (SIV) antibodies, selectively inhibited both anti-HIV CTL and CTL against uninfected CD4+ T cells. Alloantigen-specific CTL and NK cells from either HIV-infected individuals or controls were unaffected by 1F7. Prolonged incubation of CD8+ T cells from HIV-infected individuals with 1F7 induces apoptosis, which was shown to be reflected functionally in reduced total CTL activity and in especially reduced CTL activity against uninfected CD4+ lymphocytes. The selective reactivity of 1F7 with certain CD8+ CTL could be applied towards the modulation of CD8+ T cell responses involved in AIDS pathogenesis.     

CD11b (Mac-1): a marker for CD8+ cytotoxic T cell activation and memory in virus infection.

We have found that CD11b, a cell surface integrin of macrophages, granulocytes, and NK cells, is expressed by a subset of CD8+ T cells that include both the active virus-specific CTL and the virus-specific memory CTL populations. CD8+CD11b+ cells comprise less than 3% of naive mouse splenocytes, but after lymphocytic choriomeningitis virus (LCMV) infection increase by 9- to 12-fold by the peak (day 8) of the virus-specific CTL response. Depletion of day-8 splenocytes with anti-Mac-1 and C' or enrichment by sorting for CD11b+ or CD8+CD11b+ spleen cells demonstrated that LCMV-specific CTL are CD11b+. The CD11b+ subpopulation also contained the bulk of the IL-2-responsive CD8+ cells. MEL-14, a homing marker down-regulated on activated T cells, was down-regulated on the majority of CD8+ cells that became CD11b+. Less than 1% of LCMV-immune splenic lymphocytes expressed CD11b. Antibody and C' depletion of this population severely impaired the ability of immune splenocytes to respond to in vitro secondary stimulation with LCMV-infected peritoneal macrophages, but did not affect the generation of a primary allospecific CTL response in MLC. Mixing of CD8-depleted and CD11b-depleted LCMV-immune splenocytes failed to restore the ability of these cells to mount a virus-specific memory CTL response, indicating that a cell coexpressing CD8 and CD11b is essential for this response. As determined by limiting dilution analysis, the precursors for the LCMV-specific memory CTL response were enriched in the CD11b+ population of LCMV-immune splenocytes. CD11b stained far fewer CD8+ splenocytes from naive mice than did CD44 (Pgp-1), and among immune splenocytes it identified a small subpopulation of CD44hi cells, indicating that CD11b may be the best single marker available for discriminating between naive and memory CD8+ T cells.     

CD4+CD25+ regulatory T cells attenuate the phosphatidylinositol 3-kinase/Akt pathway in antigen-primed immature CD8+ CTLs during functional maturation

This study was designed to determine the role of CD25(+)CD4(+) regulatory T (Tr) cells in CTL maturation and effector functions using a murine CTL line and in vitro MLC. Tr cells inhibited CTL functional maturation, but had no effect on CTL effector functions. In CD4(+) responder T cell-depleted MLC supplemented with IL-2, Tr cells suppressed mature CTL generation only when added within the first 2 days of culture. Tr cells down-regulated levels of active Akt, but not STAT5 or ZAP70 in Ag-primed immature CTLs. Down-regulation of active Akt was accompanied by a reduction in CTL cell size and IL-2Ralpha expression. In Tr cell-depleted MLC, CTLs were generated that exhibited high levels of nonspecific cytotoxicity. Our in vitro findings suggest that Tr cells regulate functional CTL maturation to generate optimal Ag-specific immune responses through the control of the PI3K/Akt pathway.     

Function of the CD4 and CD8 molecules on human cytotoxic T lymphocytes: regulation of T cell triggering

The function of the T cell differentiation antigens CD4 (Leu-3/T4) and CD8 (Leu-2/T8) on human cytotoxic T lymphocytes (CTL) is presently seen only in conjugate formation between CTL and target cell via class II or class I MHC antigens rather than in the later killing steps. In this study, human CD4+ and CD8+ CTL clones were used to investigate the effects of monoclonal antibodies against these differentiation antigens on nonspecific triggering of cytotoxicity. Cytotoxicity was induced either by antibodies against the CD3 (T3) antigen or by the lectins Con A and PHA. Anti-CD4 or anti-CD8 antibodies specifically inhibited all types of cytotoxicity of CD4+ or CD8+ CTL, respectively, regardless of the specificity of the CTL for class I or class II HLA antigens and regardless of whether target cells expressed class I or class II antigens. These results are incompatible with an exclusive role of the CD4 and CD8 molecules in MHC class recognition and are discussed with respect to a function as negative signal receptors for these molecules on CTL.     

IL-18 prevents the development of chronic graft-versus-host disease in mice

The development of chronic graft-versus-host disease (GVHD), which is induced by the transfer of DBA/2 spleen cells into (C57BL/6 x DBA/2)F1 (BDF1) mice, is closely related to diminished donor anti-host CTL activity and host B cell hyperactivation. Therefore, an approach which activates donor CD8+ T cells or suppresses donor CD4+ T cell-host B cell interaction may have clinical utility in the treatment of chronic GVHD. We have previously demonstrated that IL-18 induces the development of naive CD8+ T cells into type I effector cells in DBA/2 anti-BDF1 MLC. In this paper we examined the effect of IL-18 administration on the development of chronic GVHD in mice. The treatment was started before or after the onset of clinical evidence of the disease. Regardless of the treatment schedule, IL-18 significantly decreased immunological parameters indicative of chronic GVHD, such as elevated serum IgG antinuclear Abs, IgG1, and IgE levels, and host B cell numbers and their activation. Importantly, IL-18-treated mice did not show the same acute GVHD-like symptoms reported for IL-12 treatment, because there was no weight loss, death, or severe immunodeficiency as indicated by a decrease in IL-2 and IFN-gamma production by Con A-stimulated spleen cells. In contrast, IL-18 treatment partially but significantly restored the production of these cytokines. Data further suggested that these IL-18-mediated therapeutic effects may be due to the induction of donor CD8+ CTL, the decrease in donor CD4+ T cell numbers, and a down-regulation of host B cell MHC class II expression. Thus, our results suggest that IL-18 has beneficial effects in the prevention and treatment of chronic GVHD.     

IL-21 influences the frequency, phenotype, and affinity of the antigen-specific CD8 T cell response

IL-21, a newly described cytokine belonging to the IL-2 gamma-chain receptor cytokine family (that includes IL-2, IL-7, and IL-15), has been described as an important regulator of the cellular immune response. In this study, the role of IL-21 in the generation of a human Ag-specific CD8+ T cell response is characterized by tracking a rare, but measurable population of self-Ag-specific T cells in vitro. Autologous dendritic cells pulsed with the melanoma antigen recognized T cells 1 self-peptide were used to stimulate CD8+ T cells from HLA-A2+ healthy donors and melanoma patients. We demonstrate that exposure to IL-21 increased the total number of MART-1-specific CD8+ T cells that could be elicited by >20-fold and, at the clonal level, enriched for a population of high-affinity CD8+ T cells with a peptide dose requirement more than 1 log(10)-fold less than their untreated counterparts. Phenotypic analysis of T cells from IL-21-treated cultures revealed a unique population of CD45RO+ CD28(high) CD8+ T cells, a phenotype that was stable for at least 4 wk after IL-21 exposure. These CD28(high) CD8+ T cells produced IL-2 upon Ag stimulation and represent potential helper-independent CTLs. Our studies demonstrate a significant role for IL-21 in the primary Ag-specific human CTL response and support the use of IL-21 in the ex vivo generation of potent Ag-specific CTLs for adoptive therapy or as an adjuvant cytokine during in vivo immunization against tumor Ags.     

The role of CD4+ cells in sustaining lymphocyte proliferation during lymphocytic choriomeningitis virus infection.

The murine immune response to lymphocytic choriomeningitis virus (LCMV) infection involves the activation of CD8+, class I MHC-restricted and virus-specific CTL. At times coinciding with CTL activation, high levels of IL-2 gene expression and production occur, the IL-2R is expressed, and T cell blastogenesis and proliferation are induced. We have previously found that, although both CD4+ and CD8+ T cell subsets transcribe IL-2, the CD4+ subset appears to be the major producer of IL-2 whereas the CD8+ subset appears to be the major proliferating population when the subsets are separated after activation in vivo. The studies presented here were undertaken to examine the contribution made by the CD4+ subset to lymphocyte proliferation in vivo. Responses to LCMV infection were examined in intact mice and in mice depleted of CD4+ or CD8+ subsets by antibody treatments in vivo. Protocols were such that in vivo treatments with anti-CD4 or anti-CD8 depleted the respective subset by greater than 90%. In situ hybridizations demonstrated that the IL-2 gene was expressed in non-B lymphocytes isolated from either CD4+ cell-depleted or CD8+ cell-depleted mice on day 7 post-infection with LCMV. When placed in culture, however, cells from CD8+ cell-depleted mice produced significantly higher levels of detectable IL-2 than did cells isolated from CD4+ cell-depleted mice on day 7 post-infection. IL-2 was apparently produced in vivo in mice depleted of either CD4+ or CD8+ cells, as expression of the gene for the p55 chain of the IL-2R, IL-2 responsiveness, and lymphocyte proliferation were observed with cells isolated from both sets of mice. Lymphocyte proliferation was shown to be sustained in mice depleted of CD4+ cells in vivo by three criteria: 1) non-B lymphocytes isolated from infected mice depleted of CD4+ cells underwent more DNA synthesis than did those isolated from uninfected mice or from infected mice depleted of CD8+ cells; 2) leukocyte yields were expanded during infection of CD4+ cell-depleted mice; and 3) CD8+ cell numbers were increased during infection of CD4+ cell-depleted mice. The majority of non-B lymphocytes having the characteristics of blast lymphocytes was recovered in the CD8+ populations isolated from infected CD4+ cell-depleted mice. These findings suggest that the requirement for the CD4+ subset to sustain CD8+ lymphocyte proliferation in vivo is limited, and that CD4+ and CD8+ cell types can function independently in many aspects of their responses to viral infections.     

Postoperative administration of interleukin-12 significantly enhances the anti-tumor immune response of MBT-2 bladder cancer bearing mice

This study, using the MBT-2 murine bladder tumor model, mainly investigated the role of interleukin-12 (IL-12) in the specific antitumor immune response of a tumor-bearing host when systemically administrated after surgery. Day 17 tumor-bearing mice (D17TBM) along with non-tumor bearing naive mice were treated with daily intraperitoneal (i.p.) injection of IL-12 (0.25 microg/mouse) from day 18 to day 24 for a total of 7 doses. Their splenocytes were obtained on Day 31 for natural killer cells (NK), lymphokine activated killer cells (LAK) and cytotoxic T lymphocyte (CTL) activity assay and lymphocyte subsets phenotypic analysis. The tumor suppression effect of systemic IL-12 administration was evaluated based on the tumor outgrowth of the higher number of tumor cells rechallenged 24 hours after resectioning of the primary tumor. After evaluation on Day 31, the result of in vitro cytotoxicity assay revealed that systemic administration of IL-12 mainly enhanced the splenic LAK and CTL activities in non-tumor-primed naive mice, and the NK activity in tumor-primed D17TBM, respectively. However, in vivo administration of IL-12 with or without IL-2 failed to upgrade the proportions of either CD4+ CD44+ or CD8+ CD44+ T cells subsets in the spleens and regional inguinal lymph nodes (LNs) of both the D17TBM and naive mice. However, the splenic CD8+ CD44+ T-cell subset in the IL-12-treated D17TBM increased prominently after further culturing in the presence of IL-2 400 units/ml plus IL-12 25 ng/ml for 4 days. The fact that systemic administration of IL-12 significantly suppressed the outgrowth of Day-18 challenged tumor cells, especially in D17TBM, clearly indicates that the established specific antitumor immunity in tumor-primed D17TBM was efficiently augmented. From the results of this study, we conclude that, after surgical resection of a primary tumor, systemic administration of IL-12 can be an effective adjuvant therapy because it demonstrates a significant augmentation effect on the tumor-specific immune response in the tumor-primed host.     

CD4-positive T lymphocytes are required for the generation of the primary but not the secondary CD8-positive cytolytic T lymphocyte response to herpes simplex virus in C57BL/6 mice.

To understand the cellular basis for recovery from HSV infection, it is critical to identify functional interactions between HSV-specific T lymphocyte subpopulations involved in the generation of the optimal response. To this end, the requirement for CD4+ (L3T4+) T lymphocytes in the development of the primary and secondary CD8+ (Lyt-2+) cytolytic T lymphocyte (CTL) response following HSV infection in C57BL/6 mice was investigated. It was found that chronic depletion of CD4+ cells in vivo by treatment with the mAb GK1.5, which resulted in greater than 95% depletion of peripheral CD4+ T lymphocytes in treated animals, caused a profound decrease in the levels of cytolytic activity obtained during the primary response in the draining popliteal lymph nodes of mice responding to infection in the hind footpads. However, treatment did not affect the levels of in vivo secondary CTL activity in the popliteal lymph nodes, nor the in vitro secondary response in the spleen. The decreased CTL activity observed during the primary response was not due to an inability to prime HSV-specific CTL precursors (CTLp), as full cytolytic activity was obtained following culture of lymphocytes in the presence of exogenous IL-2 and antigen, and the response could be reconstituted by treatment with recombinant IL-2 in vivo. Analysis of the secondary CTL response in the spleen indicated that CD4+ cells were not required for either the generation or maintenance of this aspect of the response. However, blockade of IL-2 utilization by CTL using anti-IL-2R antibodies indicated that this lymphokine was absolutely essential for secondary CTL expansion in vitro. Finally, mice that had been infected 12 months previously exhibited a decreased ability to generate secondary HSV-specific CTL in vitro following CD4-depletion in vivo. Taken together, these results suggest two distinct stages of CTL development during the response: an early primary stage dependent upon the presence of CD4+ cells, and a later, CD4-independent stage operative during the secondary response, which decays with time postinfection.     

Localization of Fas ligand in cytoplasmic granules of CD8+ cytotoxic T lymphocytes and natural killer cells: participation of Fas ligand in granule exocytosis model of cytotoxicity

Fas ligand (FasL) has been implicated in cytotoxic T lymphocyte (CTL)- and natural killer (NK) cell-mediated cytotoxicity. In the present study, we investigated the localization of FasL in murine CTL and NK cells. Immunocytochemical staining showed that FasL was stored in cytoplasmic granules of CD8+ CTL clones and in vivo activated CTL and NK cells, where perforin and granzyme A also resided. Immunoelectron microscopy revealed that FasL was localized on outer membrane of the cytoplasmic granules, while perforin was localized in internal vesicles. Western blot analysis showed that the membrane-type FasL of 40 kDa was stored in CD8+ CTL clones but not in CD4+ CTL clones. By utilizing a granule exocytosis inhibitor (TN16), we demonstrated that FasL translocated onto cell surface upon degranulation of anti-CD3-stimulated CD8+ CTL clones. Moreover, TN16 markedly inhibited the FasL-mediated cytotoxicity by CD8+ T cell clones and NK cells. These results suggested a substantial contribution of FasL to granule exocytosis-mediated target cell lysis by CD8+ CTL and NK cells.     

Either B7 costimulation or IL-2 can elicit generation of primary alloreactive CTL

B7-1 and B7-2 are important costimulatory molecules in the activation of T cell immunity. We have used mice made genetically deficient in either or both B7 molecules to determine the role of B7 molecules in activation of primary alloreactive CTL. The absence of either B7-1 or B7-2 did not alter generation of CTL from unfractionated lymphocytes, but the absence of B7-2 greatly decreased CTL generation from purified CD8+ responder cells. However, if B7-1 was induced on the stimulating cells then CTL generation was restored to wild-type levels. Absence of both B7-1 and B7-2 from MLR using whole splenocytes resulted in a profound reduction in generation of CTL. This could completely be reversed by the addition of IL-2. B7 molecules could directly costimulate CD8+ cells, as purified CD8+ cells developed into mature CTL when stimulated with wild-type APC, but not with B7-deficient APC. Again, IL-2 could drive CTL generation from purified CD8+ cells, even in the absence of B7 molecules. Taken together, these results demonstrate an important role for B7 costimulation in CTL generation.