CD8+ cell activation to a major mastocytoma rejection antigen,P815AB: requirement for tum− or helper peptides in priming for skin test reactivity to a P815AB-related peptide

Delayed-type hypersensitivity (DTH) responses, mediated by CD8⁺ cells and detected by skin test assay, occur in sensitized mice in response to challenge with class I-restricted antigenic peptides of mutagenized (tum^?) P815 mastocytoma cells. In contrast, a nonapeptide related to a tumor rejection antigen, P815AB, failed in this study to elicit DTH after sensitization of mice with irradiated tumor cells or adoptive transfer of P815AB-pulsed dendritic cells. Unresponsiveness, however, could be overcome by immunization with tumor cells co-expressing P815AB and tum^? antigens. When used for cell pulsing in vitro, a mixture of P815AB and tum^? peptides was also highly effective in inducing anti-P815AB reactivity, as was the combined use of P815AB and class II-restricted peptides of tetanus toxin or Plasmodium berghei circumsporozoite protein. While the effector phase of the CD8⁺ cell-mediated DTH to P815AB was unaffected by the ablation of CD4⁺ cells, the same treatment, or neutralization of IFN-γ, negated the induction of reactivity if it occurred at the time of sensitization. Thus, defective activation of CD4⁺ cells may contribute to the poor immunogenicity of P815AB. Besides providing an insight into the mechanisms of anti-tumor protection induced by tum^? cells, these data offer useful information for the design of vaccination strategies against identified tumor antigens.     

Paralysis of B7 co-stimulation through the effect of viral IL-10 on T cells as a mechanism of local tolerance induction

The Epstein-Barr virus (EBV) encodes an open reading frame with significant homology to the cellular IL-10 gene. This viral IL-10 (vIL-10) might enable EBV to evade antiviral T cells. We employed transfectants of a murine tumor cell line (P815) to investigate whether vIL-10 interferes with the first (antigenic) or second (co-stimulatory) signal of T cell activation. Untransfected P815 cells caused tumors in syngeneic DBA/2 mice after s.c. inoculation. In contrast, transfectants that provided either a strong antigenic stimulus (P815-K^b cells) or a strong co-stimulatory signal (P815-B7 cells) were rejected. Injection of double-transfected P815 cells expressing K^b and secreting high levels of vIL-10 (P815-K^b -vIL-10) did not result in tumor growth. We then investigated whether vIL-10 could paralyse co-stimulation by B7 under the same conditions. Therefore P815-B7 cells were mixed with vIL-10-secreting P815-K^b cells and co-injected into DBA/2 animals. Most of these mice developed a tumor. Explanted tumor cells expressed the B7 molecule but not the K^b antigen. These observations in vivo were mirrored by experiments in vitro: vIL-10 could induce T cell tolerance towards P815-B7 cells but not P815-K^b cells. Taken together our results suggest that vIL-10 acts directly on T cells to inhibit co-stimulatory signals mediated via B7 receptors such as CD28 or CTLA-4.     

Role of cellular density,in vitro,in anti-tumor activity of CFA-treated and immunized cells

Incorporation of tritiated deoxythymidine (³HdT) into DNA was used to measure growth, in vitro, of P815 tumor cells admixed with spleen and peritoneal effector cells. At a high tumor cell density (1×10⁵ cells per dish), using anti-theta and anti-macrophage sera, T-cells and macrophages from the peritoneum of immunized mice could be identified as cells possessing anti-tumor activity. A nonspecific inhibition by normal effector cells, which occurred at the high tumor cell density, did not occur at a lower tumor cell density (1×10⁴ cells per dish). Therefore, the effects of immunization and Freund's adjuvant treatment on the anti-tumor activity of effector cells were determined more accurately when normal cells were no longer inhibitory. Thus, experimental variables dealing with cellular density (cells/mm² of the culture vessel surface) and effector: tumor cell ratios play an important role in the anti-proliferative capacity of effector cells.     

The role of IL-4 and IL-10 cytokines in controlling an anti-tumor response in vivo

The effect of systemic administration of anti-inflammatory cytokines (IL-4 and IL-10) on the development and maintenance of an anti-tumor rejection response in vivo was studied by following the growth patterns of P815.B7 tumors on B6D2F1 [(C57BI/6 x DBA/2)F1] mice. The anti- P815.B7 rejection response was found to be T cell dependent, involving both CD4 and CD8 cells. IL-4 treatment resulted in a compromised rejection response; IL-10 treatment alone had little or no effect. These results demonstrate that treatment with an anti-inflammatory cytokine can compromise an otherwise effective anti-tumor rejection response. For the anti-inflammatory cytokine IL-4, the immunosuppressive effects of the cytokine appear to outweigh any possible anti-tumor activities as have been reported using tumor cells genetically altered to produce IL-4. Relatively high systemic doses of IL-10, in contrast, were not immunosuppressive and, when given in combination with IL-4, countered the IL-4 suppressive effect. Pathologically, IL-4 treatment led to splenomegaly characterized by a marked increase in neutrophils and NK activity. The possible linkages between neutrophils, NK activity and IL-12 are discussed.      

Localization of two cytotoxic T lymphocyte epitopes and three anchoring residues on a single nonameric peptide that binds to H-2Ld and is recognized by cytotoxic T lymphocytes against mouse tumor P815

Mouse mastocytoma P815 expresses tumor antigens P815A and P815B encoded by a single gene called P1A and carried by a single peptide named P1A 35–43 (NH₂-Leu-Pro-Tyr-Leu-Gly-Trp-Leu-Val-Phe-COOH). P1A 35–43 is presented to anti-P815A and anti-P815B cytotoxic T lymphocytes (CTL) by major histo-compatibility complex (MHC) H-2L^d molecules. In order to determine the individual role played by each amino acid residue of P1A 35-43 in binding to H-2L^d and in recognition by anti-A and anti-B T cell receptors (TcR), a series of P1A 35-43 peptides substituted by alanine at single positions was synthesized and tested for binding to H-2L^d and for CTL recognition. Binding to H-2L^d was estimated by measuring the ability of the peptide to up-regulate cell surface expression of H-2L^d. We found that three residues were important for interaction of P1A 35-43 with H-2L^d. Two of them, Pro at position 2 and Phe at position 9 were consistent with the described H-2L^d binding motif. A third residue, Trp at position 6, was also required for effective MHC binding of the tumor antigen. CTL sensitization assays showed that alanine substitution at position 7 (Leu) or at position 8 (Val) dramatically affected peptide recognition by anti-A CTL while positions 3 (Tyr) and 4 (Leu) were critical for recognition by anti-B CTL. We conclude that Pro², Trp⁶ and Phe⁹ constitute the anchor residues of P1A 35-43 to H-2L^d, whereas the dipeptidyl sequences Tyr³-Leu⁴ and Leu⁷-Val⁸ form the core epitopes recognized by the TcR of anti-P815B and anti-P815A CTL, respectively.     

Interleukin-12 and B7.1 co-stimulation cooperate in the induction of effective antitumor immunity and therapy of established tumors

Interleukin-12 (IL-12) promotes specific and long-lasting anti-tumor immunity mediated by T cells in a variety of murine tumor models. IL-12 also synergizes with B7.1 (CD80) co-stimulation to induce proliferation and cytokine production by both human and murine T cells in vitro. We evaluated the combined antitumor efficacy of IL-12 and B7.1 gene delivery in two apparently poorly immunogenic tumor models (TS/A and MCA207). In both of these models, expression of B7.1 and production of IL-12 in the inoculum led to improved anti-tumor immunity, with up to 80% long-term tumor-free animals (vs 0– 20% of mice remaining tumor free when inoculated with either B7.1- or IL-12-transfected tumors alone). Tumor-free mice were capable of rejecting a subsequent rechallenge with the wild-type tumor in 66% of the cases. Cooperativity was dependent upon the level of IL-12 secreted by engineered cells. IL-12 delivery required B7 expression of therapeutic effects to be observed in these models. Vaccines provided at a site distal to a control, non-transfected tumor slowed (TS/A) or abrogated (MCA207) the progression of wild-type tumors. The synergistic anti-tumor effects associated with combined application of B7.1- and IL-12-transfected tumors were partially negated by systemic administration of the CD28-B7.1/B7.2 antagonist CTLA4-Ig or by inoculation with neutralizing antibodies directed against murine interferon-γ or tumor necrosis factor-α, two cytokines elicited in response to IL-12 stimulation. These data support the potential clinical utility of combined gene therapy using IL-12- and B7.1-engineered autologous cells (tumor or fibroblasts) as a vaccine to elicit specific anti-tumor immunity.     

Heterogeneous effects of B7-1 and B7-2 in the induction of both protective and therapeutic anti-tumor immunity against different mouse tumors

Experimental mouse tumors are classified as intrinsically immunogenic when, after a single injection into syngeneic mice as nonreplicating cell vaccines, they elicit a protective immune response against a subsequent lethal challenge. Tumors that do not retain this residual immunogenicity are defined as poorly immunogenic or nonimmunogenic. The expression of the B7-1 co-stimulatory molecule on immunogenic tumors can further increase their capacity to induce a T cell-dependent anti-tumor immunity, whereas it has limited effects on nonimmunogenic tumors. Recently, B7-2, a second molecule with an apparently similar co-stimulatory activity, has been cloned. In this report, we compare the efficiency of nonreplicating cells from one immunogenic and two nonimmunogenic mouse tumors transfected with B7-1 or B7-2 in the induction of protective and curative anti-tumor immunity. Immunogenic lymphoma cells expressing B7-1 or B7-2 are equally effective in both protecting against a subsequent challenge and curing established tumors. By contrast, nonimmunogenic adenocarcinoma and melanoma cells expressing B7-2 provide superior protective immunity, and only B7-2⁺ adenocarcinoma cells induce an efficient curative immunity. CD8⁺ and polymorphonuclear cells, but not CD4⁺ T cells, are critically involved in the rejection of the adenocarcinoma elicited by both B7-1⁺ and B7-2⁺ vaccines. These data indicate that B7-1 and B7-2 are not redundant co-stimulatory molecules and that, in these experimental models, B7-2 is superior to B7-1 in the induction of an efficient immunity when the immunogenicity of a tumor is a limiting factor.     

Use of a skin test assay to determine tumor-specific CD8+ T cell reactivity

We have observed delayed-type hypersensitivity (DTH) reactions in immunized mice challenged subcutaneously with class I-binding peptides related to rejection antigens recognized by cytotoxic T lymphocytes on mutagenized (tum^?) variants of mastocytoma P815. As observed by skin test in virally infected mice challenged with viral peptides, the intrafootpad injection of tum^? peptides resulted in a dose-dependent DTH that peaked at approximately 24 h. The response was mediated by CD8⁺ cells and could be induced by previous vaccination of mice with live tumor cells, intrasplenic deposition of the eliciting peptide, or adoptive transfer with peptide-pulsed syngeneic dendritic cells. These sensitization procedures resulted in an immunologically specific footpad reaction detectable for up to 2–6 months after priming. The evaluation by DTH in cancer patients of long-lived CD8⁺ anti-tumor T cell responses following local challenge with tumor-specific peptides may be of great interest in human immunotherapy trials involving immunization against identified tumor antigens.     

Adoptive Transfer of Low Numbers of CD4+ T Cells into SCID Mice chronically treated with Soluble IL-4 Receptor does not prevent Engraftment of IL-4-Producing T Cells

After intravenous injection of 10⁵ purified, lymph node (LN)-derived dm2 (H-2^d/L^d) CD4⁺ T cells into young C.B-17 scidjscid (severe combined immunodeficiency, SCID) mice (H-2^d/L^d+), the transplanted L^d- T cells show a selective pattern of engraftment: they repopulate the spleen, the lamina propria of the small intestine and the mesenteric LN (but not other peripheral LN) of the immunodeficient host. CD4⁺ cells repopulating different lymphoid organs of the SCID recipient mice produce interleukin-2 (IL-2) and interleukin-4 (IL-4) in response to polyclonal stimulation in vitro. Some evidence has recently been provided that cytokines (e.g. IL-4) present at the site of antigen stimulation in vivo decisively influence the pattern of cytokines expressed by T cells activated at these sites. We therefore asked if neutralization of IL-4 by chronic treatment of SCID mice with high doses of recombinant soluble IL-4 receptor (sIL-4R) changes the IL-4 or IL-2 expression pattern of CD4⁺ T cells adoptively transferred into young SCID recipients. Transplanted SCID mice were chronically treated with two different, recombinant murine sIL-4R proteins. The experimental series further included groups of transplanted SCID mice treated with a recombinant human sIL-4R protein (which does not bind murine IL-4), treated with the anti-murine IL-4 monoclonal antibody (MoAb) 11B11, or non-treated. Transplanted SCID mice treated with the recombinant murine sIL-4R protein preparations displayed detectable sIL-4R serum levels, which demonstrates that the substitution therapy could maintain neutralizing serum levels of anti-IL-4 activity in SCID mice. By contrast, no serum sIL-4R levels were detectable in the sensitive ELISA readout in transplanted SCID mice which were non-treated, treated with the MoAb 11B11, or treated with the recombinant human sIL-4R protein. The efficiency and the pattern of CD4⁺ T-cell engraftment, and the lymphokine-producing phenotype of the engrafted dm2 CD4⁺ cells, was not affected by the continuous IL-4-neutralizing treatment of mice with either the MoAb 11B11 or the soluble IL-4R preparations. Hence, in contrast to the published evidence of the dramatic effect of IL-4 on the lymphokine-producing phenotype of CD4⁺ T cells stimulated in vitro or in vivo, the chronic suppression in vivo of IL-4 activity (by either different sIL4-R protein constructs, or by the anti-IL-4 MoAb 11B11) did not lead to preferential engraftment of Th1-type CD4⁺ T cells after adoptive transfer of CD4⁺ T-cell populations into an immunodeficient recipient.     

Cell-mediated anti-tumor response in the RLmale 1 system. I. T nature, H-2Dd involvement and antigenic specificity of the effector cells.

After in vivo priming and in vitro secondary stimulation by the radio-induced RL♂ 1 lymphoma cells, syngeneic BALB/c splenic lymphocytes evidenced a specific but very weak cytolytic activity against RL♂ 1 target cells. Under the same experimental conditions, spleen cells from (B6 x BALB/c)F₁ were at least 100 times more efficient. In both cases, the effector cells were cytolytic T lymphocytes (CTL). Normal H-2D^d antigens of the tumor cell surface were involved in the effector-to-target cell interaction since anti-H-2 or anti-H-2D^d antisera abolished the RL♂ 1 cytolysis by immune syngeneic CTL, whereas anti-H-2K^d were devoid of blocking activity. The testing of a series of lymphoma cells, either virus-induced or not, belonging to different H-2 haplotypes, show that the immune CTL were highly specific for RL♂ 1 target cells. Only with the P815 mastocytoma cells was a weak cross-reactivity detected by both direct tests and competition experiments. Allogeneic (H-2^k) AKR lymphoma cells which share the X1 antigen with RL♂ 1 do not react, possibly due to an H-2 restriction of the CTL activity. The CTL-reacting antigen cannot be definitely identified, since several specificities, including the serologically defined X1 antigen, are possibly involved simultaneously. The Hh antigens which could have accounted for the F₁ anti-parent reaction, appear irrelevant.     

H-2 antigen-specific cytotoxic T cells induced by concanavalin A: estimation of their relative frequency.

Specific and nonspecific lysis of DBA/2 (H-2d) mastocytoma cells, P815, by concanavalin A (Con A)-induced cytotoxic T cells was studied. In the assay for nonspecific lysis, phytohemagglutinin (PHA) was present to glue the target and killer cells together. We have presented evidence previously to show that PHA reveals only, and all, cytotoxic T cells. In the assay for specific lysis the only glue present was specific receptors on a fraction of the killer cells and surface antigens of P815. We show that when PHA was present, Con A-induced cells which were syngeneic, semi-syngeneic, or allogeneic, lysed P815 very efficiently in a 4-h 51Cr release assay. However, only Con A-induced T cells which were allogeneic and did not carry H-2d lysed P815 when the assay was carried out in the absence of PHA. In an experiment with two target cells, Con A-induced B10 (H-2b) T cells lysed B10.D2 (H-2d) targets specifically but did not lyse B10 targets, while Con A-induced B10.D2 T cells lysed B10 targets specifically but not B10.D2 targets. Furthermore, Con A-induced B6 (H-2b) T cells from normal mice lysed P815 specifically but Con A-induced B6 T cells from irradiated F1 (B6 x BALB/c) (H-2b/d) mice reconstituted with B6 bone marrow did not lyse P815 specifically. A fraction of Con A-induced T cells therefore appear to bear specific surface receptors for nonself H-2 coded structures. We describe conditions of assay and a new method of plotting the results such that nonspecific (PHA-revealed) and specific (PHA-independent) cytotoxicity can be quantitatively compared. We conclude that 1-4% of the total Con A-induced cytotoxic effector T cells are directed against any particular foreign H-2 haplotype. This is the first estimate of the relative frequency of antigen-reactive cytotoxic T cells.     

Lysis of enucleated tumor cells with allogeneic and syngeneic cytotoxic thymus-derived lymphocytes.

These studies were initiated to understand the minimal requirements for lysis of target cells by cytotoxic thymus-derived lymphocytes (CTL). In particular, the significance of the nucleus to the susceptibility of the target cell to be lysed by CTL have been studied. P815 mastocytoma cells and EL4 lymphoma cells were enucleated by cen- trifugation through a discontinuous Ficoll gradient containing 10 μg/ml of cytochalasin B. The resultant vesicles were then analyzed for susceptibility to lysis by different CTL specific for the H-2K and H-2D gene products, minor histocompatibility antigens, trinitrophenyl groups, and Sendai virus-specific antigens. The results indicate that enucleated vesicles obtained from both EL 4 and P 815 cells are susceptible to lysis by these CTL. The lysis of EL 4-enucleated vesicles (unlike P 815-enucleated vesicles), however, required 2–4 times more effector cells than were required for equivalent lysis of intact EL 4 cells. This reduced susceptibility to lysis of enucleated EL 4 vesicles was not the result of an inability of the CTL to recognize and bind the vesicles. This conclusion was suggested by the fact that both enucleated EL 4 vesicles and intact cells inhibited equally well the lysis of ⁵¹Cr-labeled concanavalin A-stimulated BALB. B spleen cells by anti-H-2^b CTL. Although enucleated vesicles obtained from both P 815 and EL 4 cells were susceptible to lysis by CTL, they exhibited a reduced capacity to “cap” their serologically defined H-2 antigens. Lysis of these enucleated vesicles by CTL has the same requirements as lysis of intact cells. This conclusion was based upon the requirement for Ca⁺⁺ and Mg⁺⁺ and also because lysis of the modified vesicles (trinitrophenyl or viral antigens) occurred in an H-2-restricted fashion. Moreover, the enucleated vesicles were susceptible to antibody and complement-mediated lysis and also lectin-dependent cell-mediated cytotoxicity.     

Modification of anti-tumor immunity by tolerogenic dendritic cells

Immunosuppressive functions of glucocorticoids (GC) can be mediated via various mechanisms, including the modulation of dendritic cells (DC). Our study investigates the effects of tolerogenic GC-treated DCs on NK and T cell anti-tumor responses in OT-1/Rag^?/? mice, expressing a transgenic TCR in CD8⁺ T cells. The effects caused by GC-treated DCs were compared to the responses to immunogenic, CpG-activated DCs. The effects of DCs on anti-tumor immune responses were analyzed using the EG7 tumor model, where the tumor cells express the peptide epitope recognized by OT-1 T cells. We observed that immunization with CpG and peptide-treated DCs protected against tumor growth by activation of NK cell response. Also, immunogenic DCs induced the expansion of cytotoxic CD8⁺OT-1 cells, expressing activation markers CD44 and CD69 and producing IFNγ. In contrast, the peptide and GC-treated DCs in OT-1 mice increased the numbers of immature Mac-1⁺CD27^? NK cells as well as Foxp3⁺ and IL-10 secreting CD8⁺OT-1 cells with suppressive properties. We conclude that the generation of tolerogenic DCs is one of many immunosuppressive mechanisms that can be induced by GC. Our study demonstrated that tolerogenic DCs modify anti-tumor immune response by suppressing NK cell activity and stimulating the formation of IL-10-secreting CD8⁺ Tregs.     

Effects of histamine type-2 receptor antagonists on indomethacin and IL-2 immunotherapy of metastasis

Histamine type-2 receptor antagonists (H-2RA) have been used chronically to prevent dyspepsia in cancer patients subjected to immunotherapy with chronic indomethacin (Indo) and intermittent IL-2 in our cancer centre. We tested the effects of these agents during immunotherapy of C3H/HeJ mice transplanted s.c. with 5 × 10⁵ C3L5 mammary adenocarcinoma cells. Tumor-transplanted mice were divided into groups receiving: (1) Indo (14 µg/ml); (2) H-2RA, i.e. (a) ranitidine at 28.6 µg/ml (Ran-lo) or 143 µg/ml (Ran-hi), or (b) famotidine (Fam) at 4.3 µg/ml, or (c) cimetidine (Cim) at 107 µg/ml, all in the drinking water on days 5–24; (3) IL-2 (1.5 × 10³ Cetus U i.p. every 8 h on days 10–14 and 20–24); (4) combinations of H-2RA + Indo; or (5) combinations of H-2RA + Indo + IL-2. Animals were killed on day 24 for examination of primary s.c. tumor growth, secondary lung metastasis and splenocyte cytotoxicity against YAC-1 lymphoma cells (⁵¹Cr release assay). Results revealed: (1) primary tumor growth was reduced in mice treated with Fam + Indo, Indo + IL-2 and any of the H-2RA + Indo + IL-2 (no differences were observed within the last two groups); (2) lung metastases decreased in mice treated with IL-2 alone, Indo + IL-2, and Indo + IL-2 + Ran-hi; (3) splenic cytotoxicity was suppressed in tumor-bearing controls, with partial restoration seen in Ran (both doses), Ran-lo + Indo, Ran-lo + Indo + IL-2, and Cim + Indo + IL-2 treated groups. Nearly complete restoration was seen in Cim, Cim + Indo, Indo + IL-2, Ran-hi + Indo + IL-2, and Fam + Indo + IL-2 groups. Thus, addition of H-2RA did not alter the overall therapeutic efficacy of the standard Indo + IL-2 tumor immunotherapy.     

Interleukin-13 is produced by activated human CD45RA+ and CD45R0+ T cells: modulation by interleukin-4 and interleukin-12

Interleukin (IL)-13 is a cytokine originally identified as a product of activated T cells. Little is known, however, about IL-13 production by human T cells and its modulation by other cytokines. Here, we show that IL-13 is produced by activated human CD4⁺ and CD8⁺ CD45R0⁺ memory T cells and CD4⁺ and CD8⁺ CD45RA⁺ naive T cells. In contrast, IL-4, which shares many biological activities with IL-13, is only produced by CD45R0⁺ T cells following activation. Analysis of intracellular cytokine production by single CD45RA⁺ and CD45R0⁺ T cells indicated that IL-13 continued to be produced for more than 24 h after stimulation, whereas IL-4 could not be detected after 24 h. These data were confirmed by measurement of specific mRNA and suggest that IL-13, unlike IL-4, but like interferon-γ (IFN-γ), is a cytokine with long-lasting kinetics. The majority of human CD45R0⁺ T cells produced IL-4 and IL-13 simultaneously. In contrast, IFN-γ protein was generally not co-expressed with IL-4 or IL-13. IL-4 added to primary cultures of highly purified peripheral blood T cells activated by the combination of anti-CD3+anti-CD28 mAb enhanced IL-13 production by CD45RA⁺ and to a lesser extent by CD45R0⁺ T cells. Under these conditions, however, IL-12 inhibited IL-13 production by CD45RA⁺ T cells and to a lesser extent by CD45R0⁺ T cells in a dose-dependent fashion. These inhibiting effects were not related to enhanced IFN-γ production induced by IL-12, since IFN-γ by itself did not affect IL-13 production. Collectively, our data indicate that IL-13 is produced by peripheral blood T cells which also produce IL-4, but not IFN-γ, and by naive CD45RA⁺ T cells which, in contrast, fail to produce IL-4. These observations, together with the long-lasting production of IL-13, suggest that IL-13 may have IL-4-like functions in situations where T cell-derived IL-4 is still absent or where its production has already been down-regulated.     

Modulation of proinflammatory cytokine production in tumour necrosis factor-alpha (TNF-α)-transgenic mice by treatment with cells engineered to secrete IL-4, IL-10 or IL-13

TNF-α is one of the major proinflammatory cytokines involved in the pathogenesis of chronic inflammatory joint disease, in human rheumatoid arthritis as well as in murine models of this disease. It was previously described that a highly destructive chronic spontaneous inflammatory arthritis develops in mice expressing a human TNF-α transgene modified with the 3′ untranslated region of β-globin. The present study investigates in this mouse model the effects of the anti-inflammatory cytokines IL-4, IL-10 and IL-13 administered in vivo on proinflammatory cytokine expression. Groups of TNF-α-transgenic mice were engrafted with xenogeneic transfected Chinese hamster ovary (CHO) fibroblasts secreting murine IL-4, IL-10 or IL-13. In vivo treatments consisted of 3 or 4 weekly engraftments, starting when the mice were 4 weeks old. Control groups of transgenic mice were engrafted with β-galactosidase gene-transfected CHO cells or injected with medium. A significant decreased expression of TNF-α transgene, endogenous mouse TNF-α and IL-1 mRNA was observed in splenocytes of mice treated for 3 or 4 weeks with CHO/IL-4 and CHO/IL-13, and, to a lesser extent, with CHO/IL-10, compared with controls. Finally, attenuation of histological scores of arthritides was statistically significant only in the group of CHO/IL-4-treated mice after 3 weeks of treatment (P < 0.05), and was not significant in any other group. These results show that IL-4, IL-10 or IL-13, administered by gene therapy, can decrease the mRNA steady state levels of both endogenous and transgenic cytokines in human TNF-α transgenic mice. In addition, IL-4 can slightly attenuate the development of arthritides in this model.     

Degradation of specificity in cytolytic T lymphocyte clones: two broad specificity, H-2-independent recognition systems, one natural killer-like, develop during culture, in addition to the clonally distributed antigen-specific receptor

Ly-2+ CBA mouse T lymphocytes stimulated with concanavalin A in limiting dilution culture produce clones of cytotoxic T lymphocytes (CTL) which, although initially specific, eventually lyse a wide range of target cells. The nature of the recognition system for this apparently "nonspecific" cytolysis was examined using a range of tumor cells as labeled targets and as cold target inhibitors. Most syngeneic and allogeneic murine tumor cells were lysed but the degree of lysis varied, even for different sublines of the same tumor. All tumor cells cold target inhibited their own lysis, and cross-inhibited lysis of other targets to varying degrees. The recognition stage of "nonspecific" cytolysis appeared to be independent of target cell H-2 expression; some H-2-negative murine target cells were lysed and some were not, but all gave cold target inhibition of "nonspecific" cytolysis. Xenogeneic tumor cells were resistant to lysis, but some nevertheless gave cold target inhibition of the "nonspecific" cytolysis of murine targets. A study of the specificity of cold target cross-inhibition revealed two distinct patterns of recognition which existed simultaneously in "nonspecific" CTL; one was like that of natural killer cells and was directed to targets such as YAC-1, the other was distinct from that of natural killer cells and was directed to targets such as P815. Thus, murine CTL may express three distinct receptors, the clonally distributed, H-2-restricted, antigen-specific T cell receptor and two different "broad-range" receptors common to most clones.     

Disaggregation and invasion of ovarian carcinoma ascites spheroids

Background

In vivo studies have recently demonstrated that interleukin 21 (IL-21) enhances the anti-tumor function of T-cells and NK cells in murine tumor models, and the combined use of IL-21 and IL-15 has resulted in prolonged tumor regression and survival in mice with previously established tumors. However, the combined anti-tumor effects of IL-21 and low dose IL-2 have not been studied even though IL-2 has been approved for human use, and, at low dose administration, stimulates the proliferation of memory T cells, and does not significantly increase antigen-induced apoptosis or regulatory T cell (Treg) expansion. This study examined whether recombinant IL-21 alone or in combination with low-dose IL-2 could improve the in vivo anti-tumor function of naïve, tumor-antigen specific CD8⁺ T cells in a gp100_25–33 T cell receptor transgenic pmel murine melanoma model.

Methods

Congenic C57BL/6 (Ly5.2) mice bearing subcutaneous B16F10 melanoma tumors were sublethally irradiated to induce lymphopenia. After irradiation naive pmel splenocytes were adoptively transferred, and mice were immunized with bone marrow-derived dendritic cells pulsed with human gp100_25–33 (hgp100_25–33). Seven days after vaccination groups of mice received 5 consecutive days of intraperitoneal administration of IL-2 alone (20 × 10³ IU), IL-21 alone (20 μg) or IL-21 and IL-2. Control animals received no cytokine therapy.

Results

IL-21 alone and IL-2 alone both delayed tumor progression, but only IL-21 significantly augmented long-term survival (20%) compared to the control group. However, combination therapy with IL-21 and IL-2 resulted in the highest long-term (>150 days) tumor-free survival frequency of 46%. Animals that were tumor-free for > 150 days demonstrated tumor-specific protection after rechallenge with B16F10 melanoma cells. At peak expansion (21 days post vaccination), the combination of IL-21 plus IL-2 resulted in a 2- to 3-fold higher absolute number of circulating tumor antigen-specific pmel CD8⁺ T cells than was stimulated by IL-2 or IL-21 alone. Pmel CD8⁺ T cells were predominantly partitioned into central memory (CD62L⁺/CD127⁺) or effector-memory (CD62L^-/CD127⁺) phenotypes by day 28-post vaccination in IL-21 + IL-2 treated mice.

Conclusion

These observations support the potential use of IL-21 and low-dose IL-2 therapy in combination with a tumor-antigen vaccine and lymphopenic conditioning in future cancer clinical trials to maintain high numbers of anti-tumor memory CD8⁺ T cells with the potential to sustain long term tumor regression and survival.     

Selective regulation of ICAM-1 and major histocompatibility complex class I and II molecule expression on epidermal Langerhans cells by some of the cytokines released by keratinocytes and T cells

Epidermal Langerhans cells (LC) are major histocompatibility complex (MHC) class II (Ia)-positive dendritic cells that act as potent antigen-presenting or accessory cells for primary and secondary T cell-dependent immune responses. Recent studies have disclosed that the morphological, functional, and phenotypic characteristics of LC are variably and drastically modulated by external stimuli both in vivo and in vitro. However, little is known of the biological significance of diverse cytokines in regulating the surface molecules of LC. To determine the regulatory properties of ICAM-1, Ia, and MHC class I (H-2K) molecules in LC, we have examined the effects of interleukin (IL)-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the expression of these molecules. Among the cytokines examined, IFN-γ markedly and reproducibly up-regulates the expression of H-2K, but not ICAM-1, in Ia⁺ LC in a time- and dose-dependent manner. TNF-α consistently up-regulates the expression of ICAM-1, but not H-2K, in a time- and dose-dependent manner. IL-10 slightly but reproducibly inhibits the expression of ICAM-1, but not H-2K, in a time- and dose-dependent manner. IL-10 potently inhibits the TNF-α-induced ICAM-1 up-regulation, but not the IFN-γ-induced H-2K up-regulation. Moreover, no cytokine consistently affects the Ia expression of LC. In addition, slight enhancing effects have been observed on H-2K expression by IL-4, and on ICAM-1 expression by IL-1α, IL-1β, or GM-CSF. The present data suggest that the selective regulation is operative in a certain cell surface moiety of LC by various cytokines. These results further facilitate our understanding of immunobiology of LC.