Lymphokine-activated tumor inhibition in mice. Ability of a nonapeptide of the human IL-1 beta to recruit anti-tumor reactivity in recipient mice

The ability of the VQGEESNDK synthetic peptide corresponding to fragment 163-171 of human IL-1 beta to trigger lymphokine-activated tumor inhibition (LATI) of a poorly immunogenic fibrosarcoma (CE-2) of BALB/c mice was compared to that of the whole IL-1 beta. Neither molecule inhibits in vitro proliferation of CE-2 cells. Administration at the tumor challenge site for 10 days of daily injections of 50 micrograms of peptide 163-171 induce a consistent, although limited, inhibition of tumor growth, whereas similar injections of 1 pg of IL-1 beta induced a more marked LATI. However, strong LATI was elicited when these injections were performed in mice challenged with tumor cells admixed at 1/10 cell ratio with nonreactive lymphocytes from CE-2-bearing mice. The L3T4+ lymphocyte subset is mainly responsible for this enhancement. This reaction is abolished when recipient mice are sub-lethally irradiated, treated with cyclosporin A, or when the reactivity of L3T4+ and asialo GM1+ cells is suppressed. A similarly efficient LATI is found on combining the daily peptide injections with that of 10 U of IL-2. LATI stemming from this association, too, is abolished when mice are irradiated or treated with anti-L3T4 antibody, whereas it is not affected by cyclosporin A or anti-asialo GM1 antibody. Finally, a tumor-specific immune memory is acquired by about 50% of mice after LATI induced by IL-1 beta or 163-171 peptide alone and by about 80% of mice after LATI induced by peptide and lymphocytes from tumor-bearing mice or peptide and IL-2. These findings could lead to the building of a molecularly defined system to induce efficient immune recognition of tumor cells by using a peptide that does not cause any of the several inflammation-associated changes induced by the whole IL-1 beta.     

Interleukin 2 activated tumor inhibition in vivo depends on the systemic involvement of host immunoreactivity

Daily local administration at the tumor challenge site of 10 injections of 10 U of recombinant interleukin 2 (IL 2) obtained from different factories induces a consistent, though limited inhibition of the growth of CE-2, a poorly immunogenic methylcholanthrene-induced tumor. By contrast, almost complete tumor inhibition is observed when these injections are performed in mice challenged with tumor cells admixed at 1:5 cell ration with nylon wool column purified lymphocytes obtained from tumor-bearing animals. The host immune system plays a fundamental role in this lymphokine-activated tumor inhibition (LATI), which is derived from the local combination of IL 2 and nonreactive lymphocytes. When the host is sublethally irradiated, or the reactivity of L3T4 and Asialo GM1 lymphocytes is suppressed by in vivo antibody treatment, in fact, LATI no longer takes place. Daily injections of antibody to murine interferon-gamma or cyclosporin A have the same effect, indicating that lymphokine release plays an important role in the recruitment of host reactivity. The morphological data show that when LATI is taking place the tumor challenge area becomes infiltrated by mononuclear cells and granulocytes (mostly eosinophils), which establish close contacts with each other and with tumor cells as determined at ultrastructural analysis. Tumor draining lymph nodes display marked expansion of cortical and paracortical areas. Lymphocyte proliferation, interferon-gamma release and cytotoxicity against CE-2 and YAC-1 target cells are greatly enhanced during LATI. Contralateral lymph nodes and the spleen also show a slight increment of these functions. In mice challenged with CE-2 tumor cells only and receiving daily IL 2 injections, these reaction functions (with the exception of interferon-gamma secretion) are also enhanced, though to a lesser extent than during LATI and only in tumor-draining lymph nodes. Last, the growth of a second contralateral tumor challenge is significantly impaired during or after LATI, showing that a persistent and effective systemic reactivity can be quickly induced in this way.     

Lymphokine-activated tumor inhibition in vivo. I. The local administration of interleukin 2 triggers nonreactive lymphocytes from tumor-bearing mice to inhibit tumor growth

CE-2 is a chemically induced tumor of low immunogenicity in syngeneic BALB/c mice. Nylon wool columns eluting lymphocytes from the spleen of mice bearing clinically evident (5-mm mean diameter) CE-2 tumors (CE-2 TB lymphocytes) do not react with CE-2 cells in vitro, nor are they able to affect their growth in vivo in a Winn-type neutralization assay at 5:1 lymphocyte:tumor cell ratio. However, they become able to inhibit CE-2 tumor growth when 20 U of interleukin 2 (IL 2) in 0.4 ml are injected daily for 10 days at the challenge site. In contrast, mice injected with CE-2 cells and IL 2 only display tumor takes and growth that are not significantly different from those in controls challenged with CE-2 cells alone. This lymphokine-activated tumor inhibition (LATI) is not a peculiarity of the CE-2 tumor-host combination, because different tumors can be inhibited in this way and various TB lymphocytes can initiate it. In these experiments, IL 2-rich 25,000 to 30,000 m.w. fractions were obtained routinely from the culture supernatants of a clone of EL-4 thymoma stimulated with phorbol myristic acetate. Equally active IL 2-rich preparations were obtained from rat spleen cells stimulated with concanavalin A, or from MLA 144 gibbon lymphosarcoma spontaneously releasing IL 2. Treatment of CE-2 TB lymphocytes with various antibody and C, with 2000 rad gamma-irradiation, or fractionation on Percoll density gradients suggested that radioresistant functions of Thy-1.2+, Lyt-1.2+, Lyt-2.2- and of asialo GM1+ cells are independently involved in LATI induction. These lymphocytes inhibit tumor growth by recruiting the radiosensitive effector mechanisms of the recipient mice required for ultimate tumor destruction. CE-2 tumor inhibition by LATI leaves a specific delayed-type hypersensitivity and an immunologic memory, resulting in rejection of a second lethal CE-2 challenge in a significant number of mice.     

Role of neutrophils and CD4+ T lymphocytes in the primary and memory response to nonimmunogenic murine mammary adenocarcinoma made immunogenic by IL-2 gene.

TS/A is a spontaneous adenocarcinoma, apparently not immunogenic in BALB/cnAnCr mice. TS/A cells are unable to stimulate a syngeneic antitumor response either in vitro or in vivo. To evaluate the immunogenic potential of IL-2-releasing neoplastic cells, we used an expression vector to introduce the cDNA coding for murine IL-2 into TS/A cells. Six clones releasing between 30 and 6800 U of IL-2/10(5) cells/ml/48 h have been isolated. Both low (30 U, B1.30) and high (6000 U, B4.6000) IL-2-releasing clone are capable of stimulating a proliferative and cytotoxic response in syngeneic cultures. While the B1.30 clone grows in 60% of syngeneic mice with a delayed pattern, the five clones that release higher levels of IL-2 are promptly rejected. Rejection is associated with neutrophil infiltration, the intensity of which is directly proportional to the amount of IL-2 released. NK cells and CD4+ lymphocytes are uninfluential, whereas CD8+ lymphocytes play only a minor role. This neutrophil-dominated rejection leaves a long-lasting, tumor-specific, T lymphocyte-mediated immune memory. For its induction, CD4+ lymphocytes are required. Their specific activation appears to depend on both the amount of IL-2 released and the granulocyte-mediated reaction that may lead to a more efficient presentation of tumor-associated Ag. These data support the notion that, after transduction of IL-2 gene, cancer cells may elicit an immune antitumor response, and stress the potential use of IL-2 as a component of new tumor vaccines.     

The influence of immunoregulatory cytokines IL-2, IL-7, and IL-15 upon activation, proliferation, and apoptosis of immune memory T-cells in vitro

The influence of immunoregulatory cytokines IL-2, IL-7, and IL-15 on the activation, proliferation, and apoptosis of different subpopulations of an immune memory T-cell (CD45RO+) in healthy donors were investigated. It was demonstrated that rIL-2 equally affected both the activation and proliferation of CD4⁺ and CD8⁺ subpopulations of memory T-cells in vitro. High concentrations of rIL-2 increased the number of CD8⁺ memory cells expressing apoptotic marker CD95. Effect of rIL-7 and rIL-15 on the activation and proliferation of cytotoxic CD8⁺ memory cells in vitro was different. CD4⁺ memory lymphocytes exhibited relative resistance to activation and proliferation by rIL-7 and rIL-15 compared to rIL-2. This can provide them with relative resistance to apoptosis, as well as create the necessary conditions for accelerated implementation of their functional capacity in the development of a secondary immune response.     

CD25+ regulatory T cell depletion augments immunotherapy of micrometastases by an IL-21-secreting cellular vaccine

IL-21 is an IL-2-like cytokine, signaling through a specific IL-21R and the IL-2R gamma-chain. Because the TS/A mammary adenocarcinoma cells genetically modified to secrete IL-21 (TS/A-IL-21) are strongly immunogenic in syngeneic mice, we analyzed their application as vaccine. In mice bearing TS/A-parental cell (pc) micrometastases, vaccination with irradiated TS/A-IL-21 cells significantly increased the animal life span, but cured only 17% of mice. Spleen cells from cured mice developed CTL activity and produced IFN-gamma in response to stimulation by the AH1 epitope of the gp70env Ag of TS/A-pc. We tested whether the low therapeutic outcome might be due to CD4+CD25+ regulatory T cells (Treg) present in TS/A-pc tumors and draining lymph nodes and whether IL-21 had any effect on these cells. Indeed, CD4+CD25+ cells suppressed IFN-gamma production by splenocytes from immune mice in response to stimulation by the AH1 peptide. Low concentrations of IL-21 (10 ng/ml) failed to reverse the inhibitory activity of CD4+CD25+ cells in an allogeneic MLR, whereas 60 ng/ml rIL-21 partially restored responder T cell proliferation. IL-21R expression on CD25- lymphocytes suggested that IL-21 could be more effective in mice depleted of CD25+ cells. Depletion of Treg cells by a single dose of anti-CD25 mAb combined with TS/A-IL-21 cell vaccine cured >70% of mice bearing micrometastases, whereas anti-CD25 mAb treatment alone had no effect. Successful combined immunotherapy required NK cells, CD8+ T cells, and IFN-gamma. In conclusion, immunotherapy of micrometastases by an IL-21-based cellular vaccine is strongly potentiated by CD25+ cell depletion.     

Phenotype and homing of CD4 tumor-specific T cells is modulated by tumor bulk

Technical difficulties in tracking endogenous CD4 T lymphocytes have limited the characterization of tumor-specific CD4 T cell responses. Using fluorescent MHC class II/peptide multimers, we defined the fate of endogenous Leishmania receptor for activated C kinase (LACK)-specific CD4 T cells in mice bearing LACK-expressing TS/A tumors. LACK-specific CD44(high)CD62L(low) CD4 T cells accumulated in the draining lymph nodes and had characteristics of effector cells, secreting IL-2 and IFN-gamma upon Ag restimulation. Increased frequencies of CD44(high)CD62L(low) LACK-experienced cells were also detected in the spleen, lung, liver, and tumor itself, but not in nondraining lymph nodes, where the cells maintained a naive phenotype. The absence of systemic redistribution of LACK-specific memory T cells correlated with the presence of tumor. Indeed, LACK-specific CD4 T cells with central memory features (IL-2(+)IFN-gamma(-)CD44(high)CD62L(high) cells) accumulated in all peripheral lymph nodes of mice immunized with LACK-pulsed dendritic cells and after tumor resection. Together, our data demonstrate that although tumor-specific CD4 effector T cells producing IFN-gamma are continuously generated in the presence of tumor, central memory CD4 T cells accumulate only after tumor resection. Thus, the continuous stimulation of tumor-specific CD4 T cells in tumor-bearing mice appears to hinder the systemic accumulation of central memory CD4 T lymphocytes.     

Regulation of interleukin-2 and interleukin-6 production from T-cells: involvement of interleukin-1 beta and transforming growth factor-beta

The effect of recombinant (r) interleukin-1 beta (rIL-1 beta) and transforming growth factor-beta (TGF-beta) on the production of interleukin-2 (IL-2) and interleukin-6 (IL-6) from an antigen-specific (LBRM-33-1A5) and an antigen-nonspecific (EL-4-NOB-1) T-cell line was investigated. rIL-1 beta induced the production of IL-2 and IL-6 from EL-4-NOB-1 cells in a dose-related manner. The LBRM-33-1A5 cells required phytohemagglutinin (PHA) in addition to rIL-1 beta in order to produce IL-2 and IL-6. IL-2 production was found to precede IL-6 production in both cell lines. No IL-2 or IL-6 production was observed by adding r murine tumor necrosis factor-alpha or r murine interferon gamma to the cells. The presence of 1 ng/ml TGF-beta reduced IL-2 and IL-6 production from both T-cell lines by more than 80%. The inhibition of IL-2 and IL-6 production was still evident by a concentration as low as 10 pg/ml of TGF-beta. rIL-1 beta and PHA also stimulated murine thymocytes to produce IL-6 which was inhibited up to 85% in the presence of 1 ng/ml TGF-beta. Taken together these results suggest that TGF-beta may suppress immune responses by inhibiting the endogenous production of IL-2 and IL-6.     

Alloantigen-activated lymphocytes from mice bearing a spontaneous "nonimmunogenic" adenocarcinoma inhibit its growth in vivo by recruiting host immunoreactivity

Nylon wool columns eluting lymphocytes from the spleen of mice bearing a clinically evident spontaneous, nonimmunogenic adenocarcinoma of recent origin (TS/A) do not display cytotoxic response, release of lymphokines, and proliferation in vitro against TS/A cells, nor do they inhibit TS/A tumor growth in a Winn-type neutralization assay in vivo. After 5-day co-culture with allogeneic spleen cells from mice differing at multiple minor histocompatibility antigens only, these lymphocytes are still noncytolytic against TS/A cells, whereas they release interferon-gamma, mediate delayed-type hypersensitivity (DTH) reactions, and inhibit TS/A tumor growth in the Winn assay. In the Winn test, alloactivated lymphocytes from TS/A tumor-bearing mice are more effective than those from normal mice on a per cell basis. The induction of this TS/A tumor inhibition ability depends on the presence in the cultures of Thy-1+ lymphocytes. The presence of Lyt-2+ lymphocytes is also important, whereas that of asialo GM1+ is not. The TS/A inhibition in vivo by alloactivated lymphocytes mostly depends on Thy-1+, Lyt-2- and asialo GM- lymphocytes, even though a few Thy- cells are also very efficient tumor inhibitors. The alloactivated lymphocytes inhibit TS/A tumor growth by recruiting the radiosensitive effector mechanisms of the recipient mice required for ultimate tumor rejection. TS/A tumor rejection leaves a specific DTH and an immunologic memory resulting in rejection of a second lethal TS/A challenge in a significant number of mice.     

Tumor rejection and immune memory elicited by locally released LEC chemokine are associated with an impressive recruitment of APCs, lymphocytes, and granulocytes

The human beta chemokine known as LEC (also called NCC-4, HCC-4, or LMC) displays chemotactic activity for monocytes and dendritic cells. The possibility that its local presence increases tumor immunogenicity is addressed in this paper. TSA parental cells (TSA-pc) are poorly immunogenic adenocarcinoma cells that grow progressively, kill both nu/nu and syngeneic BALB/c mice, and give rise to lung metastases. TSA cells engineered to release LEC (TSA-LEC) are still able to grow in nu/nu mice, but are promptly rejected and display a marginal metastatic phenotype in BALB/c mice. Rejection is associated with a marked T lymphocyte and granulocyte infiltration, along with extensive macrophage and dendritic cell recruitment. NK cells and CD4+ T lymphocytes are uninfluential in TSA-LEC cell rejection, whereas both CD8+ lymphocytes and polymorphonuclear leukocytes play a major role. An antitumor immune memory is established very quickly after rejection, since 6 days later 75% of BALB/c mice were already resistant to a TSA-pc challenge. Spleen cells from rejecting mice display specific cytotoxic activity against TSA-pc and secrete IFN-gamma and IL-2 when restimulated by TSA-pc. The ability of LEC to markedly improve recognition of poorly immunogenic cells by promoting APC-T cell cross-talk suggests that it could be an effective component of antitumor vaccines.     

IL-12 is required for induction but not maintenance of protective, memory responses to Blastomyces dermatitidis: implications for vaccine development in immune-deficient hosts

Cellular immunity mediated by T lymphocytes, in particular CD4(+) and CD8(+) type 1 (T1) cells, is the main defense against pathogenic fungi. IL-12 initiates T1 cell development and cell-mediated immunity, but it is unclear whether IL-12 contributes to the maintenance of an antifungal T1 response. In this study, we addressed the role of IL-12 for vaccine-induced memory T cell development against experimental pulmonary blastomycosis. CD4(+) T cells absolutely required IL-12 to control a live genetically engineered attenuated strain of Blastomyces dermatitidis given s.c. as a vaccine, whereas CD8(+) T cells were significantly less dependent on IL-12. Despite differential dependency of T cell subsets on IL-12 during vaccination, neither subset acquired memory immunity in the absence of IL-12. In contrast, adoptive transfer of immune CD4 T cells from wild-type mice into IL-12(-/-) mice showed that CD4(+) T1 memory cells sustained a T1 cytokine profile and remained protective over a period of 6 mo posttransfer. Similarly, memory CD8 cells elicited in IL-12(-/-) mice with killed yeast and transient rIL-12 treatment (during vaccination) remained durable and protective after animals were rested for 3 mo. In conclusion, these studies demonstrate that once CD4 and CD8 cells have acquired a protective T1 phenotype they no longer require the presence of IL-12 to maintain antifungal protective memory.     

Interleukin-2-induced production of interferon-gamma by resting human T cells and large granular lymphocytes: requirement for accessory cell factors, including interleukin-1

Between 5 and 20% of normal human lymphocytes were found to synthesize interferon-gamma (IFN-gamma) in primary cultures with recombinant interleukin-2 (rIL-2). After 22 hr, IFN-gamma-producing cells included CD5+ T lymphocytes, CD16+ large granular lymphocytes (LGL), and a population of CD5-, CD16- blast cells. Only a small proportion (0-7%) of IFN-gamma-synthesizing cells expressed HLA-DR. The production of IFN-gamma by all rIL-2-responding lymphocyte subsets was shown to require the presence of DR+ accessory cells, probably including nonadherent, esterase-negative monocytes and/or dendritic cells. Accessory cell function in lymphocyte preparations depleted of DR+ cells, or in purified (greater than or equal to 95%) suspensions of LGL, was fully replaced either by addition of 2% autologous, adherent monocytes or by monocyte culture supernatant. The activity of monocyte supernatant was greatly reduced by treatment with antiserum specific for human interleukin-1 beta (IL-1 beta), although a combination of rIL-1 beta and rIL-2 failed to stimulate IFN-gamma production in DR- lymphocytes. These results indicate that rIL-2-induced IFN-gamma synthesis in both T cells and LGL requires the synergistic activity of IL-1, and possibly of one or more other monokines, as yet unidentified.     

Different sensitivity to interleukin 4 of interleukin 2- and interferon alpha-induced CD69 antigen expression in human resting NK cells and CD3+, CD4-, CD8- lymphocytes.

The effect of rIL-4 on CD69 antigen expression induced by rIL-2 or by rINF-alpha on human resting NK cells and CD3+, CD4-, CD8- T lymphocytes has been investigated. rIL-4 drastically inhibited CD69 antigen expression induced by rIL-2 in both cell types. In contrast, rIL-4 did not alter rINF-alpha-induced CD69 antigen expression. Consistent results were obtained evaluating the cytolytic activity of NK cells against the Raji target cell line: rINF-alpha-induced lytic activity was not inhibited by rIL-4, while rIL-2-induced lytic activity was drastically inhibited. Proliferative activity of NK cells induced by rIL-2, in contrast, was only slightly reduced by rIL-4. rIL-4 did not alter the expression of the beta chain of IL-2 receptor, evaluated in NK cells by indirect immunofluorescence. Expression of the alpha chain of IL-2 receptor could not be detected in NK cells by indirect immunofluorescence. It can therefore be suggested that the selective inhibitory effect of rIL-4 on rIL-2-induced activation of NK cells is not mediated by downregulation of alpha and beta chains of IL-2 receptor.     

Modulation of CD103 expression on human colon carcinoma-specific CTL

Recent results have shown a correlation between survival and frequency of tumor-infiltrating T cells in colorectal cancer patients. However, the mechanisms controlling the ability of human T lymphocytes to infiltrate colon carcinoma remain unclear. Although, it is known that expression of the integrin CD103alpha(E)/beta(7) by intraepithelial lymphocytes controls the retention of lymphocytes in epithelial layers, very little is known about the expression of intestinal homing receptors in human T lymphocytes. In particular, it remains unknown whether expression of CD103/beta(7) by human colon cancer-specific T lymphocytes is controlled by recognition of tumor Ags and is imprinted during T cell priming, facilitating its expression during memory T cell activation. In this study, we demonstrate that expression of CD103/beta(7) in human colon carcinoma-specific CTL is synergistically enhanced by the simultaneous TGF-beta1 stimulation and Ag recognition. These results were confirmed by using a panel of human CTL clones. Finally, we show that priming of naive CD8(+) T cells in the presence of TGF-beta1 ensures up-regulation of CD103/beta(7) in recall responses, at concentrations of TGF-beta1 significantly lower than those required by memory T cells primed in the absence of TGF-beta1. These results indicate a role of TGF-beta1 during T cell priming in modulating expression of CD103/beta(7) and controlling retention of human memory CD8(+) T cells into tumor epithelium.     

Adoptive immunotherapy of a mouse colon carcinoma with recombinant interleukin-2 alone or combined with lymphokine-activated killer cells or tumor-immune lymphocytes

Summary We have used a BALB/c colonic adenocarcinoma (C-26) to evaluate the therapeutic potential of recombinant interleukin-2 (rIL-2) at high and low dosages in combination with or without lymphokine-activated killers (LAK) or tumor-specific, immune lymphocytes in either an adjuvant spontaneous or an artificial metastasis system. Most (80%) of the mice that underwent s.c. C-26 tumor excision were shown to die of spontaneous metastasis with lung involvement by 1–4 months after excision. Postsurgical systemic treatment with low-dose rIL-2 (3 × 10⁴ U/day, i.p.) increased the survival rate to 31% as compared to 21% (not significant) in excised controls while administration of high-dose rIL-2 (8 × 10⁴ U/day) led to 53% survival (P <0.01). Both LAK cells and C-26-tumor-immune lymphocytes given during rIL-2 treatment significantly increased the effects of rIL-2 at the low but not at the high-dose, with tumor-immune effectors resulting in the highest percentage (63%) of cures. When mice bearing 3-day artificial lung metastases of C-26 cells were treated with low- or high-dose rIL-2, in combination with or without LAK or tumor-immune lymphocytes, a highly significant reduction or abrogation of the number of lung foci was observed with all treatments, including those involving or tumor-immune lymphocytes alone. Assessment of survival benefit in these mice, however, showed survival prolongation, with 20% cures achieved by low-dose rIL-2 alone and up to 65% cures by LAK in combination with low-dose rIL-2. In this system of artificial metastasis high-dose rIL-2 alone increased the survival time but failed to cure the animals, and the addition of LAK was ineffective whereas that of tumor-immune lymphocytes led to 80% cure. These results suggest that tumorimmune lymphocytes are more effective than LAK when combined with rIL-2 and that caution is necessary in extrapolating findings obtained in artificial metastasis models.     

Prophylactic anti-tumor immunity against a murine fibrosarcoma triggered by the Salmonella type III secretion system

The potential of an attenuated Salmonella enterica serovar Typhimurium strain as a prophylactic anti-tumor vaccine against the murine fibrosarcoma WEHI 164 was evaluated. Tumor cells were transfected with the DNA sequence encoding the MHC class I-restricted peptide p60(217-225) from Listeria monocytogenes. BALB/c mice received a single orogastric immunization with Salmonella that translocates a chimeric p60 protein via its type III secretion system. Mice were subsequently challenged subcutaneously with p60(217-225)-expressing WEHI cells. In vivo protection studies revealed that 80% of these mice remained free of the fibrosarcoma after challenge, whereas all animals of the non-vaccinated control group did develop tumor growth. In further experiments, the distribution of tetramer-positive p60(217-225)-specific effector and memory CD8 T cells after Salmonella-based immunization and tumor application was analyzed. Costaining with CD62L and CD127 revealed a predominance of p60-specific central memory and effector memory CD8 T cells in spleens, whereas in blood samples the majority of p60-specific lymphocytes belonged to effector and effector memory CD8 T cell subsets. This is the first report demonstrating that a bacterial type III secretion system can be used for heterologous antigen delivery to induce cytotoxic effector and memory CD8 T cell responses resulting in an efficient prevention of tumor growth.     

Involvement of beta 2-integrins in the migration of human natural killer cells.

Human large granular lymphocytes with the NK cell phenotype (CD16+ or CD56+CD3-) were greatly enriched among the cells which migrated spontaneously through untreated or albumin-coated, 3-microns pore size polycarbonate filters for 1 to 8 h. Three days of rIL-2 treatment (300 IU/ml) and 3 to 5 wk of rIL-2 treatment (100 IU/ml) generated a 2.7 +/- 0.9-fold and 5.6 +/- 0.8-fold increase in cell migration, respectively. The adhesion and subsequent migration of freshly isolated NK cells was mainly mediated by CD11b/CD18, because migration could be inhibited by 80 +/- 8% anti-CD11b (Mac-1) antibodies but not with antibodies against CD11a (LFA-1) or CD11c (p150,95), the other alpha-chains of the beta 2-integrins. After rIL-2 activation, however, CD11a/CD18 was the major receptor utilized in migration, inasmuch as anti-CD11a antibody caused a 69 +/- 8% reduction in the number of migrated cells. Anti-CD11b antibody decreased migration by 43 +/- 12%, and together these antibodies inhibited migration by 82 +/- 7%. Anti-CD11a alone did not have any effect on adhesion, but CD11a/CD18 cooperated in the adhesion because anti-CD11b decreased adhesion by 40 +/- 11% and together these antibodies inhibited adhesion by 74 +/- 6%. The ability of large granular lymphocytes to rapidly utilize beta 2-integrins and unidentified ubiquitous ligands for binding and migration may be significant for their capacity to function in the first line of immune defense under highly variable conditions.     

Inflammation induces myeloid-derived suppressor cells that facilitate tumor progression

Epidemiological and experimental observations support the hypothesis that chronic inflammation contributes to cancer development and progression; however, the mechanisms underlying the relationship between inflammation and cancer are poorly understood. To study these mechanisms, we have transfected the mouse 4T1 mammary carcinoma with the proinflammatory cytokine IL-1beta to produce a chronic inflammatory microenvironment at the tumor site. Mice with 4T1/IL-1beta tumors have a decreased survival time and elevated levels of immature splenic Gr1+CD11b+ myeloid-derived cells. These myeloid suppressor cells (MSC) are present in many patients with cancer and inhibit the activation of CD4+ and CD8+ T lymphocytes. 4T1/IL-1beta-induced MSC do not express the IL-1R, suggesting that the cytokine does not directly activate MSC. Neither T or B cells nor NKT cells are involved in the IL-1beta-induced increase of MSC because RAG2-/- mice and nude mice with 4T1/IL-1beta tumors also have elevated MSC levels. MSC levels remain elevated in mice inoculated with 4T1/IL-1beta even after the primary tumor is surgically removed, indicating that the IL-1beta effect is long lived. Collectively, these findings suggest that inflammation promotes malignancy via proinflammatory cytokines, such as IL-1beta, which enhance immune suppression through the induction of MSC, thereby counteracting immune surveillance and allowing the outgrowth and proliferation of malignant cells.     

Recombinant interleukin-1 beta inhibits elastin formation by a neonatal rat lung fibroblast subtype

The effect of recombinant interleukin-1 beta (rIL-1 beta) on elastin accumulation by lipid-laden interstitial cells (LIC) derived from neonatal rat lung was examined. The LIC, a fibroblast subtype, synthesized large amounts of elastin which was deposited into the extracellular matrix. This elastin was alkali-resistant and had an amino acid composition typical of adult rat elastin. Treatment of lipid-laden interstitial cell cultures with rIL-1 beta at 100 pg/ml caused a dramatic decrease in elastin accumulation as assessed by hot alkali treatment and transmission electron micrographs of the cell cultures. Tropoelastin formation was selectively decreased by rIL-1 beta relative to other proteins. Steady state levels of elastin mRNA were slightly decreased by rIL-1 beta at 5 pg/ml and markedly decreased by rIL-1 beta at 50 pg/ml or greater. The addition of indomethacin had no effect on rIL-1 beta-induced decreases in elastin mRNA levels. Inhibiting protein synthesis with cycloheximide blocked the effect of rIL-1 beta on elastin mRNA levels. The level of alpha 1(I) collagen mRNA was decreased by rIL-1 beta, but only at concentrations higher than that needed to induce a decrease in elastin mRNA. These data indicate that rIL-1 beta decreased steady state levels for elastin mRNA and elastin accumulation and can selectively regulate the accumulation of elastin and collagen.     

Comparison between crude Interleukin-2 and recombinant Interleukin-2 in maintaining killing activity of cultured lymphocytes

Peripheral blood lymphocytes, regional lymph node lymphocytes or malignant effusion lymphocytes from cancer patients were incubated with crude IL-2 (cIL-2) for 13 days. These effectors, which frequently expressed IL-2 receptor (IL-2R), proliferated well and possessed augmented killing activity against fresh autologous tumor cells and K562. However, when recombinant IL-2 (rIL-2) was added for the last 4 days of culture instead of cIL-2, IL-2R expression and killing activity against fresh autologous tumor cells decreased significantly (P<0.05). Phenotypic analysis indicated that cIL-2 significantly promoted the expansion of the cytotoxic population (CD8⁺ .11b^–)(P<0.05). The decreases in killing activity and IL-2R expression were restored by 0.004% PHA plus rIL-2, but not in the presence of rIFN-, rIL-1, rIL-l, rIL-4 or rIL-6. PHA-free cIL-2 maintained killing activity, but not IL-2R expression.We conclude that some factors in cIL-2 and a low dose of PHA-P are necessary for the maintenance of killing activity and IL-2R expression of cultured lymphocytes in the late phase of culture.