role, but instead appear to play a protective role, which has most clearly been illustrated in the case of IL-22. On the other hand, mice deficient in RORgT a critical master regulator of the Th17 lineage, or in IL-23 a cytokine with complicated but important role in Th17 responses, are protected from several types of autoimmune diseases, including EAE ad colitis. This raises a question as to the specific mechanism and effector T cells types involved in these pathogenic responses. We will discuss our recent findings suggesting a potentially dichotomous role of Th17 responses in the development of colitis, as well as the possible innate immune signaling pathways involved in their generation in the gut. Finally, it is increasingly appreciated that commensal flora plays an important role in the activation of various T cell effector responses in the gut [4]. Commensal bacteria can contribute to the initiation of immune responses for two reasons: First, they provide a source of ligands for various pattern recognition receptors, such as TLRs, which is necessary for the activation of T cell responses when other regulatory mechanisms are intact. Second, commensal bacteria provide a source of nonself antigens, which can be recognized by naïve T cells in the intestine, and these T cells can potentially differentiate into pathogenic effector T cells. It is becoming clear from recent studies, that the commensal composition may play a critical role in affecting the type of T cell responses that can develop in the intestine. Therefore, a possible contribution of commensal flora on the differential induction of pathogenic and protective T cell responses in the intestine will also be discussed.

An increasing body of evidence suggests that disordered inflammation within the tumor microenvironment is responsible for tumor progression. We hypothesize that much of this disordered inflammation is a result of release of Damage Associated Molecular Pattern molecules. DAMP molecules, including high-mobility group box-1 (HMGB1), are released by stressed or necrotic tumor cells. We have previously shown that the DAMP receptor, RAGE is present on tumor cells where it mediates resistance to chemotherapy thereby promoting autophagy and limiting apoptosis. Here we investigated whether RAGE expression on tumor cells and natural killer (NK) cells would lead to differences in the efficacy of NK-mediated killing.

To study the role of Natural Killer (NK) cells in Leishmania infection, peritoneal macrophages from BALB/c mice were infected with Leishmania (Leishmania) amazonensis promastigotes and incubated with interleukin-2 (IL-2)-activated NK (A-NK) cells at different ratios of A-NK cells to infected macrophages (5:1, 1:1, 0.2:1). The A-NK cells were added either together with the parasites (0-h group) or 24 h later (24-h group). Morphological studies of the cultures revealed predominance of parasitic debris within macrophages that were in close contact with A-NK cells and the decrease in parasite recovery was directly proportional to the A-NK cell concentration used. Interferon-gamma (IFN-gamma) and IL-12 were detected in the supernatant at levels proportional to the A-NK cell concentration used. No significant difference was observed between the groups with respect to NO levels in the culture supernatant. When A-NK cells were added directly to the L. (L.) amazonensis promastigote cultures, the parasite recovery decreased proportional to the number of A-NK cells added. In vivo studies demonstrated smaller lesion sizes in animals inoculated with both parasites and A-NK cells compared with parasites alone. Histopathology of the skin lesions from animals receiving A-NK cells together with the parasites showed moderate parasitism and a nodular inflammatory infiltrate formed by mononuclear cells and a few vacuolized macrophages. In contrast, animals inoculated only with the parasites showed a highly parasitized dermis with infiltration of intensely vacuolized macrophages. These results demonstrate the role of A-NK cells in parasite lysis and in resistance of macrophages to L. (L.) amazonensis in the early phase of infection.

Recent reports demonstrate that natural killer (NK) cells and dendritic cells (DC) support each other's activity in a positive feedback. We observed that activated NK cells induce the maturation of DCs into stable type-1 polarized DCs (DC1), characterized by up to 100-fold enhanced ability to produce IL-12p70 in response to subsequent interaction with Th cells. DC1 induction depends on NK cell-produced IFN-gamma and TNF-alpha, with a possible involvement of additional factors. DC1, induced by NK cells or by NK cell-related soluble factors, are stable, resistant to tumor-related suppressive factors, and show strongly enhanced ability to induce Th1 and CTL responses. In analogy to resting T cells, the induction of "helper" function of NK cells relies on a two-signal activation paradigm. While NKG2D-dependent tumor cell recognition is sufficient to induce the cytotoxic "effector" function of NK cells, the induction of "NK cell help" requires additional signals from type-1 IFNs, products of virally-infected cells, or from IL-2. Compared to non-polarized DCs currently-used in clinical trials, DC1s act as superior inducers of anti-cancer CTL responses during in vitro sensitization. The current data provides rationale for the clinical use of DC1s in cancer and chronic infections (such as HIV), as a new generation DC-based vaccines, uniquely combining fully mature DC status with an elevated, rather than "exhausted" ability to produce bioactive IL-12p70. We are currently implementing stage I/II clinical trials, testing the effectiveness of DC1s induced by NK cells or by NK cell-related factors, as therapeutic vaccines against melanoma.

Natural killer (NK) cells and dendritic cells (DCs), two important components of the immune system, can exchange bidirectional activating signals in a positive feedback. Myeloid DCs, the cell type specialised in the presentation of antigen and initiation of antigen-specific immune responses, have recently been documented to be involved in supporting innate immunity, promoting the production of cytokines and cytotoxicity of INK cells, and enhancing their tumouricidal activity. Natural interferon-producing cells/plasmacytoid DCs (IPCs/PDCs) play an additional role in NK cell activation. Reciprocally, NK cells, traditionally considered to be major innate effector cells, have also recently been shown to play immunoregulatory 'helper' functions, being able to activate DCs and to enhance their ability to produce pro-inflammatory cytokines, and to stimulate T helper (Th) 1 and cytotoxic T lymphocyte (CTL) responses of tumour-specific CD4(+) and CD8(+) T cells. Activated NK cells induce the maturation of myeloid DCs into stable type-1 polarised DCs (DC1), characterised by up to a 100-fold enhanced ability to produce IL-12p70 in response to subsequent interaction with Th cells. In addition, the ability of NK cells to kill tumour cells may facilitate the generation of tumour-related antigenic material, further accelerating the induction of tumour-specific immunity. DC1, induced by NK cells or by NK cell-related soluble factors, are stable, resistant to tumour-related suppressive factors, and demonstrate a strongly enhanced ability to induce Th1 and CTL responses in human in vitro and mouse in vivo models. Compared with the standard mature DCs that are used in clinical trials at present, human NK cell-induced DC1s act as superior inducers of anticancer CTL responses during in vitro sensitisation. This provides a strong rationale for the combined use of NK cells and DCs in the immunotherapy of patients with cancer and patients with chronic infections that are resistant to standard forms of treatment. Stage I/II clinical trials that are being implemented at present should allow evaluation of the immunological and clinical efficacy of combined NK-DC therapy of melanoma and other cancers.

In murine models, therapeutic efficacy of adoptive immunotherapy (AIT) of cancer with lymphokine activated killer (LAK) cells is seen only when applied together with substantial doses of interleukin-2 (IL-2), probably because this cytokine is imperative for both motility and viability of the LAK cells. We wanted to investigate whether IL-2 in addition mediates an immunostimulatory activation and expansion of endogenous effector cells contributing to tumor regression. Using an immunoperoxidase technique, we have been able to longitudinally analyze the accumulation of tumor infiltrating lymphocytes expressing the pan-T cell/activated lymphocyte phenotype (Thy1.2), the natural killer (NK) cell phenotype (AsGM,) as well as the cytotoxic T (CD8) cell phenotype within experimental established B16 pulmonary melanoma metastases in C57BL/6 mice during the first 48 h after high dose IL-2 monotherapy. Whereas a substantial and selective infiltration of AsGM1+ lymphocytes in tumor tissue was seen (262 and 937 cells per sq.mm malignant tissue at 0 and 48 h, respectively), only a minor increase in accumulation of CD8+ cells was seen (106 and 171 cells per sq.mm tumor tissue at 0 and 48 h, respectively). The addition of adoptive transfer with lymphokine-activated adherent NK (A-NK) cells to the high-dose IL-2 treatment resulted in more than a 1.5 fold increase in infiltrating AsGM1+ cells compared to IL-2 therapy alone (1520 compared to 937 AsGM1+ cells per sq.mm malignant tissue). No substantial accumulation of CD8+ cells was observed in this setting either. In contrast, the treatment with high dose IL-2 together with adoptive transfer of mitogen-stimulated, lymphokine-activated T killer (T-LAK) cells increased the infiltration of CD8+ cells 10-fold compared to IL-2 monotherapy (2078 compared to 171 CD8+ cells per sq.mm malignant tissue, respectively). Interestingly, infiltration of both endogenous and exogenous cells continued over time, since the effector-to-tumor cell ratio in metastatic tissue dramatically increased from 1:8 and 1:6 at 16 h to 1:3 and 1:2 at 48 h after adoptive transfer of A-NK and T-LAK cells, respectively. These data underline the longevity of LAK cells in vivo and highlight the importance of IL-2 treatment in recruiting endogenous immune cells to tumor areas.

Direct contact between lymphokine‐activated killer (LAK) cells and tumour cells is believed to be imperative for initiating tumour cell lysis in vitro as well as in vivo. In order to optimize adoptive immunotherapy (AIT) it is therefore desirable to identify the LAK cell subtype, which ensures maximal infiltration of tumours as well as a substantial cytotoxic reactivity. In this report we have compared short‐ and long‐term cultured murine adherent natural killer (A‐NK) cells and mitogen‐stimulated, lymphokine‐activated T‐killer (T‐LAK) cells with respect to their proliferative potential, cytotoxicity, requirement for interleukin‐2 (IL‐2) and ability to infiltrate B16 pulmonary metastases following adoptive transfer. We found that short‐term (5 days) cultured A‐NK and T‐LAK cells both showed a substantial accumulation of tumour tissues. However, A‐NK cells gradually lost this ability during in vitro culture whereas T‐LAK cells cultured for as long as 20 days retained their ability to infiltrate metastases as efficiently as their short‐term cultured counterparts. Moreover, the low requirement of IL‐2 by T‐LAK cells to achieve maximal infiltration of tumours sharply contrasted with the excessive doses necessary to ensure maximal infiltration by A‐NK cells. In conclusion, these data demonstrate that short‐term cultured LAK cells of both NK‐ and T‐cell origin are able to infiltrate B16 pulmonary metastases effectively. Importantly, the T cells retain this ability for a considerably longer time and require much less IL‐2 support than do A‐NK cells, making T‐LAK cells attractive for AIT.

Murine lymphokine-activated natural killer (A-NK) cells are able to migrate to and accumulate in tumor metastases. However, the exact migratory pattern is as yet unknown. In the present study, we have investigated the migration from the vasculature towards malignant tissues of various effector cells. Our results indicate that murine A-NK cells seem to be arrested for an extended period of time in the microvasculature and also, although infrequently, to adhere to the endothelial lining of larger vessels close to tumor tissues before extravasation. While murine T lymphokine-activated killer and rat A-NK cells accumulate significantly in the subendothelial areas of larger venules in normal tissues, no such accumulation is observed with respect to murine A-NK cells. Electron microscopy reveals that the murine A-NK cells undergo an extreme deformation during extravasation and tumor infiltration. Furthermore, the cells are shown to be in an activated stage probably facilitating their migration, and hence, the elimination of tumor cells.

To better understand immune responses to brain tumors and to develop possible approaches for immunotherapy, we have investigated the leukocyte populations infiltrating the rat 9L gliosarcoma. By immunocytochem-ical analyses of the cells infiltrating the tumor, we observed a substantial number of cells expressing natural killer cell receptor protein 1 (NKR-P1), a marker expressed only on rat lymphocytes capable of non-MHC-restricted cytotoxicity. Previous investigations have determined the existence of three populations of NKR-P1+ lymphocytes in normal rats, including NKR-P1bright/T-cell receptor (TCR)-/CD3-/CD5- (approximately 5-15%), NKR-P1dim-/TCRalphabeta+/CD3+/CD5+ (approximately 1-5%), and NKR-P1dim/TCRgammadelta+/CD3+/CD5+ (approximately 0.5-2%). By one-parameter flow cytometry, it was determined that NKR-P1+ cells constituted 30-60% of the lymphocytes in 9L tumors. Among splenic lymphocytes or peripheral blood leukocytes, NKR-P1bright cells are 1.5-4.5 times more numerous than NKR-P1dim cells. In striking contrast, NKR-p1dim cells were 4-5 times more numerous than NKR-P1bright cells among lymphocytes isolated from 9L tumors. Using quantitative analyses of laser confocal microscopic scans, we determined that NKR-P1dim cells were approximately 4 times as numerous as NKR-P1bright cells in situ, confirming flow cytometric findings. By two-color now cytometric analyses, it was observed that approximately 5-10% of the cells were NKR-p1bright/CD5-/TCR-, a phenotype representative of NK cells. Also, approximately 11-25% of the cells were NKR-P1dim/CD5+/TCR+ cells, corresponding to the T-cell subset with non-MHC-restricted lytic function. In addition, we observed a cell population among 9L-derived lymphocytes with a NKR- p1dim/CD5-/TCR- phenotype (approximately 15-25%). Cells of this phenotype have not been reported previously, and most likely represent NK cells down-modulated for expression of NKR-P1. Alternatively, they might represent cells of unknown origin or cells down-modulated for expression of T-cell markers in the microenvironment of 9L tumors. We also compared the lytic capacity of NKR-P1+ populations derived from normal animals and from 9L gliosarcomas. In these experiments, it was determined that, although cells isolated from 9L tumors had some capacity to lyse tumor target cells, they were clearly less efficient than cells isolated from normal splenocytes. Cumulatively, these data suggest that there is selective localization of cells capable of mediating antitumor responses in 9L, but that tumor-associated factors may down-regulate their function and expression of NKR-P1.

An in vivo model of liver metastasis induced by human gastric carcinoma was established in nude mice and used for locoregional or systemic immunotherapy with a subset of human A-natural killer (NK) cells defined previously. A single intrasplenic (i.s.) delivery of A-NK cells (1 x 10(7)) and interleukin 2 (IL-2; 60,000 international units, twice a day for 5 days, i.p.) to animals with 3-day established liver metastases, but not IL-2 alone, resulted in rapid (within 24 h) elimination of the majority of metastases and significantly improved survival. A single i.s. or i.v. transfer of these effector cells and IL-2 significantly prolonged survival of the mice with 3-day established metastases (P < 0.03 and P < 0.02, respectively) compared with untreated mice. Using 51Cr-labeled A-NK cells, it was determined that, at best, 75% of 1 x 10(7) cells delivered i.s., and up to 50% of those delivered i.v. were found in the liver 30 min-4 h later. Using image analysis with Di-O dye-labeled A-NK cells, 60-100% of A-NK cells delivered i.s. or i.v. were detected in the liver 24 h later. By light microscopy, 3-day liver metastases were mostly intravascular, but some had already begun to spread into liver tissue. When rhodamine- or Di-O dye-labeled A-NK cells were injected i.s. or i.v. to study their distribution in the liver, they were detectable by confocal fluorescence microscopy in tumor-free tissue and in association with tumor cells 12-24 h after transfer. No evidence for selective localization of A-NK cells to liver metastases was obtained; many A-NK cells were randomly distributed in tissue and not associated with visible metastases. However, confocal fluorescence and electron microscopy showed some A-NK cells to be in cell-to-cell contact with tumor cells, both in the blood vessel and liver tissue. These results indicate that a majority of human A-NK cells transferred i.s. or i.v. to mice with liver metastases have the capacity to migrate to the liver and to enter liver tissue and tumor metastases in vivo. The presence of these effector cells even in a modest number in the liver leads to elimination of most, but not all, metastases and to significantly prolonged survival of animals treated with A-NK cells and IL-2.

Adoptive immunotherapy with interleukin 2 (IL-2) and lymphokine activated killer (LAK) cells or plastic adherence-enriched LAK cells has produced dramatic reductions in the number of metastatic lesions in several animal systems. In clinical settings, LAK cell therapy has also been successful, with complete or partial responses in 20–30% of the patients with advanced cancer, especially malignant melanoma and renal cell carcinoma. If the anti-neoplastic effect of activated killer cells is dependent on direct contact with the tumor cells, the optimal therapeutic environment would exist when high numbers of the most cytotoxic LAK cell type are present in the in the tumor. Injection of LAK cells by the intravenous (i.v.) route results in direct delivery of these cells to the lungs and lung metastases since all i.v. injected cells initially reach the lungs. In order to infiltrate liver metastases, LAK cells must first traverse the lung vasculature and the capillary bed of the intestinal tract.

Assessment of the tissue distribution of adoptively transferred adherent lymphokine-activated killer A-LAK) cells by use of51Cr indicated that these effector cells, after an initial phase in the lungs, distributed in high numbers to liver and spleen (30% and 10% of injected dose, respectively). However, when this experiment was repeated with125IdUrd as cell label, fewer than 2% and 0.5% of the injected cells distributed into liver and spleen respectively. To analyse this discrepancy, we compared the tissue distribution of51Cr- and125IdUrd-labelled A-LAK cells with that indicated by alternative direct visual methods for identification of the injected cells, such as fluorescent dyes (rhodamine and H33342) or immunohistochemical staining of asialo-GM1-positive cells. The number of i. v. injected A-LAK cells found in the liver by all visual methods ranged from 1% to 5% of the injected dose, supporting the data obtained with125IdUrd, whereas 25%–30% of the51Cr label was consistently found in this organ. Autoradiography of the liver 24 h after i. v. injection of51Cr-labelled cells revealed a background activity that was four- to fivefold higher than the control level, indicating substantial non-specific accumulation in the liver of51Cr released from A-LAK cells. We conclude that51Cr cannot be reliably used in investigations of cell traffic to the liver because of non-specific accumulation of the51Cr label, particularly in this organ. In contrast, labelling with125IdUrd or rhodamine and immunohistochemical staining of asialo-GM1-positive cells appear to be reliable and essentially equivalent methods for investigations of the fate of adoptively transferred A-LAK cells. Using these methods, we found that only few A-LAK cells redistribute to the liver upon i. v., i. e. systemic, injection, whereas 40%–50% of locally (intraportally) injected A-LAK cells remain in the liver for at least 24 h.

While close contact between lymphokine-activated killer (LAK)/adherent, lymphokine-activated killer (A-LAK) cells and tumor cells is believed to be a prerequisite for initiating the events leading to tumor cell lysis, clear evidence for the ability of these effector cells to infiltrate tumors or tumor metastases in vivo still has to be obtained. In the present study, we report that a significant fraction of adoptively transferred A-LAK cells, labeled with fluorochromes for identification, accumulates in lung and liver metastases of the B16 melanoma, the MCA 102 sarcoma and the Lewis lung carcinoma lines. Thus, 5- to 10-fold higher numbers of A-LAK cells were found in the malignant lesions compared to the surrounding normal tissue. The infiltration seemed very heterogeneous after intravenous injection of moderate numbers of A-LAK cells (15 x 10(6)). However, after adoptive transfer of 45 million A-LAK cells, an A-LAK cell/tumor cell ratio higher than 1:1 in most metastases was observed. Surprisingly, approximately 5% of the lung metastases seemed totally resistant to infiltration even though neighboring metastases were highly infiltrated.While substantial infiltration of lung metastases was seen after iv. injection, significant infiltration of liver metastases was seen only after intraportal injection of the A-LAK cells indicating impaired traffic of intravenous injected A-LAK cells through the lung capillaries.These results present direct evidence that A-LAK cells, upon a proper route of administration, have the potential to migrate to and heavily infiltrate metastases from murine tumors of different origin.