B-cell clusters at the invasive margin associate with longer survival in early-stage oral-tongue cancer patients.

In oral-cancer, the number of tumor-infiltrating lymphocytes (TILs) associates with improved survival, yet the prognostic value of the cellular composition and localization of TILs is not defined. We quantified densities, localizations, and cellular networks of lymphocyte populations in 138 patients with T1-T2 primary oral-tongue squamous cell carcinoma treated with surgical resections without any perioperative (chemo)radiotherapy, and correlated outcomes to overall survival (OS). Multiplexed in-situ immunofluorescence was performed for DAPI, CD4, CD8, CD20, and pan-cytokeratin using formalin-fixed paraffin-embedded sections, and spatial distributions of lymphocyte populations were assessed in the tumor and stroma compartments at the invasive margin (IM) as well as the center of tumors. We observed a high density of CD4, CD8, and CD20 cells in the stroma compartment at the IM, but neither lymphocyte densities nor networks as single parameters associated with OS. In contrast, assessment of two contextual parameters within the stroma IM region of tumors, i.e., the number of CD20 cells within 20 µm radii of CD20 and CD4 cells, termed the CD20 Cluster Score, yielded a highly significant association with OS (HR 0.38; p = .003). Notably, the CD20 Cluster Score significantly correlated with better OS and disease-free survival in multivariate analysis (HR 0.34 and 0.47; p = .001 and 0.019) as well as with lower local recurrence rate (OR: 0.13; p = .028). Taken together, our study showed that the presence of stromal B-cell clusters at IM, in the co-presence of CD4 T-cells, associates with good prognosis in early oral-tongue cancer patients.

Case Report: Adequate T and B Cell Responses in a SARS-CoV-2 Infected Patient After Immune Checkpoint Inhibition.

After the COVID-19 outbreak, non-evidence based guidelines were published to advise clinicians on the adjustment of oncological treatment during this pandemic. As immune checkpoint inhibitors directly affect the immune system, concerns have arisen about the safety of immunotherapy during this pandemic. However, data on the immune response in oncology patients treated with immunotherapy are still lacking. Here, we present the adaptive immune response in a SARS-CoV-2 infected patient who was treated with immune checkpoint inhibitors for advanced renal cell cancer. To evaluate the immune response in this patient, the number of T cells and their major subsets were measured according to expression of markers for co-signalling, maturation, and chemotaxis at baseline, during therapy, and during the SARS-CoV-2 infection. In addition, plasma samples were analyzed for IgM and IgG antibodies and the ability of these antibodies to neutralise SARS-CoV-2. Despite several risk factors for an impaired immune response to SARS-CoV-2, both T- and B-cell responses were observed. Moreover, after treatment with immune checkpoint inhibitors, a sufficient cellular and humoral immune response was achieved in this SARS-CoV-2 infected patient. These findings warrant renewed discussion on withholding of immune checkpoint inhibitors during an ongoing COVID-19 pandemic.

Breast cancer genomics and immuno-oncological markers to guide immune therapies.

There is an increasing awareness of the importance of tumor - immune cell interactions to the evolution and therapy responses of breast cancer (BC). Not surprisingly, numerous studies are currently assessing the clinical value of immune modulation for BC patients. However, till now durable clinical responses are only rarely observed. It is important to realize that BC is a heterogeneous disease comprising several histological and molecular subtypes, which cannot be expected to be equally immunogenic and therefore not equally sensitive to single immune therapies. Here we review the characteristics of infiltrating leukocytes in healthy and malignant breast tissue, the prognostic and predictive values of immune cell subsets across different BC subtypes and the various existing immune evasive mechanisms. Furthermore, we describe the presence of certain groups of antigens as putative targets for treatment, evaluate the outcomes of current clinical immunotherapy trials, and finally, we propose a strategy to better implement immuno-oncological markers to guide future immune therapies in BC.

Intra-tumoral production of IL18, but not IL12, by TCR-engineered T cells is non-toxic and counteracts immune evasion of solid tumors.

Adoptive therapy with engineered T cells shows promising results in treating patients with malignant disease, but is challenged by incomplete responses and tumor recurrences. Here, we aimed to direct the tumor microenvironment in favor of a successful immune response by local secretion of interleukin (IL-) 12 and IL-18 by sadministered T cells. To this end, we engineered T cells with a melanoma-specific T cell receptor (TCR) and murine IL-12 and/or IL-18 under the control of a nuclear-factor of activated T-cell (NFAT)-sensitive promoter. These T cells produced IL-12 or IL-18, and consequently enhanced levels of IFNγ, following exposure to antigen-positive but not negative tumor cells. Adoptive transfer of T cells with a TCR and inducible (i)IL-12 to melanoma-bearing mice resulted in severe, edema-like toxicity that was accompanied by enhanced levels of IFNγ and TNFα in blood, and reduced numbers of peripheral TCR transgene-positive T cells. In contrast, transfer of T cells expressing a TCR and iIL-18 was without side effects, enhanced the presence of therapeutic CD8+ T cells within tumors, reduced tumor burden and prolonged survival. Notably, treatment with TCR+iIL-12 but not iIL-18 T cells resulted in enhanced intra-tumoral accumulation of macrophages, which was accompanied by a decreased frequency of therapeutic T cells, in particular of the CD8 subset. In addition, when administered to mice, iIL-18 but not iIL-12 demonstrated a favorable profile of T cell co-stimulatory and inhibitory receptors. In conclusion, we observed that treatment with T cells engineered with a TCR and iIL18 T cells is safe and able to skew the tumor microenvironment in favor of an improved anti-tumor T cell response.

Retronectin-assisted retroviral transduction of primary human T lymphocytes under good manufacturing practice conditions: tissue culture bag critically determines cell yield.

BACKGROUND: For our clinical immunogene therapy study for the treatment of renal cell carcinoma (RCC) patients, we had developed a protocol for gene transduction and expansion of human T cells in compliance with good manufacturing practice (GMP) criteria. Critical to our successful clinical-scale transductions of patient T cells was the use of Retronectin in combination with Lifecell X-foldtrade mark cell culture bags. METHODS: In our current study, we evaluated two alternative types of bags for the Retronectin-mediated retroviral transduction of human T cells: the Miltenyi DC-generation bag and the Takara CultiLife Spin bag. RESULTS: In static transductions, but not in spinoculation, the DC-generation bags and CultiLife Spin bags performed as well as Lifecell X-foldtrade mark bags in Retronectin-assisted retroviral transduction of human T cells with respect to transduction efficiency, lymphocyte subset composition and lymphocyte function. However, both types of bags performed less well than Lifecell X-foldtrade mark cell culture bags in terms of cell yield. DISCUSSION: Adjusted numbers of cells at the start of transduction should be used when using the Miltenyi or Takara bags in order to compensate for the lower cell yield following transduction.

Retroviral vectors for clinical immunogene therapy are stable for up to 9 years.

Recombinant retroviruses are one of the most commonly used gene transfer vehicles for therapeutic gene delivery. The stability of viral vectors upon long-term storage, anticipated to be short lived, is expected to impact timeline and financial course of clinical immunogene therapy. However, to date little is known about vector stability. Therefore, we analyzed the stability of retroviral vectors produced in culture supernatants (RTVsup) for ex vivo gene therapy upon long-term storage. We have generated RTVsups derived from two packaging cell lines, PG13 and Phoenix(Ampho). Both lines produced murine leukemia virus-derived SFG-scFv(G250)-CD4gamma vector, which were pseudotyped with the gibbon ape leukemia virus envelope and amphotropic envelope, respectively. The supernatants were stored at -80 or -196 degrees C. To date, the PG13-derived RTVsups have been evaluated over a period of 9 years (1998-2007). In addition, a clinical batch of Phoenix(Ampho)-derived RTVsup has been evaluated over a period of 5 years (2002-2007). Here, we show that both RTVsups, when stored up to 9 and 5 years, respectively, do not show any sign of decay in their capacity to functionally transduce primary human T cells. These data provide evidence that in terms of 'life expectancy' the production and storage of clinical batches of RTVsup for gene therapy warrants the corresponding professional and financial risks.

Phoenix-ampho outperforms PG13 as retroviral packaging cells to transduce human T cells with tumor-specific receptors: implications for clinical immunogene therapy of cancer.

We have designed a transgene that encodes a scFv(G250) chimeric receptor, which is specific for carboxyanhydrase IX (G250-ligand, G250L), a molecule overexpressed by renal cell cancer (RCC). Retroviral transduction of this transgene into primary human T lymphocytes confers these cells with specific functional responses towards G250L-positive RCC cells. In preparation of a clinical phase (I/II) study in RCC patients, we set up a protocol for gene transduction and expansion of primary human T cells. For this purpose, we directly compared two packaging cell lines, that is, the GALV-pseudotyped MLV producing cell line PG13, and the MLV-A-producing cell line Phi-NX-Ampho (a.k.a. Phoenix-A). We generated and characterized stable scFv(G250)-positive clones of both PG13 and Phoenix cells and optimized the retrovirus production conditions. Transductions of primary human T cells yielded 30-60% scFv(G250)+ T cells using PG13-derived retrovirus versus up to 90% scFv(G250)+ T cells using Phoenix-derived retrovirus. The median number of transgene integrations per scFv(G250)+ T cell differed only 1.5-fold as determined by real-time PCR (mean number of integrations per T cell 2.6 and 3.7 for PG13 and Phoenix-based transductions, respectively). In addition, T cells transduced with Phoenix-derived retrovirus showed, on a per cell basis, 10-30% higher levels of scFv(G250)-mediated TNFalpha production and cytolysis of G250L+ RCC cells than T cells transduced with PG13-derived retrovirus. The improved functional transduction efficiency together with a limited increase in the number of integrations per recipient cell, made us select Phoenix clone 58 for our clinical immunogene therapy study.

Parallel detection of transduced T lymphocytes after immunogene therapy of renal cell cancer by flow cytometry and real-time polymerase chain reaction: implications for loss of transgene expression.

We have started a phase I/II immunogene therapy study of metastatic renal cell cancer (RCC), using autologous T lymphocytes transduced ex vivo with a gene encoding a single-chain receptor based on the monoclonal antibody (mAb) G250 [scFv(G250)]. G250 recognizes carbonic anhydrase IX, which is overexpressed by RCC cells. We have developed and validated flow cytometric and real-time polymerase chain reaction (PCR) assays to quantitatively detect transduced T cells in patient blood. The flow assay was based on staining with the anti-G250 idiotype mAb NuH82 and showed a sensitivity of 0.06% scFv(G250)(1) cells within CD3(1) T cells. The real-time PCR method showed a sensitivity of 14 copies of scFv(G250) DNA per 100 ng of total DNA, which enabled detection of 0.008% scFv(G250)(1) T cells within leukocytes. Both assays were further validated for their specificity and reproducibility. When applied to blood samples from three RCC patients treated with intravenous infusions of scFv(G250)(1) T cells, the kinetics of scFv(G250)(1) T cell counts as detected by flow cytometry were similar to those detected by real-time PCR, although PCR allowed detection of transduced T cells over a longer period of time (i.e., for patient 3, 7 versus 32 days, respectively). Interestingly, follow-up studies of patient 3 demonstrated that the number of circulating scFv(G250)(1) T cells remained fairly constant during the first 7 days posttreatment, whereas the number of gene copies increased during the same period of time. These results suggest loss of scFv(G250) membrane expression on adoptive transfer, which would have important implications for the antitumor efficacy of this form of immunogene therapy.

Redirecting human CD4+ T lymphocytes to the MHC class I-restricted melanoma antigen MAGE-A1 by TCR alphabeta gene transfer requires CD8alpha.

Adoptive immunotherapy involving the transfer of autologous tumor or virus-reactive T lymphocytes has demonstrated its effectiveness in the eradication of cancer and virally infected cells. Clinical trails and in vitro studies have focused on CD8+ cytotoxic T-cell receptor (TCR) alphabeta lymphocytes since these cells directly kill virally infected- and tumor cells after antigen-specific recognition via their TCR alphabeta. However, increasing evidence suggests that induction of sustained immunity against cancer and viral infections depends on the presence of tumor- or virus-specific CD4+ T lymphocytes, which are restricted by MHC class II. Here, we show that these MHC class II-restricted CD4+ T lymphocytes can efficiently be redirected to MHC class I-restricted tumor cells by retroviral introduction of an HLA-A1/MAGE-A1-specific chimeric two-chain TCR ValphaCalphazeta/VbetaCbetazeta (tcTCR/zeta). However, TCR-transduced CD4+ T lymphocytes were only able to specifically bind to HLA-A1/MAGE-A1 complexes and respond to HLA-A1+/MAGE-A1+ melanoma cells when the CD8alpha gene was cointroduced. These CD4+/CD8alpha+/TCR(POS) T lymphocytes produce IFN-gamma, TNFalpha and IL-2 when specifically stimulated via the introduced TCR with immobilized HLA-A1/MAGE-A1 complexes or HLA-A1+/MAGE-A1+ melanoma cells. Furthermore, introduction of the CD8alpha gene into TCR(POS) T lymphocytes rendered these T lymphocytes cytotoxic for HLA-A1+/MAGE-A1+ melanoma cells. These results demonstrate that human CD4+ T lymphocytes when genetically grafted with an HLA-A1/MAGE-A1-specific TCR and CD8alpha are induced to kill and produce cytokines upon specific interaction with the relevant melanoma cells. Hence, CD4+ T lymphocytes, in addition to CD8+ T lymphocytes, may be critical effector cells for adoptive immuno-gene therapy to generate a sustained tumor-specific immune response in cancer patients.

Adoptive immuno-gene therapy of cancer with single chain antibody [scFv(Ig)] gene modified T lymphocytes.

Adoptive transfer of antigen-specific T cells has recently shown therapeutic successes in the treatment of viral infections and tumors. T cells specific for the antigen of interest can be generated in vitro, and adoptively transferred back to provide patients with large numbers of immune-competent T cells. Adoptive T cell therapy, however, is a patient-tailored treatment that unfortunately is not universally applicable to treat viral infections and tumors. We and others have demonstrated that the transfer of genes encoding antigen-specific receptors into T cells (i.e., genetic retargeting) represents an attractive alternative to induce antigen-specific immunity. Currently, we evaluate this concept in a clinical protocol to treat patients with metastatic renal cell cancer (RCC) using autologous RCC-specific gene-modified T lymphocytes.

A critical role for interleukin 18 in primary and memory effector responses to Listeria monocytogenes that extends beyond its effects on Interferon gamma production.

The stimulation of interferon (IFN)-gamma by interleukin (IL)-12 has been shown to provide protection from intracellular pathogens such as Listeria monocytogenes. Tumor necrosis factor (TNF) is also a major player in the resolution of Listeria infections and is suggested to have more global effects than can be explained by the induction of IFN-gamma alone. Since IL-18 synergizes with IL-12 to induce IFN-gamma production by natural killer and T helper (Th)1 cells, we determined its role in responses to Listeria. IL-18 appeared to be even more potent than either IL-12 or IFN-gamma for protection against this pathogen and IL-18 enhanced bacterial clearance in the complete absence of IFN-gamma. Indeed IL-18 was comparable to TNF in its ability to resolve the infection and showed a lowered protective capacity in the absence of TNF. Moreover, IL-18 induced macrophages to secrete both TNF and nitric oxide after a Listeria infection. IL-18 was also essential for optimal IFN-gamma production by antigen-specific T cells. Therefore, IL-18 operates via its effects on both the innate immune response, including macrophages, as well as on Th1 cells, to protect against Listeria.

Two novel IL-1 family members, IL-1 delta and IL-1 epsilon, function as an antagonist and agonist of NF-kappa B activation through the orphan IL-1 receptor-related protein 2.

IL-1 is of utmost importance in the host response to immunological challenges. We identified and functionally characterized two novel IL-1 ligands termed IL-1delta and IL-1epsilon. Northern blot analyses show that these IL-1s are highly abundant in embryonic tissue and tissues containing epithelial cells (i.e., skin, lung, and stomach). In extension, quantitative real-time PCR revealed that of human skin-derived cells, only keratinocytes but not fibroblasts, endothelial cells, or melanocytes express IL-1delta and epsilon. Levels of keratinocyte IL-1delta are approximately 10-fold higher than those of IL-1epsilon. In vitro stimulation of keratinocytes with IL-1beta/TNF-alpha significantly up-regulates the expression of IL-1epsilon mRNA, and to a lesser extent of IL-1delta mRNA. In NF-kappaB-luciferase reporter assays, we demonstrated that IL-1delta and epsilon proteins do not initiate a functional response via classical IL-1R pairs, which confer responsiveness to IL-1alpha and beta or IL-18. However, IL-1epsilon activates NF-kappaB through the orphan IL-1R-related protein 2 (IL-1Rrp2), whereas IL-1delta, which shows striking homology to IL-1 receptor antagonist, specifically and potently inhibits this IL-1epsilon response. In lesional psoriasis skin, characterized by chronic cutaneous inflammation, the mRNA expression of both IL-1 ligands as well as IL-1Rrp2 are increased relative to normal healthy skin. In total, IL-1delta and epsilon and IL-1Rrp2 may constitute an independent signaling system, analogous to IL-1alphabeta/receptor agonist and IL-1R1, that is present in epithelial barriers of our body and takes part in local inflammatory responses.

Cytokine networking in lungs of immunocompetent mice in response to inhaled Aspergillus fumigatus.

Cytokine networking in the lung in response to inhaled Aspergillus fumigatus was assessed using a murine model of primary pulmonary aspergillosis in immunocompetent Crl:CF-1 mice. Inhalation of virulent A. fumigatus (6 x 10(6) CFU) resulted in the induction of interleukin 18 (IL-18), tumor necrosis factor alpha (TNF-alpha), IL-12, and gamma interferon (IFN-gamma) protein in bronchoalveolar lavage fluid and/or lung tissue. Induction of immunoreactive IL-18 preceded induction of TNF-alpha protein, which preceded induction of immunoreactive IL-12 and IFN-gamma. Real-time reverse transcriptase (RT) PCR analysis of infected lung tissue demonstrated that induction of IL-18 protein also preceded induction of pulmonary TNF-alpha, IL-12, and IFN-gamma mRNAs. Mice were subsequently treated with cytokine-specific neutralizing monoclonal antibodies (MAbs) to the IL-18 receptor (anti-IL-18R MAb), TNF-alpha (anti-TNF-alpha MAb), IL-12 (anti-IL-12 MAb), and/or IFN-gamma (anti-IFN-gamma MAb), and effects on intrapulmonary cytokine activity and growth of A. fumigatus were assessed in infected lung homogenates. Simultaneous neutralization of IL-12 and IL-18 resulted in decreased levels of immunoreactive TNF-alpha, while neutralization of IL-18, TNF-alpha, or IL-12 alone or of IL-18 and IL-12 together resulted in decreased levels of immunoreactive IFN-gamma. Simultaneous neutralization of IL-12 and IL-18 or neutralization of TNF-alpha alone or in combination with IL-12, IL-18, or IFN-gamma also resulted in a significant increase in A. fumigatus CFU in lung tissue. Taken together, these results demonstrate that endogenous IL-18, IL-12, and TNF-alpha, through their modulatory effects on both intrapulmonary cytokine activity and growth of A. fumigatus, play key roles in host defense against primary pulmonary aspergillosis.

A phage display selected fab fragment with MHC class I-restricted specificity for MAGE-A1 allows for retargeting of primary human T lymphocytes.

The clinical benefit of adoptive transfer of MHC-restricted cytotoxic T lymphocytes(CTL) for the treatment of cancer is hampered by the low success rate to generate antitumor CTLs. To bypass the need for tumor-specific CTL, we developed a strategy that allows for grafting of human T lymphocytes with MHC-restricted antigen specificity using in vitro selected human Fab fragments fused to the Fc(epsilon)RI-gamma signaling molecule. Retroviral introduction of a Fab-based chimeric receptor specific for MAGE-A1/HLA-A1 into primary human T lymphocytes resulted in binding of relevant peptide/MHC complexes. Transduced T lymphocytes responded to native MAGE-A1/HLA-A1POS target cells by specific cytokine production and cytolysis. Therefore, peptide/MHC-specific Fab fragments represent new alternatives to TCR to confer human T lymphocytes with tumor specificity, which provides a promising rationale for developing immunogene therapies.

[Dutch Collaborative Epilepsy Surgery Program: reduction of seizures, operative complications and tapering of medication in 338 patients, 1973-1998]. / Het Nederlandse epilepsiechirurgieprogramma: aanvalsreductie, operatieve complicaties en vermindering van medicatie bij 338 patiënten, 1973-1998.

OBJECTIVE: To determine the results of surgical treatment in patients with drug-resistant epilepsy, referred to the Dutch Epilepsy Surgery Program, who were treated in the University Medical Centre Utrecht, the Netherlands, in the period January 1973-December 1998. DESIGN: Retrospective descriptive. METHOD: A total of 338 patients were operated on; 269 underwent temporal lobe resection, 41 extratemporal resection, 12 a functional hemispherectomy and 10 callosotomy. Six patients were treated with vagus nerve stimulation. For seven of the patients no follow-up data was available. RESULTS: After a minimum follow-up of 1 year class I or class II results (in accordance with the University of California in Los Angeles classification (UCLA) where class I = seizure-free and class II < or = 3 seizures per year) were obtained in 91% of patients who underwent temporal lobe resections, 67% of patients who underwent extratemporal resections, 81% of patients who underwent functional hemispherectomy and 10% of patients who underwent anterior callosotomy. In five of these patients an improvement in their behaviour occurred. Of the 6 patients who underwent vagus nerve stimulation only I experienced a beneficial seizure reduction (UCLA class III). Transient physical complications occurred in 4% of the patients treated and permanent damage in 1%. Postoperative psychiatric complications occurred almost exclusively following temporal resections; in 11% of which 7% de novo. After 4 postoperative years this had decreased to 5%. In a group of 143 patients who were seizure-free for 2 or more years, post-surgery medication was tapered in 75 cases, stopped in 33 cases and remained unchanged in 35 cases. The relapse rate following a tapering or stopping of the medication was 30% and with unchanged medication 17%. Although the majority of patients were once again seizure-free upon restarting the medication, a significant number continued to experience seizures. CONCLUSION: For a number of carefully selected epilepsy patients with intractable seizures, surgery is a successful treatment with few serious complications.

Immunomodulatory role of endogenous interleukin-18 in gamma interferon-mediated resolution of replicative Legionella pneumophila lung infection.

The in vivo role of endogenous interleukin-18 (IL-18) in modulating gamma interferon (IFN-gamma)-mediated resolution of replicative Legionella pneumophila lung infection was assessed using a murine model of Legionnaires' disease. Intratracheal inoculation of A/J mice with virulent bacteria (10(6) L. pneumophila organisms per mouse) resulted in induction of IL-18 protein in bronchoalveolar lavage fluid (BALF) and intrapulmonary expression of IL-18 mRNA. Real-time quantitative RT-PCR analysis of infected lung tissue demonstrated that induction of IL-18 in BALF preceded induction of IL-12 and IFN-gamma mRNAs in the lung. Blocking intrapulmonary IL-18 activity by administration of a monoclonal antibody (MAb) to the IL-18 receptor (anti-IL-18R MAb) prior to L. pneumophila infection inhibited induction of intrapulmonary IFN-gamma production but did not significantly alter resolution of replicative L. pneumophila lung infection. In contrast, blocking endogenous IL-12 activity by administration of anti-IL-12 MAb) alone or in combination with anti-IL-18R MAb inhibited induction of intrapulmonary IFN-gamma and resulted in enhanced intrapulmonary growth of the bacteria within 5 days postinfection. Taken together, these results demonstrate that IL-18 plays a key role in modulating induction of IFN-gamma in the lung in response to L. pneumophila and that together with IL-12, IL-18 regulates intrapulmonary growth of the bacteria.

IL-18 receptors, their role in ligand binding and function: anti-IL-1RAcPL antibody, a potent antagonist of IL-18.

IL-18 is critical in eliciting IFN-gamma production from Th1 cells both in vitro and in vivo. Th1 cells have been implicated in the pathogenesis of autoimmune disorders, making antagonists of IL-18 promising therapeutics. However, specificity and binding characteristics of IL-18R components have only been superficially explored. In this study, we show that IL-1R related protein 1 (IL-1Rrp1) and IL-1R accessory protein-like (IL-1RAcPL) confer responsiveness to IL-18 in a highly specific (no response to other IL-1 ligands) and unique manner (no functional pairing with other IL-1Rs and IL-1R-like molecules). Cotransfection with both receptor components resulted in expression of both low and high affinity binding sites for IL-18 (K:(d) of 11 and 0.4 nM, respectively). We prepared anti-IL-1RAcPL mAb TC30-28E3, which, in contrast to soluble R proteins, effectively inhibited the IL-18-induced activation of NF-kappaB. Quantitative PCR showed that Th1 but not Th2 cells are unique in that they coexpress IL-1Rrp1 and IL-1RAcPL. mAb TC30-28E3 inhibited IL-18-induced production of IFN-gamma by Th1 cells, being at least 10-fold more potent than anti-IL-18 ligand mAb. This study shows that IL-1RAcPL is highly specific to IL-18, is required for high affinity binding of IL-18, and that the anti-IL-1RAcPL mAb TC30-28E3 potently antagonizes IL-18 responses in vitro, providing a rationale for the use of anti-IL-1RAcPL Abs to inhibit Th1-mediated inflammatory pathologies.

Human keratinocytes are major producers of IL-18: predominant expression of the unprocessed form.

The cytokine network in the skin is a tightly regulated system in which IL-1 isoforms, as well as their receptors and antagonists have a central role. The recently discovered IL-1 isoform IL-18 (also known as interferon gamma-inducing factor (IGIF) or IL-1gamma), promotes IFN-gamma expression by T cells in concert with IL-12. Because IFN-gamma plays an important role in many inflammatory skin diseases by facilitating the development of Th1 cells, it is important to elucidate the role of mediators which regulate the production of this cytokine. We demonstrate that human keratinocytes constitutively express IL-18 at the mRNA as well as at the protein level. The protein was mainly expressed intracellularly in the 24 kD unprocessed pro-form, but was also secreted. Histochemistry revealed a diffuse staining of IL-18 in the epidermis of normal skin, which is in line with our in vitro data. Furthermore, we show that the level of IL-18 expressed in freshly isolated normal human epidermal cells, whether or not containing HLA-DR+ cells, significantly exceeded the expression levels of other cell types such as monocytes and bronchial epithelial cells. Finally, our results show that stimulation of the keratinocyte cell line HaCaT with PMA LPS or IL-1beta, does not significantly affect intracellular or released (pro) IL-18 levels. These experiments show for the first time that human keratinocytes relative to monocytes, PBMC or leukocytes produce a considerably larger amount of pro-IL-18, which is also readily released. High constitutive levels of IL-18 may contribute to the skewing towards a Th1-like environment, which is apparent in many human inflammatory skin diseases.

Computational identification, cloning, and characterization of IL-1R9, a novel interleukin-1 receptor-like gene encoded over an unusually large interval of human chromosome Xq22.2-q22.3.

The Interleukin-1 receptor (IL-1R) and Toll signaling pathways share the evolutionarily conserved Toll homology domain (THD), which is a critical component in the signaling cascade of the host defense responses to infection and inflammation. Our initial genomic database searches uncovered a novel THD signature sequence between DNA markers DXS87 and DXS366. The feasibility of subsequently applying a coordinated computational approach, including various exon-finding programs, homology-based searches, and receptor profile searches, in revealing the exons encoding this novel IL-1R family member is described. IL-1R9 shows restricted expression in fetal brain and is highly homologous to IL1RAPL (A. Carrie et al., 1999 Nat. Genet. 23: 25-31), which is reportedly involved in nonsyndromic X-linked mental retardation. These genes are scattered over separate genomic intervals in excess of 1.0 Mb and encode receptors with extended C-terminal tails. In our functional NF-kappaB reporter assays, IL1RAPL, IL-1R9, or versions lacking the extended C-terminal sequences failed in responding either to IL-1 directly or to IL-18 when various permutations of IL-18R ectodomain chimeras were fused to their cytoplasmic domains. Evolutionary sequence analyses reinforce our conclusion that these novel orphan receptors probably form a functionally distinct subset of the IL-1R superfamily.

IL-1 beta and IFN-gamma induce the regenerative epidermal phenotype of psoriasis in the transwell skin organ culture system. IFN-gamma up-regulates the expression of keratin 17 and keratinocyte transglutaminase via endogenous IL-1 production.

Skin biopsies from healthy human skin and non-lesional skin from patients with psoriasis were cultured for 24 h and stimulated with interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma) in a skin organ culture model and the induction of the psoriasiform regenerative epidermal phenotype was analysed using immunostaining. In the presence of IL-1 beta, the psoriasiform regenerative epidermal phenotype was clearly induced. This involved strong up-regulation of the expression of keratin 16, keratin 17, and keratinocyte transglutaminase (TGk) in the suprabasal layers, strong up-regulation and a shift of the expression of keratin 5 and integrin beta 1 from the basal to suprabasal keratinocytes, and induction of the expression of ICAM-1 and HLA-DR on basal keratinocytes. The effects of IL-1 beta in the organ cultures of normal skin could be completely neutralized by anti-IL-1 polyclonal antibodies. The effects of IFN-gamma in healthy and non-lesional psoriatic skin were qualitatively similar to those of IL-1 beta. The IFN-gamma-induced epidermal expression of keratin 17 and TGk could be completely blocked by culturing the biopsies in the presence of IL-1ra or anti-IL-1 antibodies, while the induction of HLA-DR and ICAM-1 was not inhibited. The induction of the psoriasiform regenerative epidermal phenotype by IFN-gamma is partially mediated via endogenous epidermal IL-1.