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European initiative to improve knowledge, treatment and survival of haemophagocytic syndromes in children

Final Report Summary - CUREHLH (European initiative to improve knowledge, treatment and survival of haemophagocytic syndromes in children)

Executive summary:

Haemophagocytic Lymphohistiocytosis (HLH) is a life threatening disorder of hyperinflammation. We have reached substantial progress in determining hallmarks of the poorly understood pathophysiology, in improving diagnostics to speed up the identification of hereditary disease and of high risk patients, and in preparing new treatment options.

Experimental basic research in vivo and in vitro:
None of various mouse strains with genetic defects developed HLH spontaneously, indicating that an exogenous trigger is needed for disease induction. After infection with Lymphocytic Choriomeningitis Virus (LCMV), three outcomes of HLH development could be determined:
i.) no HLH development, if virus was rapidly cleared,
ii.) transient HLH, if virus clearance was delayed and
iii.) severe, self-perpetuating HLH, if virus persisted.

We conclude antigen persistence is mandatory for HLH development. Most importantly, the studies of the mouse models indicate HLH as a threshold disease: Some mouse models did not develop HLH after infection with different viral triggers, as subtle differences in cytolytic effector functions of T cells in mice determine the disease. Granule morphology was normal and granules were able to reach the immunological synapse in different genetic deficiencies, indicating that the corresponding defects act late in the secretory process. Hermansky-Pudlak syndrome type 2 patients displayed a reduction of the CD56bright NK cell subset and a complete absence of NKT cells, and differentiation of monocyte-derived dendritic cells showed abnormal maturation, explaining recurrent viral infections and the propensity to develop tumours. Analysis of cytokine profiles in human and murine HLH serum samples suggest a role of IL15/Il15Ralpha in HLH. This is supported by the finding that Il15 is required for the expression of granzyme and perforin.

Diagnostics:

In a genotype/phenotype correlation of 84 patients with familial HLH (FHL) 3, two disruptive mutations (in comparison to one or none) lead to a significantly earlier onset of disease, even though no functional differences could be detected by natural killer cell (NK) degranulation and NK cell activity measurement. Age at onset was 4.1 months in FHL3 and 12.2 months in FHL4 (15 patients), as compared to the previously published data on FHL2 with 3 months (p=0,001). CNS disease was observed in 36% of FHL2, 60% of FHL3, and 31% of FHL4.

468 samples referred for the evaluation of HLH were analysed in four different laboratories using common protocols for the analysis of degranulation and cytotoxicity. The NK CD107 granule release assay was the most sensitive and specific test to identify genetic disorders of degranulation. Measurement after IL-2 stimulation, cytotoxicity and CTL degranulation assays were useful confirmative assays. The resulting diagnostic algorithm now allows early identification of patients with genetic disease with high reliability. This is an important advance in patient management, since the decision for life-saving hematopoietic stem cell transplantation (HSCT) can be taken earlier.

Therapy:

9 parameters predicting outcome of induction therapy could be identified in 232 patients. These finding may serve as a future basis for treatment stratification. In 210 haematopoetic stem cell transplantations, the use of haploidentical donors and active disease at HSCT were associated with higher mortality. Busulfan containing conditioning was a significant reisk factor for veno-occlusive disease. IFN-g antibodies were shown to be an effective HLH treatment in Rab27a, perforin, and syntaxin11 deficient mice. They constitute a potential future option for HLH treatment in humans.

Future:

Future research will have to focus on the exploitation of the new therapeutic options, further improvement of HSCT protocols, identification of the remaining undiscovered defects, and further characterisation of the exact pathophysiology underlying the known genetic deficiencies.

Project Context and Objectives:

Haemophagocytic lymphohistiocytosis (HLH) is a rare and life-threatening immune disease of childhood which is fatal without treatment. The condition is characterised by a severe disturbance of the immune homeostasis. In familial forms, with genetic defects in Perforin (PRF1), UNC13D, Syntaxin 11 (STX11), and UNC18B, HLH is the main manifestation. PRF1- and UNC13D- mutations are associated with impairment of lymphocyte cytotoxicity. The consequences of mutations in STX11 are not clear. At the beginning of CUREHLH, in approx. 30-40 % of patients with suspected hereditary disease a genetic defect could not be identified.

Several other genetic diseases with distinct clinical features (Griscelli syndrome, Chédiak-Higashi syndrome, Hermansky-Pudlak syndrome) and x-linked syndromes (XLP, XIAP) have a predisposition to develop HLH.

The pathophysiology of HLH is poorly understood, and research is hampered by the small number of patients in each centre and the restricted access to material. Survival has improved but still a considerable percentage of patients dies.

Pathophysiology is studied in mouse models and in vitro systems:

Unsolved questions are the importance of triggering factors (nature, magnitude, and persistence), the interaction of immune cells and soluble factors to maintain the exaggerated immune response, the role of cytolytic granule trafficking in STX11 mutations and unknown genetic defects, and the influence of cytokines in the pathological immune response.

Among the MOUSE MODELS, the lymphocytic choriomenigitis virus (LCMV)-infected perforin knock-out (prf-/-) mouse is a known model for HLH. Different LCMV-variants as triggers are evaluated regarding their potential to induce the clinical and immunological picture of HLH. LCMV is also used to infect other mice with defined genetic defects (beige, pearl and ashen mice) in which susceptibility to experimental HLH is not yet known. In mouse strains which do not develop HLH, the number of cytotoxic T cells and the infectious trigger is varied using TCR transgenic mice and different LCMV strains and recombinant pathogens expressing LCMV-GP in order to evaluate their relative contribution to HLH development. The role of antigen presentation and antigen persistence for the induction and maintenance of HLH is analysed. It is investigated if prolonged antigen presentation after infection with a specific LCMV variant is required to maintain HLH. The influence of cytokines on the expansion of LCMV-specific T-cells is another focus. Double-deficient mice for perforin and for certain cytokine receptors are generated, infected with virus and compared for disease expression.

The syntaxin 11 knock-out mouse (Stx-/-) is established as a new mouse model for HLH. The mice are characterised phenotypically, histologically, and immunologically before infection with LCMV. Furthermore the susceptibility to LCMV-infection and the induction of HLH is investigated. A dose-finding study evaluates the optimal dose for the induction of HLH. Mice generated from flox/conditioned syntaxin deficient mice crossed with transgenic mice expressing CRE under different promoters allow the analysis of the impact of a STX11- defect in different cell populations.

The pearl mouse is a known mouse model for Hermansky-Pudlak syndrome type 2 (HPS2) but an HLH phenotype has not been described so far. Perforin- and NK-cell deficiencies are associated with HLH in other genetic defects. Therefore the number, phenotype and functional properties of natural killer (NK-) cells in pearl mice are evaluated. The influence of the infection with murine cytomegalovirus (MCMV), which is a known inducer of interferon-gamma (IFN-gamma), on T- and NK-cell activation, perforin expression and cytokine secretion are tested.

IN VITRO SYSTEMS focus on the generation of T- and NK-cell-lines from patients with various defects and from Stx-/- mice. The trafficking of cytotoxic granules to the immunological synapse is studied in Stx-/- cells, defining the stage at which the secretion is blocked. Another aim is the identification of new proteins required for cytotoxicity in patients with yet unknown defects.

The contribution of cytokines to the pathogenesis of HLH is evaluated using cells derived from Il-15- and IL15R-deficient mice. The expression and secretion of cytotoxic granule-specific proteins is analysed. In addition, the cytokine profile of HLH-patients and Stx-/-mice is evaluated.

Translational research encompasses genetic/advanced diagnostics, improved treatment, and survival

Knowledge is lacking about the influence of the known and unknown genetic mutations on the clinical expression, including the immune function, and course of disease, and how to best incorporate knowledge from the genetic diseases into a diagnostic algorithm, including NK cell function studies. Characteristics of non-responders need to be identified to early initiate salvage therapy. Data are also lacking on the factors influencing outcome of stem cell transplantation (HSCT).

GENETICS encompasses a large geno-phenotype analysis in patients with STX 11 mutations, UNC13D mutations, and unknown mutations. Several parameters are evaluated; patients with STX11 and UNC13D mutations are compared to a published cohort of patients with PRF mutations. Data collection is performed with a standardised documentation sheet, stored in a specific data base and subsequently analysed.

To improve the rapid DIAGNOSIS in patients with HLH a protocol for a standardised diagnostic algorithm is implemented in the diagnostic centres of the partners. In a large cohort referred for the evaluation of HLH, the tests are evaluated regarding their potential to predict specific genetic defects and to early discriminate genetic and acquired disease. Deficient function of NK cells is a hallmark of HLH. In two specialised laboratories various functional assays for NK cells are used to characterise the patients with regard to the extent of cytotoxic impairment and over-production of inflammatory cytokines.

Treatment failure in HLH either occurs early during chemoimmunotherapy or during stem cell transplantation. Thus, the work package IMPROVED TREATMENT AND SURVIVAL pursues 3 aims:

In the first project, in a large cohort of patients treated on the international HLH studies a variety of factors are analysed to identify patients with risk factors for poor response to therapy already early in the course of treatment to facilitate switch to alternative treatment strategies. A documentation sheet is prepared, data are collected and stored for evaluation.

In the second project, patients transplanted for HLH are analysed retrospectively for risk factors regarding outcome of stem cell transplantation, and for several factors related to longterm survival and remission, such as transplant-related mortality, incomplete chimaerism and long-term sequelae.

The third project deals with experimental therapy in HLH: two mouse models are used in which an antibody against IFN- gamma is administered. IFN-gamma is considered the key cytokine in the pathology of HLH. In the first step, the experimental HLH model is set up with LCMV-infected prf-/- and ashen mice. Afterwards, the mice are treated with different doses of neutralising antibodies against IFN-gamma in order to achieve an optimal effect on disease severity and course.

The project DISSEMINATES the results within and beyond the consortium.

The MANAGEMENT covers administrative and financial aspects and ensures efficient implementation of the work and monitoring of the work progress.
Project Results:

MOUSE MODELS

Role of different triggers for the induction of HLH in perforin-/- mice

Perforin-deficient (PKO) mice do not develop HLH spontaneously. However, after infection with lymphocytic choriomeningitis virus (LCMV), severe haemophagocytic lymphohistiocytosis (HLH) is induced within 12-20 days and mice have to be eliminated due to dramatic weight loss. Which features of LCMV infection are critical for HLH induction was so far unclear. Various LCMV isolates that differ in their cell tropism, their spread into peripheral organs and in their replication kinetics are available. We performed low dose infection experiments with these LCMV isolates which differ in the extent and duration of antigen-specific T cell responses and in the induced cytokine milieu, but are cleared in C57BL/6 mice. C57BL/6 mice develop a strong CTL response and are able to control viral infection without symptoms of HLH. In contrast, HLH is induced in PKO mice after infection with a low dose of the various LCMV isolates (analysis of HLH parameters on day 12 after infection). Surprisingly, HLH was most severe after infection of PKO mice with the least virulent LCMV isolate Arm, whereas infection with the highly virulent isolate LCMV-DOCILE induced a mild and transient form of HLH and most importantly led to survival of PKO mice. Interestingly, DOCILE infected PKO mice displayed antigen-specific CD8 T cells that were limited in IFNg expression and impaired in the up-regulation of the activation marker KLRG-1 and therefore restricted in their differentiation into short lived effector cells (SLECs). At later time points after infection these T cells expressed exhaustion markers (PD-1, LAG-3 and CD137) and were further impaired in their effector functions. These findings demonstrate that LCMV-DOCILE infection of PKO mice has a profound effect on the activation and differentiation of the antigen-specific T cells at early time points, leading to a less activated effector status and a reduced HLH progression. An explanation for this phenomenon may be the extremely high viral replication of DOCILE and an early spread to peripheral organs that may favor exhaustive differentiation of antigen-specific T cells. In summary, we describe an intriguing experimental situation, where all LCMV isolates cause persistent infection of PKO mice, but induce a different clinical course of HLH, because the differentiation of antigen-specific T cells to full effector functions in the presence of persisting antigen varies with the LCMV isolate used.

In more than 50% of HLH cases in humans the trigger for disease is unknown. Therefore the contribution of host and environmental factors for the onset and the severity of this disease remain to be determined. Interestingly, spontaneous development of HLH was not observed in mouse models with corresponding genetic defects. However, if infected with LCMV such mice exhibit chronically activated T cells, are unable to eliminate the virus and develop HLH symptoms. To further evaluate the impact of the infectious trigger on HLH induction, infection experiments were performed in PKO mice with vaccinia virus (VV), vesicular stomatitis virus (VSV) and pneumovirus of the mouse (PMV). Preliminary results indicate, that during these infections the pathogens are controlled via perforin-independent mechanisms and therefore PKO mice so far did not develop HLH symptoms. On the other side, infection of PKO mice with the escape variant LCMV 8.7 carrying a point mutation in the immunodominant CTL epitope, developed HLH symptoms although this virus isolate induces a weaker CTL response. However, control of LCMV 8.7 infection is perforin-dependent and CTL in PKO mice are not able to eradicate the virus. This leads again to a situation of highly activated T cells in the presence of persisting virus.

To characterise the impact of CD8 T cell frequencies on the threshold of disease induction, we increased the frequencies of specific T cell in two situations: first in PKO mice, that develop severe and lethal HLH and second in a sub-threshold situation, beige mice, that do not develop HLH after LCMV-WE infection.

PKO mice were crossed with TCR (T cell receptor) transgenic 318 mice that express a LCMV-specific TCR on their CD8 T cells. In 318.PKO mice about 50 % of the CD8 T cells are specific for LCMV. PKO and even more pronounced 318.PKO mice (with increased frequency of specific T cells) developed a severe form of HLH. The typical clinical symptoms of HLH, were detectable in 318.PKO mice already on day 8 after LCMV-infection in comparison to PKO mice. Thus, in the PKO model of HLH, an increase in the frequency of specific T cells leads to a more severe clinical outcome of disease. This indicates that activated LCMV-specific perforin-deficient T cells are a critical effector population for HLH induction and the number of specific T cells has a direct correlate in disease progression.

In the sub-threshold model of beige mice, T cell frequencies were increased by adoptive transfer of specific CD8 T cells. Interestingly increased T cell frequencies in these mice did not induce HLH symptoms after LCMV infection, because beige CTL were able to eliminate virus.

Role of antigen-presenting cells and duration of antigen presentation for HLH pathogenesis in perforin knockout mice

The role of antigen persistence and the impact of professional antigen-presenting cells (APC) on the development of HLH were analysed. In a first experimental approach, we evaluated the correlation of antigen persistence/clearance versus the induction and severity of HLH disease. For this, C57BL/6 (wt), beige, souris, pearl, ashen and PKO mice, which differ in the genetic defects of their cytolytic effector mechanisms, were infected with LCMV-WE and monitored for symptoms of HLH. C57BL/6 and beige mice did not develop HLH symptoms, whereas HLH manifested in pearl, ashen, souris and PKO mice. Of note, pearl mice developed a mild and transient form of HLH, whereas souris, ashen and PKO mice exhibited a severe HLH phenotype after LCMV infection. The important finding of these experiments was, that the different outcome concerning HLH development correlated with virus persistence/clearance in the various mouse strains. In C57BL/6 and beige mice, CD8 T cells were functional and completely eradicated the virus with no HLH symptoms detectable. Pearl mice developed a transient HLH, which correlated with a delayed virus clearance. In contrast, CD8 T cells in souris, ashen and PKO mice were highly activated but not able to control viral infection, leading to persistant LCMV infection and development of severe HLH.

Pathogenesis of HLH in syntaxin 11 deficient mice

To characterise the basic immunological phenotype of Stx11-deficient mice, single cell suspension from different primary and secondary lymphoid organs were analysed by fluorescence activated cell sorting (FACS) and compared to WT C57BL/6 mice at one participating institution. The deficiency in STX11 had no impact on the development and differentiation of several immune cells. Normal numbers and percentages of CD4+ and CD8+ T cells were observed in thymus, spleen and lymph nodes as compared to WT mice. B cell development was independent of Stx11 deficiency, as no differences in the phenotype or total numbers of B cells were observed in spleen and lymph nodes. Similar results were observed for NK cells and antigen-presenting cells, where no differences were detected between WT and KO mice.

To test the susceptibility of Stx11-deficient mice to develop an HLH syndrome upon LCMV infection, these mice received a single intraperitoneal dose of LCMV and disease development was monitored. Our data show that Stx11-/- mice develop a HLH syndrome characterised by a general worsening of the general condition, loss of body weight and a significant drop of the body temperature. Stx11-deficient mice also present other features of HLH such as severe pancytopenia and tissue infiltration of activated CD8 T cells and macrophages. We also detected high serum levels of IFNg and inflammatory cytokines. Importantly, none of the STX11-/- mice died during the observation time (up to 5 weeks after LCMV infection). Thus, our data show that Stx11-/- mice represent a suitable animal model for the study of FHL4.

Previous data show that Stx11 expression on T cells was very low or undetectable, suggesting that FHL4 could be triggered by a different mechanism and not necessarily by a defect in cytotoxic activity. To address this issue, we analysed, in vitro, the role of Stx11-deficiency in different isolated cell populations. CD8 T cells were purified from spleen of WT and Stx11-deficient mice and degranulation and cytolytic functions were analysed. Degranulation activity was monitored after activation with anti-CD3. Whereas most of the WT cells stained positive for surface CD107 expression, Stx11-defficient CD8+ T cells presented a significant reduction in CD107 surface labeling. This defect also correlates with a decreased cytotoxic activity compared to control cells. Hence, our data show that syntaxin 11 is involved in degranulation process and that a defect in syntaxin 11 impairs cytotoxic effector functions in murine CD8+ T cells, indicating that the defective cytotoxic activity in T cells observed in Stx11-/- mice should play a role in the development of HLH syndrome.

Two other partners analysed in collaboration Stx-/- mice for the development of HLH symptoms after infection with LCMV-WE. A detailed analysis at d12 post infection showed that Stx-/- mice developed severe HLH, comparable to PKO mice, with profound loss of body weight, drop in ear temperature, leucopenia, thrombocytopenia and reduction in red blood cell counts. Furthermore liver enzymes, ferritin, soluble CD25 and triglyceride levels in sera of Stx-/- mice were elevated and signs of haemophagocytosis detectable in spleen and liver. However, first experiments revealed also subtle differences in the clinical symptoms between PKO- and Stx-/- mice after LCMV infection. Although the two mouse strains initially manifested similar clinical symptoms, loss of body weight continued in PKO mice (and animals had to be eliminated) whereas Stx-/- mice stabilised and survived. Analysis of immunological parameters showed that frequencies of antigen-specific T cells and the capacity to up-regulate the activation marker KLRG-1 were comparable in Stx-/- and PKO mice. However, antigen-specific CD8 T cells expressed lower levels of IFNg in Stx-/- mice and these mice had higher viral titers in lung and kidney, when compared to PKO mice. Following Stx-/- mice for 180 days they were not able to increase their body weight and exhibited a kind of chronic wasting syndrome.

At another partner, the immune system development of Stx11-/- mice was examined. Compared with WT mice, Stx11-/- mice exhibited no differences in cell numbers, cell population percentages and expression of specific cell surface molecules and activation markers in spleen and lymph nodes. B- and T-cell differentiation in the bone marrow and thymus were also comparable between WT and Stx11-/- mice. Additionally, a histologic examination of lymphoid tissues such as spleen, lymph nodes and thymus, from WT and Stx11-/- mice showed similar structural features. Thus, this suggests that the deletion of Stx11 has no overt effect on the phenotype of the mice in relation to immune system development and immune cell populations in lymphatic tissues. In addition, there were no abnormal signs of activation or hyperproliferation of T cells or histiocytes (DCs, MF), indicating that Syntaxin 11 deficiency is not changing the activation status of cells. Also after stimulation with T cell specific stimulators (mitogen ConA or CD3) or B cell stimulator (LPS), Stx 11-/- and wt cells proliferate with a similar efficiency. Therefore, our results indicate, that Syntaxin 11 deficiency does not cause global changes in immune cell distribution and functionality.

Impact of other genetic HLH defects on the threshold for the induction of HLH

HLH is a genetically heterogeneous disease with defects in effector molecules, like perforin, and in genes that are involved in biogenesis, transport and release of lytic granules. Mice with different defects in the cytoloytic effector machinery were analysed for their susceptibility for HLH development under identical infection conditions. For this beige and souris mice with different mutations in the lyst gene, pearl mice with mutations in the AP3 gene, ashen mice with mutation in the Rab27A gene and PKO mice (deficient in the perforin gene) were infected with LCMV-WE and compared with syngeneic C57BL/6 mice. No HLH development was observed in C57BL/6 and beige mice. Pearl mice developed a mild HLH (detectable at d8 after infection) but recovered thereafter without any symptoms of HLH at later time points. In contrast severe HLH, as a self-perpetuating disorder, was detectable in PKO, souris and ashen mice after LCMV infection. Typically these mice lost body weight, had decreased body temperature (measured in the ear), exhibited leucopenia and thrombocytopenia, developed increased levels of liver enzymes, triglycerides, soluble CD25 and ferritin in the serum and showed signs of haemophagocytosis in spleen and liver.

The different development of HLH in beige and souris mice was analysed in more detail since in both mouse strains the same gene was mutated (lyst) and NK cell degranulation and cytotoxicity was impaired to a comparable extent. Beige and souris mice are models for the human Chédiak-Higashi syndrome (CHS) with two different mutations in the Lyst gene. One mouse strain -the beige mice- carry a deletion of three nucleotides predicting an inframe deletion of one amino acid (isoleucin) at position 3741 in the WD40 domain of the Lyst protein. The other mouse strain -souris mice- carry a donor splice site mutation in intron 27 with a T to A transversion, which is predicted to cause skipping of exon 27, thereby destroying the reading frame. This finally creates a premature stop codon that would truncate the protein after amino acid 2482. After infection with LCMV-WE the souris mice, but not beige mice, developed HLH symptoms like weight loss and elevated levels of ferritin, sCD25 and serum levels of IFN-gamma. Of note, weight loss in souris mice started 1-2 days later and was less severe than in PKO mice. Furthermore, a massive infiltration of macrophages with pronounced haemophagocytosis in the liver and elevated liver enzymes were exclusively observed in souris mice. In summary, LCMV infection induced the full clinical picture of HLH in souris but not in beige mice.

To determine to which extend differences in the cytotoxic capacity of effector cells contribute to the different outcomes of disease in these two Lyst-mutant mice, we analysed NK and CTL in more detail. NK cell activity after poly(I:C) injection was impaired in both mouse strains with respect to degranulation and cytotoxic activity, when compared to wt mice. Degranulation activity of CTL after LCMV infection, as measured by CD107a surface expression, was reduced in beige mice, when compared to wt mice and even more pronounced in souris mice. Surprisingly, CTL cytotoxicity was comparable in wt, beige and souris mice when measured in a standard 51Cr-release assay, using target cells loaded with high concentration of antigen. However, differences in lytic activity of CTL between the mouse strain became apparent when the 51Cr-release assay was done with target cells loaded with physiological antigen concentration or infected with virus. In such a setting, beige CTL also showed decreased cytotoxicity compared to wt CTL. However, souris CTL again displayed a significantly more pronounced cytotoxicity defect than beige CTL. To evaluate whether such in vitro differences in CTL activity were relevant for in vivo situations, a short-term protection assay was performed, in which virus control is strictly perforin-dependent. We could demonstrate that beige CTL were able to control the infection under these experimental conditions, while souris CTL were not. This finding confirms results from HLH experiments with low dose LCMV-WE in beige mice which were able to control the infection until d12 after infection and no spread of virus to peripheral organs was found. In contrast, souris mice could not control viral infection and were persistently infected with high virus titers in peripheral organs.

In summary the data demonstrate that HLH is a threshold disease that is determined by subtle differences in CTL cytotoxicity. Beige mice represent a sub-threshold situation where antigen-specific CTL controlled LCMV infection and HLH was not induced. In contrast, souris mice with the more severe defect in CTL cytotoxicity, were not able to control the LCMV infection and developed the full clinical picture of HLH. These findings may have important implications for our understanding of the variable HLH development and progression in patients with different mutations in the same gene.

At another partner, LCMV infected ashen mice first backcrossed on B6 background were also analysed for the development of HLH. Ashen mice developed the clinical, biological and histopathological manifestation of HLH which characterised Griscelli syndrome. Compared to perforin KO mice, ashen mice showed a substantially better survival and less sever manifestations of HLH, in terms of pancytopenia and inflammatory cytokine production. This study thus demonstrates that LCMV-infected ashen mice represent a relevant murine model of Human GS2.

In order to evaluate the susceptibility of HPS2 patients to hemophagocytic syndrome, we have evaluated the immune function of pearl mice that lack expression of beta3 subunit of AP3 complex. In addition, we have assessed the biological response of pearl mice to infection by MCMV.

Given the role of NK cells in MCMV infection, we examined if there were any changes in NK cell subpopulations in spleen cells of AP3 and wild type mice before and after infection. No changes were observed in the percentage of DX5+NK1.1- DX5+NK1.1+ DX5-NK1.1+. Membrane CD1d expression was higher in all AP3 NK cells subpopulations compared to wild type. NKT cell numbers were determined by staining with both a-GalCer-loaded CD1d tetramers and an anti-TCRb chain Ab. The percentages of tetramer-positive cells were comparable in both mice strains. To focus on NKT cells response, splenocytes from AP3 and wild type mice were stimulated in vitro with 100 ng/ml of a-GalCer and supernatants were tested for IFNg production. An increase in IFNg levels was observed in a-GalCer treated splenocytes but with no differences between AP3 and wild type.

Impact of cytokines on T cell responsiveness and HLH pathogenesis

Goal of the project was to dissect the cytokine-mediated mechanisms (focus: IL-15, IL-21) that control CD8+ T and/or NK cell cytotoxic granules generation, trafficking, and degranulation, so as to understand how abnormalities in this system may impair T and NK cell cytotoxicity in HLH patients and mouse models. Therefore we have focused on the investigation of the:
- cytokine-mediated regulation of expression of cytotoxic granules-specific proteins;
- cytokine-induced regulation of CD8+/NK cell cytotoxicity;
- cytokine profile in sera from HLH patients.

Cytokine-mediated regulation of expression of cytotoxic granules-specific proteins and cytotoxicity:

Receptors for IL-2 and IL-15 are expressed in NK cells at various developmental stages and in vitro studies indicate that IL-2 and IL-15 can support NK cell differentiation and survival. However, how cytokines influence NK cell cytotoxicity and cytotoxic granule content has still to be elucidated. For instance IL-21 is a member of the cytokine family consisting of IL-2, IL-4, IL-7, IL-9 and IL-15 and it is mainly produced by T cells. IL-21 acts on a variety of immune and non-immune cells and with regard to NK cell function IL-21 is both an activating and suppressing cytokine. At P4, we first analysed the transcriptome of flow sorted resting WT and IL-15Ralpha-deficient, and either IL-15 or IL-21-activated murine WT NK cells by using Affymetrix gene expression microarrays.

IL-15 has no effect on Gzma and Prf1 pools, but it increases GzmB mRNA. Gzma mRNA expression is not affected by IL-21 treatment, instead Gzmb and Prf1 mRNAs are downregulated. Based on those results we decided to stimulate WT and IL-15Ralpha-deficient splenocytes with IL-15 (50 ng/ml) 6 days and to assess cytotoxicity by release of cytoplasmic lactate dehydrogenase in vitro from NK cells added to YAC-1 cells at various effector/target ratios. Our results show that IL-15Ralpha-deficient NK cells display an impaired cytotoxic activity compared to wild type.

This might be partially due to a lack of pre-existing pool of Granzyme A in IL-15Ralpha-deficient NK cells. Indeed, even after 6 days of IL-15 stimulation in vitro, IL-15Ralpha-deficient NK cells do not express Granzyme A even not at the protein level. Instead Granzyme B is expressed and its expression is IL-15Ralpha chain independent. Conversely IL-2/15 receptor beta, a chain shared by IL-2 and IL-15 cytokines, has an important role in Granzyme B expression after IL-15 treatment. Furthermore, we have analyzed the phenotype of WT and IL-15Ralpha-deficient NK cells founding that the latter are not fully differentiated into mature NK cells. Thus, our findings suggest that the IL-15Ralpha chain is required for NK cells' maturation and cytotoxic activity as well as Granzyme A expression, but dispensable for Granzyme B induction. Moreover IL-15/IL-15R alpha-mediated signaling is required for the generation of pre-existing pool of Granzyme A in NK cells. These results indicate that the role of the IL-15Ralpha chain in the regulation of NK cell cytotoxicity might be due to an association between NK cell granula content and the expression of the IL-15Ralpha chain.

Role of IL-15 in the enhancement of survival and inhibition of apoptosis for the uncontrolled proliferation of T cells:

IL-15 is a cytokine which is required for the homeostasis of CD8+ T cells and NK cells. However, IL-15 deficient mice do display only a 50% and not a total reduction of the CD8+ T cell population. Therefore we were interested in understanding whether IL-15 specifically sustains the proliferation and survival of a selective CD8+ T cell subpopulation. In the last years most attention has been given to CD4+CD25+ regulatory T cells. Most recently, however, CD8+ regulatory T cells have witnessed a renaissance as important immunoregulators with a predominantly immunosuppressive function in pathologies such as autoimmune and infectious diseases and cancer. Thus we have investigated whether IL-15 promotes survival of CD8+ regulatory T cells with the perspective purpose to understand if in HLH the CD8+ regulatory T cell population is impaired. We could indeed demonstrate the IL-15 sustain the survival and activity of a both a mouse and a human memory-like suppressive CD8+ T cell population which results reduced in number in Il-15 deficient mice.

Analysis of cytokine spectrum in murine and human samples:

sCD25 is one of the targets used to diagnose HLH patients. Moreover, LCMV infection in the prf-/- mouse have demonstrated the importance of excessive IFN gamma secretion for the pathophysiology of HLH. It is well known that cytokines orchestrate NK and CD8+ T cell development and/or maturation into fully functional cytotoxic cells. To investigate new cytokines as possible targets for the diagnosis and treatment HLH we have investigated the cytokine signature in the sera of HLH mouse models as well as in human samples sera from HLH patients.

Sera from different mouse models (Perforin-deficient, Beige, Leaky SCID, Wild type mice) infected with LCMV were analysed for a panel of Cytokines (IL-1alpha, IL-1beta, IL-2, sIL2Ra, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15/IL-15Ra complex, IL-17, IL-21, eotaxin, G-CSF, GM-CSF, IFN-gamma, KC, MCP-1 (MCAF), MIP-1alpha, MIP-1beta, RANTES, TNF-alpha).

Sera from three different mice models (Perforin-deficient, Beige, Wild type mice) infected with LCMV were analysed for the IL-15/IL-15Ra complex and IL-21. The results do not show a clear difference among the three strains used.

In addition, human samples from patients with HLH, MAS, EBV, unknown defect and healthy donors were collected and tested to investigate their cytokine content (IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, hIL-15, IL-17, G-CSF, GM-CSF, IFN-gamma, MCP-1 (MCAF), MIP-1beta, TNF-alpha). The results of the investigations did not point out to the role of new cytokines, between the one analysed, in the modulation of disease activity; our results confirm the previous reports in the literature. However, new and interesting is to report that IL15 can be detected in the serum of MAS and also in genetic HLH. The significance of the findings and the correlation to selective cytotoxic defects and macrophage hyperactivation need to be defined in the future.

IN VITRO SYSTEMS

Regulation of polarised secretion from immune cells (CTL and NK) in genetic HLH

CTL lines were generated in vitro from PHA blasts stimulated with irradiated allogeneic buffy coat, PHA and IL2 and CD8 expressing clones derived from single cells, to examine protein expression and localisation of the secretory apparatus. These cells were examined for cytotoxic effector function against P815 target cells bound by anti-CD3 (redirected lysis) for human cells. Mouse CTL from syntaxin 11 deficient mice were raised against irradiated allogeneic (BALB/c) targets and cytotoxicity assayed against P815 target cells. Cytotoxicity was measured by LDH release from targets or by degranulation of CD107a from CTL using the FACS analyser.

CTL were analysed from patients with clinically likely FHL where mutations were identified in FHL 4 or 5 or where no mutations could be identified in FHL2-5, Rab27a or LYST. In CTL derived from patients with FHL4 and 5, CTL were all capable of killing normally in vitro. This is consistent with data from NK which are grown in IL2 in which killing activity is normal. CTL from FHL5 patients showed reduced protein levels of syntaxin 11 as detected by western blotting, suggesting that these protein physiologically form a complex. In CTL from mice lacking syntaxin 11 cytotoxicity and degranulation were reduced but not absent.

C ytokine contribution to HLH pathogenesis

This topic is addressed in the section on murine models.

Role of AP-3 complex in differentiation of NK and dendritic cells and in disease pathogenesis

We have analysed by flow cytometry the distribution of NK subsets by evaluating the expression of the surface markers CCR7, ChemR23, and CXCR1 by CD56+ NK cells. In both HPS2 patients, we observed an important reduction of CD56bright NK cell subset, as compared to NK cells from normal controls; this subset of CD56dim NK cells showed also expression of CD16. We observed that CXCR1 was expressed by CD56dim NK cells a lower density in cells of HPS2 patients (Figure 3B), while CCR7 was not detectable in CD56+ NK cells from the two siblings. In addition, quantification of the percentage of CD56+/CD3- cells showed a lower number of NK cells in HPS2 patients (Pt1 7%, Pt2 4%) as compared to age-matched subjects.

In the last 12 months of the project, our research group has identified a subject with the clinical and immunological features of Hermansky-Pudlak syndrome. After excluding the known causes of partial albisnim associated to immunodeficiency, we have identified by exome analysis a new disease gene. Investigation of NK subsets in this novel patient has shown impairment of cytotoxic activity and abnormal trafficking of lytic granules. These studies have been performed without any additional personnel, but have required a limited increase in the expenses of reagents.

NKT cells have been defined as lymphocytes that express both TCR and NK1.1 that may have important immune regulatory functions. It has been reported that AP3-deficient mice have a significantly reduced NKT cell population, suggesting that the generation of the endogenous ligand that selects NKT cells may also be AP3-dependent.

In order to evaluate the number of NKT cells, freshly isolated PBMC from HPS2 patients and from a healthy donor were stained with PBS57-loaded CD1d tetramer. Despite our efforts we could only identify a number of positive events that did not excide those identified using the unloaded tetramer as the negative control. The same results were obtained when staining the cells with an antibody against the CDR3 region of the invariant chain that characterises NKT cells. The reduction of NKT cell numbers in HPS2 patients are consistent with an important role for the adaptor protein AP3 in positive selection of NKT cells.

GENETICS AND ADVANCED DIAGNOSTICS

Genotype-phenotype correlation

In this project, we have analysed the presenting features of patients with FHL according to their genetic diagnosis. As a first step, we have defined the set of characteristics to be analysed and data were collected on patients with FHL 3 and FHL4. The collected data were analysed for completeness and consistency; in some cases lacking data had been traced or asked to be confirmed. Once control of the quality of the data was completed, the data-base was frozen.

A total of 84 patients with FHL3 from 69 unrelated families were analysed. Of them 36 families were homozygous and 33 were compound heterozygous for UNC13D mutations. A total of 54 different mutations were identified: 19 missense, 14 deletions or insertions, 12 nonsense, 9 splice errors; of them, 15 were novel mutations. Patients with FHL3 due to two distruptive mutations had a significantly younger age at diagnosis than patients with at least one missense mutation.

Functional studies showed that granule release capacity was significantly reduced in 29/30 patients tested; at a quantitative evaluation, deltaCD107a in the 17 patients tested was significantly inferior to healthy controls; deltaCD107a values were not significantly different between the 3 groups of patients (i.e. those with one, two or no disruptive mutation). Reduced or absent NK activity was found in 44/45 patients tested; quantitative evaluation of LU in the 17 patients tested was significantly inferior than in healthy controls; Mean LU values were not significantly different between the 3 groups of patients.

A total of 15 patients with FHL4 were identified from 13 unrelated families; they were from Germany and Sweden, with no cases diagnosed in Italy. Ethnic origin of the families was Turkish, n=12 or Lebanese, n=1. Male:female ratio was 1.5:1. Median age at the diagnosis was 12.2 months (range, 9 days to 7.6 years; Quartiles, 6.5 12.2 32.2 months).

All patients were homozygous. Only 3 different mutations were observed: two deletions causing a fs: c.369_370delAG;c.374_376delCGC and c.110delC p.T37RfsX25; one stop mutation: c.802C>T p.Q268X. The 11 patients homozygous for the most comon mutation c.369_370delAG;c.374_376delCGC had an age at the diagnosis ranging between 3 and 70 months.

The diagnosis of HLH is based on a set of clinical and laboratory criteria. In this context, a reduction of NK cell cytotoxicity is the only functional immunological parameter that has so far been systematically evaluated. However, the protocols used to study NK cell cytotoxicity are labor-intensive and many patients with HLH have few circulating NK cells. Therefore, reduced NK cell cytotoxicity can reflect a reduced frequency rather than reduced function of these cells. Also, the NK cell cytotoxicity assay cannot reliably discriminate between primary and secondary HLH.

Apart from perforin-deficiency (FHL-2), all FHL forms genetically defined so far are due to defects in genes involved in transport and exocytosis of lytic granules. We therefore evaluated diagnostic assays that allow quantification of lytic granule exocytosis by human lymphocytes in the diagnosis of patients with FHL. These previously established degranulation assays as well as cytotoxicity assays and assays for intracellular perforin expression were standardised between 4 diagnostic laboratories in a consensus conference. To assess inter-center variability, samples from 4 healthy blood donors were sent to all participating laboratories in November 2009 and the results were compared. These round robin tests revealed a good inter-center reproducibility.

The performance of these assays was evaluated in 468 patients referred to one of the 4 participating laboratories for diagnostic evaluation of HLH during the study period. 72 of 73 patients with FHL3-5 and 9 of 10 patients with Griscelli or Chediak-Higashi syndrome had abnormal NK cell degranulation, while it was normal in 8 of 12 patients with FHL-2, in all 15 patients with XLP and 42 of 56 patients with secondary HLH. The additional investigation of NK cell degranulation after IL-2 stimulation and of CTL degranulation assays further improved the specificity of the assays. Active disease or immunosuppressive therapy did not impair the assay performance. CD107 based degranulation assays proved to be robust and sensitive assays for the rapid diagnosis of FHL. Identification of patients with one of the known forms of FHL is now possible within 2-3 days with a significant impact on necessary treatment decisions.

The following conclusions can be drawn:

- Degranulation of unstimulated NK cells is the most sensitive and specific assay for diagnosis of genetic disorders of degranulation.
- NK CD107less than 5% predicts FHL with high likelihood
- In patients with NK CD107 5-10%, poor recovery after IL-2 stimulation and/or impaired CTL degranulation suggest genetic disease, while normal recovery suggests 2° HLH (but does not exclude 1° HLH)
- Cytotoxicity assays are useful as confirmative assays, but are not needed for primary analysis of patients with HLH.

All data have been generated and compiled and the manuscript figures have been discussed with all contributors. The manuscript is in preparation.

Characterisation of HLH patients by NK cell assays

New data were collected concerning the function of the NK cell receptors, which can vary in the different groups of HLH patients, eventually affecting NK cell function.

In addition to the main diagnostic assays based on NK cells (i.e. the perforin expression assay and the degranulation assay in resting or activated NK cells upon co-culture with K562), we found that exocytosis triggered by engagement of different NK cell receptors was defective or severely impaired in all patients tested with FHL3, FHL4, FHL5, or GS2. This highlights how activation pathways through many different receptors converge for exocytosis of lytic granules and is in contrast to some data previously reported by other centers.

THERAPY

To identify prognostic factors for pre-transplant treatment failure

About 20-30 percent of HLH patients do not respond well to therapy and die before reaching HSCT. It is important to identify these patients early, possibly already at diagnosis or during the first weeks of treatment in order to have a chance to alter their therapeutic strategy. Approval by the ethics committee was obtained. Data on clinical parameters from before treatment start and early into therapy were collected on documentation sheets, stored and analysed using an SPSS statistic program database.

In total, 232 patients were eligible for inclusion in the study. Analysis of the data showed that several clinical parameters that were related to pre-transplant treatment failure could be identified, some presumably more related to multi-organ failure of terminally ill patients, and some more related to the immunological hyper-activated state that characterises HLH. Overall, nine predictors from the pre-treatment time point were identified. Hyperbilirubinemia and jaundice were strong risk factors for pre-transplant death, as were hyperferritinemia and pleocytosis. Moreover, three predictors from the time-point two weeks into treatment were also identified. Furthermore, non-improvement of parameters within the first two weeks of treatment was also identified as associated with increased risk for fatal outcome. We conclude that there are clinical parameters that may serve as prognostic factors. In patients without confirmed homozygous mutations or a known familial disease, these predictors may help risk stratification and guide treatment decisions in HLH patients. Future directions include validation of these predictors, possibly in a prospective study.

To develop experimental remission-inducing treatment of HLH

In order to develop a strategy to treat experimental HLH, the use of blocking antibodies anti-IFNg was evaluated in different murine models. In a first part, this study was focused in the treatment of perforin- and Rab27a-deficient mice, whereas in later experiments we tried to lower the dose of anti-IFNg antibody required to obtain remission of the disease and to expand this therapy to other models of experimental HLH.

HLH was induced in perforin and Rab27a-deficient mice and anti-IFNg therapy was started when the mice showed multiple features of HLH, allowing us to evaluate the therapeutic potential of the anti-IFNg therapy. In this setup, the mice were treated with several injections of monoclonal antibody and the efficacy of anti-IFNg was determined by comparing with a control group of mice receiving control isotypic antibody.

To identify factors influencing the outcome of haematopoietic stem cell transplantation

This retrospective analysis is based on available data on patients who had received a haematopoeitic stem cell transplantation (HSCT) due to haemophagocytic lymphohistiocytosis (HLH) in a 15 year- period between June 1994 and June 2009. Data was collected on standardised record forms from four different HLH-reference centres in Europe.

One of the major goals in rare diseases in general and in this project in particular, is combining efforts in order to gather more clinical data. This study is so far the largest multicentre study on HSCT in HLH patients including 186 patients. Most prior reports are single or multicentre experiences on patients who have received conventional Busulfan-based conditioning showing survival rates from 60-70%. Recent small case series on reduced-intensity conditioning (RIC) for HSCT in HLH patients have indicated a better survival (75-100%) compared to historic controls who had received myeloablative conditioning (MAC) resulting in an even worse survival rate of 50%.

Most patients in our study received a MAC including Busulfan and were transplanted at a median age of one year. The overall survival rate was favorable (69 %). There was no significant difference between patients who received grafts from matched related and unrelated or mismatched unrelated donors. However, patients receiving haploidentical grafts fared much worse. Furthermore, patients with active disease at start of HSCT had a higher probability to die than those with inactive disease. Primary graft failure occurred in only 10% of the transplantations. In 35% of the HSCTs the development of a veno-occlusive disease (VOD) was reported. Busulfan -containing MAC was associated with a significant higher risk for VOD compared to alternative/RIC regimens (p = 0.0012) but VOD was not a major cause of death. Nevertheless 80% of the fatalities occurred early within the first 6 months (median 3 months) indicating a high transplant-related mortality.

Potential Impact:

Project CUREHLH focused on a rare immunological disease in which pathophysiology is incompletely understood, diagnosis is difficult and therefore often missed, and which still has a high rate of mortality in spite of treatment. Moreover, this disease is a paradigm for the deleterious consequences of impaired immune function in the control of infectious agents and for immune surveillance in general.

Potential impact on knowledge and management of the disease

It has been a matter of debate whether HLH is triggered by a viral infection in all instances. Interestingly, in none of the mouse models the animals developed HLH spontaneously, indicating that an exogenous trigger (infection) is indeed needed. Five mouse models of HLH (perforin knock-out, beige, souri, pearl, ashen), infected with LCMV, showed that the development of HLH correlated highly with viral clearance: no HLH if the virus was cleared rapidly; transient HLH if virus clearance was delayed; and severe HLH if the virus persisted. This suggests that persistence of antigen is mandatory for the development of HLH by keeping T cells in a highly activated state. However, not in all situations with antigen persistence HLH necessarily emerges: Perforin knock-out mice, infected with a highly virulent LCMV strain (DOCILE), paradoxically developed an attenuated phenotype. This reflected T-cell exhaustion with less activated effector cells.

Hypercytokinemia characterizes HLH and is responsible for the clinical symptoms.

Whereas INFgamma is thought to be important for disease pathology, the role of other cytokines is not known. In human sera, interleukin-15, which regulates CTL and NK cell development, maturation and survival, was found to be elevated. Experiments in the interleukin-15-Ra knock-out mouse showed that interleukin15 is needed for granzyme A and perforin expression.

The results suggest that also in humans interleukin 15 and its receptor may be important for the development and maintenance of HLH. Interference with this pathway may be a potential therapeutic possibility and will have to be tested in mice.

In work package 2 cell lines from patients with HLH were derived in order to examine protein expression and localization of the secretory apparatus. Screening of protein expression for confirmation of genetic HLH was found to be necessary in some patients with mutations affecting splicing.

In patients with mutations in syntaxin 11, who have impaired degranulation, polarization towards the immunological synapse was shown not to be affected. Eleven patients with impaired killing by cytotoxic cells, in whom mutations in all known HLH genes had been excluded, were found to have normal polarization.

These results have important implications for the diagnosis and management of HLH: i) protein expression may be required to confirm diagnosis of HLH; ii) there are other killing defects outside the granule exocytosis pathway.

Hermansky-Pudlak syndrome type II (HPSII) is an extremely rare immune deficiency in which HLH has been described. Little is known about the pathophysiologic consequences of the mutation in the ADTB3A gene. Cooperation between several partners of the CUREHLH project now allowed important insights into the pathophysiology of HPSII. In patients a reduction of certain NK cell subsets and of NKT cells, and impaired maturation of dendritic cells was found. The reduction of NKT cell number in HPSII patients suggests an important role for the adaptor protein AP3 in positive selection of NKT cells. In a mouse model for HPSII, for the first time it was shown that transient HLH could be induced using LCMV infection. In contrast, infection with another virus (mouse cytomegalovirus) resulted only in immune activation without HLH, in line with other HLH mouse models in which not every virus led to the clinical picture of HLH. By silencing the AP3 complex it could be shown that in HPSII the mutation itself does not influence perforin expression, as was suggested previously, but that the altered level of perforin mRNA expression is probably due to the maturation state of NK cells.

These studies explain the recurrent viral infections and the development of tumours in patients with HPSII. In addition, they are important for understanding the pathophysiology of HPSII, demonstrating altered numbers and maturation states of immune effector cells, and ruling out an influence of the mutation on perforin expression.

HLH still remains a difficult disease to diagnose on the basis of clinical symptoms only. Thus diagnosis and treatment is often delayed with potential disastrous consequences for the patient. Impaired NK cell activity has been considered the gold standard for diagnosis in patients with symptoms of HLH. However, the test is not only time consuming and not widely available, but additionally cannot reliably discriminate between genetic and acquired cases.

One major achievement of CUREHLH is the implementation of a novel diagnostic algorithm between 4 laboratories. NK cell degranulation with and without addition of interleukin-2 was able to correctly identify all patients with a genetic defect in the exocytosis pathway. Moreover, this assay could differentiate between genetic and acquired HLH. CTL degranulation and cytotoxicity were used as additional tests. Protein expression of perforin, SAP and XIAP was used to exclude a genetic defect in the respective genes. This comprehensive approach facilitates the diagnosis of genetic HLH within 2-3 days.

These results will have a major impact on the speediness and correctness of diagnosis and on planning of therapy, especially stem cell transplantation for patients with genetic HLH. Additionally, confirmatory mutation analysis can now focus on the most likely gene(s) which results in considerable cost saving. For the first time a uniform and comparable diagnostic approach for HLH is now used in several European countries.

High levels of INFgamma, secreted by T-lymphocytes, play a central role in the pathophysiology of HLH. INFgamma activates macrophages which in turn secrete several other inflammatory cytokines. Hypercytokinemia leads to the clinical picture of HLH and may have fatal consequences if not stopped in time. Therapy of HLH, as used in contemporary protocols, relies on immunosuppressive, immunomodulatory and cytostatic agents. However, even with rapid diagnosis there are a substantial proportion of patients who do not respond to treatment and die before stem cell transplantation (SCT) or undergo SCT in a poor clinical condition. Before CUREHLH prognostic factors for treatment response and new treatment approaches were not available.

These results have a major impact on the treatment of patients with HLH, regarding treatment stratification and early switch to salvage therapy in patients with adverse prognostic factors. Most important, the experiments in mice open the perspective to use INFgamma in clinical studies in the future.

Potential socioeconomic impact and wider societal implication

Genetic HLH is a paradigm for an uncontrolled immune activation in response to an infectious trigger. Thus increased knowledge of the pathophysiology of this disease may also allow important insights on the function of the immune system in general, including pathophysiology and management of infectious diseases. In addition, NK cells and cytotoxic T-lymphocytes play an important role in surveillance of neoplastic cells and in immune homeostasis. Strictly regulated interactions between immune cells prevent that "self" antigens are attacked. This regulatory process fails in autoimmune diseases. The molecular mechanisms necessary for the regulated action of NK cells and T-cells which has been studied in CUREHLH, is therefore also of interest for cancer surveillance and the pathophysiology of autoimmune diseases.

The perforin dependent pathway, relying on the intact function of lytic granule exocytosis, was shown to be essential for adequate control of certain viruses in all HLH mouse models. A lesson from this project is that slight differences in cytotoxicity may lead to a completely different clinical picture. Depending on the cytotoxic defect and on the strain of the virus, several outcomes were possible: no disease and clearance of the virus; mild disease with delayed clearance of virus; mild disease with persisting virus due to T cell exhaustion; severe disease with persistent viral infection with and without death. It can be assumed that these results also apply to patients with HLH. In addition, the individual capacity of the immune cells and the type of virus will also determine the outcome of a viral infection in patients without HLH. This may explain the occurrence of HLH or persistent virus infections in apparently healthy individuals without a genetic defect in the above pathway. The (probably multifactorial) mechanisms which lead to an acquired impairment of immune function, have still to be elucidated.

A highly interesting phenomenon, not yet described in HLH mouse models, was the paradox development of mild HLH with virus persistence with a highly virulent strain of LCMV. Characterization of the T-cells was compatible with T-cell exhaustion. This has also been described in patients with AIDS and may be a mechanism for the development of other chronic virus infections.

Perforin is not only necessary for adequate control of viruses; it also plays a role in immune surveillance. There is sufficient evidence that patients with some forms of genetic HLH who live longer are at risk for developing malignancies. These include especially malignant lymphomas; howeve,r certain subtypes of acute leukaemia have also been linked to perforin mutations. In addition, perforin deficiency has been shown to be associated with autoimmune diseases in mouse models and in humans. Thus the study of the function and interaction of the molecules of the exocytotic pathway which leads to the delivery of perforin to the target cell is not only of interest for patients with HLH, but also for the medical community in general.

Formerly the diagnosis of genetic HLH had to rely on the clinical course, on another affected family member or on a genetic defect, proven by time-consuming mutation analysis. Stem cell transplantation (SCT) in these patients was initiated late and many patients meanwhile had CNS relapses with long term neurological sequelae. The rapid diagnostic approach as worked out in CUREHLH allows for an early search for a suitable donor, will spare costs for unnecessary long treatment, and will reduce the time at risk for relapse before SCT. This hopefully will result in fewer patients with chronic neurological handicaps. In addition, mutation analysis can now be restricted to the most likely genes.

Main dissemination activities and exploitation results

Many public results of CUREHLH have already been widely disseminated, including publication in peer reviewed journals, presentations at national and international congresses, workshops and media presentations. Reports of public results at the end of the study were sent to the national parent organization for histiocytoses in the country of partner 1 and to the international Histiocyte Society who operates a web site for physicians and parents on histiocytic diseases. This summary will be made available also to the other partners to disseminate results in the respective countries.

Project website: http://www.uke.de/curehlh

Prof. Dr Gritta Janka
Department of Paediatric Haematology and Oncology
University Medical Centre
Martinistrasse 52
20246 Hamburg
janka@uke.de

Dr Kai Lehmberg
Department of Paediatric Haematology and Oncology
University Medical Centre
Martinistrasse 52
20246 Hamburg
k.lehmberg@uke.de

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