Perforin-dependent Cytotoxicity Research Articles

Preclinical Assessment for Gene Addition Strategy in Familial Hemophagocytic Lymphohistiocytosis Related to Munc 13-4 Deficiency

Introduction: Familial Hemophagocytic Lymphohistiocytosis (FHL) comprises a group of rare genetic disorders with defect cytotoxicity in T and NK cells predisposing to severe hyperinflammation with the clinical hallmark of the Hemophagocytic LymphoHistiocytosis (HLH) syndrome. The uncontrolled proliferation of activated T-CD8 lymphocytes and macrophages infiltrates lymphoid tissues, bone marrow, and multiple organs including the brain. Thus, if untreated full-scale FHL is usually fatal. FHL3 is due to mutations in the UNC13D gene coding for Munc 13-4 protein, one of the components of the perforin-dependent cytotoxicity apparatus. This condition accounts for 30% to 35 % of all genetic cases of HLH. Hematopoietic stem and progenitor cell (HSPC) transplantation, which is the only curative treatment for FHL3 to date, remains difficult even when a compatible donor is available because of the patients' critical inflammatory background. In this context, gene therapy could be a promising therapeutic option, especially for those patients without HLA-compatible donor. Method: The feasibility and efficacy of gene addition strategy for FHL3 have been previously demonstrated in mouse models. We then translated this approach to human cells using a third-generation self-inactivated lentivirus vector pseudo-typed with a conventional vesicular stomatitis virus-G (VSVG) expressing a codon-optimized UNC13D gene under the transcriptional control of EF1 alpha (LV-EF1a-UNC13D). HSPC or CD3+ T-cells were transduced with a GMP-like-grade LV-EF1a-UNC13D lentiviral vector. We studied the restoration of cytotoxic function. Results: Our GMP transduction protocol led to efficient transcription of optimized mRNA codon and expression of Munc 13-4 protein as evidenced by RT qPCR and western blot, respectively. Interestingly, we show restored cytotoxicity activity after CD34+ differentiation toward CD3+ T-cells and NK cells. The vector copy number is compatible with a clinical approach. In vivo experiments are ongoing to validate the absence of genotoxicity in this setting. Discussion conclusion: Our results support the feasibility and efficacy of UNCD13D gene transfer into hematopoietic stem and progenitor cells or T cells from Munc 13-4 deficient patients. Alternatively, gene therapy of HSPCs could be combined with transduced autologous T-cells infusion. The first attempts regarding this option are encouraging. UNC13D gene addition strategy could dramatically improve the care of FHL3 patients lacking an HLA-genoidentical donor.

Effects of a Small-Molecule Perforin Inhibitor in a Mouse Model of CD8 T Cell-Mediated Neuroinflammation.

Alteration of the blood-brain barrier (BBB) at the interface between blood and CNS parenchyma is prominent in most neuroinflammatory diseases. In several neurologic diseases, including cerebral malaria and Susac syndrome, a CD8 T cell-mediated targeting of endothelial cells of the BBB (BBB-ECs) has been implicated in pathogenesis. In this study, we used an experimental mouse model to evaluate the ability of a small-molecule perforin inhibitor to prevent neuroinflammation resulting from cytotoxic CD8 T cell-mediated damage of BBB-ECs. Using an in vitro coculture system, we first identified perforin as an essential molecule for killing of BBB-ECs by CD8 T cells. We then found that short-term pharmacologic inhibition of perforin commencing after disease onset restored motor function and inhibited the neuropathology. Perforin inhibition resulted in preserved BBB-EC viability, maintenance of the BBB, and reduced CD8 T-cell accumulation in the brain and retina. Therefore, perforin-dependent cytotoxicity plays a key role in the death of BBB-ECs inflicted by autoreactive CD8 T cells in a preclinical model and potentially represents a therapeutic target for CD8 T cell-mediated neuroinflammatory diseases, such as cerebral malaria and Susac syndrome.

Reduced CTL motility and activity in avascular tumor areas.

Patchy infiltration of tumors by cytotoxic T cells (CTLs) predicts poorer prognosis for cancer patients. The factors limiting intratumoral CTL dissemination, though, are poorly understood. To study CTL dissemination in tumors, we histologically examined human melanoma samples and used mice to image B16-OVA tumors infiltrated by OT-I CTLs using intravital two-photon microscopy. In patients, most CTLs concentrated around peripheral blood vessels, especially in poorly infiltrated tumors. In mice, OT-I CTLs had to cluster around tumor cells to efficiently kill them in a contact-and perforin-dependent manner and cytotoxicity was strictly antigen-specific. OT-I CTLs as well as non-specific CTLs concentrated around peripheral vessels, and cleared the tumor cells around them. This was also the case when CTLs were injected directly into the tumors. CTLs crawled rapidly only in areas within 50µm of flowing blood vessels and transient occlusion of vessels immediately, though reversibly, stopped their migration. In vitro, oxygen depletion and blockade of oxidative phosphorylation also reduced CTL motility. Taken together, these results suggest that hypoxia limits CTL migration away from blood vessels, providing immune-privileged niches for tumor cells to survive. Normalizing intratumoral vasculature may thus synergize with tumor immunotherapy.

Cancer risk in relatives of patients with a primary disorder of lymphocyte cytotoxicity: a retrospective cohort study

Mutations in genes for perforin-dependent lymphocyte cytotoxicity are associated with haemophagocytic lymphohistiocytosis, a rare disease of severe hyperinflammation that typically becomes evident in early childhood. It has been suggested that individuals with hypomorphic biallelic mutations in genes associated with haemophagocytic lymphohistiocytosis are at increased risk of developing haematological malignancies. We aimed to assess whether relatives of patients with primary haemophagocytic lymphohistiocytosis (ie, heterozygous carriers of these mutated genes) were more likely to develop cancer. In this retrospective cohort study, we used a multigeneration registry to identify relatives (parents and grandparents) of 79 Swedish children (<15 years) with primary haemophagocytic lymphohistiocytosis diagnosed between 1971 and 2011. For each relative, we randomly selected eight matched individuals from the Swedish total population registry, stratified for sex, birth year, and birth region. Relatives and matched controls were cross-linked with the Swedish Cancer Registry to establish cancer incidence rate. We then calculated the incidence rate ratio between first-degree and second-degree relatives and the matched controls. Additionally, we assessed natural-killer-cell-mediated cytotoxicity in a subgroup of first-degree relatives using standard 4 h (51)Cr assay and flow cytometry quantification of the upregulation of surface CD107a. We identified 346 first-degree and second-degree relatives from 67 families (67 mothers, 66 fathers, 106 grandmothers, and 107 grandfathers) and 2768 matched controls. Median follow-up was 49 years, range 0-54 years. By death or last follow-up (Dec 31, 2012), first-degree relatives had a significantly increased incidence rate of malignancies than did controls (incidence rate per 1000 person-years 2.78 [95% CI 1.42-4.15] vs 1.56 [1.16-1.95]; incidence rate ratio 1.79 [95% CI 1.06-3.03]; p=0.030). Mothers had a particularly increased risk (incidence rate per 1000 person-years 4.43 [95% CI 1.99-6.87] vs 1.60 [1.08-2.11]; incidence rate ratio 2.78 [95% CI 1.48-5.21]; p=0.0014), whereas no difference was found between fathers and controls (1.24 [0.00-2.51] vs 1.52 [0.89-2.15]; 0.82 [0.29-2.29]; p=0.70) or between grandparents and controls (7.24 [5.44-9.04] vs 6.36 [5.70-7.03]; 1.14 [0.88-1.48]; p=0.33). Functional analysis of heterozygous carriers of mutations associated with haemophagocytic lymphohistiocytosis could not show significantly reduced lymphocyte cytotoxicity. Heterozygous mutations in genes associated with haemophagocytic lymphohistiocytosis might be a new risk factor for cancer. The increased risk of cancer might imply haploinsufficiency of cytotoxic lymphocyte-mediated immunosurveillance of cancer in carriers of these mutations. Our findings might support intensified screening for malignancies in relatives of patients with haemophagocytic lymphohistiocytosis. Swedish Children's Cancer Foundation, Swedish Research Council, Histiocytosis Association, Swedish Cancer Society, Swedish Cancer and Allergy Foundation, Mary Béve Foundation, Karolinska Institutet Research Foundation, Stockholm County Council (ALF-project).

Inflammasomes Coordinate Pyroptosis and Natural Killer Cell Cytotoxicity to Clear Infection by a Ubiquitous Environmental Bacterium

Defective neutrophils in patients with chronic granulomatous disease (CGD) cause susceptibility to extracellular and intracellular infections. Microbes must first be ejected from intracellular niches to expose them to neutrophil attack, so we hypothesized that inflammasomes detect certain CGD pathogens upstream of neutrophil killing. Here, we identified one such ubiquitous environmental bacterium, Chromobacterium violaceum, whose extreme virulence was fully counteracted by the NLRC4 inflammasome. Caspase-1 protected via two parallel pathways that eliminated intracellular replication niches. Pyroptosis was the primary bacterial clearance mechanism in the spleen, but both pyroptosis and interleukin-18 (IL-18)-driven natural killer (NK) cell responses were required for liver defense. NK cells cleared hepatocyte replication niches via perforin-dependent cytotoxicity, whereas interferon-γ was not required. These insights suggested a therapeutic approach: exogenous IL-18 restored perforin-dependent cytotoxicity during infection by the inflammasome-evasive bacterium Listeria monocytogenes. Therefore, inflammasomes can trigger complementary programmed cell death mechanisms, directing sterilizing immunity against intracellular bacterial pathogens.

Perforin-dependent direct cytotoxicity in natural killer cells induces considerable knockdown of spontaneous lung metastases and computer modelling-proven tumor cell dormancy in a HT29 human colon cancer xenograft mouse model.

BackgroundFor long, natural killer (NK) cells have been suspected to play a critical role in suppressing the development of spontaneous metastases in cancer patients. Despite a wide range of studies it remains unclear so far to what extent primary tumor growth together with formation of distant metastases and NK cell activity influence each other.MethodsTo precisely investigate the role of NK cells with a perforin-deficiency in cancer growth and metastasis formation, human HT29 colon cancer cells were subcutaneously grafted into pore forming protein and recombination activating gene 2 double knock out (pfp/rag2) mice and in recombination activating gene 2 only knock out (rag2) mice both with black six background. Both mice lack B and T cell functions due to the absence of rag2.ResultsPrimary tumors developed in 16/16 in pfp/rag2 and 20/20 rag2 mice. At sacrifice primary tumor weight did not differ significantly. However, tumors grew faster in pfp/rag2 mice (50 days) than in pfp/rag2 mice (70 days). Circulating tumor cells (CTC) in murine blood were nearly three times higher in pfp/rag2 (68 cells/ml) than in rag2 mice (24 cells/ml). Lung metastases occurred frequently in pfp/rag2 mice (13/16) and infrequently in rag2 mice (5/20). The mean number of metastases was 789 in pfp/rag2 mice compared to 210 in rag2 mice. Lung metastases in pfp/rag2 mice consisted of 10–100 tumor cells while those in rag2 mice were generally disseminated tumor cells (DTCs).Computer modelling showed that perforin-dependent killing of NK cells decelerates the growth of the primary tumour and kills 80% of CTCs. Furthermore, perforin-mediated cytotoxicity hampers the proliferation of the malignant cells in host tissue forcing them to stay dormant for at least 30 days.ConclusionThe results exactly quantified the effect of perforin-dependent direct cytotoxicity of NK cells on HT29 on primary tumor growth, number of CTCs in the blood and the number of metastases. The largest effects were seen in the number of mice developing spontaneous lung metastases and the mean number of lung metastases. Hence, perforin-mediated cytotoxicity used for direct killing by NK cells is more important than indirect killing by secretion of death-inducing ligands by NK cells.

TNFα and IFNγ but not perforin are critical for CD8 T cell-mediated protection against pulmonary Yersinia pestis infection.

Septic pneumonias resulting from bacterial infections of the lung are a leading cause of human death worldwide. Little is known about the capacity of CD8 T cell-mediated immunity to combat these infections and the types of effector functions that may be most effective. Pneumonic plague is an acutely lethal septic pneumonia caused by the Gram-negative bacterium Yersinia pestis. We recently identified a dominant and protective Y. pestis antigen, YopE69–77, recognized by CD8 T cells in C57BL/6 mice. Here, we use gene-deficient mice, Ab-mediated depletion, cell transfers, and bone marrow chimeric mice to investigate the effector functions of YopE69–77-specific CD8 T cells and their relative contributions during pulmonary Y. pestis infection. We demonstrate that YopE69–77-specific CD8 T cells exhibit perforin-dependent cytotoxicity in vivo; however, perforin is dispensable for YopE69–77-mediated protection. In contrast, YopE69–77-mediated protection is severely impaired when production of TNFα and IFNγ by CD8 T cells is simultaneously ablated. Interestingly, TNFα is absolutely required at the time of challenge infection and can be provided by either T cells or non-T cells, whereas IFNγ provided by T cells prior to challenge appears to facilitate the differentiation of optimally protective CD8 T cells. We conclude that cytokine production, not cytotoxicity, is essential for CD8 T cell-mediated control of pulmonary Y. pestis infection and we suggest that assays detecting Ag-specific TNFα production in addition to antibody titers may be useful correlates of vaccine efficacy against plague and other acutely lethal septic bacterial pneumonias.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Etoposide Selectively Ablates Activated T Cells To Control the Immunoregulatory Disorder Hemophagocytic Lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is an inborn disorder of immune regulation caused by mutations affecting perforin-dependent cytotoxicity. Defects in this pathway impair negative feedback between cytotoxic lymphocytes and APCs, leading to prolonged and pathologic activation of T cells. Etoposide, a widely used chemotherapeutic drug that inhibits topoisomerase II, is the mainstay of treatment for HLH, although its therapeutic mechanism remains unknown. We used a murine model of HLH, involving lymphocytic choriomeningitis virus infection of perforin-deficient mice, to study the activity and mechanism of etoposide for treating HLH and found that it substantially alleviated all symptoms of murine HLH and allowed prolonged survival. This therapeutic effect was relatively unique among chemotherapeutic agents tested, suggesting distinctive effects on the immune response. We found that the therapeutic mechanism of etoposide in this model system involved potent deletion of activated T cells and efficient suppression of inflammatory cytokine production. This effect was remarkably selective; etoposide did not exert a direct anti-inflammatory effect on macrophages or dendritic cells, and it did not cause deletion of quiescent naive or memory T cells. Finally, etoposide's immunomodulatory effects were similar in wild-type and perforin-deficient animals. Thus, etoposide treats HLH by selectively eliminating pathologic, activated T cells and may have usefulness as a novel immune modulator in a broad array of immunopathologic disorders.

Spectrum Of Clinical and Laboratory Findings In Adult Patients With Hemophagocytic Lymphohistiocytosis: Significance Of Ferritin Levels

BackgroundHemophagocytic lymphohistiocytosis(HLH) is a rare frequently fatal group of immunodeficiencies that mostly present with cytopenias and infections. It is characterized by an immune dysregulation that results in excessive cytokine expression, phagocytosis of blood cells by activated macrophages, and end-organ damage. There are familial forms (“primary HLH”), typically presenting in childhood, and sporadic forms (“secondary HLH”), often presenting in adults. While primary HLH is often limited to mutations affecting perforin dependent cytotoxicity, limited data are available on secondary HLH which is associated with conditions, such as malignancy and infection, without identifiable genetic abnormalities.We reviewed diagnoses of HLH in adult treated at Barnes-Jewish Hospital in St. Louis since 1998. We hypothesized that serum ferritin – which is elevated in most HLH cases – can help in differentiating HLH etiology and might predict outcome. MethodsWe identified 56 adult patients with HLH seen during inpatient and outpatient visits using HLH ICD-9 billing code 288.4. Clinical and laboratory/hematopathology records were reviewed; 12 cases were excluded from the analysis – 9 for missing data and 3 for insufficient diagnosis of HLH. We followed the 2004 Histiocyte Society Criteria for HLH. All results were presented as median plus range, mean +/- standard deviation, or percentages as indicated. We compared clinical and laboratory parameters between HLH groups by using the Student T-test, Mann-Whitney, or Chi square test, where appropriate. Predictive/prognostic factors were evaluated by univariate analysis. Results were considered significant at p <0.05. All statistical analyses were carried out using SPSS software, version 17 (SPSS Inc., Chicago, IL). ResultsAmong the included 44 cases, the median age was 45 years (range, 20-80); 21 patients were male. Fever and splenomegaly were seen in 77.3% and 52.3% of patients, respectively. The most common laboratory abnormalities during hospitalization were hyperferritinemia (95.3%; median 17,414; range 225-684,000), pancytopenia/bicytopenia (72.7%), elevated triglycerides (52.5%; median 267.5; range 71-857), and low plasma fibrinogen (30.8%). Pathology revealed the characteristics hemophagocytosis of HLH in most cases. The recognized causes of HLH were: 29.5% malignancies (B-cell lymphoma [n=3], T-cell lymphoma [n=6], acute myeloid leukemia [n=1], myeloproliferative/myelodysplastic syndromes [n=2], metastatic breast cancer [n=1]), 25% viral (HCV [n=5], HIV [n=2], HBV [n=1], EBV [n=1], CMV [n=1], influenza [n=1]), 11.4% rheumatologic (systemic rheumatoid arthritis [n=3], Still's disease [n=1], lupus-like syndrome [n=1]), 4.5% bacterial sepsis, 4.5% post solid organ transplantation (liver [n=1], lung [n=1]), 2.27% fungal infection, 2.27% histoplasma infection, 20.4% unknown. Corticosteroids were frequently used in treatment; few patients received cyclosporine, etoposide, intravenous immunoglobulins, or stem cell transplantation. Thirteen patients died, 9 (69%) of septic shock.The cohort was then dichotomized based on disease etiology into malignancy- and infection-associated HLH; this resulted in 13 and 15 cases in each category, respectively. Patients with malignancy-associated HLH had higher serum ferritin levels than those with infection-associated HLH but the different was not significant (84,592.8 +/- 185,009.4 vs. 23,262 +/- 19,090.5 ng/mL) (p = 0.23). Triglycerides were higher in the infection-associated HLH group compared to the other group with a difference approaching significance (358.6 +/- 201.7 vs. 233.8 vs. 124.6; p = 0.076). Overall, patient with higher ferritin levels were more likely to have a poor prognosis. At a cutoff ferritin level of 60,000 ng/mL, 62.5% of patients with higher levels died compared to 22.8% who died while having lower ferritin levels (p = 0.042). ConclusionHLH in the adults is associated with a wide spectrum of underlying diseases. It has a high mortality rate, particularly in patients with markedly elevated ferritin levels. Further studies are needed to develop prognostic criteria for outcomes in adult HLH. Analysis of additional cases is ongoing and will be presented at the meeting. Disclosures:No relevant conflicts of interest to declare.

Gene Therapy for Hemophagocytic Lymphohistiocytosis (HLH): Fixing a Criticial ‘Circuit Breaker' in the Immune System.

AbstractAbstract 3158In addition to its well-known role in host defense, perforin-dependent cytotoxicity also has a critical immune regulatory function. Genetic defects of perforin underlie many cases of hemophagocytic lymphohistiocytosis (HLH), a fatal disorder of extreme immune activation. While the initial treatment of HLH is largely focused on suppressing dangerous inflammation, hematopoietic cell transplantation (HCT) is required for long-term correction of the underlying immune regulatory defect. Unfortunately, mortality after HCT in patients with HLH is unusually high for a non-malignant disorder, ranging from 20 to 50%.We developed a model of HLH in which perforin deficient (prf-/-) mice develop all of the features of HLH after challenge with lymphocytic choriomeningitis virus (LCMV). Recent studies from our group have demonstrated that transplantation of wild type (WT) bone marrow into prf-/- mice rescues them from the subsequent development of HLH after challenge with LCMV. In this model, perforin-dependent immune regulation appears to function in trans, with a critical threshold of 10% WT chimerism required to protect animals from HLH. This low threshold is consistent with clinical reports of very limited numbers of patients with long term mixed chimerism after HCT who appear to be protected from HLH recurrence. These data have also provided a clear target for gene therapy efforts.We have begun to test the feasibility of autologous gene correction using lentiviral vectors in this model system. Hematopoietic stem and progenitor cells (HSC) from prf-/- mice were transduced with lentivirus vectors expressing perforin under the control of ubiquitous or tissue-specific promoters. When transduced HSC were transplanted into lethally irradiated prf-/- mice, we have observed levels of in vivo gene correction ranging from 10–80%, depending on the promoter and transduction protocols utilized. We find that the transduced CD8 T cell and NK cell progeny display significant correction of their cytotoxic defects. Challenge of gene corrected prf-/- mice with LCMV has revealed significant correction of the underlying immune regulatory defect and protection from the development of HLH. However, reestablishing normal perforin-dependent immune regulation with gene corrected prf-/- HSC’s appears to be somewhat less efficient than with mixed WT:prf-/- chimerism. Thus, further optimization of perforin gene control by lentiviral vectors appears to be warranted. Results of ongoing studies related to vector optimization and reestablishing physiologic perforin-mediated immune regulation will be presented.Because normal perforin expression in only a fraction of lymphocytes is sufficient to rescue individuals from HLH, this disorder is an attractive target for gene therapy approaches. Demonstration of long term gene correction and rescue from HLH in our animal model will pave a path for translational efforts testing gene therapy for HLH in clinical trials. Disclosures:No relevant conflicts of interest to declare.

IL-15 restores defective NK anti-fungal activity in a primary Hemophagocytic lymphohistiocytosis Patient (164.1)

Abstract Hemophagocytic lymphohistiocytosis (HLH) is a rare yet potentially fatal clinical syndrome caused by various congenital defects that result in defective perforin-dependent cytotoxicity of NK cells. This leads to increased T cell activation and proliferation and excessive cytokine production including IL-1, IL-6, IFN-γ, TNF-α and GM-CSF that causes sustained activation of macrophages that is manifest as extensive inflammation and tissue damage. One study showed that 50% of deaths from familial HLH were due to invasive fungal infections. We sought to investigate whether NK cells from a patient with HLH have defective anti-fungal activity and seek a potential therapeutic approach. NK cells (CD56+/CD3-) were isolated from a two-month-old boy with c1090-1091del of the perforin gene resulting in p.Leu364&amp;gt;Stop consistent with the FHL-2 form of HLH. Defective NK killing was observed for both Cryptococcus neoformans and Candida albicans. Remarkably, the defective anti-Candida activity of NK cells could be restored within 24 hours by IL-15 treatment that was similar to the enhanced anti-Candida activity of NK cells from healthy donors. We speculate that IL-15 enhances antifungal activity by stimulating NK cells to employ an alternate microbicidal effector mechanism. This result is the first to show that IL-15 can restore defective cytotoxic activity of NK cells from an HLH patient, and further studies might be considered to explore the mechanism and potential form of immunotherapy.

Gene Therapy for Hemophagocytic Lymphohistiocytosis (HLH): Fixing a Critical ‘Circuit Breaker’ in the Immune System

In addition to its well-known role in host defense, perforin-dependent cytotoxicity also has a critical immune regulatory function. Genetic defects of perforin underlie many cases of hemophagocytic lymphohistiocytosis (HLH), a disorder of extreme immune activation. While the initial treatment of HLH is largely focused on suppressing dangerous inflammation, HCT is required to correct the underlying immune defect. Unfortunately, mortality after HCT in patients with HLH is unusually high for a non-malignant disorder, ranging from 20 to 50%. We previously reported that transplantation of wild type (WT) bone marrow into perforin deficient (prf-/-) mice rescues them from the subsequent development of HLH after viral challenge with lymphocytic choriomeningitic virus (LCMV). In this model, perforin-dependent immune regulation appears to function in trans, with a critical threshold of 10% WT chimerism required to protect animals from HLH. This low threshold is consistent with reports of very limited numbers of patients with long term mixed chimerism after HCT who appear to be protected from HLH recurrence. These data have also provided a clear target for gene therapy efforts. We have begun to test the utility of autologous gene correction using lentivirus vectors in this model system. Hematopoietic stem and progenitor cells (HSC) from prf-/- mice were transduced with lentivirus vectors expressing perforin under the control of ubiquitous or tissue-specific promoters. When transduced HSC were transplanted into lethally irradiated prf-/- mice, we have observed levels of in vivo gene correction ranging from 10-80%, depending on the promoter and transduction protocols utilized. We find that the transduced NK cell progeny display significant correction of their cytotoxic defects. Challenge of gene corrected mice with LCMV infection is ongoing. The ability of gene corrected HSC to reestablish a normal pattern of cytokine production, T cell activation, and protection from HLH after LCMV infection will be presented. Because normal perforin expression in only a fraction of lymphocytes is sufficient to rescue individuals from HLH, this disorder is an attractive target for gene therapy approaches. Demonstration of long term gene correction and rescue from HLH in our animal model will pave a path for translational efforts testing gene therapy for HLH in clinical trials. Supported by NIH-R01HL091769.

Perforin is a critical physiologic regulator of T-cell activation

AbstractIndividuals with impaired perforin-dependent cytotoxic function (Ctx−) develop a fatal inflammatory disorder called hemophagocytic lymphohistiocytosis (HLH). It has been hypothesized that immune hyperactivation during HLH is caused by heightened infection, defective apoptosis/responsiveness of Ctx− lymphocytes, or enhanced antigen presentation. Whereas clinical and experimental data suggest that increased T-cell activation drives HLH, potential abnormalities of T-cell activation have not been well characterized in Ctx− hosts. To define such abnormalities and to test these hypotheses, we assessed in vivo T-cell activation kinetics and viral loads after lymphocytic choriomeningitis virus (LCMV) infection of Ctx− mice. We found that increased T-cell activation occurred early during infection of Ctx− mice, while they had viral burdens that were identical to those of WT animals, demonstrating that T-cell hyperactivation was independent of viral load. Furthermore, cell transfer and signaling studies indicated that increased antigenic stimulation, not a cell-intrinsic defect of responsiveness, underlay heightened T-cell activation in vivo. Finally, direct measurement of viral antigen presentation demonstrated an increase in Ctx− mice that was proportional to abnormal T-cell activation. We conclude that perforin-dependent cytotoxicity has an immunoregulatory role that is distinguishable from its pathogen clearance function and limits T-cell activation in the physiologic context by suppressing antigen presentation.

CD8+ regulatory T cells in solid organ transplantation

Recent advances in phenotypic identification of CD8 T cells with regulatory properties has allowed better definition of this subset of CD8 Tregs (also called CD8 T suppressor cells in the past). Analysis of their potential contribution in transplantation has attracted most of the interest in the past few years. A better understanding of the mechanisms of action and role of antigenic specificity of the different subtypes of CD8 Tregs and their potential in solid organ transplantation will bring considerable advances in the field. This review will focus and summarize the potential of this subset in cell-based treatments of human transplantation and highlight major discovery in the last months. Identification of markers such as PD-1 has allowed better definition of the different CD8 Tregs subsets. Recent animal and human studies have identified mechanisms used by antigen-specific CD8 Treg cells in suppressing anti-allograft immune responses such as perforin-dependent cytotoxicity. New strategies of ex-vivo stimulation (through the T-cell receptor or not) of CD8 Tregs have shown future direction for solid organ transplantation. CD8 Treg cells represent a subset of regulatory T cells with great potential for future therapeutic application and researchers are moving ahead for a better understanding of their action in antigraft immune response suppression.

Hepadnaviral infection augments hepatocyte cytotoxicity mediated by both CD95 ligand and perforin pathways

Recently, we documented that hepatocytes can eliminate contacted cells via the CD95 ligand (CD95L)-CD95 pathway and that they are also equipped in perforin and granzyme B and can eradicate other cells via the granule exocytosis mechanism. The aim of this study was to assess whether hepadnaviral infection modifies hepatocyte-mediated cell killing. Primary hepatocytes from woodchucks with progressing or resolved hepadnaviral hepatitis and hepatocyte lines transfected with woodchuck hepatitis virus (WHV) genes were examined for cytotoxic effector activity against cell targets susceptible to CD95L and/or perforin-dependent killing. Hepatocytes from healthy animals served as controls. Actively progressing and resolved hepadnaviral hepatitis is associated with a significantly greater capacity of hepatocytes to kill contacted cells. Both hepatocyte CD95L- and perforin-dependent cytotoxicity were augmented. Hepatocytes transfected with WHV X gene, but not those with complete WHV genome or virus envelope or core gene, transcribed significantly more CD95L and perforin and killed cell targets more efficiently. Exposure to interferon-gamma profoundly enhanced hepatocyte cell killing. Hepatocyte cytotoxic potential is significantly augmented during and following resolution of active hepadnaviral hepatitis. Hepatocyte cytotoxic activity may contribute to both liver physiological functions and the pathogenesis and progression of liver disease, including viral hepatitis.

Pancreatic lipase-related protein 2 (PLRP2) induction by IL-4 in cytotoxic T lymphocytes (CTLs) and reevaluation of the negative effects of its gene ablation on cytotoxicity

Pancreatic lipase-related protein 2 (PLRP2) is induced by IL-4 in vitro in cytotoxic T lymphocyte (CTL) clones and CTLs from immunized wild-type (WT) PLRP2(+/+) are more cytotoxic than PLRP2(-/-) CTLs, suggesting to previous investigators that the lipase PLRP2 might support CTL functions. Here, we further evaluate PLRP2 in CTLs. We found that PLRP2 was optimally induced in splenocytes by 3.5 x 10(-8) M IL-4 by day 6 after activation and was restricted to CD8(+) T cells. PLRP2 mRNA was detected inconsistently (and at low levels) after activation in the presence of IL-2. Cytotoxicity in 4 h (51)Cr assays of WT CTLs was approximately 3-fold the activity of PLRP2(-/-) CTLs cultured with IL-4 and, with IL-2, was unexpectedly approximately 2 fold the activity of PLRP2(-/-) CTLs. Thus, PLRP2 gene ablation affected short-term (perforin-dependent) cytotoxicity, even under the IL-2 conditions. Other variables failed to account for the reduced cytotoxicity. Granzyme B levels, activation markers, and CD8(+) T cell frequencies were similar for WT vs. PLRP2(-/-) CTLs (with either cytokine). Addition of rPLRP2 to IL-4 induced PLRP2(-/-) CTLs (or to cytotoxic granule extracts) failed to increase lysis, suggesting that the missing mediator is more than released PLRP2. Cytotoxicity of WT and PLRP2(-/-) CTLs was similar in 2-day tumor survival assays with IL-4, which can be mediated by perforin-independent mechanisms. We conclude that extracellular PLRP2 lipase is unable to directly augment the cytotoxicity that was lost by PLRP2 ablation and that after reevaluation, the question of what is PLRP2's role in CD8 T cells is still unanswered.

NKG2A Inhibits Invariant NKT Cell Activation in Hepatic Injury

Activation of invariant NKT (iNKT) cells in the liver is generally regarded as the critical step for Con A-induced hepatitis, and the role of NK cell receptors for iNKT cell activation is still controversial. In this study we show that blockade of the NKG2A-mediated inhibitory signal with antagonistic anti-NKG2A/C/E mAb (20d5) aggravated Con A-induced hepatitis in wild-type, Fas ligand (FasL)-mutant gld, and IL-4-deficient mice even with NK cell and CD8 T cell depletion, but not in perforin-, IFN-gamma-, or IFN-gamma- and perforin-deficient mice. Consistently, 20d5 pretreatment augmented serum IFN-gamma levels and perforin-dependent cytotoxicity of liver mononuclear cells following Con A injection, but not their FasL/Fas-dependent cytotoxicity. However, blockade of NKG2A-mediated signals during the cytotoxicity effector phase did not augment cytotoxic activity. Activated iNKT cells promptly disappeared after Con A injection, whereas NK1(-) iNKT cells, which preferentially expressed CD94/NKG2A, predominantly remained in the liver. Pretreatment with 20d5 appeared to facilitate disappearance of iNKT cells, particularly NK1(-) iNKT cells. Moreover, Con A-induced and alpha-galactosylceramide-induced hepatic injury was very severe in CD94/NKG2A-deficient DBA/2J mice compared with CD94/NKG2A-intact DBA/2JJcl mice. Overall, these results indicated that a NKG2A-mediated signal negatively regulates iNKT cell activation and hepatic injury.

Natural killer cell mediated cytotoxicity of tumor cells initiated by neem leaf preparation is associated with CD40–CD40L–mediated endogenous production of interleukin-12

Neem leaf preparation (NLP) was found to activate natural killer (NK) cells (CD56(+)CD3(-)) to enhance their cytotoxic ability to tumor cells and stimulate the release of interleukin-12 (IL-12) from macrophages from healthy individuals and head-and-neck squamous cell carcinoma patients. NLP upregulated cytotoxic (CD16(+) and CD56(dim)) NK cells, and the cytotoxicity of NK-sensitive K562 cells by NLP-stimulated peripheral blood mononuclear cells decreased significantly after IL-12 neutralization. This NK-mediated cytotoxicity was manifest by upregulation of IL-12-dependent intracellular expression of the perforin-granzyme B system. Moreover, NK cytotoxic function was abolished after use of concanamycin A, a perforin inhibitor, but not by brefeldin A, a Fas inhibitor, confirming the participation of the perforin-granzyme B system. In addition NLP upregulated the expression of CD40 in CD14(+) monocytes and CD40L in CD56(+) lymphocytes. Neutralization of CD40 and CD40L in NLP-stimulated peripheral blood mononuclear cells culture resulted in significant downregulation of IL-12 release and cytotoxicity of NK cells, demonstrating the role of a CD40-CD40L interaction in the observed functions. Signals involved in the NLP-induced release of IL-12, and thereby induction of NK cell cytotoxicity, are mediated by activating p38MAPK pathway, but not through the ERK1/2 signaling pathway. Overall the results suggest that NLP effects NK cellular cytotoxicity by CD40-CD40L-mediated endogenous production of IL-12, which critically controls perforin-dependent tumor cell cytotoxicity.

Residual active granzyme B in cathepsin C–null lymphocytes is sufficient for perforin-dependent target cell apoptosis

Cathepsin C activates serine proteases expressed in hematopoietic cells by cleaving an N-terminal dipeptide from the proenzyme upon granule packaging. The lymphocytes of cathepsin C–null mice are therefore proposed to totally lack granzyme B activity and perforin-dependent cytotoxicity. Surprisingly, we show, using live cell microscopy and other methodologies, that cells targeted by allogenic CD8+ cytotoxic T lymphocyte (CTL) raised in cathepsin C–null mice die through perforin-dependent apoptosis indistinguishable from that induced by wild-type CTL. The cathepsin C–null CTL expressed reduced but still appreciable granzyme B activity, but minimal granzyme A activity. Also, in contrast to mice with inactivation of both their granzyme A/B genes, cathepsin C deficiency did not confer susceptibility to ectromelia virus infection in vivo. Overall, our results indicate that although cathepsin C clearly generates the majority of granzyme B activity, some is still generated in its absence, pointing to alternative mechanisms for granzyme B processing and activation. Cathepsin C deficiency also results in considerably milder immune deficiency than perforin or granzyme A/B deficiency.