Patients and mice with deficiency in the SNARE protein SYNTAXIN-11 have a secondary B cell defect.

SYNTAXIN-11 (STX11) is a SNARE protein that mediates the fusion of cytotoxic granules with the plasma membrane at the immunological synapses of CD8 T or NK cells. Autosomal recessive inheritance of deleterious STX11 variants impairs cytotoxic granule exocytosis, causing familial hemophagocytic lymphohistiocytosis type 4 (FHL-4). In several FHL-4 patients, we also observed hypogammaglobulinemia, elevated frequencies of naive B cells, and increased double-negative DN2:DN1 B cell ratios, indicating a hitherto unrecognized role of STX11 in humoral immunity. Detailed analysis of Stx11-deficient mice revealed impaired CD4 T cell help for B cells, associated with disrupted germinal center formation, reduced isotype class switching, and low antibody avidity. Mechanistically, Stx11-/- CD4 T cells exhibit impaired membrane fusion leading to reduced CD107a and CD40L surface mobilization and diminished IL-2 and IL-10 secretion. Our findings highlight a critical role of STX11 in SNARE-mediated membrane trafficking and vesicle exocytosis in CD4 T cells, important for successful CD4 T cell-B cell interactions. Deficiency in STX11 impairs CD4 T cell-dependent B cell differentiation and humoral responses.

Gene editing of hematopoietic stem cells restores T-cell response in familial hemophagocytic lymphohistiocytosis.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder characterized by a life-threatening cytokine storm and immunopathology. Familial HLH type 3 (FHL3) accounts for approximately 30% of all inborn HLH cases worldwide. It is caused by mutations in the UNC13D gene that result in impaired degranulation of cytotoxic vesicles and hence compromised T-cell- and natural killer-cell-mediated killing. Current treatment protocols, including allogeneic hematopoietic stem cell (HSC) transplantation, still show high mortality. OBJECTIVE: We sought to develop and evaluate a curative genome editing strategy in the preclinical FHL3 Jinx mouse model. Jinx mice harbor a cryptic splice donor site in Unc13d intron 26 and develop clinical symptoms of human FHL3 upon infection with lymphocytic choriomeningitis virus (LCMV). METHODS: We employed clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technology to delete the disease-causing mutation in HSCs and transplanted Unc13d-edited stem cells into busulfan-conditioned Jinx recipient mice. Safety studies included extensive genotyping and chromosomal aberrations analysis by single targeted linker-mediated PCR sequencing (CAST-Seq)-based off-target analyses. Cure from HLH predisposition was assessed by LCMV infection. RESULTS: Hematopoietic cells isolated from transplanted mice revealed efficient gene editing (>95%), polyclonality of the T-cell receptor repertoire, and neither signs of off-target effects nor leukemogenesis. Unc13d transcription levels of edited and wild-type cells were comparable. While LCMV challenge resulted in acute HLH in Jinx mice transplanted with mock-edited HSCs, Jinx mice grafted with Unc13d-edited cells showed rapid virus clearance and protection from HLH. CONCLUSIONS: Our study demonstrates that transplantation of CRISPR-Cas edited HSCs supports the development of a functional polyclonal T-cell response in the absence of genotoxicity-associated clonal outgrowth.

Breast Cancer Stem Cell-Derived Tumors Escape from γδ T-cell Immunosurveillance In Vivo by Modulating γδ T-cell Ligands.

There are no targeted therapies for patients with triple-negative breast cancer (TNBC). TNBC is enriched in breast cancer stem cells (BCSC), which play a key role in metastasis, chemoresistance, relapse, and mortality. γδ T cells hold great potential in immunotherapy against cancer and might provide an approach to therapeutically target TNBC. γδ T cells are commonly observed to infiltrate solid tumors and have an extensive repertoire of tumor-sensing mechanisms, recognizing stress-induced molecules and phosphoantigens (pAgs) on transformed cells. Herein, we show that patient-derived triple-negative BCSCs are efficiently recognized and killed by ex vivo expanded γδ T cells from healthy donors. Orthotopically xenografted BCSCs, however, were refractory to γδ T-cell immunotherapy. We unraveled concerted differentiation and immune escape mechanisms: xenografted BCSCs lost stemness, expression of γδ T-cell ligands, adhesion molecules, and pAgs, thereby evading immune recognition by γδ T cells. Indeed, neither promigratory engineered γδ T cells, nor anti-PD-1 checkpoint blockade, significantly prolonged overall survival of tumor-bearing mice. BCSC immune escape was independent of the immune pressure exerted by the γδ T cells and could be pharmacologically reverted by zoledronate or IFNα treatment. These results pave the way for novel combinatorial immunotherapies for TNBC.

IFNγ binding to extracellular matrix prevents fatal systemic toxicity.

Interferon-γ (IFNγ) is an important mediator of cellular immune responses, but high systemic levels of this cytokine are associated with immunopathology. IFNγ binds to its receptor (IFNγR) and to extracellular matrix (ECM) via four positively charged C-terminal amino acids (KRKR), the ECM-binding domain (EBD). Across evolution, IFNγ is not well conserved, but the EBD is highly conserved, suggesting a critical function. Here, we show that IFNγ lacking the EBD (IFNγΔKRKR) does not bind to ECM but still binds to the IFNγR and retains bioactivity. Overexpression of IFNγΔKRKR in tumors reduced local ECM binding, increased systemic levels and induced sickness behavior, weight loss and toxicity. To analyze the function of the EBD during infection, we generated IFNγΔKRKR mice lacking the EBD by using CRISPR-Cas9. Infection with lymphocytic choriomeningitis virus resulted in higher systemic IFNγΔKRKR levels, enhanced sickness behavior, weight loss and fatal toxicity. We conclude that local retention of IFNγ is a pivotal mechanism to protect the organism from systemic toxicity during prolonged immune stimulation.

Adoptive T cell therapy cures mice from active hemophagocytic lymphohistiocytosis (HLH).

Primary hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome caused by impaired lymphocyte cytotoxicity. First-line therapeutic regimens directed against activated immune cells or secreted cytokines show limited efficacy since they do not target the underlying immunological problem: defective lymphocyte cytotoxicity causing prolonged immune stimulation. A potential rescue strategy would be the adoptive transfer of ex vivo gene-corrected autologous T cells. However, transfusion of cytotoxicity-competent T cells under conditions of hyperinflammation may cause more harm than benefit. As a proof-of-concept for adoptive T cell therapy (ATCT) under hyperinflammatory conditions, we transferred syngeneic, cytotoxicity-competent T cells into mice with virally triggered active primary HLH. ATCT with functional syngeneic trigger-specific T cells cured Jinx mice from active HLH without life-threatening side effects and protected Perforin-deficient mice from lethal HLH progression by reconstituting cytotoxicity. Cured mice were protected long-term from HLH relapses. A threshold frequency of transferred T cells with functional differentiation was identified as a predictive biomarker for long-term survival. This study is the first proof-of-concept for ATCT in active HLH.

Immunopathology caused by impaired CD8+ T-cell responses.

Recent findings indicate that many immunopathologies are at their roots a consequence of impaired immune responses ("too little" immunity) and not the result of primarily exaggerated immune responses ("too much" immunity). We have summarized this conceptional view as "IMPATH paradox." In this review, we will focus on impaired immune reactions in the context of CD8+ T-cell-mediated immunopathologies. In particular, we will exemplify this concept in two disease models: Virus-triggered primary hemophagocytic lymphohistiocytosis, an inflammatory syndrome caused by genetically impaired cytolytic functions of T cells, and viral hepatitis, where T-cell exhaustion is a major underlying mechanism for impaired effector functions. In both situations, T cells fail to eliminate the source of immune stimulation, which usually serves as an important negative feedback loop curtailing immune reactions. Persistent antigen presentation by APCs and/or infected cells results in continuous stimulation causing chronic inflammation and immunopathology mediated by residual T-cell functions. Hence, immune stimulation or reconstitution rather than immune suppression may be strategies for therapeutic interventions.

Eomes cannot replace its paralog T-bet during expansion and differentiation of CD8 effector T cells.

The two T-box transcription factors T-bet and Eomesodermin (Eomes) are important regulators of cytotoxic lymphocytes (CTLs), such as activated CD8 T cells, which are essential in the fight against intracellular pathogens and tumors. Both transcription factors share a great degree of homology based on sequence analysis and as a result exert partial functional redundancy during viral infection. However, the actual degree of redundancy between T-bet and Eomes remains a matter of debate and is further confounded by their distinct spatiotemporal expression pattern in activated CD8 T cells. To directly investigate the functional overlap of these transcription factors, we generated a new mouse model in which Eomes expression is under the transcriptional control of the endogenous Tbx21 (encoding for T-bet) locus. Applying this model, we demonstrate that the induction of Eomes in lieu of T-bet cannot rescue T-bet deficiency in CD8 T cells during acute lymphocytic choriomeningitis virus (LCMV) infection. We found that the expression of Eomes instead of T-bet was not sufficient for early cell expansion or effector cell differentiation. Finally, we show that imposed expression of Eomes after acute viral infection promotes some features of exhaustion but must act in concert with other factors during chronic viral infection to establish all hallmarks of exhaustion. In summary, our results clearly underline the importance of T-bet in guiding canonical CTL development during acute viral infections.

NK1.1+ innate lymphoid cells in salivary glands inhibit establishment of tissue-resident memory CD8+ T cells in mice.

NK1.1+ cells found in salivary glands (SG) represent a unique cell population of innate lymphoid cells (ILC) with characteristics of both conventional NK cells and ILC1. Here, we demonstrate that these NK1.1+  cells limit the accumulation and differentiation of virus-specific tissue-resident memory CD8+ T cells (TRM  cells) in SG of mice infected with lymphocytic choriomeningitis virus (LCMV). The negative regulation of LCMV-specific CD8+ TRM  cells by NK1.1+  cells in SG is independent of NKG2D, NKp46, TRAIL, and perforin. Moreover, analysis of NKp46iCre+ Eomesfl/fl mice revealed that Eomes-dependent conventional NK cells are dispensable for negative regulation. Since the SG are prone to autoimmune reactions, regulation of TRM  cells by tissue-resident ILC may be particularly important to prevent immunopathology in this organ.

Trigger-dependent differences determine therapeutic outcome in murine primary hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is a hyperinflammatory syndrome affecting patients with genetic cytotoxicity defects. Perforin-deficient (PKO) mice recapitulate the full clinical picture of FHL after infection with lymphocytic choriomeningitis virus (LCMV). Hyperactivated CD8+ T cells and IFN-γ have been identified as the key drivers of FHL and represent targets for therapeutic interventions. However, the response of patients is variable. This could be due to trigger-dependent differences in pathogenesis, which is difficult to address in FHL patients, since the trigger frequently escapes detection. We established an alternative FHL model using intravenous infection of PKO mice with murine CMV (MCMV)Smith . PKO mice developed acute FHL after both infections and fulfilled HLH diagnostic criteria accompanied by excessive IFN-γ production by disease-inducing T cells, that enrich in the BM. However, direct comparison of the two infection models disclosed trigger-dependence of FHL progression and revealed a higher contribution of CD4 T cells and NK cells to IFN-γ production after MCMV infection. Importantly, therapeutic intervention by IFN-γ neutralization or CD8+ T-cell depletion had less benefit in MCMV-triggered FHL compared to LCMV-triggered FHL, likely due to MCMV-induced cytopathology. Thus, the context of the specific triggering viral infection can impact the success of targeted immunotherapeutic HLH control.

Retroviral UNC13D Gene Transfer Restores Cytotoxic Activity of T Cells Derived from Familial Hemophagocytic Lymphohistiocytosis Type 3 Patients In Vitro.

Familial hemophagocytic lymphohistiocytosis (FHL) is a group of life-threatening, autosomal recessive disorders of severe hyperinflammation. FHL type 3 (FHL-3) accounts for about 30% of FHL cases. It is characterized by mutations in the UNC13D gene that give rise to functionally impaired or absent Munc13-4 protein, resulting in impaired secretion of lytic granules by cytotoxic lymphocytes. Etoposide-based therapy is currently used as the standard of care that results in around 60% 5-year survival, illustrating the need for novel treatment approaches. Key problems include treatment toxicity and failure to induce or maintain remission of the hyperinflammation. Instead of immunosuppression, transplantation of autologous gene-corrected T cells can be envisaged as an approach to restore the impaired immune reaction. This study established a protocol that enabled hyperactivated, FHL-3 patient-derived T cells to be cultured and a codon-optimized UNC13D expression cassette to be delivered by either alpha- or gamma-retroviral gene transfer. The data demonstrate that the established protocol can be applied to FHL-3 patient cells with various genetic backgrounds and that gamma-retroviral UNC13D transfer restored expression of functional Munc13-4, as well as degranulation capacity and cell-mediated cytotoxicity of those patient-derived CD8+ T cells. Furthermore, the study shows that the co-introduction of a truncated low-affinity nerve growth factor receptor coding sequence enabled the therapeutic effect to be optimized by enriching transduced cells in a Good Manufacturing Practice-compliant manner. In conclusion, this study lays the foundation for an adaptive immune cell therapy approach aiming at immunological stabilization of FHL-3 patients with autologous, immune-competent T cells prior to hematopoietic stem-cell transplantation.

Regulatory T cells characterized by low Id3 expression are highly suppressive and accumulate during chronic infection.

Foxp3+ regulatory T (Treg) cells are broadly divided into naive-like and activated Treg cells, however recent studies suggest further Treg cell heterogeneity. Treg cells contribute to impaired T cell responses in chronic infections, but the role of specific Treg cell subpopulations in viral infections is not well defined. Here, we report that activated Treg cells are separated into two transcriptionally distinct subpopulations characterized by low or high expression of the transcriptional regulator Id3. Id3lo Treg cells are a highly suppressive Treg cell subpopulation, expressing elevated levels of immunomodulatory molecules and are capable of broadly targeting T cell responses. Viral infection and interleukin-2 promote the differentiation of Id3hi into Id3lo Treg cells and during chronic infection Id3lo Treg cells are the predominant Treg cell population. Thus, our report provides a framework, in which different activated Treg cell subpopulations specifically affect immune responses, possibly contributing to T cell dysfunction in chronic infections.

Immunological phenotype of the murine Lrba knockout.

Biallelic mutations in the human lipopolysaccharide responsive beige-like anchor (LRBA) gene lead to a primary immunodeficiency known as LRBA deficiency, characterized by a broad range of clinical manifestations including autoimmunity, organomegaly, hypogammaglobulinemia and recurrent infections. Considering the phenotypic heterogeneity in patients and the severity of the disease, our aim was to assess the role of LRBA in immune cells and to understand the underlying pathomechanisms through the study of a Lrba knockout (Lrba-/-) mouse model. LRBA-deficient mice did not show severe clinical or immunological signs of disease, either at steady state under specific-pathogen-free conditions, after vaccination with T-dependent and T-independent antigens, or in the context of acute infections with lymphocytic choriomeningitis virus (LCMV) or Salmonella Typhimurium. Although Lrba-/- mice were able to produce normal serum immunoglobulin M (IgM) and IgG and to mount a specific immune response after immunization, they showed elevated serum and secretory basal IgA levels. LRBA was dispensable for B- and T-cell development, as well as for in vitro B-cell proliferation, survival, isotype switching and plasmablast differentiation. Interestingly, Lrba-/- mice displayed decreased cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) expression by regulatory T cells and activated conventional CD4+ and CD8+ T lymphocytes, reduced frequency of peritoneal B-1a cells along with diminished interleukin-10 production and increased percentages of T follicular helper cells in Peyer's patches, but without developing overt signs of autoimmunity. Our findings expand the role of LRBA in immune regulatory mechanisms previously reported in patients, and suggest a novel role in IgA production that is crucial for the protection of mucosal surfaces and gut-associated immune tolerance.

Id3 Controls Cell Death of 2B4+ Virus-Specific CD8+ T Cells in Chronic Viral Infection.

Sustained Ag persistence in chronic infection results in a deregulated CD8(+) T cell response that is characterized by T cell exhaustion and cell death of Ag-specific CD8(+) T cells. Yet, the underlying transcriptional mechanisms regulating CD8(+) T cell exhaustion and cell death are poorly defined. Using the experimental mouse model of lymphocytic choriomeningitis virus infection, we demonstrate that the transcriptional regulator Id3 controls cell death of virus-specific CD8(+) T cells in chronic infection. By comparing acute and chronic infection, we showed that Id3 (-) virus-specific CD8(+) T cells were less abundant, whereas the absolute numbers of Id3 (+) virus-specific CD8(+) T cells were equal in chronic and acute infection. Phenotypically, Id3 (-) and Id3 (+) cells most prominently differed with regard to expression of the surface receptor 2B4; although Id3 (-) cells were 2B4(+), almost all Id3 (+) cells lacked expression of 2B4. Lineage-tracing experiments showed that cells initially expressing Id3 differentiated into Id3 (-)2B4(+) cells; in turn, these cells were terminally differentiated and highly susceptible to cell death under conditions of persisting Ag. Enforced Id3 expression specifically increased the persistence of 2B4(+) virus-specific CD8(+) T cells by decreasing susceptibility to Fas/Fas ligand-mediated cell death. Thus, our findings reveal that the transcriptional regulator Id3 promotes the survival of virus-specific CD8(+) T cells in chronic infection and suggest that targeting Id3 might be beneficial for preventing cell death of CD8(+) T cells in chronic infection or for promoting cell death of uncontrolled, hyperactive CD8(+) T cells to prevent immunopathology.

The viral context instructs the redundancy of costimulatory pathways in driving CD8(+) T cell expansion.

Signals delivered by costimulatory molecules are implicated in driving T cell expansion. The requirements for these signals, however, vary from dispensable to essential in different infections. We examined the underlying mechanisms of this differential T cell costimulation dependence and found that the viral context determined the dependence on CD28/B7-mediated costimulation for expansion of naive and memory CD8(+) T cells, indicating that the requirement for costimulatory signals is not imprinted. Notably, related to the high-level costimulatory molecule expression induced by lymphocytic choriomeningitis virus (LCMV), CD28/B7-mediated costimulation was dispensable for accumulation of LCMV-specific CD8(+) T cells because of redundancy with the costimulatory pathways induced by TNF receptor family members (i.e., CD27, OX40, and 4-1BB). Type I IFN signaling in viral-specific CD8(+) T cells is slightly redundant with costimulatory signals. These results highlight that pathogen-specific conditions differentially and uniquely dictate the utilization of costimulatory pathways allowing shaping of effector and memory antigen-specific CD8(+) T cell responses.

T cell expansion is the limiting factor of virus control in mice with attenuated TCR signaling: implications for human immunodeficiency.

Defining the minimal thresholds for effective antiviral T cell immunity is important for clinical decisions in immunodeficient patients. TCR signaling is critical for T cell development, activation, and effector functions. In this article, we analyzed which of these TCR-mediated processes is limiting for antiviral immunity in a mouse strain with reduced expression of SLP-76 (twp mice). Despite severe T cell activation defects in vitro, twp mice generated a normal proportion of antiviral effector T cells postinfection with lymphocytic choriomeningitis virus (LCMV). Twp CD8(+) T cells showed impaired polyfunctional cytokine production, whereas cytotoxicity as the crucial antiviral effector function for LCMV control was normal. The main limiting factor in the antiviral response of twp mice was impaired T cell proliferation and survival, leading to a 5- to 10-fold reduction of antiviral T cells at the peak of the immune response. This was still sufficient to control infection with the LCMV Armstrong strain, but the more rapidly replicating LCMV-WE induced T cell exhaustion and viral persistence. Thus, under conditions of impaired TCR signaling, reduced T cell expansion was the limiting factor in antiviral immunity. These findings have implications for understanding antiviral immunity in patients with T cell deficiencies.

Helicobacter pylori protects oncogenically transformed cells from reactive oxygen species-mediated intercellular induction of apoptosis.

Malignant transformation of gastric epithelial cells by chronic Helicobacter pylori infection is caused by several mechanisms including attraction of reactive oxygen species (ROS)-producing neutrophils and cytotoxin-associated antigen A-mediated dysplastic alterations. Here we show that H.pylori protects transformed cells from ROS-mediated intercellular induction of apoptosis. This potential control step in oncogenesis depends on the HOCl and NO/peroxynitrite (PON) signaling pathways. Helicobacter pylori-associated catalase and superoxide dismutase (SOD) efficiently cooperate in the inhibition of HOCl and the NO/PON signaling pathways. Helicobacter pylori catalase prevents HOCl synthesis through decomposition of hydrogen peroxide. Helicobacter pylori-associated SOD interferes with the crucial interactions between superoxide anions and HOCl, as well as superoxide anions and NO. The ratio of bacteria to malignant cells is critical for sufficient protection of transformed cells. Low concentrations of H.pylori more efficiently inhibited ROS-mediated destruction of transformed cells when compared with high concentrations of bacteria. Our data demonstrate the critical role of H.pylori antioxidant enzymes in the survival of transformed cells, modulating an early step of oncogenesis that is distinct from the transformation process per se.

Coinhibitory suppression of T cell activation by CD40 protects against obesity and adipose tissue inflammation in mice.

BACKGROUND: Costimulatory cascades such as the CD40L-CD40 dyad enhance immune cell activation and inflammation during atherosclerosis. Here, we tested the hypothesis that CD40 directly modulates traits of the metabolic syndrome in diet-induced obesity in mice. METHODS AND RESULTS: To induce the metabolic syndrome, wild-type or CD40(-/-) mice consumed a high-fat diet for 20 weeks. Unexpectedly, CD40(-/-) mice exhibited increased weight gain, impaired insulin secretion, augmented accumulation of inflammatory cells in adipose tissue, and enhanced proinflammatory gene expression. This proinflammatory and adverse metabolic phenotype could be transplanted into wild-type mice by reconstitution with CD40-deficient lymphocytes, indicating a major role for CD40 in T or B cells in this context. Conversely, therapeutic activation of CD40 signaling by the stimulating antibody FGK45 abolished further weight gain during the study, lowered glucose levels, improved insulin sensitivity, and suppressed adipose tissue inflammation. Mechanistically, CD40 activation decreased the expression of proinflammatory cytokines in T cells but not in B cells or macrophages. Finally, repopulation of lymphocyte-free Rag1(-/-) mice with CD40(-/-) T cells provoked dysmetabolism and inflammation, corroborating a protective role of CD40 on T cells in the metabolic syndrome. Finally, levels of soluble CD40 showed a positive association with obesity in humans, suggesting clinical relevance of our findings. CONCLUSIONS: We present the surprising finding that CD40 deficiency on T cells aggravates whereas activation of CD40 signaling improves adipose tissue inflammation and its metabolic complications. Therefore, positive modulation of the CD40 pathway might describe a novel therapeutic concept against cardiometabolic disease.