Early induction of cytokine release syndrome by rapidly generated CAR T cells in preclinical models.

Cytokine release syndrome (CRS) is a significant side-effect of conventional chimeric antigen receptor (CAR) T-cell therapy. To facilitate patient accessibility, short-term (st) CAR T cells, which are administered to patients only 24 h after vector exposure, are in focus of current investigations. Their impact on the incidence and severity of CRS has been poorly explored. Here, we evaluated CD19-specific stCAR T cells in preclinical models. In co-culture with tumor cells and monocytes, stCAR T cells exhibited anti-tumoral activity and potent release of CRS-related cytokines (IL-6, IFN-γ, TNF-α, GM-CSF, IL-2, IL-10). When administered to NSG-SGM3 mice, stCAR T cells, but not conventional CAR T cells, induced severe acute adverse events within 24 h, including hypothermia and weight loss, as well as high body scores, independent of the presence of tumor target cells. Human (IFN-γ, TNF-α, IL-2, IL-10) and murine (MCP-1, IL-6, G-CSF) cytokines, typical for severe CRS, were systemically elevated. Our data highlight potential safety risks of rapidly manufactured CAR T cells and suggest NSG-SGM3 mice as sensitive model for their preclinical safety evaluation.

AAV vectors displaying bispecific DARPins enable dual-control targeted gene delivery.

Precise delivery of genes to therapy-relevant cells is crucial for in vivo gene therapy. Receptor-targeting as prime strategy for this purpose is limited to cell types defined by a single cell-surface marker. Many target cells are characterized by combinations of more than one marker, such as the HIV reservoir cells. Here, we explored the tropism of adeno-associated viral vectors (AAV2) displaying designed ankyrin repeat proteins (DARPins) mono- and bispecific for CD4 and CD32a. Cryo-electron tomography revealed an unaltered capsid structure in the presence of DARPins. Surprisingly, bispecific AAVs transduced CD4/CD32a double-positive cells at much higher efficiencies than single-positive cells, even if present in low amounts in cell mixtures or human blood. This preference was confirmed when vector particles were systemically administered into mice. Cell trafficking studies revealed an increased cell entry rate for bispecific over monospecific AAVs. When equipped with an HIV genome-targeting CRISPR/Cas cassette, the vectors prevented HIV replication in T cell cultures. The data provide proof-of-concept for high-precision gene delivery through tandem-binding regions on AAV. Reminiscent of biological products following Boolean logic AND gating, the data suggest a new option for receptor-targeted vectors to improve the specificity and safety of in vivo gene therapy.

CAR Gene Delivery by T-cell Targeted Lentiviral Vectors is Enhanced by Rapamycin Induced Reduction of Antiviral Mechanisms.

Lentiviral vectors (LV) have become the dominant tool for stable gene transfer into lymphocytes including chimeric antigen receptor (CAR) gene delivery to T cells, a major breakthrough in cancer therapy. Yet, room for improvement remains, especially for the latest LV generations delivering genes selectively into T cell subtypes, a key requirement for in vivo CAR T cell generation. Toward improving gene transfer rates with these vectors, whole transcriptome analyses on human T lymphocytes are conducted after exposure to CAR-encoding conventional vectors (VSV-LV) and vectors targeted to CD8+ (CD8-LV) or CD4+ T cells (CD4-LV). Genes related to quiescence and antiviral restriction are found to be upregulated in CAR-negative cells exposed to all types of LVs. Down-modulation of various antiviral restriction factors, including the interferon-induced transmembrane proteins (IFITMs) is achieved with rapamycin as verified by mass spectrometry (LC-MS). Strikingly, rapamycin enhances transduction by up to 7-fold for CD8-LV and CD4-LV without compromising CAR T cell activities but does not improve VSV-LV. When administered to humanized mice, CD8-LV results in higher rates of green fluorescent protein (GFP) gene delivery. Also in vivo CAR T cell generation is improved in kinetics and tumor control, however to a moderate extent, leaving room for improvement by optimizing the rapamycin administration schedule. The data favor multi-omics approaches for improvements in gene delivery.

CD62L as target receptor for specific gene delivery into less differentiated human T lymphocytes.

Chimeric antigen receptor (CAR)-expressing T cells are a complex and heterogeneous gene therapy product with variable phenotype compositions. A higher proportion of less differentiated CAR T cells is usually associated with improved antitumoral function and persistence. We describe in this study a novel receptor-targeted lentiviral vector (LV) named 62L-LV that preferentially transduces less differentiated T cells marked by the L-selectin receptor CD62L, with transduction rates of up to 70% of CD4+ and 50% of CD8+ primary T cells. Remarkably, higher amounts of less differentiated T cells are transduced and preserved upon long-term cultivation using 62L-LV compared to VSV-LV. Interestingly, shed CD62L neither altered the binding of 62L-LV particles to T cells nor impacted their transduction. The incubation of 2 days of activated T lymphocytes with 62L-LV or VSV-LV for only 24 hours was sufficient to generate CAR T cells that controlled tumor growth in a leukemia tumor mouse model. The data proved that potent CAR T cells can be generated by short-term ex vivo exposure of primary cells to LVs. As a first vector type that preferentially transduces less differentiated T lymphocytes, 62L-LV has the potential to circumvent cumbersome selections of T cell subtypes and offers substantial shortening of the CAR T cell manufacturing process.

Highly Efficient Generation of Transgenically Augmented CAR NK Cells Overexpressing CXCR4.

Natural killer (NK) cells are a noteworthy lymphocyte subset in cancer adoptive cell therapy. NK cells initiate innate immune responses against infections and malignancies with natural cytotoxicity, which is independent of foreign antigen recognition. Based on these substantive features, genetically modifying NK cells is among the prime goals in immunotherapy but is currently difficult to achieve. Recently, we reported a fully human CAR19 construct (huCAR19) with remarkable function in gene-modified T-cells. Here, we show efficient and stable gene delivery of huCAR19 to primary human NK cells using lentiviral vectors with transduction efficiencies comparable to those achieved with NK cell lines. These huCAR19 NK cells display specific and potent cytotoxic activity against target cells. To improve homing of NK cells to the bone marrow, we augmented huCAR19 NK cells with the human CXCR4 gene, resulting in transgenically augmented CAR NK cells (TRACKs). Compared to conventional CAR NK cells, TRACKs exhibit enhanced migration capacity in response to recombinant SDF-1 or bone marrow stromal cells while retaining functional and cytolytic activity against target cells. Based on these promising findings, TRACKs may become a novel candidate for immunotherapeutic strategies in clinical applications.

In Vivo Generation of CAR T Cells Selectively in Human CD4+ Lymphocytes.

T cells modified with CD19-specific chimeric antigen receptors (CARs) result in significant clinical benefit for leukemia patients but constitute a challenge for manufacturing. We have recently demonstrated the in vivo generation of CD19-CAR T cells using the CD8-targeted lentiviral vector (CD8-LV). In this study, we investigated the in vivo generation of CD4+ CAR T cells using CD4-targeted LV (CD4-LV). Administration of CD4-LV into NSG mice transplanted with human peripheral blood mononuclear cells (PBMCs) led to 40%-60% of human CD4+ lymphocytes being CAR positive while CD8+ cells remained CAR negative. CAR+ T cells displayed a T helper 1 (Th1)/Th2 phenotype, which was accompanied by CD19+ B cell elimination. Intravenous administration of CD4-LV into NSG mice reconstituted with human CD34+ cells induced CAR expression and B cell elimination within 2-3 weeks post-injection. Preclinical analysis in a tumor mouse model revealed that mice administered CD4-LV exhibited faster and superior tumor cell killing compared to mice injected with CD8-LV alone or as a mixture with CD4-LV. Further analysis suggests that CD4+CAR+ cells may outperform CD8+CAR+ cells, especially at a high burden of target antigen, mainly since CD8 cells are more prone to exhaustion. This first description of in vivo-generated CD4+ CAR T cells supports their importance for cellular therapy.

In vivo generated human CAR T cells eradicate tumor cells.

Chimeric antigen receptor (CAR) T cells are in prime focus of current research in cancer immunotherapy. Facilitating CAR T cell generation is among the top goals. We have recently demonstrated direct in vivo generation of human CD19-CAR T cells by targeting CD8+ cells using lentiviral vectors (LVs). The anti-tumor potency of in vivo generated CAR T cells was assessed in human PBMC-transplanted NSG mice carrying i.v. injected CD19+ Nalm-6 tumor cells. A single injection of CD8-targeted LV delivering CD19-CAR was sufficient to completely eliminate the tumor cells from bone marrow and spleen, whereas control animals contained high levels of CD19+ cells. Tumor elimination was due to in vivo generated CAR+ cells. Notably, these were not only composed of T lymphocytes but also included CAR+ natural killer cells (NK and NKT). This is the first demonstration of tumor elimination by in vivo generated human CAR T cells.

GluA4-Targeted AAV Vectors Deliver Genes Selectively to Interneurons while Relying on the AAV Receptor for Entry.

Selective gene delivery into subtypes of interneurons remains an important challenge in vector development. Adeno-associated virus (AAV) vector particles are especially promising for intracerebral injections. For cell entry, AAV2 particles are supposed to attach to heparan-sulfate proteoglycans (HSPGs) followed by endocytosis via the AAV receptor (AAVR). Here, we assessed engineered AAV particles deficient in HSPG attachment but competent in recognizing the glutamate receptor 4 (GluA4, also known as GluRD or GRIA4) through a displayed GluA4-specific DARPin (designed ankyrin repeat protein). When injected into the mouse brain, histological evaluation revealed that in various regions, more than 90% of the transduced cells were interneurons, mainly of the parvalbumin-positive subtype. Although part of the selectivity was mediated by the DARPin, the chosen spleen focus-forming virus (SFFV) promoter had contributed as well. Further analysis revealed that the DARPin mediated selective attachment to GluA4-positive cells, whereas gene delivery required expression of AAVR. Our data suggest that cell selectivity of AAV particles can be modified rationally and efficiently through DARPins, but expression of the AAV entry receptor remains essential.

Tumor-Specific Delivery of Immune Checkpoint Inhibitors by Engineered AAV Vectors.

Immune checkpoint inhibitors (ICIs) can block distinct receptors on T cells or tumor cells thus preventing T cell inactivation and tumor immune escape. While the clinical response to treatment with ICIs in cancer patients is impressive, this therapy is often associated with a number of immune-related adverse events. There is therefore a need to explore innovative strategies of tumor-specific delivery of ICIs. Delivery of therapeutic proteins on a genetic level can be accomplished with viral vectors including those derived from adeno-associated virus (AAV). Here, we assessed the tumor-targeted Her2-AAV, a receptor-targeted AAV vector binding to the tumor antigen Her2/neu for cell entry, as vehicle for ICI gene delivery. Initially, we packaged the coding sequence of a scFv-Fc fusion protein directed against mouse programmed cell death protein-1 (PD-1) into Her2-AAV. Upon transduction of Her2/neu+ RENCA cells, AAV-encoded αPD-1 was readily detectable in the cell culture supernatant and revealed specific binding to its target antigen. In vivo, in BALB/c mice bearing subcutaneous RENCA-Her2/neu tumors, Her2-AAV mediated specific gene delivery into tumor tissue upon intravenous administration as verified by luciferase gene transfer and in vivo imaging thus demonstrating unimpaired tumor-targeting by Her2-AAV vectors in immunocompetent animals. When delivering the αPD-1 gene, levels of ICI were similar in tumor tissue for Her2-AAV and AAV2 but substantially reduced in liver for Her2-AAV. When combined with chemotherapy a tendency for reduced progression of tumor growth was documented for Her2-AAV treated mice. To get closer to the clinical situation, AAV constructs that deliver the complete coding sequence of the therapeutic antibody nivolumab which is directed against human PD-1 were generated next. The AAV-Nivolumab constructs were expressed and released from transduced MDA-MB-453 cells in vitro and from RENCA-Her2/neu cells upon intratumoral as well as intravenous administration in vivo. Antibody processing and expression levels were further improved through optimization of construct design. In conclusion, we provide proof-of-principle for redirecting the biodistribution of ICIs from liver and serum to tumor tissue by the use of engineered AAV vectors. This strategy can be easily combined with other types of immunotherapeutic concepts.

In vivo generation of human CD19-CAR T cells results in B-cell depletion and signs of cytokine release syndrome.

Chimeric antigen receptor (CAR) T cells brought substantial benefit to patients with B-cell malignancies. Notwithstanding, CAR T-cell manufacturing requires complex procedures impeding the broad supply chain. Here, we provide evidence that human CD19-CAR T cells can be generated directly in vivo using the lentiviral vector CD8-LV specifically targeting human CD8+ cells. Administration into mice xenografted with Raji lymphoma cells and human peripheral blood mononuclear cells led to CAR expression solely in CD8+ T cells and efficacious elimination of CD19+ B cells. Further, upon injection of CD8-LV into mice transplanted with human CD34+ cells, induction of CAR T cells and CD19+ B-cell depletion was observed in 7 out of 10 treated animals. Notably, three mice showed elevated levels of human cytokines in plasma. Tissue-invading CAR T cells and complete elimination of the B-lymphocyte-rich zones in spleen were indicative of a cytokine release syndrome. Our data demonstrate the feasibility of in vivo reprogramming of human CD8+ CAR T cells active against CD19+ cells, yet with similar adverse effects currently notorious in the clinical practice.

Vitamin A-Retinoic Acid Signaling Regulates Hematopoietic Stem Cell Dormancy.

Dormant hematopoietic stem cells (dHSCs) are atop the hematopoietic hierarchy. The molecular identity of dHSCs and the mechanisms regulating their maintenance or exit from dormancy remain uncertain. Here, we use single-cell RNA sequencing (RNA-seq) analysis to show that the transition from dormancy toward cell-cycle entry is a continuous developmental path associated with upregulation of biosynthetic processes rather than a stepwise progression. In addition, low Myc levels and high expression of a retinoic acid program are characteristic for dHSCs. To follow the behavior of dHSCs in situ, a Gprc5c-controlled reporter mouse was established. Treatment with all-trans retinoic acid antagonizes stress-induced activation of dHSCs by restricting protein translation and levels of reactive oxygen species (ROS) and Myc. Mice maintained on a vitamin A-free diet lose HSCs and show a disrupted re-entry into dormancy after exposure to inflammatory stress stimuli. Our results highlight the impact of dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity. VIDEO ABSTRACT.

DNA-damage response gene GADD45A induces differentiation in hematopoietic stem cells without inhibiting cell cycle or survival.

Hematopoietic stem cells (HSCs) maintain blood cell production life-long by their unique abilities of self-renewal and differentiation into all blood cell lineages. Growth arrest and DNA-damage-inducible 45 alpha (GADD45A) is induced by genotoxic stress in HSCs. GADD45A has been implicated in cell cycle control, cell death and senescence, as well as in DNA-damage repair. In general, GADD45A provides cellular stability by either arresting the cell cycle progression until DNA damage is repaired or, in cases of fatal damage, by inducing apoptosis. However, the function of GADD45A in hematopoiesis remains controversial. We revealed the changes in murine HSC fate control orchestrated by the expression of GADD45A at single cell resolution. In contrast to other cellular systems, GADD45A expression did not cause a cell cycle arrest or an alteration in the decision between cell survival and apoptosis in HSCs. Strikingly, GADD45A strongly induced and accelerated the differentiation program in HSCs. Continuous tracking of individual HSCs and their progeny via time-lapse microscopy elucidated that once GADD45A was expressed, HSCs differentiate into committed progenitors within 29 hours. GADD45A-expressing HSCs failed to long-term reconstitute the blood of recipients by inducing multilineage differentiation in vivo. Importantly, γ-irradiation of HSCs induced their differentiation by upregulating endogenous GADD45A. The differentiation induction by GADD45A was transmitted by activating p38 Mitogen-activated protein kinase (MAPK) signaling and allowed the generation of megakaryocytic-erythroid, myeloid, and lymphoid lineages. These data indicate that genotoxic stress-induced GADD45A expression in HSCs prevents their fatal transformation by directing them into differentiation and thereby clearing them from the system.

STAT5-regulated microRNA-193b controls haematopoietic stem and progenitor cell expansion by modulating cytokine receptor signalling.

Haematopoietic stem cells (HSCs) require the right composition of microRNAs (miR) for proper life-long balanced blood regeneration. Here we show a regulatory circuit that prevents excessive HSC self-renewal by upregulation of miR-193b upon self-renewal promoting thrombopoietin (TPO)-MPL-STAT5 signalling. In turn, miR-193b restricts cytokine signalling, by targeting the receptor tyrosine kinase c-KIT. We generated a miR-193b knockout mouse model to unravel the physiological function of miR-193b in haematopoiesis. MiR-193b(-/-) mice show a selective gradual enrichment of functional HSCs, which are fully competent in multilineage blood reconstitution upon transplantation. The absence of miR-193b causes an accelerated expansion of HSCs, without altering cell cycle or survival, but by decelerating differentiation. Conversely, ectopic miR-193b expression restricts long-term repopulating HSC expansion and blood reconstitution. MiR-193b-deficient haematopoietic stem and progenitor cells exhibit increased basal and cytokine-induced STAT5 and AKT signalling. This STAT5-induced microRNA provides a negative feedback for excessive signalling to restrict uncontrolled HSC expansion.

Single-Stranded DNA-Binding Transcriptional Regulator FUBP1 Is Essential for Fetal and Adult Hematopoietic Stem Cell Self-Renewal.

The ability of hematopoietic stem cells (HSCs) to self-renew is a prerequisite for the establishment of definitive hematopoiesis and life-long blood regeneration. Here, we report the single-stranded DNA-binding transcriptional regulator far upstream element (FUSE)-binding protein 1 (FUBP1) as an essential factor of HSC self-renewal. Functional inactivation of FUBP1 in two different mouse models resulted in embryonic lethal anemia at around E15.5 caused by severely diminished HSCs. Fetal and adult HSCs lacking FUBP1 revealed an HSC-intrinsic defect in their maintenance, expansion, and long-term blood reconstitution, but could differentiate into all hematopoietic lineages. FUBP1-deficient adult HSCs exhibit significant transcriptional changes, including upregulation of the cell-cycle inhibitor p21 and the pro-apoptotic Noxa molecule. These changes caused an increase in generation time and death of HSCs as determined by video-microscopy-based tracking. Our data establish FUBP1 and its recognition of single-stranded genomic DNA as an important element in the transcriptional regulation of HSC self-renewal.

The functional interplay between the t(9;22)-associated fusion proteins BCR/ABL and ABL/BCR in Philadelphia chromosome-positive acute lymphatic leukemia.

The hallmark of Philadelphia chromosome positive (Ph(+)) leukemia is the BCR/ABL kinase, which is successfully targeted by selective ATP competitors. However, inhibition of BCR/ABL alone is unable to eradicate Ph(+) leukemia. The t(9;22) is a reciprocal translocation which encodes not only for the der22 (Philadelphia chromosome) related BCR/ABL, but also for der9 related ABL/BCR fusion proteins, which can be detected in 65% of patients with chronic myeloid leukemia (CML) and 100% of patients with Ph+ acute lymphatic leukemia (ALL). ABL/BCRs are oncogenes able to influence the lineage commitment of hematopoietic progenitors. Aim of this study was to further disclose the role of p96(ABL/BCR) for the pathogenesis of Ph(+) ALL. The co-expression of p96(ABL/BCR) enhanced the kinase activity and as a consequence, the transformation potential of p185(BCR/ABL). Targeting p96(ABL/BCR) by RNAi inhibited growth of Ph(+) ALL cell lines and Ph(+) ALL patient-derived long-term cultures (PD-LTCs). Our in vitro and in vivo stem cell studies further revealed a functional hierarchy of p96(ABL/BCR) and p185(BCR/AB)L in hematopoietic stem cells. Co-expression of p96(ABL/BCR) abolished the capacity of p185(BCR/ABL) to induce a CML-like disease and led to the induction of ALL. Taken together our here presented data reveal an important role of p96(ABL/BCR) for the pathogenesis of Ph(+) ALL.

Exit from dormancy provokes DNA-damage-induced attrition in haematopoietic stem cells.

Haematopoietic stem cells (HSCs) are responsible for the lifelong production of blood cells. The accumulation of DNA damage in HSCs is a hallmark of ageing and is probably a major contributing factor in age-related tissue degeneration and malignant transformation. A number of accelerated ageing syndromes are associated with defective DNA repair and genomic instability, including the most common inherited bone marrow failure syndrome, Fanconi anaemia. However, the physiological source of DNA damage in HSCs from both normal and diseased individuals remains unclear. Here we show in mice that DNA damage is a direct consequence of inducing HSCs to exit their homeostatic quiescent state in response to conditions that model physiological stress, such as infection or chronic blood loss. Repeated activation of HSCs out of their dormant state provoked the attrition of normal HSCs and, in the case of mice with a non-functional Fanconi anaemia DNA repair pathway, led to a complete collapse of the haematopoietic system, which phenocopied the highly penetrant bone marrow failure seen in Fanconi anaemia patients. Our findings establish a novel link between physiological stress and DNA damage in normal HSCs and provide a mechanistic explanation for the universal accumulation of DNA damage in HSCs during ageing and the accelerated failure of the haematopoietic system in Fanconi anaemia patients.

Cytokine-regulated GADD45G induces differentiation and lineage selection in hematopoietic stem cells.

The balance of self-renewal and differentiation in long-term repopulating hematopoietic stem cells (LT-HSC) must be strictly controlled to maintain blood homeostasis and to prevent leukemogenesis. Hematopoietic cytokines can induce differentiation in LT-HSCs; however, the molecular mechanism orchestrating this delicate balance requires further elucidation. We identified the tumor suppressor GADD45G as an instructor of LT-HSC differentiation under the control of differentiation-promoting cytokine receptor signaling. GADD45G immediately induces and accelerates differentiation in LT-HSCs and overrides the self-renewal program by specifically activating MAP3K4-mediated MAPK p38. Conversely, the absence of GADD45G enhances the self-renewal potential of LT-HSCs. Videomicroscopy-based tracking of single LT-HSCs revealed that, once GADD45G is expressed, the development of LT-HSCs into lineage-committed progeny occurred within 36 hr and uncovered a selective lineage choice with a severe reduction in megakaryocytic-erythroid cells. Here, we report an unrecognized role of GADD45G as a central molecular linker of extrinsic cytokine differentiation and lineage choice control in hematopoiesis.

Incomplete cytokinesis and re-fusion of small mononucleated Hodgkin cells lead to giant multinucleated Reed-Sternberg cells.

Multinucleated Reed-Sternberg (RS) cells are pathognomonic for classical Hodgkin lymphoma (HL), and their presence is essential for diagnosis. How these giant tumor cells develop is controversial, however. It has been postulated that RS cells arise from mononucleated Hodgkin cells via endomitosis. Conversely, continuous single-cell tracking of HL cell lines by long-term time-lapse microscopy has identified cell fusion as the main route of RS cell formation. In contrast to growth-induced formation of giant Hodgkin cells, fusion of small mononuclear cells followed by a size increase gives rise to giant RS cells. Of note, fusion of cells originating from the same ancestor, termed re-fusion, is seen nearly exclusively. In the majority of cases, re-fusion of daughter cells is preceded by incomplete cytokinesis, as demonstrated by microtubule bonds among the cells. We confirm at the level of individual tracked cells that giant Hodgkin and RS cells have little proliferative capacity, further supporting small mononuclear Hodgkin cells as the proliferative compartment of the HL tumor clone. In addition, sister cells show a shared propensity for re-fusion, providing evidence of early RS cell fate commitment. Thus, RS cell generation is related neither to cell fusion of unrelated Hodgkin cells nor to endomitosis, but rather is mediated by re-fusion of daughter cells that underwent mitosis. This surprising finding supports the existence of a unique mechanism for the generation of multinuclear RS cells that may have implications beyond HL, given that RS-like cells are frequently observed in several other lymphoproliferative diseases as well.

Immune modulation by Fas ligand reverse signaling: lymphocyte proliferation is attenuated by the intracellular Fas ligand domain.

Fas ligand (FasL) not only induces apoptosis in Fas receptor-bearing target cells, it is also able to transmit signals into the FasL-expressing cell via its intracellular domain (ICD). Recently, we described a Notch-like proteolytic processing of FasL that leads to the release of the FasL ICD into the cytoplasm and subsequent translocation into the nucleus where it may influence gene transcription. To study the molecular mechanism underlying such reverse FasL signaling in detail and to analyze its physiological importance in vivo, we established a knockout/knockin mouse model, in which wild-type FasL was replaced with a deletion mutant lacking the ICD. Our results demonstrate that FasL ICD signaling impairs activation-induced proliferation in B and T cells by diminishing phosphorylation of phospholipase C γ, protein kinase C, and extracellular signal-regulated kinase 1/2. We also demonstrate that the FasL ICD interacts with the transcription factor lymphoid-enhancer binding factor-1 and inhibits lymphoid-enhancer binding factor-1-dependent transcription. In vivo, plasma cell numbers, generation of germinal center B cells, and, consequently, production of antigen-specific immunoglobulin M antibodies in response to immunization with T cell-dependent or T cell-independent antigen are negatively affected in presence of the FasL ICD, suggesting that FasL reverse signaling participates in negative fine-tuning of certain immune responses.