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Titanium dioxide (in the form of E171) is a ubiquitous excipient in tablets and capsules for oral use. In the coating of a tablet or in the shell of a capsule the material disperses visible and UV light so that the contents are protected from the effects of light, and the patient or caregiver cannot see the contents within. It facilitates elegant methods of identification for oral solid dosage forms, thus aiding in the battle against counterfeit products. Titanium dioxide ensures homogeneity of appearance from batch to batch fostering patient confidence. The ability of commercial titanium dioxide to disperse light is a function of the natural properties of the anatase polymorph of titanium dioxide, and the manufacturing processes used to produce the material utilized in pharmaceuticals. In some jurisdictions E171 is being considered for removal from pharmaceutical products, as a consequence of it being delisted as an approved colorant for foods. At the time of writing, in the view of the authors, no system or material which could address both current and future toxicological concerns of Regulators and the functional needs of the pharmaceutical industry and patients has been identified. This takes into account the assessment of materials such as calcium carbonate, talc, isomalt, starch and calcium phosphates. In this paper an IQ Consortium team outlines the properties of titanium dioxide and criteria to which new replacement materials should be held.

Here we report that the murine Tox gene encodes two proteins from a single mRNA, and we investigate the mechanism of production and function of these proteoforms. The annotated thymocyte selection–associated HMG-box protein (TOX) coding sequence is predicted to produce a 526-aa protein (TOXFL). However, Western blots reveal two bands. We found that the lower band consists of an N-terminally truncated variant of TOX (TOXΔN), whereas the slower-migrating band is TOXFL. The TOXΔN proteoform is alternatively translated via leaky ribosomal scanning from an evolutionarily conserved translation initiation site downstream of the annotated translation initiation site. When expressed exogenously from a cDNA in murine CD8 T cells or HEK cells, or endogenously from the murine Tox locus, both forms are translated, although the ratio of TOXFL/TOXΔN significantly varies with cellular context. This includes regulation of proteoform production during development of murine CD4 T cells in the thymus, where the positive selection of CD4+CD8+ cells and subsequent differentiation to CD4+CD8lo transitional and CD4SP cell subsets is associated with both an increase in total TOX protein and increased TOXΔN production relative to TOXFL. Finally, we found that sole expression of TOXFL had a greater effect on gene regulation during chronic stimulation of murine CD8 T cells in culture mimicking exhaustion than did TOXΔN, including uniquely regulated cell cycle and other genes.

Giant multilocular prostatic cystadenoma (GMC) is an extremely rare, benign tumor seen in both adult and pediatric males. The neoplasm originates from prostatic tissue and is typically found within the rectovesical pouch, varying in both size and morphology. Microscopically, GMC contains both glandular and cystic prostatic tissue lined by cuboidal and columnar epithelium. Symptoms often arise once the pelvic mass begins to obstruct the surrounding structures and organs, although invasion into surrounding tissue is unlikely. Common symptoms include abdominal pain, urinary retention, and dysuria. The standard treatment for GMC is surgical removal of the mass with good outcomes and only 1 known case of recurrence. Here we present the case of a 14-year-old male with GMC—the youngest patient reported to date—who presented with abdominal pain, difficulty voiding, and hydroureteronephrosis.

Tumour-specific CD8 T cell dysfunction is a differentiation state that is distinct from the functional effector or memory T cell states1–6. Here we identify the nuclear factor TOX as a crucial regulator of the differentiation of tumour-specific T (TST) cells. We show that TOX is highly expressed in dysfunctional TST cells from tumours and in exhausted T cells during chronic viral infection. Expression of TOX is driven by chronic T cell receptor stimulation and NFAT activation. Ectopic expression of TOX in effector T cells in vitro induced a transcriptional program associated with T cell exhaustion. Conversely, deletion of Tox in TST cells in tumours abrogated the exhaustion program: Tox-deleted TST cells did not upregulate genes for inhibitory receptors (such as Pdcd1, Entpd1, Havcr2, Cd244 and Tigit), the chromatin of which remained largely inaccessible, and retained high expression of transcription factors such as TCF-1. Despite their normal, ‘non-exhausted’ immunophenotype, Tox-deleted TST cells remained dysfunctional, which suggests that the regulation of expression of inhibitory receptors is uncoupled from the loss of effector function. Notably, although Tox-deleted CD8 T cells differentiated normally to effector and memory states in response to acute infection, Tox-deleted TST cells failed to persist in tumours. We hypothesize that the TOX-induced exhaustion program serves to prevent the overstimulation of T cells and activation-induced cell death in settings of chronic antigen stimulation such as cancer.

Exhausted CD8+ T (Tex) cells in chronic infections and cancer have limited effector function, high co-expression of inhibitory receptors and extensive transcriptional changes compared with effector (Teff) or memory (Tmem) CD8+ T cells. Tex cells are important clinical targets of checkpoint blockade and other immunotherapies. Epigenetically, Tex cells are a distinct immune subset, with a unique chromatin landscape compared with Teff and Tmem cells. However, the mechanisms that govern the transcriptional and epigenetic development of Tex cells remain unknown. Here we identify the HMG-box transcription factor TOX as a central regulator of Tex cells in mice. TOX is largely dispensable for the formation of Teff and Tmem cells, but it is critical for exhaustion: in the absence of TOX, Tex cells do not form. TOX is induced by calcineurin and NFAT2, and operates in a feed-forward loop in which it becomes calcineurin-independent and sustained in Tex cells. Robust expression of TOX therefore results in commitment to Tex cells by translating persistent stimulation into a distinct Tex cell transcriptional and epigenetic developmental program.

Immunotherapies targeting the PD-1/PD-L1 axis are now a mainstay in the clinical management of multiple cancer types, however, many tumors still fail to respond. CCL2 is highly expressed in various cancer types and has been shown to be associated with poor prognosis. Inhibition or blockade of the CCL2/CCR2 signaling axis has thus been an area of interest for cancer therapy. Here we show across multiple murine tumor and metastasis models that CCR2 antagonism in combination with anti-PD-1 therapy leads to sensitization and enhanced tumor response over anti-PD-1 monotherapy. We show that enhanced treatment response correlates with enhanced CD8+ T cell recruitment and activation and a concomitant decrease in CD4+ regulatory T cell. These results provide strong preclinical rationale for further clinical exploration of combining CCR2 antagonism with PD-1/PD-L1-directed immunotherapies across multiple tumor types especially given the availability of small molecule CCR2 inhibitors and antibodies., Investigating signalling induced by the cytokine CCL2 as a therapeutic target, Tu et al demonstrate that blockade of the CCL2 receptor, CCR2 enhances CD8+ T cell recruitment and activation and the therapeutic efficacy of PD-1 inhibition in tumours.

Harly et al. show that early innate lymphoid progenitors (EILPs) are a developmental intermediate between all-lymphoid progenitors (ALPs) and ILC precursors (ILCps) and identify requirements for their generation and further differentiation., Early innate lymphoid progenitors (EILPs) have recently been identified in mouse adult bone marrow as a multipotential progenitor population specified toward innate lymphoid cell (ILC) lineages, but their relationship with other described ILC progenitors is still unclear. In this study, we examine the progenitor–successor relationships between EILPs, all-lymphoid progenitors (ALPs), and ILC precursors (ILCps). Functional, bioinformatic, phenotypical, and genetic approaches collectively establish EILPs as an intermediate progenitor between ALPs and ILCps. Our work additionally provides new candidate regulators of ILC development and clearly defines the stage of requirement of transcription factors key for early ILC development.

Innate lymphoid cells (ILCs) are immune cells that lack specific antigen receptors but possess similar effector functions as T cells. Interestingly, ILCs and T cells express many of the same transcription factors. One such factor is TOX, a sequence non-specific nuclear transcriptional regulator that is crucial for development of both CD4 T cells and ILCs. TOX-deficient mice have significantly reduced number of early innate lymphoid progenitors (EILP) and ILC progenitors (ILCP). Consequently, the mature NK cells, ILC1, ILC2 and ILC3 subsets are greatly reduced in Tox−/− mice. Interestingly ILC3 numbers in gut are not reduced by the absence of TOX. We have discovered that TOX2, another member of the TOX family of HMG-box proteins, is highly expressed in ILC precursors. We generated Tox2−/− mice and found they have reduced ILC3 in gut; however, ILC3 cells in spleen or lymph node are intact. We also observed ILCP in fetal gut and adult gut were significantly reduced in Tox2−/−mice suggesting TOX2 is required for gut ILC3 development. However, ILCP numbers in adult gut of Tox−/− mice were unaffected. We are currently performing single-cell RNA-sequencing on gut ILC3 from Tox2−/− and Tox−/− mice to transcriptionally characterize these cells and to identify the heterogeneity within the ILC3 population. Taken together, our results indicate the existence of at least two pathways of ILC development: a central pathway in bone marrow and fetal liver that is TOX dependent; and a peripheral pathway of development of ILC3 in gut that requires TOX2.

Introduction Post-transplant cyclophosphamide has allowed alloHSCT using haploidentical donors. PTCy is very effective at reducing GVHD-causing T cells, but it has the unwanted effect of eliminating Natural Killer cells. We have developed a new reduced-intensity haploHSCT transplant conditioning regimen based on an a regimen developed for fully-matched donors with lymphoma: bendamustine 130 mg/m2 IV daily, and fludarabine 30 mg/m2 IV daily, for 3 days, +/- rituximab. We made additional modifications designed to improve NK immune recovery: 1.) no mycophenolate, 2.) no routine G-CSF, 3.) early tacrolimus taper starting on day +60, 4.) pre-emptive NK-enriched donor lymphocyte infusion on Day +8. Objectives Conduct a prospective clinical trial to evaluate the safety of bendamustine-fludarabine conditioning, PTCy, NK cell add-back, and short course tacrolimus for patients with myeloma or lymphoma undergoing haploHSCT. Assess clinical outcomes over the first year after transplantation. Efficacy Endpoints: Death or engraftment failure at D+30, death or severe chronic extensive GVHD at D+100. Test rate of survival, engraftment, and GVHD. Measure reconstitution of adaptive and innate lymphocyte subsets. Measure NK KIR repertoire recovery and assess for ‘missing self’. Methods This is a Phase I, single center, single cohort, open-label, proof-of-concept clinical trial to evaluate the safety of the BFRHaplo regimen (Figure 1) and preemptive CD56-selected DLI following PTCy for adults with myeloma or lymphoma. Results We have safety data on six patients (Table 1). Recovery of neutrophils and platelets is prompt. No primary or secondary graft failures have occurred. No patients have developed Grade III-IV or steroid-refractory acute GVHD. No organ toxicities, and no alkylator-alkylator toxicity from bendamustine – PTCy, have been observed. No instances of either veno-occlusive disease or transplant-related thrombotic microangiopathy, and no deaths, serious infections, or re-hospitalizations. One patient relapsed and is alive without GVHD receiving salvage therapy. We used single-step CD56 selection to prepare CD56-enriched donor lymphocyte infusions (Table 2). The one-step CD56 selection method has the attractive property of preserving CD3+/CD56+ cells, including NK/T and gdT. The target cell dose (> 1 million CD56+ cells/kg and Conclusion The trial will establish whether BFRHaplo, and the infusion of cytotoxic NK and some T cells (

Infections are thought to trigger CD8(+) cytotoxic T lymphocyte (CTL) responses during autoimmunity. However, the transcriptional programs governing the tissue-destructive potential of CTLs remain poorly defined. In a model of central nervous system (CNS) inflammation, we found that infection with lymphocytic choriomeningitis virus (LCMV), but not Listeria monocytogenes (Lm), drove autoimmunity. The DNA-binding factor TOX was induced in CTLs during LCMV infection and was essential for their encephalitogenic properties, and its expression was inhibited by interleukin-12 during Lm infection. TOX repressed the activity of several transcription factors (including Id2, TCF-1, and Notch) that are known to drive CTL differentiation. TOX also reduced immune checkpointsensitivity by restraining the expression of the inhibitory checkpoint receptor CD244 on the surface of CTLs, leading to increased CTL-mediated damage in the CNS. Our results identify TOX as a transcriptional regulator of tissue-destructive CTLs in autoimmunity, offering a potential mechanistic link to microbial triggers.

Type 2 innate lymphoid cells (ILC2) share cytokine and transcription factor expression with CD4+ Th2 cells, but functional diversity of the ILC2 lineage has yet to be fully explored. Here, we show induction of a molecularly distinct subset of activated lung ILC2, termed ILC210. These cells produce IL-10 and downregulate some pro-inflammatory genes. Signals that generate ILC210 are distinct from those that induce IL-13 production, and gene expression data indicate that an alternative activation pathway leads to the generation of ILC210. In vivo, IL-2 enhances ILC210 generation and is associated with decreased eosinophil recruitment to the lung. Unlike most activated ILC2, the ILC210 population contracts after cessation of stimulation in vivo, with maintenance of a subset that can be recalled by restimulation, analogous to T-cell effector cell and memory cell generation. These data demonstrate the generation of a previously unappreciated IL-10 producing ILC2 effector cell population., Type 2 innate lymphoid cells (ILC2) are thought to be a uniform population of effector cells that produce IL-5 and IL-13. Here, the authors shown that, in mice, IL-33 can alternatively activate these cells to generate a molecularly distinct IL-10-producing subset designated ILC210.

TOX is a member of an evolutionarily conserved DNA-binding protein family and is expressed in several immune-relevant cell subsets. Here, we review the key role of TOX in regulating development of CD4 T cells, natural killer cells and lymphoid tissue inducer cells, the latter responsible for the generation of lymph nodes. Although the exact molecular mechanism of action of TOX remains to be elucidated, the role of TOX in establishment of gene programs in the thymus and the potential of TOX as a regulator of E protein activity are discussed.

The factors that regulate thymic development of the CD4+ T cell gene program remain poorly defined. The transcriptional regulator ThPOK is a dominant factor in CD4+ T cell development, which functions primarily to repress the CD8 lineage fate. Previously, we showed that nuclear protein TOX is also required for murine CD4+ T cell development. In this study, we sought to investigate whether the requirement for TOX was solely due to a role in ThPOK induction. In apparent support of this proposition, ThPOK upregulation and CD8 lineage repression were compromised in the absence of TOX, and enforced ThPOK expression could restore some CD4 development. However, these “rescued” CD4 cells were defective in many aspects of the CD4+ T cell gene program, including expression of Id2, Foxo1, and endogenous Thpok, among others. Thus, TOX is necessary to establish the CD4+ T cell lineage gene program, independent of its influence on ThPOK expression.

Tumor-specific CD8 T-cells in cancers enter a state of dysfunction characterized by the expression of inhibitory receptors and failure to produce effector cytokines and cytotoxic molecules. Here we identify the nuclear factor, Thymocyte selection-associated HMG box protein, TOX, as a master regulator of tumor-specific T-cell dysfunction. TOX is uniquely expressed in dysfunctional CD8 T-cells from mouse and human tumors but absent in functional T-cells. TOX expression is driven by continuous TCR stimulation and NFAT activity. Forced expression of TOX in functional effector T-cells was sufficient to induce a transcriptional program of dysfunction through the concerted expression of genes encoding numerous inhibitory receptors and dysfunction-associated transcription factors. Notably, TOX-deficient tumor-infiltrating T-cells did not upregulate inhibitory receptors such as PD1, LAG3, CD38, or CD39 and maintained high TCF1 expression. Surprisingly, despite their normal, “non-exhausted” phenotype, TOX-deficient T-cells failed to make effector cytokines, suggesting that loss of effector function in tumor-specific T-cells is uncoupled from inhibitory receptor expression. Furthermore, TOX-deficient T-cells failed to persist in tumors, ultimately undergoing activation-induced cell death. We propose that the TOX-induced transcriptional program of hyporesponsiveness is a physiologic negative feedback mechanism that prevents overstimulation; thus TOX is absolutely required for T-cell survival in the setting of chronic antigen stimulation as in cancers. Citation Format: Andrew C. Scott, Steven Camara, Peter Lauer, Alexandra Synder, Dmitriy Zamarin, Tyler Walther, Olivier Levy, Michael Glickman, Jonathan Kaye, Mary Philip, Andrea Schietinger. Thymocyte selection-associated HMG box protein TOX is a master regulator of tumor-specific T-cell dysfunction [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A215.

Coinhibitory pathways are thought to act in later stages of an adaptive immune response, but whether coinhibition contributes to early innate immunity is unclear. We show that engagement of the newly discovered coinhibitory receptor B and T lymphocyte attenuator (BTLA) by herpesvirus entry mediator (HVEM) is critical for negatively regulating early host immunity against intracellular bacteria. Both HVEM−/− and BTLA−/−, but not LIGHT−/−, mice are more resistant to listeriosis compared with wild-type mice, and blockade of the BTLA pathway promotes, while engagement inhibits, early bacterial clearance. Differences in bacterial clearance were seen as early as 1 day postinfection, implicating the initial innate response. Therefore, innate cell function in BTLA−/− mice was studied. We show that innate cells from BTLA−/− mice secrete significantly more proinflammatory cytokines upon stimulation with heat-killed Listeria. These results provide the first evidence that a coinhibitory pathway plays a critical role in regulating early host innate immunity against infection.

Summary: The thymus is responsible for the production of CD4+ helper and CD8+ cytotoxic T cells, which constitute the cellular arm of the immune system. These cell types derive from common precursors that interact with thymic stroma in a T-cell receptor (TCR)-specific fashion, generating intracellular signals that are translated into function-specific changes in gene expression. This overall process is termed positive selection, but it encompasses a number of temporally distinct and possibly mechanistically distinct cellular changes, including rescue from apoptosis, initiation of cell differentiation, and commitment to the CD4+ or CD8+ T-cell lineage. One of the puzzling features of positive selection is how specificity of the TCR controls lineage commitment, as both helper and cytolytic T cells utilize the same antigen-receptor components, with the exception of the CD4 or CD8 coreceptors themselves. In this review, we focus on the signals required for positive selection, particularly as they relate to lineage commitment. Identification of genes encoding transcriptional regulators that play a role in T-cell development has led to significant recent advances in the field. We also provide an overview of nuclear factors in this context and, where known, how their regulation is linked to the same TCR signals that have been implicated in initiating and regulating positive selection.

T cell development is dependent on the integration of multiple signaling pathways, although few links between signaling cascades and downstream nuclear factors that play a role in thymocyte differentiation have been identified. We show here that expression of the HMG box protein TOX is sufficient to induce changes in coreceptor gene expression associated with β-selection, including CD8 gene demethylation. TOX expression is also sufficient to initiate positive selection to the CD8 lineage in the absence of MHC–TCR interactions. TOX-mediated positive selection is associated with up-regulation of Runx3, implicating CD4 silencing in the process. Interestingly, a strong T cell receptor–mediated signal can modify this cell fate. We further demonstrate that up-regulation of TOX in double positive thymocytes is calcineurin dependent, linking this critical signaling pathway to nuclear changes during positive selection.

Type-2 innate lymphoid (ILC2) cells share cytokine and transcription factor expression with CD4+ Th2 cells, but it is unknown if this ILC subtype undergoes functional diversification. Here, we report in vivo induction of a molecularly distinct subset of activated lung ILC2 cells, termed ILC210 cells, that produce IL-10 and downregulate genes associated with inflammation. Signals that generate ILC210 cells were distinct from those that induced IL-13 production and, along with transcriptome data, suggested an alternative pathway of activation. In vivo, IL-2 greatly enhanced ILC210 cell generation by IL-33 and was associated with decreased eosinophil recruitment to the lung. Unlike the majority of activated ILC2 cells, the ILC210 cell population underwent contraction following cessation of IL-33 stimulation in vivo, but with maintenance of a subpopulation of cells that can be recalled by restimulation, analogous to T cell effector cell and memory cell generation. Together, these data demonstrate previously unappreciated heterogeneity in the ILC2 cell response.

Diverse innate lymphoid cell (ILC) subtypes have been defined on the basis of effector function and transcription factor expression. ILCs derive from common lymphoid progenitors, although the transcriptional pathways that lead to ILC-lineage specification remain poorly characterized. Here we found that the transcriptional regulator TOX was required for the in vivo differentiation of common lymphoid progenitors into ILC lineage-restricted cells. In vitro modeling demonstrated that TOX deficiency resulted in early defects in the survival or proliferation of progenitor cells, as well as ILC differentiation at a later stage. In addition, comparative transcriptome analysis of bone marrow progenitors revealed that TOX-deficient cells failed to upregulate many genes of the ILC program, including genes that are targets of Notch, which indicated that TOX is a key determinant of early specification to the ILC lineage.

Compared with that of naive CD8+ T cells, the homeostatic population expansion of naive CD4+ T cells in a lymphopenic environment is limited. New data indicate that this difference is caused by high systemic concentrations of IL-7, which inhibit the function of dendritic cells.

Recent studies have demonstrated the utility of synthetic combinatorial libraries for the rapid identification of peptide ligands that stimulate clonotypic populations of T cells. Here we screen a decapeptide combinatorial library arranged in a positional scanning format with two different clonotypic populations of CD4+ T cells to identify peptide epitopes that stimulate proliferative responses by these T cells in vitro. An extensive collection of mimic peptide sequences was synthesized and used to explore the fine specificity of TCR/peptide/MHC interactions. We also demonstrate that many of these deduced ligands are not only effective immunogens in vivo, but are capable of inducing T cell responses to the original native ligands used to generate the clones. These results have significant implications for considerations of T cell specificity and the design of peptide vaccines for infectious disease and cancer using clinically relevant T cell clones of unknown specificity.

The T cell costimulatory protein LIGHT and coinhbitory protein BTLA share a common ligand, HVEM. Now CD160 is also shown to bind HVEM and deliver a potent inhibitory signal to CD4 T cells.

The cellular and molecular events that drive early innate lympoid cell (ILC) development remain poorly understood. We show that transcription factor TCF-1 is required for the efficient generation of all known adult ILC subsets and their precursors. Using novel reporter mice, we identified a new subset of early ILC progenitors (EILP) that expressed high amounts of TCF-1. EILP lacked efficient T and B lymphocyte potential, but efficiently gave rise to NK cells and all known adult helper-ILC lineages, indicating that they are the earliest identified ILC-committed progenitors. Our results suggest that upregulation of TCF-1 expression denotes the earliest stage of ILC fate specification. The discovery of EILP provides a basis to decipher additional signals that specify the ILC fate.

LFA-1 is a β2 integrin that plays well-characterized roles in adhesion of T lymphocytes to APC, T cell-mediated cytolysis, and leukocyte-endothelial cell interactions. Although it is clear that LFA-1 must undergo affinity or avidity changes to bind its cellular ligand ICAM-1, the intracellular signaling pathways involved are not well characterized. Here, we show that the Ras-mitogen-activated protein kinase (MAPK) signaling pathway is also involved in TCR-activated LFA-1 adhesion. Expression of a dominant negative form of p21ras in a thymocyte cell line inhibits, while constitutively active p21ras both enhances and sustains, subsequent TCR-triggered adhesion to isolated ICAM-1. However, the Ras/MAPK pathway alone is not sufficient for activating T cell LFA-1, as inhibition of both downstream MAPK/extracellular regulated kinase kinase (MEK) activity and phosphatidylinositol 3-kinase activity is required for complete inhibition of adhesion.

NF-κB transcription factors are known to regulate the expression of a number of genes involved in T cell activation and function. Some evidence has suggested that they also play a role in T cell development. However, the role of NF-κB in Ag-induced thymocyte differentiation has not been directly addressed to date. Here we critically examine this role by employing DPK, a CD4+CD8+ thymocyte line that undergoes differentiation upon TCR engagement in a process that closely mimics positive selection. Expression of a degradation-resistant form of IκBα in DPK cells results in constitutive inhibition of NF-κB activity. We find that in the absence of NF-κB activity, MHC-peptide-induced differentiation of DPK is blocked. Furthermore, differentiation induced by a nonphysiologic stimulus, anti-TCR Ab, is greatly reduced. Altogether, our data indicate a requirement for NF-κB in the developmental changes associated with positive selection.

There is little known about the regulation of gene expression during TCR-mediated differentiation of immature CD4+8+ (double positive) thymocytes into mature T cells. Using the DPK CD4+8+thymocyte precursor cell line, we demonstrate that the early growth response-1 gene (Erg-1), encoding a zinc finger transcription factor, is rapidly upregulated after TCR stimulation. We also report that Egr-1 is expressed by a subset of normal double positive thymocytes in the thymic cortex, as well by a majority of medullary single positive thymocytes. Expression of Egr-1 is dramatically reduced in the thymus of major histocompatibility complex knockout mice, but can be induced by anti-CD3 antibody stimulation of isolated thymocytes from these animals. These and other data suggest that high level expression of Egr-1 in the thymus is a consequence of selection. A similar pattern of expression is found for family members Egr-2 and Egr-3. Using the DPK cell line, we also demonstrate that expression of Egr-1, 2, and 3 is dependent upon ras activation, as is the initiation of differentiation to a single positive cell. In contrast, the calcineurin inhibitor cyclosporin A, which inhibits DPK cell differentiation as well as positive selection, inhibits expression of Egr-2 and Egr-3, but not Egr-1. The identification of the Egr family in this context represents the first report of a link between the two known signaling pathways involved in positive selection and downstream transcriptional regulators.

Thymocyte selection-associated HMG box factor (TOX) is a DNA-binding factor required for development of CD4 T cells, natural killer T cells, and T regulatory cells. Here we document that both NK cell development and lymphoid tissue organogenesis are inhibited in the absence of TOX. We find that development of lymphoid tissue inducer cells, a rare subset of specialized cells that plays an integral role in lymphoid tissue organogenesis, requires TOX. Tox is highly upregulated in immature NK cells in the bone marrow, consistent with the loss of mature NK cells in the absence of this nuclear protein. Thus, multiple cell lineages in the immune system share a TOX-dependent step for development.

To study the processes of thymic development, we have established transgenic mice expressing and alpha/beta T cell antigen receptor (TCR) specific for cytochrome c associated with class II major histocompatibility complex (MHC) molecules. The transgenic TCR chains are expressed by most of the thymocytes in these mice, and these cells have been shown to efficiently mature in association with Ek- and Ab-encoded class II MHC molecules. This report describes a characterization of the negative selection of these transgenic thymocytes in vivo that is associated with the expression of As molecules. Negative selection by As molecules appears to result in the deletion of a late stage of CD4/CD8 double-positive thymocytes in that there is a virtual absence of transgenic TCR bearing CD4 single-positive thymocytes. This phenotype is accompanied by the appearance of CD4/CD8 double-negative thymocytes and peripheral T cells that are functionally antigen reactive. The process of negative selection has also been investigated using an in vitro culture system. Upon presentation of cytochrome c by Eb-expressing nonthymic antigen-presenting cells, there occurs an antigen dose-dependent deletion of the majority of CD4/CD8 double-positive thymocytes. In contrast, presentation of Staphylococcal enterotoxin A by Eb in vitro results in minimal deletion of double-positive thymocytes. In addition, we use this in vitro model to examine the effects of cyclosporin A on negative selection. In contrast to its effects on mature T cells, and the findings of others in vivo, cyclosporin A does not inhibit antigen-induced deletion of double-positive thymocytes. Finally, a comparison of the antigen dose responses for thymocyte deletion and for peripheral T cell activation indicates that double-positive thymocyte recognition is more sensitive than mature T cells to antigen recognition.

B- and T-lymphocyte attenuator (BTLA, CD272) is a polymorphic molecule belonging to the Ig superfamily (SF) that attenuates BCR and TCR-mediated signaling, and thereby functions as a negative regulator of lymphocyte activation. Herein, we report an anti-murine BTLA mAb (clone 4G12b) that remarkably detects protein expression on BTLA(low) cells such as naive CD4(+) cells, CD8(+) T cells, dendritic cells (DC), as well as in NKT cells and for the first time, we found BTLA expression on DX5(dim) and DX5(bright) subsets of non-T NK cells in both C57BL/6 (B6) and BALB/c BTLA allelic variants. Anti-BTLA 4G12b mAb binds to an overlapping epitope to that recognized by anti-BTLA 6A6 mAb, but in contrast to the concept widely accepted of blocking activity of 6A6 mAb, surprisingly neither 4G12b nor 6A6 mAbs impeded murine HVEM-mIgG(2a).Fc recombinant fusion protein from interacting with BTLA-expressing cells. Lastly, in vivo administration of anti-BTLA 4G12b mAb induced a profound and lasting down-modulation of BTLA expression that led to BTLA receptor internalization with the potential utility of shutting down BTLA expression at any stage during the course of the immune response in both B6 and BALB/c strains of mice.

The immunodeficient state associated with adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiency may result from the selective phosphorylation by thymus-derived lymphocytes of the ADA substrate deoxyadenosine and the PNP substrate deoxyguanosine, leading to the intracellular trapping of toxic deoxyribonucleoside triphosphates. Agents such as deoxycytidine might be able to favourably modify the immunodeficient state by inhibiting deoxyribonucleoside phosphorylation. Deficiencies of other nucleotide catabolic enzymes, if selectively expressed by lymphocytes, might also lead to immunodeficiency via nucleoside trapping in lymphoid tissues. Purine deoxyribonucleoside analogues, either alone or in combination with ADA inhibitors, may have value as lymphospecific antimetabolites.

CD8+ cytotoxic and CD4+ helper/inducer T cells develop from common thymocyte precursors that express both CD4 and CD8 molecules. Upon T cell receptor signaling, these cells initiate a differentiation program that includes complex changes in CD4 and CD8 expression, allowing identification of transitional intermediates in this developmental pathway. Little is known about regulation of these early transitions or their specific importance to CD4 and CD8 T cell development. Here, we show a severe block at the CD4loCD8lo transitional stage of positive selection caused by loss of the nuclear HMG box protein TOX. As a result, CD4 lineage T cells, including regulatory T and CD1d-dependent natural killer T cells, fail to develop. In contrast, functional CD8+ T cells develop in TOX-deficient mice. Our data suggest that TOX-dependent transition to the CD4+CD8lo stage is required for continued development of class II major histocompatibility complex–specific T cells, regardless of ultimate lineage fate.