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Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment of patients with nonmalignant or malignant blood disorders. Its success has been limited by graft-versus-host disease (GVHD). Current systemic nontargeted conditioning regimens mediate tissue injury and potentially incite and amplify GVHD, limiting the use of this potentially curative treatment beyond malignant disorders. Minimizing systemic nontargeted conditioning while achieving alloengraftment without global immune suppression is highly desirable. Antibody-drug-conjugates (ADCs) targeting hematopoietic cells can specifically deplete host stem and immune cells and enable alloengraftment. We report an anti-mouse CD45-targeted-ADC (CD45-ADC) that facilitates stable murine multilineage donor cell engraftment. Conditioning with CD45-ADC (3 mg/kg) was effective as a single agent in both congenic and minor-mismatch transplant models resulting in full donor chimerism comparable to lethal total body irradiation (TBI). In an MHC-disparate allo-HSCT model, pretransplant CD45-ADC (3 mg/kg) combined with low-dose TBI (150 cGy) and a short course of costimulatory blockade with anti-CD40 ligand antibody enabled 89% of recipients to achieve stable alloengraftment (mean value: 72%). When CD45-ADC was combined with pretransplant TBI (50 cGy) and posttransplant rapamycin, cyclophosphamide (Cytoxan), or a JAK inhibitor, 90% to 100% of recipients achieved stable chimerism (mean: 77%, 59%, 78%, respectively). At a higher dose (5 mg/kg), CD45-ADC as a single agent was sufficient for rapid, high-level multilineage chimerism sustained through the 22 weeks observation period. Therefore, CD45-ADC has the potential utility to confer the benefit of fully myeloablative conditioning but with substantially reduced toxicity when given as a single agent or at lower doses in conjunction with reduced-intensity conditioning.

Innate lymphoid cells (ILCs) play an important role in immunity, inflammation, and tissue remodeling and their dysregulation is implicated in autoimmune and inflammatory disorders. We analyzed the impact of daclizumab, a humanized monoclonal anti-CD25 antibody, on circulating natural killer (NK) cells and ILCs in a cohort of multiple sclerosis patients. An increase in CD56bright NK cells and CD56hiCD16intermediate transitional NK cells was observed. No significant change in total ILCs or major ILC subpopulations was observed. These results refine our understanding of the impact of daclizumab on innate lymphoid cell populations.

For patients with refractory or high-risk hematologic malignancies, like acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and acute lymphoblastic leukemia (ALL), allogeneic hematopoietic stem cell transplant (Allo-HSCT) is a potentially curative approach. Morbidities and mortality associated with current conditioning regimens limit the use of this curative procedure. As a result, many eligible patients do not consider transplant and 2/3 of those transplanted are only able to tolerate a reduced intensity conditioning regimen, which is associated with increased relapse rates (Scott, J Clin Onc 2017). Thus, there is an urgent need for a safer and more effective conditioning agent with improved disease control. Our targeted antibody drug conjugate (ADC) approach is designed to improve the safety of current conditioning protocols by specifically depleting CD45+ cells. We developed a novel anti-human CD45-targeted short half-life ADC conjugated to amanitin (AM). CD45 is the ideal target for allo-HSCT conditioning because it is expressed on all hematopoietic cells (except erythrocytes, plasma cells and platelets), and most hematologic malignancies. Given their targeted specificity, anti-CD45-AM can potentially provide dual benefit to leukemia patients by combining effective conditioning for HSCT with depletion of target-bearing tumor cells. To demonstrate our engineered short half-life anti-human CD45-AM has anti-leukemic activity we tested it in human leukemic xenograft murine models. A panel of models were evaluated to mimic untreated and refractory disease; AML PDX models (from treatment naive and relapsed post allogeneic HCT patients), ALL cells from an immortalized cell line (REH-Luc), T-ALL patient-derived xenograft (PDX) model (from a patient progressing post DHAP chemotherapy). In the REH-Luc model, single doses of anti-CD45-AM were well tolerated, and cytoreductive resulting in delayed tumor growth compared to vehicle (PBS), isotype-AM, or standard of care (SoC) doxorubicin. Anti-CD45-AM treatment in the PDX AML, and T-ALL significantly decreased peripheral tumor burden resulting in delayed tumor growth compared to vehicle, isotype-AM, and comparable to 2 clinically validated standards of care (Ara-C, and dexamethasone respectively; figure 1). As designed for the transplant indication, the ADC had a reduced half-life compared to wild type antibody controls (16 vs 79h). These data in humanized murine xenograft models demonstrate that short half-life CD45-AM ADCs are potent targeted anti-leukemia agents. Together with prior reports demonstrating the potency of anti-CD45-AM as conditioning agents, these non-genotoxic ADCs may be useful in reducing disease burden and inducing durable remissions in patients after transplant particularly those who receive reduced intensity conditioning that are at high risk of relapse.

Fumarate-containing pharmaceuticals are potent therapeutic agents that influence multiple cellular pathways. Despite proven clinical efficacy, there is a significant lack of data that directly defines the molecular mechanisms of action of related, yet distinct fumarate compounds. We systematically compared the impact of dimethyl fumarate (DMF), monomethyl fumarate (MMF) and a mixture of monoethyl fumarate salts (Ca++, Mg++, Zn++; MEF) on defined cellular responses. We demonstrate that DMF inhibited NF-κB-driven cytokine production and nuclear translocation of p65 and p52 in an Nrf2-independent manner. Equivalent doses of MMF and MEF did not affect NF-κB signaling. These results highlight a key difference in the biological impact of related, yet distinct fumarate compounds.

Vaccination strategies for protection against a number of respiratory pathogens must induce T-cell populations in both the pulmonary airways and peripheral lymphoid organs. In this study, we show that pulmonary immunization using plasmid DNA formulated with the polymer polyethyleneimine (PEI-DNA) induced antigen-specific CD8(+) T cells in the airways that persisted long after antigen local clearance. The persistence of the cells was not mediated by local lymphocyte proliferation or persistent antigen presentation within the lung or airways. These vaccine-induced CD8(+) T cells effectively mediated protective immunity against respiratory challenges with vaccinia virus and influenza virus. Moreover, this protection was not dependent upon the recruitment of T cells from peripheral sites. These findings demonstrate that pulmonary immunization with PEI-DNA is an efficient approach for inducing robust pulmonary CD8(+) T-cell populations that are effective at protecting against respiratory pathogens.

Despite the low and short expression of secondary Ag, prime-boost immunizations using homologous or heterologous vectors are capable of amplifying memory CD8+ T cells. This is mainly attributed to the rapid presentation of Ag by APCs and the high proliferative capacity of memory CD8+ T cells. Nevertheless, certain viruses and vectors often require prolonged Ag presentation for optimal T cell priming, and the influence of such a prolonged presentation during secondary immune induction is not clear. To address this issue, we primed and boosted mice intradermally (i.d.) with plasmid DNA that was recently reported to require prolonged Ag presentation for maximal CD8+ T cell priming. Although functional memory CD8+ T cells were present in the mice after i.d. priming, the secondary CD8+ T cell response elicited was limited and reached a similar level of that observed during priming. The initial levels of secondary Ag expressed in the boosted mice were sufficient to prime CD8+ T cell response in naive hosts, suggesting that lower Ag load alone does not explain the limited secondary immune responses observed. Removal of the injection site 5 or 10 days after i.d. boosting immunization resulted in diminished Ag presentation and no expansion of memory CD8+ T cells. In fact, Ag-presenting activity following boost occurred mainly two weeks postimmunization, a time when the Ag was no longer expressed in situ. These findings suggest that when the boosting vector triggers prolonged Ag presentation, the lack of synchronicity between Ag accessibility and Ag presentation limits secondary immune responses.

The duration of Ag expression in vivo has been reported to have a minimal impact on both the magnitude and kinetics of contraction of a pathogen-induced CD8+ T cell response. In this study, we controlled the duration of Ag expression by excising the ear pinnae following intradermal ear pinnae DNA immunization. This resulted in decreased magnitude, accelerated contraction and differentiation, and surprisingly greater secondary CD8+ T cell responses. Furthermore, we found delayed and prolonged Ag presentation in the immunized mice; however, this presentation was considerably decreased when the depot Ag was eliminated. These findings suggest that the magnitude and the contraction phase of the CD8+ T cell response following intradermal DNA immunization is regulated by the duration rather than the initial exposure to Ag.

The prevalent view of thymic epithelial differentiation and Aire activity holds that Aire functions in terminally differentiated medullary thymic epithelial cells (MTECs) to derepress the expression of structural tissue-restricted Ags, including pancreatic endocrine hormones. An alternative view of these processes has proposed that Aire functions to regulate the differentiation of immature thymic epithelial cells, thereby affecting tissue-restricted Ag expression and negative selection. In this study, we demonstrate that Aire impacts several aspects of murine MTECs and provide support for this second model. Expression of transcription factors associated with developmental plasticity of progenitor cells, Nanog, Oct4, and Sox2, by MTECs was Aire dependent. Similarly, the transcription factors that regulate pancreatic development and the expression of pancreatic hormones are also expressed by wild-type MTECs in an Aire-dependent manner. The altered transcriptional profiles in Aire-deficient MTECs were accompanied by changes in the organization and composition of the medullary epithelial compartment, including a reduction in the medullary compartment defined by keratin (K) 14 expression, altered patterns of K5 and K8 expression, and more prominent epithelial cysts. These findings implicate Aire in the regulation of MTEC differentiation and the organization of the medullary thymic compartment and are compatible with a role for Aire in thymic epithelium differentiation.

Although thymic ectopy has long been recognized in humans, the functional activity or potential immunological significance of this thymic tissue is unknown. In this study, we describe murine thymic ectopy, cervical thymic tissue that possesses the same general organization as the thoracic thymus, that is able to support T cell differentiation, and that can export T cells to the periphery. Unexpectedly, the pattern of autoantigen expression by ectopic thymic tissue differs from that of the thoracic thymus, raising the possibility that these two thymic environments may project different versions of self.

Mycobacteria, the etiological agents of tuberculosis and leprosy, have coevolved with mammals for millions of years and have numerous ways of suppressing their host's immune response. It has been suggested that mycobacteria may contain genes that reduce the host's ability to elicit CD8 + T cell responses. We screened 3,290 mutant Mycobacterium bovis bacillus Calmette Guerin (BCG) strains to identify genes that decrease major histocompatibility complex (MHC) class I presentation of mycobacterium-encoded epitope peptides. Through our analysis, we identified 16 mutant BCG strains that generated increased transgene product-specific CD8 + T cell responses. The genes disrupted in these mutant strains had disparate predicted functions. Reconstruction of strains via targeted deletion of genes identified in the screen recapitulated the enhanced immunogenicity phenotype of the original mutant strains. When we introduced the simian immunodeficiency virus (SIV) gag gene into several of these novel BCG strains, we observed enhanced SIV Gag-specific CD8 + T cell responses in vivo . This study demonstrates that mycobacteria carry numerous genes that act to dampen CD8 + T cell responses and suggests that genetic modification of these genes may generate a novel group of recombinant BCG strains capable of serving as more effective and immunogenic vaccine vectors.

Live attenuated nonpathogenic Mycobacterium bovis bacillus Calmette-Guérin (BCG) mediates long-lasting immune responses, has been safely administered as a tuberculosis vaccine to billions of humans, and is affordable to produce as a vaccine vector. These characteristics make it very attractive as a human immunodeficiency virus (HIV) vaccine vector candidate. Here, we assessed the immunogenicity of recombinant BCG (rBCG) constructs with different simian immunodeficiency virus (SIV) gag expression cassettes as priming agents followed by a recombinant replication-incompetent New York vaccinia virus (NYVAC) boost in rhesus macaques. Unmutated rBCG constructs were used in comparison to mutants with gene deletions identified in an in vitro screen for augmented immunogenicity. We demonstrated that BCG-SIV gag is able to elicit robust transgene-specific priming responses, resulting in strong SIV epitope-specific cellular immune responses. While enhanced immunogenicity was sustained at moderate levels for >1 year following the heterologous boost vaccination, we were unable to demonstrate a protective effect after repeated rectal mucosal challenges with pathogenic SIVmac251. Our findings highlight the potential for rBCG vaccines to stimulate effective cross-priming and enhanced major histocompatibility complex class I presentation, suggesting that combining this approach with other immunogens may contribute to the development of effective vaccine regimens against HIV.

Recombinant Mycobacterium bovis bacillus Calmette-Guerin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8+ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT) cells to enhance priming of CD8+ T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag). We found that the incorporation of the synthetic NKT activating glycolipid α-galactosylceramide (α-GC) into rBCG-SIV gag significantly enhanced CD8+ T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of α-GC to enhance CD8+ T cell responses to rBCG were compared in both wild type and partially humanized mice that express human CD1d molecules in place of mouse CD1d. These studies identified an α-GC analogue known as 7DW8-5, which has previously been used successfully as an adjuvant in non-human primates, as a promising compound for enhancing immunogenicity of antigens delivered by rBCG.vectors. Our findings support the incorporation of synthetic glycolipid activators of NKT cells as a novel approach to enhance the immunogenicity of rBCG-vectored antigens for induction of CD8+ T cell responses. The glycolipid adjuvant 7DW8-5 may be a promising candidate for advancing to non-human primate and human clinical studies for the development of HIV vaccines based on rBCG vectors.

While immunological memory has long been considered the province of T- and B- lymphocytes, it has recently been reported that innate cell populations are capable of mediating memory responses. We now show that an innate memory immune response is generated in mice following infection with vaccinia virus, a poxvirus for which no cognate germline-encoded receptor has been identified. This immune response results in viral clearance in the absence of classical adaptive T and B lymphocyte populations, and is mediated by a Thy1+ subset of natural killer (NK) cells. We demonstrate that immune protection against infection from a lethal dose of virus can be adoptively transferred with memory hepatic Thy1+ NK cells that were primed with live virus. Our results also indicate that, like classical immunological memory, stronger innate memory responses form in response to priming with live virus than a highly attenuated vector. These results demonstrate that a defined innate memory cell population alone can provide host protection against a lethal systemic infection through viral clearance., Author Summary Immunological memory is a hallmark of adaptive immunity and provides the basis for our ability to become ‘immune’ to pathogens to which we have previously been exposed, and provides the basis for vaccination. For decades, the paradigm held that only the classical adaptive lymphocytes were capable of forming and maintaining protective immunological memory. Recently, several papers have shown the capacity of an innate cell population, a subset of natural killer (NK) cells, to exhibit certain aspects of immunological memory. Here we show that innate memory forms in response to infection with vaccinia virus and resides in a discrete subset of NK cells. We further demonstrate that this innate memory provides significant host protection against a subsequent systemic infection with a lethal dose of vaccinia virus, in some cases resulting in the complete clearance of detectable virus. We also demonstrate that priming with live, replicating virus stimulates innate memory more robustly than a highly attenuated vector. These findings shed new light on this emergent area of immunology, and hold significant implications for harnessing innate memory as part of creating novel vaccination strategies.

Although mucosal CD8+T-cell responses are important in combating mucosal infections, the generation of such immune responses by vaccination remains problematic. In the present study, we evaluated the ability of plasmid DNA to induce local and systemic antigen-specific CD8+T-cell responses after pulmonary administration. We show that the pulmonary delivery of plasmid DNA formulated with polyethyleneimine (PEI-DNA) induced robust systemic CD8+T-cell responses that were comparable in magnitude to those generated by intramuscular (i.m.) immunization. Most importantly, we observed that the pulmonary delivery of PEI-DNA elicited a 10-fold-greater antigen-specific CD8+T-cell response in lungs and draining lymph nodes of mice than that of i.m. immunization. The functional evaluation of these pulmonary CD8+T cells revealed that they produced type I cytokines, and pulmonary immunization with PEI-DNA induced lung-associated antigen-specific CD4+T cells that produced higher levels of interleukin-2 than those induced by i.m. immunization. Pulmonary PEI-DNA immunization also induced CD8+T-cell responses in the gut and vaginal mucosa. Finally, pulmonary, but not i.m., plasmid DNA vaccination protected mice from a lethal recombinant vaccinia virus challenge. These findings suggest that pulmonary PEI-DNA immunization might be a useful approach for immunizing against pulmonary pathogens and might also protect against infections initiated at other mucosal sites.

While it is well established that CD8(+) T cells generated in the absence of CD4(+) T cells mediate defective recall responses, the mechanism by which CD4(+) T cells confer help in the generation of CD8(+) T-cell responses remains poorly understood. To determine whether CD4(+) T-cell-derived IL-21 is an important regulator of CD8(+) T-cell responses in help-dependent and -independent viral infections, we examined these responses in the IL-21Rα(-/-) mouse model. We show that IL-21 has a role in primary CD8(+) T-cell responses and in recall CD8(+) T-cell responses in help-dependent viral infections. This effect is due to a direct action of IL-21 in enhancing the proliferation of virus-specific CD8(+) T cells and reducing their TRAIL expression. These findings indicate that IL-21 is an important mediator of CD4(+) T-cell help to CD8(+) T cells.

Heterogeneous epithelial populations comprising the thymic environment influence early and late stages of T-cell development. The processes that regulate the differentiation of thymic epithelium and that are responsible for this heterogeneity are not well understood, although mesenchymal/epithelial interactions are clearly involved. Here, we show that targeted expression by thymocytes of an fibroblast growth factor receptor-2IIIb (FGFR2IIIb) ligand, FGF10, profoundly alters the differentiation and function of thymic epithelium (TE). Reconstitution of irradiated lckFGF10 mice with normal bone marrow restores normal thymic organization and function, while wild-type mice reconstituted with lckFGF10 bone marrow recapitulates some of the thymic alterations seen in lckFGF10 mice. We also demonstrate that interference with FGFR2IIIb signaling in the thymus with a soluble FGFR2IIIb dominant-negative fusion protein leads to precocious reductions in thymic size and cellularity that resemble age-related thymic involution. These findings indicate that TE compartments are dynamically maintained and that FGF signals are involved in this process. Developmental Dynamics 236:3459–3471, 2007. © 2007 Wiley-Liss, Inc.

Although putative thymic epithelial progenitor cells have been identified, the developmental potential of these cells, the extent of medullary thymic epithelium (mTEC) heterogeneity, and the mechanisms that mediate the expression of a wide range of peripheral tissue-restricted Ags (TRAs) by mTECs remain poorly defined. Here we have defined several basic properties of the mTEC population that refine our understanding of these cells and impose important constraints for any model of mTEC differentiation and function. We report here that mTECs from adult mice are mitotically active, implying continual turnover, differentiation, and replacement of mTEC populations in the adult thymus. The mTEC population in adult thymus expresses transcription factors implicated in the maintenance of multipotential progenitor cell populations, suggesting that epithelial progenitors in the adult thymus may not be restricted to a thymic fate. mTECs also express multiple transcription factors required for the specification of multiple epithelial lineages in peripheral tissues. Thus, expression of some TRAs by mTECs may represent coordinated gene expression that reflects alternate programs of epithelial differentiation among mTECs. Analysis of TRA expression in individual and small pools of sorted mTECs show that mTECs are highly heterogeneous; each individual mTEC expresses a limited spectrum of TRAs, and the frequency of mTECs that express any individual TRA is quite low (>0.4–2%). Collectively, these findings suggest that the differentiation of mTECs can involve some of the developmental programs used by other epithelial lineages and that expression of some TRAs by mTECs may reflect this activity.

The role of central tolerance induction has recently been revised after the discovery of promiscuous expression of tissue-restricted self-antigens in the thymus. The extent of tissue representation afforded by this mechanism and its cellular and molecular regulation are barely defined. Here we show that medullary thymic epithelial cells (mTECs) are specialized to express a highly diverse set of genes representing essentially all tissues of the body. Most, but not all, of these genes are induced in functionally mature CD80hi mTECs. Although the autoimmune regulator (Aire) is responsible for inducing a large portion of this gene pool, numerous tissue-restricted genes are also up-regulated in mature mTECs in the absence of Aire. Promiscuously expressed genes tend to colocalize in clusters in the genome. Analysis of a particular gene locus revealed expression of clustered genes to be contiguous within such a cluster and to encompass both Aire-dependent and –independent genes. A role for epigenetic regulation is furthermore implied by the selective loss of imprinting of the insulin-like growth factor 2 gene in mTECs. Our data document a remarkable cellular and molecular specialization of the thymic stroma in order to mimic the transcriptome of multiple peripheral tissues and, thus, maximize the scope of central self-tolerance.

Here we demonstrate that keratinocyte growth factor (KGF) and FGFR2IIIb signaling can affect development and function of thymic epithelium (TE) and that αβ-lineage thymocytes contribute to intrathymic levels of KGF. Thymocyte expression of KGF is developmentally regulated, being undetectable in CD3−4−8− thymocytes and expressed at highest levels by mature CD4 or CD8 thymocytes. Exposure of thymocyte-depleted fetal thymic lobes to KGF resulted in reduced thymic epithelial expression of class II major histocompatibility complex (MHC), invariant chain (Ii), and cathepsin L (CatL) molecules involved in thymocyte-positive selection and also stimulated expression of the cytokines interleukin 6 (IL-6) and thymic stromal-derived lymphopoietin (TSLP), while having little effect on IL-7 or stem cell factor expression. Within intact fetal thymic organ culture (FTOC), exogenous KGF impairs the generation of CD4 thymocytes. Two lines of evidence point to responsiveness of the medullary TE compartment to KGF and FGFR2IIIb signaling. First, the medullary compartment is expanded in intact FTOC exposed to KGF in vitro. Second, in the RAG-deficient thymus, where the thymocytes do not express detectable levels of KGF message, the hypoplastic medullary TE compartment can be expanded by administration of recombinant KGF in vivo. This expansion is accompanied by restoration of the normal profile of medullary TE–associated chemokine expression in the RAG2−/−thymus. Collectively, these findings point to a role for KGF and FGFR signaling in the development and function of thymic epithelium.

Eosinophils contain many preformed cytokines and chemokines, which are stored in specific granules along with cationic granule proteins. Mobilization and release of these granule contents can be selective and mediated by vesicular transport. We have developed a sensitive method to detect and quantitate eosinophil vesicular transport-mediated release of specific eosinophil proteins. Our EliCell assay is based on microscopic observations of individual viable eosinophils embedded in an agarose matrix that contains immobilized antibody to the protein of interest. Following stimulation of eosinophils, released protein is bound by the capture antibody at its site of release and is detected by a fluorochrome-conjugated detection antibody. We have validated this assay by evaluating interferon-gamma-induced release of RANTES from eosinophils. Extracellularly released RANTES was visualized as focal immunoflourescent staining and was quantitated by scoring the numbers of eosinophils releasing RANTES and by measuring the fluorescent intensity over individual eosinophils. In comparison with ELISA assays of RANTES released into supernatant fluids by interferon-gamma-stimulated eosinophils, EliCell assays were more sensitive enabling detection of RANTES release at earlier times and at lower levels of interferon-gamma stimulation. The EliCell assay provides a sensitive method to study the regulated release of eosinophil-derived cytokines, chemokines and other granule proteins.

Comment on Gray et al, page [3777][1] The relative radioresistance of thymic epithelium has led to the assumption that this population is static and exhibits little turnover during postnatal life. Recent studies demonstrating significant proliferation by thymic epithelial cells in the postnatal