Geographical analysis of seroprevalence of Ehrlichia spp., Anaplasma spp., Borrelia burgdorferi and Dirofilaria immitis, in clinics and dog shelters in different Mexican states.

This study aimed to determine the seroprevalence and geographical distribution of Ehrlichia spp., Anaplasma spp., Borrelia burgdorferi and Dirofilaria immitis in dogs in Mexico, including owned dogs from veterinary clinics with regular medical care and shelter dogs. The Mexican territory was divided into eight geographical regions; 22 out of 32 states were included; 110 veterinary clinics and 53 dog shelters participated. SNAP® 4Dx Plus® (IDEXX® Laboratories) was used to detect antibodies against Ehrlichia spp., Anaplasma spp., Borrelia burgdorferi and Dirofilaria immitis antigens. A total of 3522 apparently healthy dogs were tested, 1648 from clinics and 1874 from shelters. The highest seroprevalence of infection/exposure was found for Ehrlichia spp. (30.9%), followed by Anaplasma spp. (14.6%), D. immitis (5.3%) and B. burgdorferi (0.1%). Significantly more positive dogs were older than 3 years. Regarding differences between facility types, there were only differences for D. immitis which was more prevalent in clinics than in shelters (OR â€‹= â€‹1.97; 95% CI: 1.45-2.69; P â€‹< â€‹0.0001). Co-infections were detected in 38.4% of the positive samples. Dogs from Mexican states located on the Atlantic and the Pacific coast were significantly more at risk for Ehrlichia spp. and Anaplasma spp. infections than dogs from interior states. Dogs in Atlantic coastal states were more at risk for Dirofilaria immitis infection.

Cell-based artificial APC resistant to lentiviral transduction for efficient generation of CAR-T cells from various cell sources.

BACKGROUND: Adoptive cell therapy with chimeric antigen receptor T cells (CAR-T) has become a standard treatment for patients with certain aggressive B cell malignancies and holds promise to improve the care of patients suffering from numerous other cancers in the future. However, the high manufacturing cost of CAR-T cell therapies poses a major barrier to their broader clinical application. Among the key cost drivers of CAR-T production are single-use reagents for T cell activation and clinical-grade viral vector. The presence of variable amounts of contaminating monocytes in the starting material poses an additional challenge to CAR-T manufacturing, since they can impede T cell stimulation and transduction, resulting in manufacturing failure. METHODS: We created K562-based artificial antigen-presenting cells (aAPC) with genetically encoded T cell stimulation and costimulation that represent an inexhaustible source for T cell activation. We additionally disrupted endogenous expression of the low-density lipoprotein receptor (LDLR) on these aAPC (aAPC-ΔLDLR) using CRISPR-Cas9 gene editing nucleases to prevent inadvertent lentiviral transduction and avoid the sink effect on viral vector during transduction. Using various T cell sources, we produced CD19-directed CAR-T cells via aAPC-ΔLDLR-based activation and tested their in vitro and in vivo antitumor potency against B cell malignancies. RESULTS: We found that lack of LDLR expression on our aAPC-ΔLDLR conferred resistance to lentiviral transduction during CAR-T production. Using aAPC-ΔLDLR, we achieved efficient expansion of CAR-T cells even from unpurified starting material like peripheral blood mononuclear cells or unmanipulated leukapheresis product, containing substantial proportions of monocytes. CD19-directed CAR-T cells that we produced via aAPC-ΔLDLR-based expansion demonstrated potent antitumor responses in preclinical models of acute lymphoblastic leukemia and B-cell lymphoma. CONCLUSIONS: Our aAPC-ΔLDLR represent an attractive approach for manufacturing of lentivirally transduced T cells that may be simpler and more cost efficient than currently available methods.

Reducing Ex Vivo Culture Improves the Antileukemic Activity of Chimeric Antigen Receptor (CAR) T Cells.

The success of chimeric antigen receptor (CAR)-mediated immunotherapy in acute lymphoblastic leukemia (ALL) highlights the potential of T-cell therapies with directed cytotoxicity against specific tumor antigens. The efficacy of CAR T-cell therapy depends on the engraftment and persistence of T cells following adoptive transfer. Most protocols for T-cell engineering routinely expand T cells ex vivo for 9 to 14 days. Because the potential for engraftment and persistence is related to the state of T-cell differentiation, we hypothesized that reducing the duration of ex vivo culture would limit differentiation and enhance the efficacy of CAR T-cell therapy. We demonstrated that T cells with a CAR-targeting CD19 (CART19) exhibited less differentiation and enhanced effector function in vitro when harvested from cultures at earlier (day 3 or 5) compared with later (day 9) timepoints. We then compared the therapeutic potential of early versus late harvested CART19 in a murine xenograft model of ALL and showed that the antileukemic activity inversely correlated with ex vivo culture time: day 3 harvested cells showed robust tumor control despite using a 6-fold lower dose of CART19, whereas day 9 cells failed to control leukemia at limited cell doses. We also demonstrated the feasibility of an abbreviated culture in a large-scale current good manufacturing practice-compliant process. Limiting the interval between T-cell isolation and CAR treatment is critical for patients with rapidly progressing disease. Generating CAR T cells in less time also improves potency, which is central to the effectiveness of these therapies. Cancer Immunol Res; 6(9); 1100-9. ©2018 AACR.

Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia.

Tolerance to self-antigens prevents the elimination of cancer by the immune system1,2. We used synthetic chimeric antigen receptors (CARs) to overcome immunological tolerance and mediate tumor rejection in patients with chronic lymphocytic leukemia (CLL). Remission was induced in a subset of subjects, but most did not respond. Comprehensive assessment of patient-derived CAR T cells to identify mechanisms of therapeutic success and failure has not been explored. We performed genomic, phenotypic and functional evaluations to identify determinants of response. Transcriptomic profiling revealed that CAR T cells from complete-responding patients with CLL were enriched in memory-related genes, including IL-6/STAT3 signatures, whereas T cells from nonresponders upregulated programs involved in effector differentiation, glycolysis, exhaustion and apoptosis. Sustained remission was associated with an elevated frequency of CD27+CD45RO-CD8+ T cells before CAR T cell generation, and these lymphocytes possessed memory-like characteristics. Highly functional CAR T cells from patients produced STAT3-related cytokines, and serum IL-6 correlated with CAR T cell expansion. IL-6/STAT3 blockade diminished CAR T cell proliferation. Furthermore, a mechanistically relevant population of CD27+PD-1-CD8+ CAR T cells expressing high levels of the IL-6 receptor predicts therapeutic response and is responsible for tumor control. These findings uncover new features of CAR T cell biology and underscore the potential of using pretreatment biomarkers of response to advance immunotherapies.

The Flipside of the Power of Engineered T Cells: Observed and Potential Toxicities of Genetically Modified T Cells as Therapy.

Autologous T cells modified to recognize novel antigen targets are a novel form of therapy for cancer. We review the various potential forms of observed and hypothetical toxicities associated with genetically modified T cells. Despite the focus on toxicities in this review, re-directed T cells represent a powerful and highly effective form of anti-cancer therapy; we remain optimistic that the common toxicities will become routinely manageable and that some theoretical toxicity will be exceedingly rare, if ever observed.

CARs in the Lead Against Multiple Myeloma.

The recent clinical success of CD19-directed chimeric antigen receptor (CAR) T cell therapy in chronic and acute leukemia has led to increased interest in broadening this technology to other hematological malignancies and solid tumors. Now, advances are being made using CAR T cell technology to target myeloma antigens such as B cell maturation antigen (BCMA), CD138, and kappa-light chain as well as CD19 on putative myeloma stem cells. To date, only a limited number of multiple myeloma patients have received CAR T cell therapy but preliminary results have been encouraging. In this review, we summarize the recently reported results of clinical trials conducted utilizing CAR T cell therapy in multiple myeloma (MM).

Chimeric Antigen Receptor T Cells: Self-Replicating Drugs for Cancer.

The chimeric antigen receptor (CAR) technology started out as a tool to understand lymphocyte biology but rapidly developed into a T cell therapeutic agent for the treatment of cancers. Here, we describe the technological advances in the field of CARs and highlight critical components of its success. Additionally, we describe how various laboratories have worked toward developing new, safer, and more potent CARs for cancer.

Distinct modes of antigen presentation promote the formation, differentiation, and activity of foxp3+ regulatory T cells in vivo.

How the formation and activity of CD4(+)Foxp3(+) regulatory T cells (Tregs) are shaped by TCR recognition of the diverse array of peptide:MHC complexes that can be generated from self-antigens and/or foreign Ags in vivo remains poorly understood. We show that a self-peptide with low (but not high) stimulatory potency promotes thymic Treg formation and can induce conventional CD4(+) T cells in the periphery to become Tregs that express different levels of the transcription factor Helios according to anatomical location. When Tregs generated in response to this self-peptide subsequently encountered the same peptide derived instead from influenza virus in the lung-draining lymph nodes of infected mice, they proliferated, acquired a T-bet(+)CXCR3(+) phenotype, and suppressed the antiviral effector T cell response in the lungs. However, these self-antigen-selected Tregs were unable to suppress the antiviral immune response based on recognition of the peptide as a self-antigen rather than a viral Ag. Notably, when expressed in a more immunostimulatory form, the self-peptide inhibited the formation of T-bet(+)CXCR3(+) Tregs in response to viral Ag, and Ag-expressing B cells from these mice induced Treg division without upregulation of CXCR3. These studies show that a weakly immunostimulatory self-peptide can induce thymic and peripheral Foxp3(+) Treg formation but is unable to activate self-antigen-selected Tregs to modulate an antiviral immune response. Moreover, a strongly immunostimulatory self-peptide expressed by B cells induced Tregs to proliferate without acquiring an effector phenotype that allows trafficking from the draining lymph node to the lungs and, thereby, prevented the Tregs from suppressing the antiviral immune response.

Viral antigen induces differentiation of Foxp3+ natural regulatory T cells in influenza virus-infected mice.

We examined the formation, participation, and functional specialization of virus-reactive Foxp3(+) regulatory T cells (Tregs) in a mouse model of influenza virus infection. "Natural" Tregs generated intrathymically, based on interactions with a self-peptide, proliferated in response to a homologous viral Ag in the lungs and, to a lesser extent, in the lung-draining mediastinal lymph nodes (medLNs) of virus-infected mice. In contrast, conventional CD4(+) T cells with identical TCR specificity underwent little or no conversion to become "adaptive" Tregs. The virus-reactive Tregs in the medLNs and the lungs of infected mice upregulated a variety of molecules associated with Treg activation, as well as acquired expression of molecules (T-bet, Blimp-1, and IL-10) that confer functional specialization to Tregs. Notably, however, the phenotypes of the T-bet(+) Tregs obtained from these sites were distinct, because Tregs isolated from the lungs expressed significantly higher levels of T-bet, Blimp-1, and IL-10 than did Tregs from the medLNs. Adoptive transfer of Ag-reactive Tregs led to decreased proliferation of antiviral CD4(+) and CD8(+) effector T cells in the lungs of infected hosts, whereas depletion of Tregs had a reciprocal effect. These studies demonstrate that thymically generated Tregs can become activated by a pathogen-derived peptide and acquire discrete T-bet(+) Treg phenotypes while participating in and modulating an antiviral immune response.

Identification of mitochondrial genome concatemers in AIDS-associated lymphomas and lymphoid cell lines.

Since most oncogenic viruses persist as extrachromosomal covalently closed circular DNA (cccDNA) in tumor cells, we developed an assay to visualize and identify cccDNA in primary lymphomas. We identified concatemers of the mitochondrial genome in all samples analyzed, but not in normal lymphocytes. One AIDS-associated lymphoma (EL) was further studied in detail as its mitochondrial genome consisted of tandem head-to-tail duplications. Insertion of C-residues was noted near the origin of replication of EL mtDNA. EL cells responded weakly to Fas-apoptotic stimulus, displayed reduced mitochondrial activity and mass, and produced higher levels of reactive oxygen intermediates. Screening of several AIDS-associated lymphomas and established lymphoid cell lines also revealed the presence of mitochondrial genome concatemers consisting of interlinked monomer molecules. Taken together, our results suggest that formation of mtDNA concatemers is associated with oncogenic transformation in lymphoid cells.

Formation of Mitochondrial Genome Concatemers as an Alternative Mechanism Promoting Oncogenic Transformation of Lymphoid Cells.

We have previously shown that AIDS-associated lymphomas and lymphoma cell lines contain mitochondrial genome concatemers not present in normal T-lymphocytes. Since cellular homeostasis and energy production rely heavily on mitochondrial DNA (mtDNA) stability, mutations in the mtDNA have long been linked to the development of various types of cancers. In most of the cases, however, neoplastically transformed cells harbor non-mutated mtDNA. Herein, we propose an alternative mechanism that shows how the formation of mitochondrial genome concatemers may promote oncogenic transformation of normal lymphoid progenitor cells when no mtDNA mutations or chromosomal aberrations are present. We detected high reactive oxygen species (ROS) levels in the lymphoma samples tested despite no identification of putative mutations in the coding mtDNA. We propose that the formation of atypical mtDNA configurations (i.e. dimers and concatemers) interferes with normal mitochondrial function. Unstable mitochondria lead to abnormal assembly and dysfunction of the oxidative phosphorylation (OXPHOS) complexes, eventually leading to oxidative stress from elevated production of intracellular ROS. ROS have been reported to activate transcription factors associated with cellular proliferation and apoptosis inhibition. Therefore, we hypothesize that formation of mitochondrial genome concatemers can augment endogenous ROS levels capable of promoting oncogenic transformation of normal lymphoid progenitor cells.

Pyrin-only protein 2 modulates NF-kappaB and disrupts ASC:CLR interactions.

NF-kappaB is pivotal for transactivation of cell-cycle regulatory, cytokine, and adhesion molecule genes and is dysregulated in many cancers, neurodegenerative disorders, and inflammatory diseases. Proteins with pyrin and/or caspase recruitment domains have roles in apoptosis, innate immunity, and inflammation. Many pyrin domain (PYD) proteins modulate NF-kappaB activity as well as participate in assembling both the perinuclear "apoptotic speck" and the pro-IL1beta/IL-18-converting inflammasome complex. "Pyrin-only" proteins (POP) are attractive as negative regulators of PYD-mediated functions and one such protein, POP1, has been reported. We report the identification and initial characterization of a second POP. POP2 is a 294 nt single exon gene located on human chromosome 3 encoding a 97-aa protein with sequence and predicted structural similarity to other PYDs. Highly similar to PYDs in CATERPILLER (CLR, NLR, NALP) family proteins, POP2 is less like the prototypic pyrin and ASC PYDs. POP2 is expressed principally in peripheral blood leukocytes and displays both cytoplasmic and nuclear expression patterns in transfected cells. TNF-alpha-stimulated and p65 (RelA)-induced NF-kappaB-dependent gene transcription is inhibited by POP2 in vitro by a mechanism involving changes in NF-kappaB nuclear import or distribution. While colocalizing with ASC in perinuclear specks, POP2 also inhibits the formation of specks by the CLR protein CIAS1/NALP3. Together, these observations demonstrate that POP2 is a negative regulator of NF-kappaB activity that may influence the assembly of PYD-dependent complexes.