Distinct Signaling of Coreceptors Regulates Specific Metabolism Pathways and Impacts Memory Development in CAR T Cells.

Chimeric antigen receptors (CARs) redirect T cell cytotoxicity against cancer cells, providing a promising approach to cancer immunotherapy. Despite extensive clinical use, the attributes of CAR co-stimulatory domains that impact persistence and resistance to exhaustion of CAR-T cells remain largely undefined. Here, we report the influence of signaling domains of coreceptors CD28 and 4-1BB on the metabolic characteristics of human CAR T cells. Inclusion of 4-1BB in the CAR architecture promoted the outgrowth of CD8(+) central memory T cells that had significantly enhanced respiratory capacity, increased fatty acid oxidation and enhanced mitochondrial biogenesis. In contrast, CAR T cells with CD28 domains yielded effector memory cells with a genetic signature consistent with enhanced glycolysis. These results provide, at least in part, a mechanistic insight into the differential persistence of CAR-T cells expressing 4-1BB or CD28 signaling domains in clinical trials and inform the design of future CAR T cell therapies.

Immunoregulation by antibody secreting cells in inflammation, infection, and cancer.

Antibody-secreting cells (ASCs) are considered work horses of the humoral immune response for their tireless effort to produce large amounts of antibodies that fulfill an array of functions in host defense, inflammation, and maintenance of homeostasis. While traditionally considered largely senescent cells, surprising recent findings demonstrate that subsets of ASCs downmodulate ongoing immune responses independent of antibody formation. Such regulatory ASCs produce IL-10 or IL-35 and are implicated in maintaining tissue and immune homeostasis. They also serve to suppress pathogenic leukocytes in infection, allergy, and inflammatory diseases that affect tissues, such as the central nervous system and the respiratory tract. Additionally, regulatory ASCs infiltrate various cancer types and restrict effective anti-tumor T cell responses. While incompletely understood, there is significant overlap in factors that control ASC differentiation, IL-10 expression by B cells and the generation of ASCs that secrete both antibodies and IL-10. In this review, we will cover the biology, phenotype, generation, maintenance and function of regulatory ASCs in various tissues under pathological and steady states. An improved understanding of the development of regulatory ASCs and their biological roles will be critical for generating novel ASC-targeted therapies for the treatment of inflammatory diseases, infection, and cancer.

Skin-Associated B Cells in Health and Inflammation.

Traditionally, the skin was believed to be devoid of B cells, and studies of the skin immune system have largely focused on other types of leukocytes. Exciting recent data show that B cells localize to the healthy skin of humans and other mammalian species with likely homeostatic functions in host defense, regulation of microbial communities, and wound healing. Distinct skin-associated B cell subsets drive or suppress cutaneous inflammatory responses with important clinical implications. Localized functions of skin-associated B cell subsets during inflammation comprise Ab production, interactions with skin T cells, tertiary lymphoid tissue formation, and production of proinflammatory cytokines but also include immunosuppression by providing IL-10. In this review, we delve into the intriguing new roles of skin-associated B cells in homeostasis and inflammation.

Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia.

Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is highly promising but requires robust T-cell expansion and engraftment. A T-cell defect in chronic lymphocytic leukemia (CLL) due to disease and/or therapy impairs ex vivo expansion and response to CAR T cells. To evaluate the effect of ibrutinib treatment on the T-cell compartment in CLL as it relates to CAR T-cell generation, we examined the phenotype and function of T cells in a cohort of CLL patients during their course of treatment with ibrutinib. We found that ≥5 cycles of ibrutinib therapy improved the expansion of CD19-directed CAR T cells (CTL019), in association with decreased expression of the immunosuppressive molecule programmed cell death 1 on T cells and of CD200 on B-CLL cells. In support of these findings, we observed that 3 CLL patients who had been treated with ibrutinib for ≥1 year at the time of T-cell collection had improved ex vivo and in vivo CTL019 expansion, which correlated positively together and with clinical response. Lastly, we show that ibrutinib exposure does not impair CAR T-cell function in vitro but does improve CAR T-cell engraftment, tumor clearance, and survival in human xenograft models of resistant acute lymphocytic leukemia and CLL when administered concurrently. Our collective findings indicate that ibrutinib enhances CAR T-cell function and suggest that clinical trials with combination therapy are warranted. Our studies demonstrate that improved T-cell function may also contribute to the efficacy of ibrutinib in CLL. These trials were registered at www.clinicaltrials.gov as #NCT01747486, #NCT01105247, and #NCT01217749.

ICOS-based chimeric antigen receptors program bipolar TH17/TH1 cells.

With the notable exception of B-cell malignancies, the efficacy of chimeric antigen receptor (CAR) T cells has been limited, and CAR T cells have not been shown to expand and persist in patients with nonlymphoid tumors. Here we demonstrate that redirection of primary human T cells with a CAR containing the inducible costimulator (ICOS) intracellular domain generates tumor-specific IL-17-producing effector cells that show enhanced persistence. Compared with CARs containing the CD3ζ chain alone, or in tandem with the CD28 or the 4-1BB intracellular domains, ICOS signaling increased IL-17A, IL-17F, and IL-22 following antigen recognition. In addition, T cells redirected with an ICOS-based CAR maintained a core molecular signature characteristic of TH17 cells and expressed higher levels of RORC, CD161, IL1R-1, and NCS1. Of note, ICOS signaling also induced the expression of IFN-γ and T-bet, consistent with a TH17/TH1 bipolarization. When transferred into mice with established tumors, TH17 cells that were redirected with ICOS-based CARs mediated efficient antitumor responses and showed enhanced persistence compared with CD28- or 4-1BB-based CAR T cells. Thus, redirection of TH17 cells with a CAR encoding the ICOS intracellular domain is a promising approach to augment the function and persistence of CAR T cells in hematologic malignancies.

Type I interferon upregulates Bak and contributes to T cell loss during human immunodeficiency virus (HIV) infection.

The role of Type I interferon (IFN) during pathogenic HIV and SIV infections remains unclear, with conflicting observations suggesting protective versus immunopathological effects. We therefore examined the effect of IFNα/ß on T cell death and viremia in HIV infection. Ex vivo analysis of eight pro- and anti-apoptotic molecules in chronic HIV-1 infection revealed that pro-apoptotic Bak was increased in CD4+ T cells and correlated directly with sensitivity to CD95/Fas-mediated apoptosis and inversely with CD4+ T cell counts. Apoptosis sensitivity and Bak expression were primarily increased in effector memory T cells. Knockdown of Bak by RNA interference inhibited CD95/Fas-induced death of T cells from HIV-1-infected individuals. In HIV-1-infected patients, IFNα-stimulated gene expression correlated positively with ex vivo T cell Bak levels, CD95/Fas-mediated apoptosis and viremia and negatively with CD4+ T cell counts. In vitro IFNα/ß stimulation enhanced Bak expression, CD95/Fas expression and CD95/Fas-mediated apoptosis in healthy donor T cells and induced death of HIV-specific CD8+ T cells from HIV-1-infected patients. HIV-1 in vitro sensitized T cells to CD95/Fas-induced apoptosis and this was Toll-like receptor (TLR)7/9- and Type I IFN-dependent. This sensitization by HIV-1 was due to an indirect effect on T cells, as it occurred in peripheral blood mononuclear cell cultures but not purified CD4+ T cells. Finally, peak IFNα levels and viral loads correlated negatively during acute SIV infection suggesting a potential antiviral effect, but positively during chronic SIV infection indicating that either the virus drives IFNα production or IFNα may facilitate loss of viral control. The above findings indicate stage-specific opposing effects of Type I IFNs during HIV-1 infection and suggest a novel mechanism by which these cytokines contribute to T cell depletion, dysregulation of cellular immunity and disease progression.

Specificity of ertapenem susceptibility screening for detection of Klebsiella pneumoniae carbapenemases.

Detection of Klebsiella pneumoniae carbapenemases (KPCs) can be nonspecific, especially when KPCs are uncommon. We determined the positive predictive value and specificity of ertapenem resistance for KPC detection in 2,696 Enterobacteriaceae isolates. The positive predictive value and specificity of ertapenem resistance for KPC detection were 74% and 99.2%, respectively.

Prevalence of CTX-M beta-lactamases in Philadelphia, Pennsylvania.

CTX-M beta-lactamases were thought to be rare in the United States, but a recent study in Texas showed that up to 70% of extended-spectrum beta-lactamase (ESBL)-containing members of the Enterobacteriaceae family were CTX-M positive (J. S. Lewis, M. Herrera, B. Wickes, J. E. Patterson, and J. H. Jorgensen, Antimicrob. Agents Chemother. 51:4015-4021, 2007). We used PCR to detect CTX-M in all 291 extended-spectrum cephalosporin-resistant gram-negative bacteria isolated in our laboratory during 2007. Thirty (48%) Escherichia coli isolates, 6 (3%) Klebsiella sp. isolates, and 7 (100%) Proteus mirabilis isolates tested were CTX-M positive, with 15% of all Enterobacteriaceae tested being positive. The E. coli CTX-M groups were I (57%), IV (37%), II (3%), and not groupable (3%); three of the group IV isolates were positive for CTX-M-18, and three of the group I isolates were positive for CTX-M-15. One of seven positive P. mirabilis isolates was in group II, with the remainder being positive for a CTX-M-25-like beta-lactamase; and 33% of the Klebsiella sp. isolates were in group I or IV, with the remainder not being in groups I to IV. CTX-M-producing bacteria were isolated from urine (n = 13), blood (n = 13), wounds (n = 12), and the respiratory tract (n = 4). All 31 CTX-M-positive isolates tested for the presence of ESBL were confirmed to produce ESBLs by the use of tests recommended by the CLSI. Pulsed-field gel electrophoresis of the CTX-M-positive isolates showed that six P. mirabilis isolates were clonal and that there were seven different E. coli clusters. Five of seven P. mirabilis isolates were from blood cultures. The CLSI tests for the confirmation of ESBL production reliably detect these isolates if both cefotaxime and ceftazidime are tested, but only about half would be classified as a possible CTX-M producers on the basis of the antibiogram alone. A new panprimer set increases the ability to detect CTX-M-producing strains. CTX-M-positive bacteria are common in our geographic region, are often invasive, and, with the exception of P. mirabilis, are multiclonal.

IgM Plasma Cells Reside in Healthy Skin and Accumulate with Chronic Inflammation.

Antibodies are key to cutaneous host defense and inflammation. Despite their importance, the mechanisms by which skin antibodies are sustained are poorly described. Here, we identified that, in addition to antibody production in lymphoid tissues, plasma cells reside in healthy mouse and human skin. In naïve mice, IgM was the predominant isotype produced in skin. Skin plasma cells developed independently of T cells and microbiota. Importantly, chronic skin inflammation promoted the massive accumulation of IgM-secreting cells, and cutaneous immunization directed both T cell-dependent and -independent antigen-specific IgM-secreting cells into skin. Unlike their counterparts in lymphoid tissues, cutaneous IgM-secreting cells were completely dependent on survival factors such as a proliferation-inducing ligand or B cell-activating factor, which were constitutively expressed and upregulated during inflammation in skin. Our data support a model in which skin plasma cells supply natural and adaptive IgM to the cutaneous environment, thereby supporting homeostatic skin barrier functions and providing defense against pathogen intrusion. Our results are also of potential relevance for manipulation of cutaneous plasma cells in inflammatory skin diseases or cutaneous plasma cell malignancies.

Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation.

Successful tumor eradication by chimeric antigen receptor-expressing (CAR-expressing) T lymphocytes depends on CAR T cell persistence and effector function. We hypothesized that CD4+ and CD8+ T cells may exhibit distinct persistence and effector phenotypes, depending on the identity of specific intracellular signaling domains (ICDs) used to generate the CAR. First, we demonstrate that the ICOS ICD dramatically enhanced the in vivo persistence of CAR-expressing CD4+ T cells that, in turn, increased the persistence of CD8+ T cells expressing either CD28- or 4-1BB-based CARs. These data indicate that persistence of CD8+ T cells was highly dependent on a helper effect provided by the ICD used to redirect CD4+ T cells. Second, we discovered that combining ICOS and 4-1BB ICDs in a third-generation CAR displayed superior antitumor effects and increased persistence in vivo. Interestingly, we found that the membrane-proximal ICD displayed a dominant effect over the distal domain in third-generation CARs. The optimal antitumor and persistence benefits observed in third-generation ICOSBBz CAR T cells required the ICOS ICD to be positioned proximal to the cell membrane and linked to the ICOS transmembrane domain. Thus, CARs with ICOS and 4-1BB ICD demonstrate increased efficacy in solid tumor models over our current 4-1BB-based CAR and are promising therapeutics for clinical testing.

Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells.

This study compared second-generation chimeric antigen receptors (CAR) encoding signaling domains composed of CD28, ICOS, and 4-1BB (TNFRSF9). Here, we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T cells with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to 3 months following a single stimulation through the T-cell receptor (TCR). Sustained numeric expansion was independent of cognate antigen and did not require the addition of exogenous cytokines or feeder cells after a single stimulation of the TCR and CD28. Results from gene array and functional assays linked sustained cytokine secretion and expression of T-bet (TBX21), EOMES, and GATA-3 to the effect. Sustained expression of the endogenous IL2 locus has not been reported in primary T cells. Sustained proliferation was dependent on CAR structure and high expression, the latter of which was necessary but not sufficient. The mechanism involves constitutive signaling through NF-κB, AKT, ERK, and NFAT. The propagated CAR T cells retained a diverse TCR repertoire, and cellular transformation was not observed. The CARs with a constitutive growth phenotype displayed inferior antitumor effects and engraftment in vivo. Therefore, the design of CARs that have a nonconstitutive growth phenotype may be a strategy to improve efficacy and engraftment of CAR T cells. The identification of CARs that confer constitutive or nonconstitutive growth patterns may explain observations that CAR T cells have differential survival patterns in clinical trials.

Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma.

Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII(+) glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376).