Functional Memory Cells Research Articles

Insights into Clonal Relationships of Putative Adaptive Natural Killer Cells (NK) in Humans, Via Mapping of Somatic Piga Mutations in Patients with Paroxysmal Nocturna Hemoglobinuria (PNH)

NK cells play a central role in innate immunity, specifically in tumor surveillance and microbial pathogen control. Recent murine models and human studies have identified subsets of NK-cells with apparent memory cell function, strongly linked to CMV infection in humans and termed "adaptive" NK. Our recent clonal tracking studies following autologous hematopoietic stem cells transplantation (HSCT) of genetically-barcoded CD34+ cells in macaques revealed distinct clonal ontogeny of a subset of NK cells within the primate equivalent of the human CD56dim population (Wu et al., Cell Stem Cell, 2014), with little clonal overlap with T-, B-lymphoid or myeloid cells, suggesting a separate precursor pool for this NK subtype. Peripheral blood CD 56bright -NK cells have been previously hypothesized to be precursors for the main population of circulating cytotoxic CD56dim cells. To further investigate NK-cell ontogeny and clonal relationships in humans we took advantage of naturally-occurring somatic mutations in the X-linked phosphatidylinositol glycan class A (PIGA) gene in patients with the hemolytic disorder paroxysmal nocturnal hemoglobinuria. This gene codes for an enzyme required for cell surface localization of glycosylphophatidylinositol (GPI)-anchored proteins, and thus loss of function mutations result in hematopoietic cells lacking GPI-anchored proteins, and red cell hemolysis. PNH patients have not been reported to have immune dysfunction and can have stable disease for many years. Membrane bound GPI anchors can be detected on any cell via flow cytometry using a labeled inactive aerolysin (FLAER), and serves as a marker for the fraction of cells comprising the PIGA mutant (GPI negative) clonal compartment. The PNH clone sizes contributing to peripheral blood cells are variable in but can reach almost 100% in some patients, and can be stable over decades. We selected 9 PNH patients with GPI negative granulocytes ranging from 5% to 98% and a median time from diagnosis of 43.7 months (15-100) for this study. NK cells were defined as CD56+/CD16+/CD3-/CD20- lymphocytes. We observed disproportionally fewer GPI negative NK cells compared to granulocytes (Fig 1), with the discrepancy most marked the major peripheral blood CD56dim population (mean 65% vs 25% GPI negative granulocytes, p = 0.0028, paired t-test), in contrast to 46% GPI negative cells in the CD56bright population (p=0.057). Due to the prolonged life span of memory T and B cells, fewer GPI negative B and particularly T-lymphocytes have been reported in PNH patients. In our cohort 3.4% of CD3+ T-cells and 13.2% of CD20+ B-cells were GPI negative (p=0.0005 and 0.0014, respectively versus granulocytes). Compared to the NK subsets, the CD56bright population showed the most significant differences (p = 0.0063 versus CD3 and p=0.0151 versus CD20). To further characterize the phenotype of the GPI positive versus negative CD56dim cells, we analyzed cells co-expressing either the terminal differential marker CD57, the inhibitory receptor NKG2A, or activating receptor NKG2C for FLAER positivity. Prior studies have suggested that the human CMV-linked adaptive NK subset is CD57+, NKG2A- and NKG2C+. The NKG2C+ CD 56dim population was highly enriched for GPI positive cells (p=0.0024 vs granulocytes, Fig 1). This profile was most prominent in CMV-IgG positive patients who had also significantly more GPI positive CD 56dim/CD57+ cells compared to granulocytes (p=0.008). Interestingly, one CMV positive patient (#5) had a complete lack of NKG2C expression, most likely due to homozygous loss of function mutation, and this patient had almost 100% GPI negative NK cells, matching his neutrophil pattern. Compared to granulocytes, NKGA2A+ or CD57+ positive CD56dim cells were also mostly GPI negative (p=0.0081 and 0.028).Circulating NK cell turnover has been estimated to be about 14 days. The PNH patients studied had documented clonal PIG-A mutations for many years. Our observation that the majority of CD56dim NK cells, specifically the NKG2C subset, are not progeny of the same progenitors producing CD56bright NK cells or myeloid cells based on clonal disparity regarding the PNH clone is suggestive of an independent, very long-lived or self-renewing NK cell progenitor for a CMV-linked CD56dim/CD57+/NKG2C+ memory NK cell compartment. These observations provide novel further insights into the human adaptive NK cell subset. [Display omitted] DisclosuresWinkler:Novartis: Research Funding; GSK: Research Funding. Townsley:Novartis: Research Funding; GSK: Research Funding.

CD39 Expression Identifies Terminally Exhausted CD8+ T Cells.

Exhausted T cells express multiple co-inhibitory molecules that impair their function and limit immunity to chronic viral infection. Defining novel markers of exhaustion is important both for identifying and potentially reversing T cell exhaustion. Herein, we show that the ectonucleotidse CD39 is a marker of exhausted CD8+ T cells. CD8+ T cells specific for HCV or HIV express high levels of CD39, but those specific for EBV and CMV do not. CD39 expressed by CD8+ T cells in chronic infection is enzymatically active, co-expressed with PD-1, marks cells with a transcriptional signature of T cell exhaustion and correlates with viral load in HIV and HCV. In the mouse model of chronic Lymphocytic Choriomeningitis Virus infection, virus-specific CD8+ T cells contain a population of CD39high CD8+ T cells that is absent in functional memory cells elicited by acute infection. This CD39high CD8+ T cell population is enriched for cells with the phenotypic and functional profile of terminal exhaustion. These findings provide a new marker of T cell exhaustion, and implicate the purinergic pathway in the regulation of T cell exhaustion.

Repeated Injections of IL-2 Break Renal Allograft Tolerance Induced via Mixed Hematopoietic Chimerism in Monkeys.

Tolerance of allografts achieved in mice via stable mixed hematopoietic chimerism relies essentially on continuous elimination of developing alloreactive T cells in the thymus (central deletion). Conversely, while only transient mixed chimerism is observed in nonhuman primates and patients, it is sufficient to ensure tolerance of kidney allografts. In this setting, it is likely that tolerance depends on peripheral regulatory mechanisms rather than thymic deletion. This implies that, in primates, upsetting the balance between inflammatory and regulatory alloimmunity could abolish tolerance and trigger the rejection of previously accepted renal allografts. In this study, six monkeys that were treated with a mixed chimerism protocol and had accepted a kidney allograft for periods of 1-10 years after withdrawal of immunosuppression received subcutaneous injections of IL-2 cytokine (0.6-3 × 10(6) IU/m(2) ). This resulted in rapid rejection of previously tolerated renal transplants and was associated with an expansion and reactivation of alloreactive pro-inflammatory memory T cells in the host's lymphoid organs and in the graft. This phenomenon was prevented by anti-CD8 antibody treatment. Finally, this process was reversible in that cessation of IL-2 administration aborted the rejection process and restored normal kidney graft function.

(Invited) Vlsi Carbon Nanotube Thin-Film Circuits

With the availability of large quantities of electronically monodisperse carbon nanotubes (CNTs) [1], homogeneous semiconducting CNT thin films can be produced through a variety of solution-based methods including vacuum filtration [2], dielectrophoresis [3], evaporation-driven self-assembly [4], aerosol jet printing [5], and inkjet printing [6,7]. Through careful integration of these thin films with high-k gate dielectrics [8], CNT thin-film transistors (TFTs) can achieve device metrics that are competitive with polycrystalline silicon and amorphous oxide semiconductors [9]. Furthermore, with appropriate device geometries, doping schemes, and gate electrode materials, threshold voltage can be tuned to enable enhancement-mode p-type and n-type TFTs, resulting in CNT CMOS logic gates with subnanowatt static power dissipation and full rail-to-rail voltage swing [10]. While these results suggest that very-large-scale integration (VLSI) should be possible for CNT thin-film circuits, further improvements are needed in CNT TFT large-area spatial homogeneity and long-term environmental stability. Towards these ends, this talk will introduce encapsulation schemes and automated measurement strategies that yield wafer-scale CNT TFT arrays with sufficiently tight control over on/off ratio, subthreshold swing, and threshold voltage for VLSI CNT thin-film circuits. Using this methodology, large-area arrays of fully functional CNT TFT static random access memory (SRAM) cells are demonstrated with desirable static noise and write margins. [1] D. Jariwala, V. K. Sangwan, L. J. Lauhon, T. J. Marks, and M. C. Hersam, “Carbon nanomaterials for electronics, optoelectronics, photovoltaics, and sensing,” Chem. Soc. Rev., 42, 2824 (2013). [2] A. Behnam, V. K. Sangwan, X. Zhong, F. Lian, D. Estrada, D. Jariwala, A. J. Hoag, L. J. Lauhon, T. J. Marks, M. C. Hersam, and E. Pop, “High-field transport and thermal reliability of sorted carbon nanotube network devices,” ACS Nano, 7, 482 (2013). [3] M. Steiner, M. Engel, Y.-M. Lin, Y. Wu, K. Jenkins, D. B. Farmer, J. J. Humes, N. L. Yoder, J.-W. T. Seo, A. A. Green, M. C. Hersam, R. Krupke, and Ph. Avouris, “High-frequency performance of scaled carbon nanotube array field-effect transistors,” Appl. Phys. Lett., 101, 053123 (2012). [4] T. A. Shastry, J.-W. T. Seo, J. J. Lopez, H. N. Arnold, J. Z. Kelter, V. K. Sangwan, L. J. Lauhon, T. J. Marks, and M. C. Hersam, “Large-area, electronically monodisperse, aligned single-walled carbon nanotube thin films fabricated by evaporation-driven self-assembly,” Small, 9, 45 (2013). [5] M. Ha, J.-W. T. Seo, P. L. Prabhumirashi, W. Zhang, M. L. Geier, M. J. Renn, C. H. Kim, M. C. Hersam, and C. D. Frisbie, “Aerosol jet printed, low voltage, electrolyte-gated carbon nanotube ring oscillators with sub-5 µs stage delays,” Nano Lett., 13, 954 (2013). [6] B. Kim, S. Jang, M. L. Geier, P. L. Prabhumirashi, M. C. Hersam, and A. Dodabalapur, “High-speed, inkjet-printed carbon nanotube/zinc tin oxide hybrid complementary ring oscillators,” Nano Lett., 14, 3683 (2014). [7] B. Kim, S. Jang, M. L. Geier, P. L. Prabhumirashi, M. C. Hersam, and A. Dodabalapur, “Inkjet printed ambipolar transistors and inverters based on carbon nanotube/zinc tin oxide heterostructures,” Appl. Phys. Lett., 104, 062101 (2014). [8] Y.-G. Ha, K. Everaerts, M. C. Hersam, and T. J. Marks, “Hybrid gate dielectric materials for unconventional electronic circuitry,” Acc. Chem. Res., 47, 1019 (2014). [9] V. K. Sangwan, R. P. Ortiz, J. M. P. Alaboson, J. D. Emery, M. J. Bedzyk, L. J. Lauhon, T. J. Marks, and M. C. Hersam, “Fundamental performance limits of carbon nanotube thin-film transistors achieved using hybrid molecular dielectrics,” ACS Nano, 6, 7480 (2012). [10] M. L. Geier, P. L. Prabhumirashi, J. J. McMorrow, W. Xu, J.-W. T. Seo, K. Everaerts, C. H. Kim, T. J. Marks, and M. C. Hersam, “Subnanowatt carbon nanotube complementary logic enabled by threshold voltage control,” Nano Lett., 13, 4810 (2013).

Abstract 2541: Differential trafficking of dendritic cells after immunization determines establishment of systemic anti-tumor responses

Abstract Systemic immune activation and memory cell generation is desired in dendritic cell (DC)-based therapies against cancers, especially with metastasis. Migration properties of DCs, however, could limit the responses regionally constrained. Clinically, DC-vaccines are often given subcutaneously (s.c.), intravenously (i.v.) or intradermally (i.d.), without monitoring the migration of DCs. In this study, we visualized trafficking of DCs labeled with In-111 oxine using a whole-body micro-SPECT imager after immunization via different routes and examined induction of immune activation and memory cell generation in mice. Bone marrow-derived DCs were used. Surprisingly, most of s.c. transferred DCs failed to migrate to the adjacent lymph nodes (LNs) and died at the injection site, irrespective of their maturation status. DCs injected i.v. were initially trapped in the lungs, but gradually migrated to the spleen, liver and, to a lesser extent, LNs. In contrast, DCs administered i.d. and intraperitoneally (i.p.) quickly entered lymphatic system and distributed to LNs. Proliferation of CMFDA labeled OT-1 CD8 T cells pre-transferred to mice receiving ovalbumin loaded DCs (OVA-DCs) indicated that i.v. and i.p. immunizations triggered stronger and more systemic expansion of effector T cells than s.c. and i.d. immunizations. To examine the establishment of long-term defense, we next immunized mice with OVA-DCs and challenged them with OVA-expressing tumors intramuscularly, contra-lateral to the s.c. and i.d. injection site. Intriguingly, i.v. immunization seemed less effective than the others in developing long-term antigen-specific immunity, despite its capability to induce an extensive acute responses. These results might suggest that difference in the microenvironment of organs where CD8 T cells encounter the antigen (i.e., spleen or LNs) controls the generation of long-lived memory cells. Collectively, immunization route would be a critical factor that determines the spatial distribution of immune activation and generation of functional memory cells in DC-based therapies. Citation Format: Noriko Sato, Kingsley Asiedu, Peter L. Choyke. Differential trafficking of dendritic cells after immunization determines establishment of systemic anti-tumor responses. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2541. doi:10.1158/1538-7445.AM2014-2541

T cell-intrinsic role of IL-6 signaling in primary and memory responses.

Innate immune recognition is critical for the induction of adaptive immune responses; however the underlying mechanisms remain incompletely understood. In this study, we demonstrate that T cell-specific deletion of the IL-6 receptor α chain (IL-6Rα) results in impaired Th1 and Th17 T cell responses in vivo, and a defect in Tfh function. Depletion of Tregs in these mice rescued the Th1 but not the Th17 response. Our data suggest that IL-6 signaling in effector T cells is required to overcome Treg-mediated suppression in vivo. We show that IL-6 cooperates with IL-1β to block the suppressive effect of Tregs on CD4(+) T cells, at least in part by controlling their responsiveness to IL-2. In addition, although IL-6Rα-deficient T cells mount normal primary Th1 responses in the absence of Tregs, they fail to mature into functional memory cells, demonstrating a key role for IL-6 in CD4(+) T cell memory formation.DOI: http://dx.doi.org/10.7554/eLife.01949.001.

Differential requirements of CD4+T‐cell signals for effector cytotoxic T‐lymphocyte (CTL) priming and functional memory CTL development at higher CD8+T‐cell precursor frequency

Increased CD8(+) T-cell precursor frequency (PF) precludes the requirement of CD4(+) helper T (Th) cells for primary CD8(+) cytotoxic T-lymphocyte (CTL) responses. However, the key questions of whether unhelped CTLs generated at higher PF are functional effectors, and whether unhelped CTLs can differentiate into functional memory cells at higher PF are unclear. In this study, ovalbumin (OVA) -pulsed dendritic cells (DC(OVA)) derived from C57BL/6, CD40 knockout (CD40(-/-)) or CD40 ligand knockout (CD40L(-/-)) mice were used to immunize C57BL/6, Ia(b-/-), CD40(-/-) or CD40L(-/-) mice, whose PF was previously increased with transfer of 1 × 10(6) CD8(+) T cells derived from OVA-specific T-cell receptor (TCR) transgenic OTI, OTI(CD40(-/-)) or OTI(CD40L(-/-)) mice. All the immunized mice were then assessed for effector and memory CTL responses. Following DC immunization, relatively comparable CTL priming occurred without CD4(+) T-cell help and Th-provided CD40/CD40L signalling. In addition, the unhelped CTLs were functional effectors capable of inducing therapeutic immunity against established OVA-expressing tumours. In contrast, the functional memory development of CTLs was severely impaired in the absence of CD4(+) T-cell help and CD40/CD40L signalling. Finally, unhelped memory CTLs failed to protect mice against lethal tumour challenge. Taken together, these results demonstrate that CD4(+) T-cell help at higher PF, is not required for effector CTL priming, but is required for functional memory CTL development against cancer. Our data may impact the development of novel preventive and therapeutic approaches in cancer patients with compromised CD4(+) T-cell functions.

IL-6 Production by Dendritic Cells Is Dispensable for CD8+Memory T-Cell Generation

Following activation, naïve CD8+ T cells will differentiate into effectors that differ in their ability to survive: some will persist as memory cells while the majority will die by apoptosis. Signals given by antigen-presenting cells (APCs) at the time of priming modulate this differential outcome. We have recently shown that, in opposition to dendritic cell (DC), CD40-activated B-(CD40-B) cell vaccination fails to efficiently produce CD8+ memory T cells. Understanding why CD40-B-cell vaccination does not lead to the generation of functional long-lived memory cells is essential to define the signals that should be provided to naïve T cells by APCs. Here we show that CD40-B cells produce very low amount of IL-6 when compared to DCs. However, supplementation with IL-6 during CD40-B-cell vaccination did not improve memory generation. Furthermore, IL-6-deficient DCs maintained the capacity to promote the formation of functional CD8+ effectors and memory cells. Our results suggest that in APC vaccination models, IL-6 provided by the APCs is dispensable for proper CD8+ T-cell memory generation.

Extremely Flexible Nanoscale Ultrathin Body Silicon Integrated Circuits on Plastic

In recent years, flexible devices based on nanoscale materials and structures have begun to emerge, exploiting semiconductor nanowires, graphene, and carbon nanotubes. This is primarily to circumvent the existing shortcomings of the conventional flexible electronics based on organic and amorphous semiconductors. The aim of this new class of flexible nanoelectronics is to attain high-performance devices with increased packing density. However, highly integrated flexible circuits with nanoscale transistors have not yet been demonstrated. Here, we show nanoscale flexible circuits on 60 Å thick silicon, including functional ring oscillators and memory cells. The 100-stage ring oscillators exhibit the stage delay of ~16 ps at a power supply voltage of 0.9 V, the best reported for any flexible circuits to date. The mechanical flexibility is achieved by employing the controlled spalling technology, enabling the large-area transfer of the ultrathin body silicon devices to a plastic substrate at room temperature. These results provide a simple and cost-effective pathway to enable ultralight flexible nanoelectronics with unprecedented level of system complexity based on mainstream silicon technology.

Abrogation of Src Homology Region 2 Domain-Containing Phosphatase 1 in Tumor-Specific T Cells Improves Efficacy of Adoptive Immunotherapy by Enhancing the Effector Function and Accumulation of Short-Lived Effector T Cells In Vivo

T cell expression of inhibitory proteins can be a critical component for the regulation of immunopathology owing to self-reactivity or potentially exuberant responses to pathogens, but it may also limit T cell responses to some malignancies, particularly if the tumor Ag being targeted is a self-protein. We found that the abrogation of Src homology region 2 domain-containing phosphatase-1 (SHP-1) in tumor-reactive CD8(+) T cells improves the therapeutic outcome of adoptive immunotherapy in a mouse model of disseminated leukemia, with benefit observed in therapy employing transfer of CD8(+) T cells alone or in the context of also providing supplemental IL-2. SHP-1(-/-) and SHP-1(+/+) effector T cells were expanded in vitro for immunotherapy. Following transfer in vivo, the SHP-1(-/-) effector T cells exhibited enhanced short-term accumulation, followed by greater contraction, and they ultimately formed similar numbers of long-lived, functional memory cells. The increased therapeutic effectiveness of SHP-1(-/-) effector cells was also observed in recipients that expressed the tumor Ag as a self-antigen in the liver, without evidence of inducing autoimmune toxicity. SHP-1(-/-) effector CD8(+) T cells expressed higher levels of eomesodermin, which correlated with enhanced lysis of tumor cells. Furthermore, reduction of SHP-1 expression in tumor-reactive effector T cells by retroviral transduction with vectors that express SHP-1-specific small interfering RNA, a translatable strategy, also exhibited enhanced antitumor activity in vivo. These studies suggest that abrogating SHP-1 in effector T cells may improve the efficacy of tumor elimination by T cell therapy without affecting the ability of the effector cells to persist and provide a long-term response.

IL-4 has a critical, mouse strain-limited role in innate memory CD8+ T cell development. (115.19)

Abstract Mice have large numbers of innate memory cells: CD8+ T cells that develop functional and phenotypic memory cell characteristics in the absence of purposeful immunization. The role of IL-4 production by iNKT cells in innate memory cell development is shown by an ~80% decrease in splenic memory phenotype CD8+ T cell number in IL-4-, IL-4Rα-, Stat6- and CD1d KO BALB/c mice and an ~1/3 normal basal IL-4 level in CD1d KOs. The stimulatory effect of IL-4 on innate memory cell development is first observed in CD8hiCD4lo thymocytes and persists through adulthood. Anti-IL-4 mAb decreases innate memory cell number in thymus and spleen in immature mice, but only in thymus in adults. In contrast to BALB/c mice, IL-4Rα deficiency has little effect on CD8+ memory phenotype T cell number in C57BL/6 mice. This BALB/c - C57BL/6 difference is not associated with appreciable differences in basal total body or thymic IL-4 expression, CD8+ T cell IL-4Rα expression or IL-4 responsiveness, but is associated with greater IL-15Rα and IL-2/15Rβ expression and IL-15 responsiveness by C57BL/6 than BALB/c CD8+ splenocytes. In 5 additional mouse strains studied, 3 resemble BALB/c and 2 resemble C57BL/6 mice with regard to the IL-4 stimulation of innate memory cell development; an IL-4 effect correlates positively with thymic memory phenotype CD8+ T cell number. Thus, IL-4 produced by thymic NKT cells has a major role in memory phenotype CD8+ T cell development that varies among mouse strains.

Demonstration of Split-Gate Type Trigate Flash Memory With Highly Suppressed Over-Erase

The functional split-gate type trigate flash memory cell transistors have successfully been fabricated for the first time, and their threshold voltage (V <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">t</sub> ) variations before and after NOR-mode program/erase cycle have systematically been compared with the stack-gate ones. It was experimentally found that split-gate type cell transistors with the same control gate length (L <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">CG</sub> ) of 176 nm show much smaller Vt distribution after erase compared to those of stack-gate ones. Moreover, the measured source-drain breakdown voltage (BV <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">DS</sub> ) is higher than 3.1 V even the L <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">CG</sub> was down to 76 nm. This indicates that the developed split-gate type trigate flash memory is very effective for scaled NOR-type flash memory with highly suppressed over-erase.

FoxP3+ regulatory CD4 T cells control the generation of functional CD8 memory

During the primary immune response, CD8 memory emerges from an environment of strong immune activation. The FoxP3+ regulatory CD4 T-cell subset (Treg) is known as a key suppressive component of the immune system. Here we report that Tregs are required for the generation of functional CD8 memory. In the absence of Tregs during priming, the resulting memory cells proliferate poorly and fail to differentiate into functional cytotoxic secondary effectors following antigen reactivation. We find that the Tregs act early, during the expansion phase of primary CD8 effectors, by fine tuning interleukin-2 exposure of CD8 memory precursors. This crucial new role of Tregs has implications for optimal vaccine development.

Antigen-specific prevention of type 1 diabetes in NOD mice is ameliorated by OX40 agonist treatment

Antigen-specific therapies are possibly the safest approach to prevent type 1 diabetes (T1D). However their clinical translation has yielded poor results and greater efforts need to be put into the development of novel strategies to ameliorate their clinical outcome. OX40 is a costimulatory molecule expressed by T cells after antigen recognition and has been implicated in the control effector but also regulatory T cells (Tregs) function in vivo. The activity of OX40 signal on Tregs function has been controversial. In this context we investigated whether an anti-OX40 agonist antibody treatment can ameliorate antigen-specific immune intervention for the prevention of T1D. We show that treatment of non-obese diabetic (NOD) mice with an OX40 agonistic antibody (OX86) reduced type 1 diabetes (T1D) incidence by inducing both CD4 +CD25 +Foxp3 + Tregs and CD4 +Foxp3 − T cells expressing the latency-associated peptide (LAP). These OX86-induced CD4 +Foxp3 −LAP + T cells also demonstrated suppressive activity in vitro. A significant increase in protection was observed when OX86 was combined with insulin B9:23 (insB9:23) peptide immunizations. Synergy resulted from an expansion of IL-10-expressing insB9:23-reactive Tregs which augmented the proportion of CD4 + T cells with in vivo suppressive activity. Consequently, CD4 + T cells purified from OX86/insB9:23 combination treatment prevented T1D development when adoptively transferred into recipient mice. These findings suggest that the requirement for OX40 signaling by antigen-induced Tregs can be dominant over its well-documented need for effector memory cell function and may have potentially important implications for improving the clinical translation of antigen-specific prevention of T1D and possibly other autoimmune disorders.

A Novel Nanoinjection Lithography (NInL) Technology and Its Application for 16-nm Node Device Fabrication

For more than 45 years, photon- and electron-sensitive materials have been used to produce pattern-transfer masks in the lithographic manufacturing of integrated circuits. With the semiconductor technology feature size continuing to shrink and the requirements of low-variability and low-cost manufacturing, optical lithography is driven to its limits. In this paper, we report a novel nanoinjection lithography (NInL) technique that employs electron-beam-assisted deposition to form pattern-transfer hard mask in a direct-write deposit approach. By scanning the 4.6-nm-diameter electron beam while injecting a suitable organometallic precursor gas around the location of e-beam and just above the substrate, we form a high-density (pitch: 40 nm) high-uniformity (3-sigma linewidth roughness: 2 nm) hard mask for subsequent etching without using proximity-effect correction techniques. Furthermore, this technique can also directly deposit a metal pattern for interconnect or a dielectric pattern without the need for separate metal or dielectric deposition, photoresist etch-mask, and etching processes. The NInL approach simplifies the hard-mask creation or even metal or dielectric pattern creation process modules from five or tens of steps to only a single step. Therefore, it saves both photomask making and wafer processing costs. In addition, room-temperature NInL deposition of conductor/dielectric materials enables the fabrication of small versatile devices and circuits. For demonstration, we fabricated a functional 16-nm six-transistor static random access memory (SRAM) cell (area: occupying only 0.039 μm2), 43% the size of the smallest previously reported SRAM cell, using the FinFET structure and a dynamic Vdd regulator approach. The NInL technique offers a new way of exploring low-volume high-value 16-nm complementary metal-oxide-semiconductor (CMOS) devices and circuit designs with minimal additional investment and obtains early access to extreme CMOS scaling.

Chronic Virus Infection Enforces Demethylation of the Locus that Encodes PD-1 in Antigen-Specific CD8 + T Cells

Functionally exhausted Tcells have high expression of the PD-1 inhibitory receptor, and therapies that block PD-1 signaling show promise for resolving chronic viral infections and cancer. By using human and murine systems of acute and chronic viral infections, we analyzed epigenetic regulation of PD-1 expression during CD8(+) Tcell differentiation. During acute infection, naive to effector CD8(+) Tcell differentiation was accompanied by a transient loss of DNA methylation of the Pdcd1 locus that was directly coupled to the duration and strength of Tcell receptor signaling. Further differentiation into functional memory cells coincided with Pdcd1 remethylation, providing an adapted program for regulation of PD-1 expression. In contrast, the Pdcd1 regulatory region was completely demethylated in exhausted CD8(+) Tcells and remained unmethylated even when virus titers decreased. This lack of DNA remethylation leaves the Pdcd1 locus poised for rapid expression, potentially providing a signal for premature termination of antiviral functions.

T Cells with Chimeric Antigen Receptors Have Potent Antitumor Effects and Can Establish Memory in Patients with Advanced Leukemia

Tumor immunotherapy with T lymphocytes, which can recognize and destroy malignant cells, has been limited by the ability to isolate and expand T cells restricted to tumor-associated antigens. Chimeric antigen receptors (CARs) composed of antibody binding domains connected to domains that activate T cells could overcome tolerance by allowing T cells to respond to cell surface antigens; however, to date, lymphocytes engineered to express CARs have demonstrated minimal in vivo expansion and antitumor effects in clinical trials. We report that CAR T cells that target CD19 and contain a costimulatory domain from CD137 and the T cell receptor ζ chain have potent non-cross-resistant clinical activity after infusion in three of three patients treated with advanced chronic lymphocytic leukemia (CLL). The engineered T cells expanded >1000-fold in vivo, trafficked to bone marrow, and continued to express functional CARs at high levels for at least 6 months. Evidence for on-target toxicity included B cell aplasia as well as decreased numbers of plasma cells and hypogammaglobulinemia. On average, each infused CAR-expressing T cell was calculated to eradicate at least 1000 CLL cells. Furthermore, a CD19-specific immune response was demonstrated in the blood and bone marrow, accompanied by complete remission, in two of three patients. Moreover, a portion of these cells persisted as memory CAR(+) T cells and retained anti-CD19 effector functionality, indicating the potential of this major histocompatibility complex-independent approach for the effective treatment of B cell malignancies.

The Major Histocompatibility Complex (MHC) of the Secondary Transplant Tissue Donor Influences the Cross-Reactivity of Alloreactive Memory Cells

Memory cells are currently thought to be a major barrier to tolerance induction in transplantation. However, whether alloreactive memory cells resulting from a primary transplant have cross-reactivity in a second transplant is unclear. Here, we used skin transplantation from BALB/c mice donors to presensitize C₅₇ BL/6 (B6) mice. One month later, several strains of mice (including BALB/c, DBA/2, NOD, C3H and B6 mice) were chosen as donors to construct a memory model of heterotopic cardiac transplantation. The higher degree of major histocompatibility complex (MHC) mismatch to sensitizing MHC resulted in longer median survival times (MSTs, BALB/c 3.63 days versus C3H 6.08 days). After 3.5 days of cardiac transplantation, compared with the BALB/c and DBA/2 groups, in the groups of NOD and C3H, the infiltration of inflammatory cells in the grafts, the proportion and proliferation of memory cells in spleens and the function of allogeneic antibodies decreased significantly. The varying degrees of MHC mismatch between the primary and secondary donors influenced the intensity of alloreactive memory cell function, the higher degree of MHC mismatch resulted in better tolerance during secondary transplantation, and these may be related to the changed activation, proliferation and function of the alloreactive memory cells.

A Unique Role of the Cholera Toxin A1-DD Adjuvant for Long-Term Plasma and Memory B Cell Development

Adjuvants have traditionally been appreciated for their immunoenhancing effects, whereas their impact on immunological memory has largely been neglected. In this paper, we have compared three mechanistically distinct adjuvants: aluminum salts (Alum), Ribi (monophosphoryl lipid A), and the cholera toxin A1 fusion protein CTA1-DD. Their influence on long-term memory development was dramatically different. Whereas a single immunization i.p. with 4-hydroxy-3-nitrophenyl acetyl (NP)-chicken γ-globulin and adjuvant stimulated serum anti-NP IgG titers that were comparable at 5 wk, CTA1-DD-adjuvanted responses were maintained for >16 mo with a half-life of anti-NP IgG ∼36 wk, but <15 wk after Ribi or Alum. A CTA1-DD dose-dependent increase in germinal center (GC) size and numbers was found, with >60% of splenic B cell follicles hosting GC at an optimal CTA1-DD dose. Roughly 7% of these GC were NP specific. This GC-promoting effect correlated well with the persistence of long-term plasma cells in the bone marrow and memory B cells in the spleen. CTA1-DD also facilitated increased somatic hypermutation and affinity maturation of NP-specific IgG Abs in a dose-dependent fashion, hence arguing that large GC not only promotes higher Ab titers but also high-quality Ab production. Adoptive transfer of splenic CD80(+), but not CD80(-), B cells, at 1 y after immunization demonstrated functional long-term anti-NP IgG and IgM memory cells. To our knowledge, this is the first report to specifically compare and document that adjuvants can differ considerably in their support of long-term immune responses. Differential effects on the GC reaction appear to be the basis for these differences.

Effector Function-Deficient Memory CD8+ T Cells Clonally Expand in the Liver and Give Rise to Peripheral Memory CD8+ T Cells

Upon adoptive transfer into histocompatible mice, naive CD8(+) T cells stimulated ex vivo by TCR+IL-4 turn into long-lived functional memory cells. The liver contains a large number of so formed memory CD8(+) T cells, referred to as liver memory T cells (T(lm)) in the form of cell clusters. The CD62L(low) expression and nonlymphoid tissue distribution of T(lm) cells are similar to effector memory (T(em)) cells, yet their deficient cytotoxicity and IFN-γ inducibility are unlike T(em) cells. Adoptive transfer of admixtures of TCR+IL-4-activated Vβ8(+) and Vβ5(+) CD8(+) T cells into congenic hosts reveals T(lm) clusters that are composed of all Vβ5(+) or Vβ8(+), not mixed Vβ5(+)/Vβ8(+) cells, indicating that T(lm) clusters are formed by clonal expansion. Clonally expanded CD8(+) T cell clusters are also seen in the liver of Listeria monocytogenes-immune mice. T(lm) clusters closely associate with hepatic stellate cells and their formation is IL-15/IL-15R-dependent. CD62L(low) T(LM) cells can home to the liver and secondary lymphoid tissues, remain CD62L(low), or acquire central memory (T(cm))-characteristic CD62L(hi) expression. Our findings show the liver as a major site of CD8(+) memory T cell growth and that T(lm) cells contribute to the pool of peripheral memory cells. These previously unappreciated T(lm) characteristics indicate the inadequacy of the current T(em)/T(cm) classification scheme and help ongoing efforts aimed at establishing a unifying memory T cell development pathway. Lastly, our finding of T(lm) clusters suggests caution against interpreting focal lymphocyte infiltration in clinical settings as pathology and not normal physiology.