FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2084787806> ?p ?o ?g. }

• W2084787806 endingPage "34892" @default.
• W2084787806 startingPage "34883" @default.
• W2084787806 abstract "Artificial antigen-presenting cells (aAPCs) are an emerging technology to induce therapeutic cellular immunity without the need for autologous antigen-presenting cells (APCs). To fully replace natural APCs, an optimized aAPC must present antigen (signal 1), provide costimulation (signal 2), and release cytokine (signal 3). Here we demonstrate that the spatial and temporal characteristics of paracrine release of IL-2 from biodegradable polymer aAPCs (now termed paAPCs) can significantly alter the balance in the activation and proliferation of CD8+ and CD4+ T cells. Paracrine delivery of IL-2 upon T cell contact with paAPCs induces significant IL-2 accumulation in the synaptic contact region. This accumulation increases CD25 (the inducible IL-2 Rα chain) on responding T cells and increases proliferation of CD8+ T cells in vitro to levels 10 times that observed with equivalent amounts of bulk IL-2. These CD8+ T cell responses critically depend upon close contact of T cells and the paAPCs and require sustained release of low levels of IL-2. The same conditions promote activation-induced cell death in CD4+ T cells. These findings provide insight into the response of T cell subsets to paracrine IL-2. Artificial antigen-presenting cells (aAPCs) are an emerging technology to induce therapeutic cellular immunity without the need for autologous antigen-presenting cells (APCs). To fully replace natural APCs, an optimized aAPC must present antigen (signal 1), provide costimulation (signal 2), and release cytokine (signal 3). Here we demonstrate that the spatial and temporal characteristics of paracrine release of IL-2 from biodegradable polymer aAPCs (now termed paAPCs) can significantly alter the balance in the activation and proliferation of CD8+ and CD4+ T cells. Paracrine delivery of IL-2 upon T cell contact with paAPCs induces significant IL-2 accumulation in the synaptic contact region. This accumulation increases CD25 (the inducible IL-2 Rα chain) on responding T cells and increases proliferation of CD8+ T cells in vitro to levels 10 times that observed with equivalent amounts of bulk IL-2. These CD8+ T cell responses critically depend upon close contact of T cells and the paAPCs and require sustained release of low levels of IL-2. The same conditions promote activation-induced cell death in CD4+ T cells. These findings provide insight into the response of T cell subsets to paracrine IL-2. Activation of naïve T cells to proliferate and acquire effector function requires, at a minimum, antigen recognition by the TCR 2The abbreviations used are: TCRT cell receptorAPCantigen-presenting cellpaAPCpolymer artificial antigen-presenting cellaAPCartificial antigen-presenting cellTregregulatory T cell. (signal 1) and costimulation by ligands that engage receptors such as CD28 (signal 2), both of which are provided by conventional antigen-presenting cells (APCs) (1Blachère N.E. Morris H.K. Braun D. Saklani H. Di Santo J.P. Darnell R.B. Albert M.L. J. Immunol. 2006; 176: 7288-7300Crossref PubMed Scopus (50) Google Scholar, 2Pardoll D.M. Nat. Rev. Immunol. 2002; 2: 227-238Crossref PubMed Scopus (337) Google Scholar). IL-2 is a canonical T cell growth factor and plays a central role in the expansion and differentiation of CD4+ and CD8+ effector T cells both in vivo and in vitro (2Pardoll D.M. Nat. Rev. Immunol. 2002; 2: 227-238Crossref PubMed Scopus (337) Google Scholar, 3Rochman Y. Spolski R. Leonard W.J. Nat. Rev. Immunol. 2009; 9: 480-490Crossref PubMed Scopus (774) Google Scholar) and may be described as signal 3. IL-2 is especially necessary for CD8+ T cell expansion, as this subset ceases IL-2 production after a few days of stimulation (4Janssen E.M. Lemmens E.E. Wolfe T. Christen U. von Herrath M.G. Schoenberger S.P. Nature. 2003; 421: 852-856Crossref PubMed Scopus (1334) Google Scholar, 5Shedlock D.J. Shen H. Science. 2003; 300: 337-339Crossref PubMed Scopus (1215) Google Scholar, 6Shrikant P. Mescher M.F. J. Immunol. 2002; 169: 1753-1759Crossref PubMed Scopus (59) Google Scholar). IL-2 is principally produced by CD4+ T cells (7Malek T.R. Annu. Rev. Immunol. 2008; 26: 453-479Crossref PubMed Scopus (775) Google Scholar). APCs, such as dendritic cells, can foster CD8+ T cell responses by facilitating IL-2 delivery from CD4+ T cells to CD8+ T cells. This is accomplished by bringing CD4+ and CD8+ lymphocytes into close contact (1Blachère N.E. Morris H.K. Braun D. Saklani H. Di Santo J.P. Darnell R.B. Albert M.L. J. Immunol. 2006; 176: 7288-7300Crossref PubMed Scopus (50) Google Scholar, 7Malek T.R. Annu. Rev. Immunol. 2008; 26: 453-479Crossref PubMed Scopus (775) Google Scholar). IL-2 delivery can also occur by synaptic transmission between T cells without the need for an APC, as in the case of homotypic T cell interactions. Synaptic transmission of IL-2, as such, has been demonstrated as a key signal for T cell cluster initiation and persistence after antigen priming (8Sabatos C.A. Doh J. Chakravarti S. Friedman R.S. Pandurangi P.G. Tooley A.J. Krummel M.F. Immunity. 2008; 29: 238-248Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). It has also been suggested that DCs may also secrete IL-2 at the site of T cell contact, and accumulation of this cytokine at the DC-T cell interface has been shown to be an important signal for T cell activation (9Granucci F. Feau S. Angeli V. Trottein F. Ricciardi-Castagnoli P. J. Immunol. 2003; 170: 5075-5081Crossref PubMed Scopus (146) Google Scholar). Thus, paracrine IL-2 delivery is a physiologically important process for effective immune responses. T cell receptor antigen-presenting cell polymer artificial antigen-presenting cell artificial antigen-presenting cell regulatory T cell. Both the timing and duration of IL-2 delivery are important determinants for the extent of T cell expansion (10D'Souza W.N. Lefrançois L. J. Immunol. 2003; 171: 5727-5735Crossref PubMed Scopus (186) Google Scholar, 11Lai Y.P. Lin C.C. Liao W.J. Tang C.Y. Chen S.C. PLoS ONE. 2009; 4: e7766Crossref PubMed Scopus (31) Google Scholar, 12Villarino A.V. Tato C.M. Stumhofer J.S. Yao Z. Cui Y.K. Hennighausen L. O'Shea J.J. Hunter C.A. J. Exp. Med. 2007; 204: 65-71Crossref PubMed Scopus (104) Google Scholar). IL-2 received from CD4+ T cells within the first few hours of CD8+ T cell stimulation is responsible for increased IFN-γ and granzyme B secretion and potent anti-tumor activity (11Lai Y.P. Lin C.C. Liao W.J. Tang C.Y. Chen S.C. PLoS ONE. 2009; 4: e7766Crossref PubMed Scopus (31) Google Scholar). IL-2 is also required at later time points in CD8+ T cell stimulation to maintain proliferation and produce robust secondary responses (10D'Souza W.N. Lefrançois L. J. Immunol. 2003; 171: 5727-5735Crossref PubMed Scopus (186) Google Scholar, 13Williams M.A. Tyznik A.J. Bevan M.J. Nature. 2006; 441: 890-893Crossref PubMed Scopus (605) Google Scholar). However, high concentrations and chronic exposure to IL-2 can negatively impact T cell stimulation by sensitizing T cells to activation-induced cell death and tolerance induction (14Krammer P.H. Arnold R. Lavrik I.N. Nat. Rev. Immunol. 2007; 7: 532-542Crossref PubMed Scopus (489) Google Scholar, 15Lenardo M.J. Nature. 1991; 353: 858-861Crossref PubMed Scopus (959) Google Scholar, 16Waldmann T.A. Nat. Rev. Immunol. 2006; 6: 595-601Crossref PubMed Scopus (847) Google Scholar, 17Steenblock E.R. Fahmy T.M. Mol. Ther. 2008; 16: 765-772Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The source of IL-2 is also relevant because activated CD4+ T cells may sometimes display FasL, which can result in CD8+ T cell death. Here we constructed a polymeric artificial antigen-presenting cell (paAPC) from biodegradable materials that is capable of providing an antigenic signal, effective costimulation, and adjustable paracrine IL-2 signals to T cells (17Steenblock E.R. Fahmy T.M. Mol. Ther. 2008; 16: 765-772Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 18Steenblock E.R. Wrzesinski S.H. Flavell R.A. Fahmy T.M. Expert Opin. Biol. Ther. 2009; 9: 451-464Crossref PubMed Scopus (51) Google Scholar). The polymer that constitutes the core of this system, poly(lactic co-glycolic acid), and its homopolymers poly(lactic acid) and poly(glycolic acid) have been used for over 30 years in various human clinical applications, including drug delivery systems. The biodegradability of the polymer and subsequent release of bioactive encapsulant allow for dissection of the spatial and temporal paracrine IL-2 delivery components and their importance in CD4+ and CD8+ T cell proliferation. In this report, we explore how local control over IL-2 delivery impacts lymphocyte activity. Given that peptide-MHC Class I complexes are recognized by CD8+ T cells whereas peptide-Class II MHC complexes are recognized by CD4+ T cells, no single peptide-MHC complex can be used to activate both T cell subsets. Therefore, in this study, we use a paAPC system presenting an antigen-independent signal equally effective on both T cell subsets, namely anti-CD3 antibody. Anti-CD3, which binds to invariant signaling proteins associated with all TCRs on both CD4+ and CD8+ T cells, is widely used as an antigen mimetic because like peptide-MHC complexes, it activates T cells by clustering TCR·CD3 complexes on the T cell surface. This approach allowed us to assess differences in the responses of these two subsets to paAPC-encapsulated IL-2 acting as a paracrine IL-2 point source. In cells lacking the ability to secrete IL-2, paracrine delivery is critical for maximum expansion. We show that CD8+ T cells proliferate vigorously in response to paracrine IL-2 delivery by paAPCs, whereas CD4+ T cell expansion is limited by apoptosis. These effects cannot be mimicked even by an addition of a 1000-fold excess of exogenous IL-2. We find that synaptic accumulation of IL-2 in the early stages of activation may be important. Additionally, a slow and sustained release of IL-2 accounts for the effects of paracrine delivery, whereas an initial short-lived burst of cytokine availability (on the order of hours) is dispensable. The slow, sustained release must occur in close proximity to T cells to achieve maximal response. Animal studies were approved by the Institutional Animal Care and Use Committee at Yale University. All animals were routinely used at 6–10 weeks of age, were maintained under specific pathogen-free conditions, and were routinely checked by the Yale University Animal Resource Center. C57BL/6 (B6) mice were obtained from Charles River Laboratories, Inc. (Wilmington, MA). Primary splenocytes were obtained from homogenized naïve mouse spleens after depletion of erythrocytes by hypotonic lysis (Acros Organics, Geel, Belgium). Splenocytes were resuspended in complete RPMI-10, consisting of RPMI 1640 (Invitrogen) supplemented with 10% FBS (Invitrogen), 2 mm l-glutamine (Invitrogen), 25 μm β-mercaptoethanol (American Bioanalytical, Natick, MA), 2% penicillin-streptomycin (Sigma Aldrich) and 1% gentamicin (Sigma Aldrich). B6 splenocytes were either used without further purification or subjected to negative immunoselection (R&D Systems, Minneapolis, MN) to produce CD8+, CD4+, or CD4+CD25- populations of at least 96% purity. Poly(lactic co-glycolic acid) 50/50 with an average molecular weight of 80 kDa was obtained from Durect Corp. (Cupertino, CA). Microparticles were fabricated using a double emulsion water-in-oil-in-water technique (Jain 2000 Biomaterials). Microparticles were surface-modified with avidin-palmitate conjugate as described previously (19Park, J., Mattessich, T., Jay, S. M., Agawu, A., Saltzman, W. M., Fahmy, T. M. (2011) J. Control. Release.Google Scholar, 20Fahmy T.M. Samstein R.M. Harness C.C. Mark Saltzman W. Biomaterials. 2005; 26: 5727-5736Crossref PubMed Scopus (166) Google Scholar). For cytokine encapsulation, 100 μg of recombinant human (rh) IL-2 (Proleukin, Chiron Corp., Emeryville, CA) was added to the poly(lactic co-glycolic acid) solution. Particles were lyophilized and stored at −20 °C until use. Controlled release profiles were obtained in 1 ml of PBS at 37 °C from 5 mg of particles. An ELISA analysis was performed to measure rhIL-2 levels (BD Biosciences). Biotinylated anti-mouse CD3ϵ (BD Biosciences) and anti-mouse CD28 (BD Biosciences) were added at 10 μg/ml to a 10 mg/ml solution of poly(lactic co-glycolic acid) particles in PBS and rotated at room temperature for 20 min. IL-2-biotin, where present, was added after 5 min of particle incubation with antibodies without washing. Particles were then washed in all cases with PBS + 1% FBS and resuspended in complete RPMI-10. IL-2 biotin was prepared by reacting rhIL-2 with biotin hydrazide followed by removal of excess biotin reagent. The activity of IL-2-biotin was verified (supplemental Fig. S2). paAPCs with bound antibody were added to C57Bl/6 splenocyte or purified T cell cultures containing 106 cells/ml at 0.5 mg/ml After incubation at 37 °C for 48 h, cell culture supernatant was assayed for IFN-γ and mouse IL-2 or rhIL-2 using ELISA (BD Biosciences). Cells were analyzed for viability after 96 h using Cell Titer Blue (Promega Corp., Madison, WI). For “released particle” studies, paAPCs were allowed to release IL-2 in PBS at 37 °C for 24 h and then washed with PBS + 1% FBS prior to coupling to biotinylated anti-CD3 and anti-CD28 for use in stimulation studies. Reported values were obtained by using dilutions of supernatant that yielded absorbance values within the linear portion of the standard curve. All reagents were added to cell culture at time zero and were not supplemented at a later time. Blocking antibodies (BD Biosciences) were added at 10 μg/ml, carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Promega, Madison, WI) was added at 20 μm, and exogenous rhIL-2 (Chiron, Emeryville, CA) was added at the indicated amount in 2 ml of culture. Cells were analyzed by flow cytometry after 96 h. Cells were stained with a 1:200 dilution of antibody in PBS + 1% FBS. All antibodies were obtained from BD Biosciences. Analysis of fluorescence was performed after gating on the lymphocyte population in side scatter versus forward scatter plots followed by gating on the CD8+ and CD4+ subsets. Annexin V and the BrdU flow kits were obtained from BD Biosciences and used according to the manufacturer's instructions. Samples were run on a BD FACScan or LSRII (BD Biosciences) and were analyzed using FlowJo 8.7 (Tree Star, Ashland, OR). Data fits were performed using GraphPad Prism 5.0. Controlled release data were fit to a two-phase exponential association model. Statistical analysis was performed in GraphPad Prism 5.0 using a one-way analysis of variance with p < 0.05 considered significant. Diffusion controls the shape of the IL-2 normalized concentration field (C*) near a paAPC. If the ambience is quiescent and the C field is steady, the diffusion equation reduces to the Laplace equation:∇2C*=0(Eq. 1) For an isolated (far from a T cell) paAPC with radius a, the concentration field is spherically symmetric, and the concentration decreases inversely with radial distance (r) from the center of the paAPC (see Fig. 1D, left panel).C*=C(r)C0=ar(Eq. 2) where C0 is the surface concentration (r = a). The normalized concentration (C*) thus has a value of unity on the surface of an isolated paAPC. When the paAPC is in proximity with a T cell, the problem is more complex. The C* field is distorted by the interaction and will depend not only on the geometric parameters such as surface-to-surface spacing and relative cell/particle size but also on kinetic parameters, including the release capacity of the paAPC and the absorption characteristics of the T cell. Because we are interested in the early stages of cell activation, when there are few, if any, high affinity IL-2 receptors, the T cell may be considered to be non-absorbing at that stage. If the paAPC releases IL-2 at a constant rate, then Equation 1 can be solved using a boundary collocation method (supplemental methods) (21Labowsky M. Rapid Commun. Mass Spectrom. 2010; 24: 3079-3091Crossref PubMed Scopus (11) Google Scholar, 22Labowsky M. Fenn J.B. de la Mora J.F. Anal. Chim. Acta. 2000; 406: 105-118Crossref Scopus (60) Google Scholar). Delivering IL-2 from a point source produces both spatial and temporal gradients. To examine the effect of spatial proximity of an IL-2 source on T cell stimulation, naïve T cells from mouse splenocytes were exposed to aAPCs presenting surface-bound anti-CD3 (signal 1) and anti-CD28 (signal 2) monoclonal antibodies. We then compared the effects when IL-2 was directly added to the culture medium (exogenous IL-2) or when it was provided by releasing paAPCs (encapsulated IL-2) under three different conditions, as shown in Fig. 1A. Because IL-2 is continually released from paAPCs, the concentration of IL-2 decreases with increasing distance from its paAPC source. The closest encounter (Fig. 1A, right panel) IL-2 is delivered by paAPCs that that are separated from T cells only by the nanoscale molecular interactions involving anti-CD3 and anti-CD28 binding to their ligands on T cells. Typically these interactions are on the order of an antigen-antibody molecular length scale (approximately 10–20 nm). In the second scenario, signal 1 and 2 stimulation was achieved with aAPCs (i.e. particles decorated with antibodies without encapsulated IL-2), and IL-2 delivery was achieved with uncoated paAPCs (Fig. 1A, center panel). In this setting, paAPCs may get near the responding T cells, but the nanoscale proximal interactions with T cells are expected to be less frequent, if not absent. In the third scenario, signal 1 and 2 interactions were achieved with aAPCs (particles with antibodies) in contact with the T cells, and IL-2 is provided by uncoated paAPCs that were placed across a transwell with a pore diameter of 0.4 μm (Fig. 1A, left panel). Released IL-2 freely diffuses from the transwell into the cell compartment. Again, in this scenario, cells and IL-2 releasing particles are far from each other on the order of many microns. The total amount of IL-2, either exogenously added or encapsulated in particles, was equal (1 ng of IL-2 in 2 ml of culture). IL-2 delivered from paAPCs enhanced T cell activation and proliferation compared with exogenous addition of IL-2 (Fig. 1B) and led to up-regulation of CD25 (C). However, IL-2 delivery was significantly less effective than paAPCs in molecular proximity to T cells. Specifically, paAPCs releasing IL-2 produced higher levels of CD25 on the surface of CD4+ and CD8+ T cells (Fig. 1C) and greater T cell activation as measured by secretion of IL-2 and IFN-γ and resulted in increased expansion of CD8+ T cells (Fig. 1B and supplemental Fig. S1). Interestingly, CD4+ T cell expansion was unaffected by distancing the IL-2 source (Fig. 1B). The results highlight the importance of colocalization of IL-2 delivery with stimulatory and costimulatory signals for efficient CD8+ T cell signaling. A T cell present in proximity to a paAPC can experience variable IL-2 gradients depending on its separation distance from the IL-2 source. To understand the diffusive transfer of IL-2 when a T cell interacts with a paAPC, it is important to examine the behavior of the concentration field during the interaction. The normalized concentration field (C*) field is spherically symmetric and decreases inversely with distance from an isolated paAPC in a quiescent environment (Fig. 1D, left panel). When a paAPC interacts with a T cell, the spherical symmetry of C* is compromised. Prior to activation, the naïve T cell can be considered as a poorly receptive to IL-2 compared with its activated state (7Malek T.R. Annu. Rev. Immunol. 2008; 26: 453-479Crossref PubMed Scopus (775) Google Scholar). If the paAPC releases IL-2 at a constant rate, then an accumulation of IL-2 occurs at the interface. The amount of interfacial IL-2 accumulation will depend on the proximity of the cells. When the cells are separated by 3 μm (Fig. 1D, center panel), the IL-2 surface concentration will be non-uniform on the T cell surface and will have a maximum C* value of 0.7 at the synaptic point, as may be seen from the underlying magnified view of the synaptic region. If an isolated paAPC has an IL-2 surface concentration of about 1.8 pm, then a C* = 0.7 means the concentration near the T cell interface would be 1.26 pm. When the T cell is separated from the paAPC by only 20 nm (an approximate distance of synaptic ligand-receptor interactions, Fig. 1D, right panel), the T cell synaptic point C* increases significantly to 2.2, which equates to an actual concentration of 3.96 pm. So, the closer the cells, the larger the interfacial IL-2 accumulation. At this point, the T cell contact point is bathed in a concentration of IL-2 that is considerably higher than what would be expected based on an isolated paAPC concentration. This accumulation effect will be even magnified if the T cell-paAPC interface flattens during the interaction, thereby increasing the synaptic contact area. The above calculation is for a non-absorbing T cell interacting with one paAPC. In our experiments, a T cell may simultaneously interact with several paAPCs, leading to the hypothesis that the cytokine concentration, because of accumulation at multiple interface points, maybe even further magnified. Transpresented IL-2 may enhance availability and binding of IL-2 to its receptor (7Malek T.R. Annu. Rev. Immunol. 2008; 26: 453-479Crossref PubMed Scopus (775) Google Scholar, 23Létourneau S. van Leeuwen E.M. Krieg C. Martin C. Pantaleo G. Sprent J. Surh C.D. Boyman O. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 2171-2176Crossref PubMed Scopus (144) Google Scholar). Given that IL-2 is released from paAPCs, it is possible that it may associate with the particle surface in a nonspecific manner and be presented rather than released, leading to the observed enhancement at nanoscale separations. To address the effect of non-diffusible, surface-presented IL-2 on T cell activation, IL-2 was biotinylated and immobilized on the surface of aAPCs without encapsulated IL-2. Biotinylation of IL-2 did not affect its functional activity (supplemental Fig. S2). Here the amount of IL-2 immobilized on paAPCs was varied from the total amount released with paAPCs to two orders of magnitude greater than that amount. As shown in Fig. 2, there was little or no enhancement in stimulation with surface-bound IL-2 aAPCs, and this result was similar to exogenous IL-2 addition (Fig. 2). Increasing the amount of IL-2 presented on the surface by two orders of magnitude (100 ng per mg of aAPC) over the amount released from paAPCs (1 ng per mg of particles) improved CD8+ T cell expansion (Fig. 2B) and T cell cytokine secretion (C) but was significantly lower than paAPC stimulation. Similarly, we observed no changes in the surface density of CD25 on T cells activated by presentation of IL-2 compared with exogenous delivery (Fig. 2D). The results demonstrate the importance of delivering IL-2 as a soluble paracrine factor, similar to its natural mode of action (24Huse M. Quann E.J. Davis M.M. Nat. Immunol. 2008; 9: 1105-1111Crossref PubMed Scopus (146) Google Scholar). Diffusion of IL-2 from a point source over time also leads to temporal gradients that may impact T cell function. IL-2 release from the paAPCs occurs in two distinct phases, a short burst phase of IL-2 (0–8 h) followed by a long sustained release phase that has both a lower slope (expressed as quantity of cytokine per time) and a smaller magnitude of cytokine released (Fig. 3A). These two phases occur because IL-2 is not homogeneously distributed in the particle, and a significant fraction is localized near the surface of the particle with the remainder entrapped in the core. Release from the surface is triggered upon aqueous exposure, and slow sustained release from the core takes place over time as the aqueous medium degrades the polymer, releasing entrapped IL-2. To investigate the importance of each phase on T cell stimulation, we stimulated cells with paAPCs that have been depleted of the burst IL-2 phase and compared the potency of these “released” particles with “fresh” particles for T cell activation and expansion. Particles denoted as released were suspended in PBS under constant rotation for 24 h to wash away the burst phase prior to T cell stimulation. The remaining IL-2 is denoted as R in Fig. 3A. Strikingly, released paAPCs expanded CD8+ T cells to a level that is 70% of that achieved with fresh paAPCs, despite the fact that released paAPCs deliver only about 20% as much IL-2 as fresh paAPCs (Fig. 3B). Cytokine secretion and up-regulation of CD25 were indistinguishable between released and fresh paAPCs (Fig. 3, C and D), signifying the importance of slow sustained paracrine delivery of IL-2 for effective stimulation. Though only a small amount of IL-2 is released during the sustained slow portion of the curve, this amount of cytokine is sufficient to mimic natural paracrine delivery. Our data point to the importance of high IL-2 interfacial transfer rates over prolonged periods as a key factor in the potency of the paAPC. We hypothesized that increasing the amount of exogenous IL-2 would at some high concentration produce similar effects to those resulting from paracrine IL-2 paAPCs. To test this hypothesis, we incubated paAPCs with T cells derived from wild-type mice and mice deficient in IL-2 production (IL-2−/−) to eliminate confounding variables introduced by the presence of other cells that may act as additional sources of IL-2. The amount of exogenous IL-2 added to the media at the start of the experiment was increased by up to 3 orders of magnitude with both cell types. Although10-fold and 100-fold higher concentrations of exogenous IL-2 increased stimulation, we found that 1000-fold more IL-2 was required to approximate the effects of paAPCs in terms of CD8+ expansion (Fig. 4A), cytokine secretion (B), and levels of CD25 on CD8+ T cells (C) in wild-type T cells. Interestingly, we did not observe this enhancement with additional exogenous IL-2 in IL-2−/− cells. Stimulation of IL-2−/− T cells was still far greater with paAPCs compared with a 1000-fold addition of exogenous IL-2 (Fig. 4, D–F). The data point to the fact that paAPCs facilitate stimulation (in the absence of other sources of IL-2) by an enhanced transfer rate and concentration gradients that cannot be mimicked by exogenous sources, even at high concentrations. Stimulation by paAPCs of a splenocyte culture in which similar fractions of CD4+ and CD8+ T cells are present results in a population of T cells that is skewed strongly toward the CD8+ phenotype (Fig. 5A). Given that CD4+ T cells are a primary source of IL-2, we dissected this preferential phenotypic expansion by purifying CD4+ and CD8+ T cell populations and stimulated them separately with paAPCs. The results paralleled splenocyte (mixed CD4+ CD8+) observations. CD4+ T cells failed to proliferate substantially under all conditions, and CD8+ T cells expanded robustly with paAPCs (Fig. 5B). Purified CD8+ and CD4+ T cells mixed at defined ratios prior to stimulation by paAPCs showed the same trend. CD8+ T cells expansion showed a linear dependence on the initial frequency and hence was directly proportional to the starting concentration of CD8+ T cells, whereas CD4+ T cells expanded moderately irrespective of initial frequency (Fig. 5C). Given that CD8+ T cells can also produce small amounts of IL-2, we measured IL-2 secretion after 48 h of culture to ascertain whether the enhanced expansion was due to increased IL-2 production from stimulated CD8+ T cells. Fig. 5D shows that the level of IL-2 production after 48 h increases with increasing percentages of CD4+ T cells, and the opposite effect is seen for IFN-γ. This rules out the possibility that stimulation of CD8+ cells significantly increases their production of IL-2 and leads to the conclusion that the enhanced proliferation of CD8+ T cells is solely due to IL-2 delivery by paAPCs. These results also indicate that the proliferative patterns of CD4+ and CD8+ T cells are not the result of killing of the CD4+ subset by CD8+ T cells (25Foulds K.E. Zenewicz L.A. Shedlock D.J. Jiang J. Troy A.E. Shen H. J. Immunol. 2002; 168: 1528-1532Crossref PubMed Scopus (326) Google Scholar) or competition for IL-2 but rather intrinsic properties of the CD8+ versus CD4+ T cells' responses to the intense IL-2 concentration gradient provided by paAPCs. The observed attenuation in the CD4+ T cell response compared with CD8+ T cells upon paracrine stimulation may be due to one or both of the following mechanisms: 1) CD4+ T cells are simply slower in activation and proliferation upon encounter with paAPCs, or 2) CD4+ T cells also activate and proliferate, but their numbers are reduced by programmed cell death. To determine whether CD4+ T cells divide in culture with paAPCs, BrdU incorporation was used to detect cellular division. Division of CD4+ and CD8+ cells was evident after 48 h in all aAPC and paAPC stimulations but was significantly higher when IL-2 was present (Fig. 6A). There was no significant difference in the percentage of CD4+ T cells engaged in cell division between the exogenous and paracrine IL-2 groups. To examine the number of divisions that occurred in each case, splenocytes were labeled with the cytoplasmic dye carboxyfluorescein succinimidyl ester prior to stimulation, and dilution of the dye was examined after 96 h. Although more CD8+ T cells were found to undergo extensive cell division after exposure to paracrine IL-2 versus exogenous IL-2 addition, CD4+ T cells showed similar but moderate cell division patterns regardless of treatment (Fig. 6B and supplemental Fig. S3), indicating that CD4+ T cells are responding to paAPCs by undergoing cell division under all conditions. Howev" @default.
• W2084787806 created "2016-06-24" @default.
• W2084787806 creator A5000930137 @default.
• W2084787806 creator A5013509770 @default.
• W2084787806 creator A5022235365 @default.
• W2084787806 creator A5028534770 @default.
• W2084787806 creator A5069757510 @default.
• W2084787806 date "2011-10-01" @default.
• W2084787806 modified "2023-10-17" @default.
• W2084787806 title "An Artificial Antigen-presenting Cell with Paracrine Delivery of IL-2 Impacts the Magnitude and Direction of the T Cell Response" @default.
• W2084787806 cites W1493822493 @default.
• W2084787806 cites W1516581843 @default.
• W2084787806 cites W1524005671 @default.
• W2084787806 cites W1543267956 @default.
• W2084787806 cites W1574076960 @default.
• W2084787806 cites W1645244049 @default.
• W2084787806 cites W1931750338 @default.
• W2084787806 cites W1963515569 @default.
• W2084787806 cites W1967173439 @default.
• W2084787806 cites W1973151707 @default.
• W2084787806 cites W1976027404 @default.
• W2084787806 cites W1983637812 @default.
• W2084787806 cites W2005154465 @default.
• W2084787806 cites W2005543808 @default.
• W2084787806 cites W2008667095 @default.
• W2084787806 cites W2027237928 @default.
• W2084787806 cites W2029939124 @default.
• W2084787806 cites W2036383793 @default.
• W2084787806 cites W2039314170 @default.
• W2084787806 cites W2048934768 @default.
• W2084787806 cites W2051972593 @default.
• W2084787806 cites W2052580681 @default.
• W2084787806 cites W2053267300 @default.
• W2084787806 cites W2067039219 @default.
• W2084787806 cites W2068442870 @default.
• W2084787806 cites W2076531990 @default.
• W2084787806 cites W2086121828 @default.
• W2084787806 cites W2095138364 @default.
• W2084787806 cites W2102551327 @default.
• W2084787806 cites W2102642734 @default.
• W2084787806 cites W2109163566 @default.
• W2084787806 cites W2111929893 @default.
• W2084787806 cites W2128825762 @default.
• W2084787806 cites W2135699588 @default.
• W2084787806 cites W2149804459 @default.
• W2084787806 cites W2152488116 @default.
• W2084787806 cites W2153142861 @default.
• W2084787806 cites W2159862555 @default.
• W2084787806 cites W2162342191 @default.
• W2084787806 cites W2164593087 @default.
• W2084787806 cites W2170287584 @default.
• W2084787806 doi "https://doi.org/10.1074/jbc.m111.276329" @default.
• W2084787806 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3186438" @default.
• W2084787806 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/21849500" @default.
• W2084787806 hasPublicationYear "2011" @default.
• W2084787806 type Work @default.
• W2084787806 sameAs 2084787806 @default.
• W2084787806 citedByCount "96" @default.
• W2084787806 countsByYear W20847878062012 @default.
• W2084787806 countsByYear W20847878062013 @default.
• W2084787806 countsByYear W20847878062014 @default.
• W2084787806 countsByYear W20847878062015 @default.
• W2084787806 countsByYear W20847878062016 @default.
• W2084787806 countsByYear W20847878062017 @default.
• W2084787806 countsByYear W20847878062018 @default.
• W2084787806 countsByYear W20847878062019 @default.
• W2084787806 countsByYear W20847878062020 @default.
• W2084787806 countsByYear W20847878062021 @default.
• W2084787806 countsByYear W20847878062022 @default.
• W2084787806 countsByYear W20847878062023 @default.
• W2084787806 crossrefType "journal-article" @default.
• W2084787806 hasAuthorship W2084787806A5000930137 @default.
• W2084787806 hasAuthorship W2084787806A5013509770 @default.
• W2084787806 hasAuthorship W2084787806A5022235365 @default.
• W2084787806 hasAuthorship W2084787806A5028534770 @default.
• W2084787806 hasAuthorship W2084787806A5069757510 @default.
• W2084787806 hasBestOaLocation W20847878061 @default.
• W2084787806 hasConcept C121332964 @default.
• W2084787806 hasConcept C126691448 @default.
• W2084787806 hasConcept C1276947 @default.
• W2084787806 hasConcept C147483822 @default.
• W2084787806 hasConcept C170493617 @default.
• W2084787806 hasConcept C203014093 @default.
• W2084787806 hasConcept C2776090121 @default.
• W2084787806 hasConcept C55493867 @default.
• W2084787806 hasConcept C7876069 @default.
• W2084787806 hasConcept C86803240 @default.
• W2084787806 hasConcept C8891405 @default.
• W2084787806 hasConcept C95444343 @default.
• W2084787806 hasConceptScore W2084787806C121332964 @default.
• W2084787806 hasConceptScore W2084787806C126691448 @default.
• W2084787806 hasConceptScore W2084787806C1276947 @default.
• W2084787806 hasConceptScore W2084787806C147483822 @default.
• W2084787806 hasConceptScore W2084787806C170493617 @default.
• W2084787806 hasConceptScore W2084787806C203014093 @default.
• W2084787806 hasConceptScore W2084787806C2776090121 @default.
• W2084787806 hasConceptScore W2084787806C55493867 @default.
• W2084787806 hasConceptScore W2084787806C7876069 @default.

• next