Impaired antigen-induced CD8+ T cell clonal expansion in aging is due to defects in antigen presenting cell function

CD8+ T cell activation depends on interaction with antigen-presenting cells (APCs) and this interaction leads to the expansion of T cells with the capacity to control infection. Using professional APCs, we demonstrate that with age, the duration of APC-T cell contact time required to achieve clonal expansion increases. Na?ve CD8+ T cells from aged mice showed no defect in antigen-induced proliferation when stimulated with APC from young mice. In contrast, CD8+ T cells from young mice exhibited reduced clonal expansion and secreted significantly lower amounts of IFN-gamma when stimulated by APCs from aged mice. The aged APCs were defective in costimulatory molecule expression and cytokine and chemokine secretion. These data indicate that defects in APC function lead to poor T cell clonal expansion and function in aging.     

Exploiting the role of endogenous lymphoid-resident dendritic cells in the priming of NKT cells and CD8+ T cells to dendritic cell-based vaccines

Transfer of antigen between antigen-presenting cells (APCs) is potentially a physiologically relevant mechanism to spread antigen to cells with specialized stimulatory functions. Here we show that specific CD8+ T cell responses induced in response to intravenous administration of antigen-loaded bone marrow-derived dendritic cells (BM-DCs), were ablated in mice selectively depleted of endogenous lymphoid-resident langerin+ CD8α+ dendritic cells (DCs), suggesting that the antigen is transferred from the injected cells to resident APCs. In contrast, antigen-specific CD4+ T cells were primed predominantly by the injected BM-DCs, with only very weak contribution of resident APCs. Crucially, resident langerin+ CD8α+ DCs only contributed to the priming of CD8+ T cells in the presence of maturation stimuli such as intravenous injection of TLR ligands, or by loading the BM-DCs with the glycolipid α-galactosylceramide (α-GalCer) to recruit the adjuvant activity of activated invariant natural killer-like T (iNKT) cells. In fact, injection of α-GalCer-loaded CD1d-/- BM-DCs resulted in potent iNKT cell activation, suggesting that this glycolipid antigen can also be transferred to resident CD1d+ APCs. While iNKT cell activation per se was independent of langerin+ CD8α+ DCs, some iNKT cell-mediated activities were reduced, notably release of IL-12p70 and transactivation of NK cells. We conclude that both protein and glycolipid antigens can be exchanged between distinct DC species. These data suggest that the efficacy of DC-based vaccination strategies may be improved by the incorporation of a systemic maturation signal aimed to engage resident APCs in CD8+ T cell priming, and α-GalCer may be particularly well suited to this purpose.     

4-1BB is superior to CD28 costimulation for generating CD8+ cytotoxic lymphocytes for adoptive immunotherapy

Artificial APCs (aAPCs) genetically modified to express selective costimulatory molecules provide a reproducible, cost-effective, and convenient method for polyclonal and Ag-specific expansion of human T cells for adoptive immunotherapy. Among the variety of aAPCs that have been studied, acellular beads expressing anti-CD3/anti-CD28 efficiently expand CD4+ cells, but not CD8+ T cells. Cell-based aAPCs can effectively expand cytolytic CD8+ cells, but optimal costimulatory signals have not been defined. 4-1BB, a costimulatory molecule expressed by a minority of resting CD8+ T cells, is transiently up-regulated by all CD8+ T cells following activation. We compared expansion of human cytolytic CD8+ T cells using cell-based aAPCs providing costimulation via 4-1BB vs CD28. Whereas anti-CD3/anti-CD28 aAPCs mostly expand naive cells, anti-CD3/4-1BBL aAPCs preferentially expand memory cells, resulting in superior enrichment of Ag-reactive T cells which recognize previously primed Ags and efficient expansion of electronically sorted CD8+ populations reactive toward viral or self-Ags. Using HLA-A2-Fc fusion proteins linked to 4-1BBL aAPCs, 3-log expansion of Ag-specific CD8+ CTL was induced over 14 days, whereas similar Ag-specific CD8+ T cell expansion did not occur using HLA-A2-Fc/anti-CD28 aAPCs. Furthermore, when compared with cytolytic T cells expanded using CD28 costimulation, CTL expanded using 4-1BB costimulation mediate enhanced cytolytic capacity due, in part, to NKG2D up-regulation. These results demonstrate that 4-1BB costimulation is essential for expanding memory CD8+ T cells ex vivo and is superior to CD28 costimulation for generating Ag-specific products for adoptive cell therapy.     

Inflammatory cytokines provide a third signal for activation of naive CD4+ and CD8+ T cells

The effects of inflammatory cytokines on naive T cells have been studied using MHC protein/peptide complexes on microspheres, thus avoiding the use of APCs whose functions may be affected by the cytokines. IL-1, but not IL-12, increased proliferation of CD4+ T cells in response to Ag and IL-2, which is consistent with effects on in vivo priming of CD4+ cells. In contrast, proliferation of CD8+ T cells to Ag and IL-2 required IL-12, and IL-12 replaced adjuvant in stimulating an in vivo response to peptide. These results support a model in which distinct inflammatory cytokines act directly on naive CD4+ and CD8+ T cells to provide a third signal, along with Ag and IL-2, to optimally activate differentiation and clonal expansion.     

Sarcoidosis is not associated with a generalized defect in T cell suppressor function

In pulmonary sarcoidosis, the marked expansion of CD4+ (helper/inducer) T cells in the alveolar structures of the lung is maintained by local IL-2 release by activated CD4+ HLA-DR+ T cells without concomitant expansion and activation of CD8+ (suppressor/cytotoxic) T cells, suggesting that sarcoid may be associated with a generalized abnormality of CD8+ T cells. Consistent with this concept, evaluation of the expression of the IL-2R on fresh lung T cells from individuals with active sarcoidosis demonstrated that 7 +/- 1% of sarcoid lung CD4+ T cells are spontaneously expressing the IL-2R compared with only 1 +/- 1% lung CD8+ T cells (p less than 0.01). However, stimulation of purified sarcoid blood CD8+ T cells with the anti-T3/TCR complex mAb OKT3 was followed by the normal expression of IL-2R (p greater than 0.1) and proliferation (p greater than 0.1). In addition, lung sarcoid CD8+ T cells responded to OKT3 similarly to normal lung CD8+ T cells and to autologous blood CD8+ T cells as regards expression of IL-2R (p greater than 0.1) and proliferation (p greater than 0.1). Finally, using CD4+ cells activated with allogenic Ag to induce, in coculture, fresh autologous CD8+ cells to suppress proliferation of fresh autologous CD4+ cells to the same Ag, sarcoid CD8+ T cells suppressed CD4+ cell proliferation in a normal fashion (p greater than 0.1). These results demonstrate that sarcoid CD8+ (suppressor/cytotoxic) T cells are competent to respond to a proliferation signal normally and can be induced to normally suppress CD4+ T cell proliferation to Ag, suggesting that the expansion of activated CD4+ T cells in pulmonary sarcoidosis is not due to a generalized abnormality of CD8+ T cells or of their suppressor T cell function.     

Cross-linking of 4-1BB up-regulates IL-13 expression in CD8(+) T lymphocytes

4-1BB, one of co-stimulatory molecules, is a member of TNF receptor superfamily and expressed on T cells upon TCR ligation. We have shown that 4-1BB is a co-stimulatory molecule enhancing cell cycle progression and inhibiting activation-induced cell death of CD8+ T cells by enhancing TCR signaling pathways. Here, we first report that the cross-linking of 4-1BB increased the expression of IL-13 mRNA and protein, and its secretion apparently via calcineurin, a Ca2+/calmodulin-dependent phosphatase. Ligation of 4-1BB with p815-m-4-1BBL evoked intracellular Ca2+ level in CD8+ T cells. CD8+ T cells express IL-13 receptor alpha1 mRNA. Incubation with anti-IL-13 blocking mAb reduced proliferation of CD8+ T cells enhanced by 4-1BB, and the treatment of CD3/4-1BB-ligated CD8+ T cells with recombinant IL-13 enhances cell proliferation, indicating that 4-1BB-induced IL-13 expression is partially responsible for the CD8+ T cell expansion in an autocrine or paracrine manner.     

A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro

Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparation of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin–biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.     

Selective delivery of augmented IL-2 receptor signals to responding CD8+ T cells increases the size of the acute antiviral response and of the resulting memory T cell pool

CD8(+) T cells respond to IL-2 produced both endogenously and by CD4(+) Th during an antiviral response. However, IL-2R signals can potentially promote CD8(+) T cell death as well as proliferation, making it unclear whether IL-2R signals provide a predominantly positive or negative effect upon CD8(+) T cell responses to viral infection. To more precisely define the direct role of IL-2R signaling on CD8(+) T cells during the response to a virus, we examined the effect of delivering augmented IL-2R signals selectively to CD8(+) T cells responding to lymphocytic choriomeningitis virus infection. Although naive CD8(+) T cells are competent to produce IL-2, CD8(+) T cells lose this capacity upon differentiation into effector CD8(+) T cells. However, effector CD8(+) T cells do retain the capacity to produce GM-CSF upon Ag stimulation. Thus, to deliver enhanced autocrine IL-2R signals to CD8(+) T cells, we established a transgenic mouse strain expressing a chimeric GM-CSF/IL-2R (GMIL2R). As GM-CSF production is Ag dependent, the GMIL2R delivers an augmented IL-2R signal exclusively to CD8(+) T cells responding to Ag. Following lymphocytic choriomeningitis virus infection, GMIL2R transgenic mice exhibited an increase in both the peak CD8(+) T cell response achieved and the size of the resulting memory pool established. Upon secondary viral challenge, the GMIL2R also enhanced the proliferative response of memory CD8(+) T cells. Thus, our findings indicate that IL-2 delivery to responding CD8(+) T cells is a limiting factor in both the acute and memory antiviral responses.     

Pathogen-specific CD8 T cell responses are directly inhibited by IL-10

Regulation of CD8 T cell expansion and contraction is essential for successful immune defense against intracellular pathogens. IL-10 is a regulatory cytokine that can restrict T cell responses by inhibiting APC functions. IL-10, however, can also have direct effects on T cells. Although blockade or genetic deletion of IL-10 enhances T cell-mediated resistance to infections, the extent to which IL-10 limits in vivo APC function or T cell activation/proliferation remains unknown. Herein, we demonstrate that primary and memory CD8 T cell responses following Listeria monocytogenes infection are enhanced by the absence of IL-10. Surface expression of the IL-10R is transiently up-regulated on CD8 T cells following activation, suggesting that activated T cells can respond to IL-10 directly. Consistent with this notion, CD8 T cells lacking IL-10R2 underwent greater expansion than wild-type T cells upon L. monocytogenes infection. The absence of IL-10R2 on APCs, in contrast, did not enhance T cell responses following infection. Our studies demonstrate that IL-10 produced during bacterial infection directly limits expansion of pathogen-specific CD8 T cells and reveal an extrinsic regulatory mechanism that modulates the magnitude of memory T cell responses.     

Recombinant HLA-DP2 binds beryllium and tolerizes beryllium-specific pathogenic CD4+ T cells

Chronic beryllium disease is a lung disorder caused by beryllium exposure in the workplace and is characterized by granulomatous inflammation and the accumulation of beryllium-specific, HLA-DP2-restricted CD4+ T lymphocytes in the lung that proliferate and secrete Th1-type cytokines. To characterize the interaction among HLA-DP2, beryllium, and CD4+ T cells, we constructed rHLA-DP2 and rHLA-DP4 molecules consisting of the alpha-1 and beta-1 domains of the HLA-DP molecules genetically linked into single polypeptide chains. Peptide binding to rHLA-DP2 and rHLA-DP4 was consistent with previously published peptide-binding motifs for these MHC class II molecules, with peptide binding dominated by aromatic residues in the P1 pocket. 9Be nuclear magnetic resonance spectroscopy showed that beryllium binds to the HLA-DP2-derived molecule, with no binding to the HLA-DP4 molecule that differs from DP2 by four amino acid residues. Using beryllium-specific CD4+ T cell lines derived from the lungs of chronic beryllium disease patients, beryllium presentation to those cells was independent of Ag processing because fixed APCs were capable of presenting BeSO4 and inducing T cell proliferation. Exposure of beryllium-specific CD4+ T cells to BeSO4 -pulsed, plate-bound rHLA-DP2 molecules induced IFN-gamma secretion. In addition, pretreatment of beryllium-specific CD4+ T cells with BeSO4-pulsed, plate-bound HLA-DP2 blocked proliferation and IL-2 secretion upon re-exposure to beryllium presented by APCs. Thus, the rHLA-DP2 molecules described herein provide a template for engineering variants that retain the ability to tolerize pathogenic CD4+ T cells, but do so in the absence of the beryllium Ag.     

Comparison of AAV/IL-7 autocrine (T cell) versus paracrine (DC) gene delivery for enhancing CTL stimulation and function

Adoptive transfer of antigen-specific cytotoxic T lymphocyte (CTL) into patients holds promise in treating cancer. Such anti-cancer CTL are stimulated by professional antigen-presenting dendritic cells (DC). We hypothesize the gene delivery of various Th1-response cytokines, such as interleukin 7 (IL-7), should further enhance CTL stimulation and activity. However, the issue as to which cell type, DC (paracrine) or the T cell (autocrine), should express a particular Th1 cytokine gene for optimal CTL stimulation has never been addressed. We used adeno-associated virus-2 (AAV) to compare delivery of IL-7 and IL-2 genes into DC or T cells and to exogenous commercial cytokines for generating robust carcinoembryonic antigen (CEA)-specific CTL. AAV/IL-7 transduction of T cells (autocrine delivery) generated CTL with the highest killing capability. Consistent with this, AAV/IL-7 delivery generated T cell populations with the highest proliferation, highest interferon γ expression, highest CD8(+):CD4(+) ratio, highest CD8(+), CD69(+) levels, and lowest CD4(+), CD25(+) (Treg) levels. These data are consistent with higher killing by the AAV/IL-7-altered CTL. These data strongly suggest that IL-7 autocrine gene delivery is optimal for CTL generation. These data also suggest Th1 cytokine autocrine versus paracrine delivery is an important issue for immuno-gene therapy and uncovers new questions into cytokine mechanism of action.     

Antigen-specific inhibition of the IL-2-driven proliferation of myelin basic protein-reactive, human T cells

This report examines the antigen-specific inhibition of the IL-2-driven proliferation of autoantigen-reactive, human T cells. Human, myelin basic protein (MBP)-reactive CD4+ cell lines and clones were isolated and maintained in culture by use of IL-2 and periodic antigen stimulation. When freshly isolated antigen-presenting cells (APC) were present, MBP induced proliferation of MBP-reactive T cell populations. However, under different culture conditions, MBP reduced the IL-2-driven proliferation of some MBP-reactive T cell populations. The inhibition of IL-2-driven proliferation did not appear to require CD8+ or OKM 1+ cells since these were not detected when inhibition studies were performed at least 9 days after the last restimulation by irradiated APC and MBP. Supraoptimal concentrations of MBP were not required for inhibition of proliferation. Some heterogeneity of response was apparent since MBP inhibited the IL-2-driven proliferation of some T cell clones while for others MBP had either no effect or produced slight enhancement of proliferation. These results demonstrate an antigen-specific, in vitro immune mechanism that reduces the IL-2-dependent proliferation of autoantigen-reactive, human T cells.     

Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB

The ex vivo priming and expansion of human cytotoxic T lymphocytes (CTLs) has potential for use in immunotherapy applications for cancer and infectious diseases. To overcome the difficulty in obtaining sufficient numbers of CTLs, we have developed artificial antigen-presenting cells (aAPCs) expressing ligands for the T-cell receptor (TCR) and the CD28 and 4-1BB co-stimulatory surface molecules. These aAPCs reproducibly activate and rapidly expand polyclonal or antigen-specific CD8(+) T cells. The starting repertoire of CD8+ T cells was preserved during culture. Furthermore, apoptosis of cultured CD8(+) T cells was diminished by this approach. This approach may have important therapeutic implications for adoptive immunotherapy.     

CD11chigh dendritic cell ablation impairs lymphopenia-driven proliferation of naive and memory CD8+ T cells

The peripheral lymphocyte pool size is governed by homeostatic mechanisms. Thus, grafted T cells expand and replenish T cell compartments in lymphopenic hosts. Lymphopenia-driven proliferation of naive CD8+ T cells depends on self-peptide/MHC class I complexes and the cytokine IL-7. Lymphopenia-driven proliferation and maintenance of memory CD8+ T cells are MHC independent, but are believed to require IL-7 and contact with a bone marrow-derived cell that presents the cytokine IL-15 by virtue of its high affinity receptor (IL-15Ralpha). In this study we show that optimal spontaneous proliferation of grafted naive and memory CD8+ T cells in mice rendered lymphopenic through gene ablation or irradiation requires the presence of CD11chigh dendritic cells. Our results suggest a dual role of CD11chigh dendritic cells as unique APC and cytokine-presenting cells.     

Expression and function of the B and T lymphocyte attenuator (BTLA/CD272) on human T cells

Co-signal receptors provide crucial activating or attenuating signals for T cells. The B and T lymphocyte attenuator (BTLA/CD272) is a third member of co-inhibitory receptors, which belongs to the CD28 immunoglobulin-superfamily. Using monoclonal antibodies (mAbs) against human BTLA, we show that BTLA is constitutively expressed on most CD4+ and CD8+ T cells and its expression progressively decreases upon T cell activation. Polarized Th1 and Th2 cells contained both BTLA-positive and BTLA-negative populations, but the extended culture diminished BTLA expression. Cross-linking BTLA with an agonistic mAb inhibited T cell proliferation and the production of the cytokines IFN-gamma and IL-10 in response to anti-CD3 stimulation. BTLA-mediated inhibition of T cell activation occurred during both primary CD4+ T cell responses and secondary CD4+ and CD8+ T cell responses, suggesting that BTLA ligation sends a constitutive "off" signal to T cells and thus might play an important role in the maintenance of T cell tolerance.     

Cutting edge: predetermined avidity of human CD8 T cells expanded on calibrated MHC/anti-CD28-coated microspheres

Cytotoxic CD8 T cells are key effectors in the immunotherapy of malignant and viral diseases. However, the lack of efficient methods for their in vitro priming and expansion has become a bottleneck to the development of vaccines and adoptive transfer strategies. Synthetic artificial APCs (aAPCs) are now emerging as an attractive tool for eliciting and expanding CTL responses. We show that, by controlling the MHC density on aAPCs, high- or low-avidity tumor-directed human CTL lines can be raised effectively in vitro if costimulation via CD28 and IL-12 is provided. Compared with low-avidity CTL lines, high-avidity CTLs need 100- to 1000-fold less peptide for activation, bind more MHC tetramers, and, as expected, are superior in recognizing tumor cell lines expressing Ag. We believe that the possibility to raise Ag-specific T cells with predetermined avidity will be crucial for the future use of aAPCs in immunotherapeutical settings.     

Cell-to-cell interactions and signals involved in the reconstitution of peripheral CD8 T(CM) and T(EM) cell pools

We here describe novel aspects of CD8⁺ and CD4⁺ T cell subset interactions that may be clinically relevant and provide new tools for regulating the reconstitution of the peripheral CD8⁺ T cell pools in immune-deficient states. We show that the reconstitution capacity of transferred isolated naïve CD8⁺ T cells and their differentiation of effector functions is limited, but both dramatically increase upon the co-transfer of CD4⁺ T cells. This helper effect is complex and determined by multiple factors. It was directly correlated to the number of helper cells, required the continuous presence of the CD4⁺ T cells, dependent on host antigen-presenting cells (APCs) expressing CD40 and on the formation of CD4/CD8/APC cell clusters. By comparing the recovery of (CD44⁺CD62L^high) T_CM and (CD44⁺CD62L^low) T_EM CD8⁺ T cells, we found that the accumulation of T_CM and T_EM subsets is differentially regulated. T_CM-cell accumulation depended mainly on type I interferons, interleukin (IL)-6, and IL-15, but was independent of CD4⁺ T-cell help. In contrast, T_EM-cell expansion was mainly determined by CD4⁺ T-cell help and dependent on the expression of IL-2Rβ by CD8 cells, on IL-2 produced by CD4⁺ T-cells, on IL-15 and to a minor extent on IL-6.     

Sustained expression of CD154 (CD40L) and proinflammatory cytokine production by alloantigen-stimulated umbilical cord blood T cells

Recent data suggests that graft-versus-host disease (GVHD) is initiated by host APCs. Blockade of CD40:CD154 interactions between APCs and T cells in vivo induces T cell tolerance to host alloantigen and dramatically reduces GVHD. Because allogeneic cord blood (CB) transplantation results in a lower incidence and severity of acute GVHD compared with bone marrow transplantation, we have investigated whether CB T cells can express CD154 in response to stimulation by allogeneic monocyte-derived dendritic cells (MDDC) and have used 5- (and 6-)carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling in combination with intracellular cytokine analysis to assess the proliferation and cytokine profiles of alloantigen-responsive cells. CB T cells stimulated with allogeneic MDDC showed stronger proliferation than adult blood T cells. Surface CD154 expression was detected in the actively dividing CFSElow populations of both the CD4+ and CD4- subsets and was brightest in cells that had divided the most. Assessment of supernatants from MDDC-stimulated CB and adult blood T cells showed no significant difference in the levels of either IFN-gamma or TNF-alpha, but CB T cell supernatants did show a significant lack of detectable IL-2. Intracellular cytokine analysis revealed that dividing CB T cells had been primed to produce IFN-gamma, TNF-alpha, and IL-2 on restimulation. Further phenotype analysis showed that 75% of CB T cells producing IFN-gamma were CD8+. These data suggest that MDDC-stimulated CB T cells express functional CD154 and provide enough costimulation for dendritic cells to prime naive CD8+ CB T cells and induce type 1 cytokine production.     

Investigation of peptide involvement in T cell allorecognition using recombinant HLA class I multimers

Alloreactive T cells are involved in injurious graft rejection and graft-vs-host disease. However, they can also evoke beneficial responses to tumor Ags restricted by foreign MHC molecules. Manipulation of these alloreactivities requires information on the basis of T cell allorecognition. The vigorous T cell response to foreign MHC molecules may arise from peptide-independent recognition of polymorphic residues of foreign MHC molecules or peptide-specific recognition of novel peptides presented by foreign MHC molecules. We investigated CD8+ T cell allorecognition using recombinant HLA class I/peptide complexes. Peptide-specific allorecognition was examined using tetramers of HLA-A*0201 representing five peptides derived from ubiquitously expressed self-proteins that are known to bind endogenously to HLA-A*0201. Distinct subsets of CD8+ T cells specific for each HLA-A*0201/peptide combination were detected within four in vitro-stimulated T cell populations specific for foreign HLA-A*0201. Peptide-independent allorecognition was investigated using artificial Ag-presenting constructs (aAPCs) coated with CD54, CD80, and functional densities of a single HLA-A*0201/peptide combination for four different peptides. None of the four T cell populations specific for foreign HLA-A*0201 were stimulated by the aAPCs, whereas they did produce IFN-gamma upon stimulation with cells naturally expressing HLA-A*0201. Thus, aAPCs did not stimulate putative peptide-independent allorestricted T cells. The results show that these alloreactive populations comprise subsets of T cells, each specific for a self-peptide presented by foreign class I molecules, with no evidence of peptide-independent components.