Chemokine programming dendritic cell antigen response: part I – select chemokine programming of antigen uptake even after maturation

Here, we report on the successful programming of dendritic cells (DCs) using selectively applied mixtures of chemokines as a novel protocol for engineering vaccine efficiency. Antigen internalization by DCs is a pivotal step in antigen uptake/presentation for bridging innate and adaptive immunity and in exogenous gene delivery used in vaccine strategies. Contrary to most approaches to improve vaccine efficiency, active enhancement of antigen internalization by DCs as a vaccine strategy has been less studied because DCs naturally down‐regulate antigen internalization upon maturation. Whereas chemokines are mainly known as signal proteins that induce leucocyte chemotaxis, very little research has been carried out to identify any additional effects of chemokines on DCs following maturation. Here, immature DCs are pre‐treated with select chemokines before intentional maturation using lipopolysaccharide (LPS). When pre‐treated with a mixture of CCL3 and CCL19 in a 7 : 3 ratio, then matured with LPS, chemokine pre‐treated DCs exhibited 36% higher antigen uptake capacity than immature DCs and 27% higher antigen‐processing capacity than immature DCs treated only with LPS. Further, CCL3 : CCL19 (7 : 3) pre‐treatment of DCs modulated MHC molecule expression and secretion of various cytokines of DCs. Collectively, DC programming was feasible using a specific chemokine combination and these results provide a novel strategy for enhancing DC‐based vaccine efficiency. In Part II, we report on the phenotype changes and antigen presentation capacity of chemokine pre‐treated murine bone marrow‐derived DCs examined in long‐term co‐culture with antigen‐specific CD4⁺ T cells.     

Requirement of the T cell receptor for antigen presentation by T lymphocytes. Effect of envelope glycoproteins of HIV-1 on antigen presentation by T cells.

We have developed CD4+, tetanus antigen-specific T cell clones that proliferate in the presence of tetanus antigen and autologous irradiated peripheral blood leucocytes (PBL) as antigen-presenting cells (APC). There have been several reports that T cells can present antigen themselves. We have used tetanus antigen-specific T cell clones to examine the effects of envelope glycoproteins of HIV-1 on processing and presentation of antigen to T cells. Cloned T cells were pre-incubated with soluble crude preparation of tetanus antigen for 4 h at 37 degrees C, irradiated, and used as APC (T-APC). These cells could present antigen, as assessed by the ability of the autologous cloned T cells to proliferate. Resting T cells and phytohaemagglutinin-activated T cell blasts from autologous PBL could not present tetanus antigen to the responder cloned T cells. Antigen presentation by T-APC was abrogated by treating cells with anti-HLA-DR but not by anti-HLA-DQ monoclonal antibodies; treatment of tetanus antigen-pulsed T-APC with anti-tetanus antibody also blocked the ability of these cells to induce proliferation in responder T cells. Antigen presentation by cloned T cells was by a chloroquine-resistant pathway. Pretreatment of T-APC with envelope glycoprotein of HIV-1, gp120, did not affect the proliferative responses of the responder T cells. These data suggest that gp120 does not inhibit the antigen-presenting function while suppressing antigen-specific responses.     

Anatomic location defines antigen presentation by dendritic cells to T cells in response to intravenous soluble antigens

In the spleen, exogenous antigen is preferentially presented by CD8alpha+CD11b- DC to CD8 T cells and by CD8alpha-CD11b+ DC to CD4 T cells. However, it is not yet clear whether the same rule applies to other secondary lymphoid organs. To address this issue, we first classified secondary lymphoid tissues into three categories based on the expression pattern of CD8alpha and CD11b in C57BL/6 and BALB/c mice: (a) spleen, (b) mesenteric lymph node (MLN) and (c) other peripheral lymph nodes (PLN). We then analyzed the OVA-specific T cell-stimulating capacity of each DC subset after intravenous injection with soluble OVA. Our results show that, regardless of tissue origin, CD8alpha-CD11b+ DC generally present OVA to CD4 T cells, a finding that held true as well for CD8alpha+CD11b+ DC in PLN. In striking contrast, CD8alpha+CD11b- DC in spleen, CD8alpha-CD11b+ DC in MLN and CD8alpha+CD11b+ DC in PLN mainly cross-present OVA to CD8 T cells in their respective tissues. Of note, CD8alpha-CD11b+ DC in MLN and CD8alpha+CD11b+ DC in PLN present OVA to both CD4 T and CD8 T cells. Therefore, the antigen-presenting capacity of each distinct DC subset is determined by its anatomic environment in combination with its surface phenotype.     

巨噬细胞型骨髓基质细胞对肿瘤抗原提呈的体外实验研究

目的　探讨骨髓基质细胞对肿瘤抗原的提呈功能。方法　小鼠骨髓贴壁细胞经 Ｇ Ｍ Ｃ Ｓ Ｆ 诱导,形成以成熟巨噬细胞为主的基质细胞,用小鼠红白血病细胞 Ｆ Ｂ Ｌ３ 肿瘤抗原刺激,然后再与 Ｆ Ｂ Ｌ３ 肿瘤抗原致敏的 Ｔ 淋巴细胞混合培养。结果　骨髓基质细胞经 Ｆ Ｂ Ｌ３ 肿瘤抗原刺激后, Ｔ Ｎ Ｆα和 Ｉ Ｌ１β的分泌水平明显升高,经抗原预激的骨髓基质细胞能特异性地刺激同种抗原致敏的 Ｔ 淋巴细胞增殖和分泌高水平的 Ｉ Ｌ２ 。单抗阻断试验发现, Ｍ Ｈ ＣⅡ类分子和 Ｂ７２ 分子的联合阻断能有效地抑制致敏 Ｔ 淋巴细胞分泌 Ｉ Ｌ２ 。结论　本实验证实骨髓基质细胞具有抗原提呈功能, Ｍ Ｈ ＣⅡ类分子和 Ｂ７２ 分子在其抗原提呈中发挥了重要作用。     

Protein kinase C delta stimulates antigen presentation by Class II MHC in murine dendritic cells

Maturation of dendritic cells (DCs) regulates protein sorting in endosomal compartments to promote the surface expression of molecules involved in T cell activation. MHC Class II complexes are mobilized to the surface via intracellular effector molecules that remain largely unknown. We here show that protein kinase C (PKC) stimulates Class II antigen surface expression, using knock-in mice that express a Class II-green fluorescent protein fusion protein as a read out. Selective inhibition of PKCdelta counteracts the ability of DCs to stimulate Class II MHC-restricted antigen-specific T cells. Activation of PKC does not affect antigen uptake, peptide loading and surface display of Class I MHC and transferrin receptor in DCs. We show that activation-induced Class II MHC surface expression is dependent on activation of PKCdelta and conclude that this event is pivotal for optimal CD4 T cell activation.     

Concerted antigen presentation by dendritic cells and B cells is necessary for optimal CD4 T-cell immunity in vivo

The relative contributions of different types of antigen presenting cells to T-cell activation, expansion and induction of effector functions are still not fully understood. In order to evaluate the roles of dendritic versus B cells during these phases of a CD4 T-cell response in vivo, we adoptively transferred major histocompatibility complex class II restricted, T-cell receptor-transgenic CD4+ T cells into transgenic mice expressing selectively the T-cell restricting class II molecules on either dendritic cells, B cells or both. Upon immunization with peptide antigen, we observed that dendritic cells were sufficient to induce activation, expansion, interleukin-2 production and germinal centre migration of antigen-specific T cells, independently of other antigen-presenting cells. In contrast, neither resting nor activated B cells had similar antigen-presenting capacities in vivo. However, in double transgenic mice where both B cells and dendritic cells were capable of presenting antigen, T cells showed increased proliferation, expansion and cytokine production in vivo. Moreover, higher antigen-specific CD4 T-cell numbers accumulated in germinal centres. Our data demonstrate that dendritic cells are sufficient to activate naive CD4 T cells in vivo, but B cells subsequently can enhance CD4 T-cell expansion further.     

Prevention of T cell activation by interference of internalized intravenous immunoglobulin (IVIg) with MHC II-dependent native antigen presentation

Activation of self-reactive CD4⁺ T cells plays a central role in the initiation and maintenance of autoimmune diseases. We recently reported that intravenous immunoglobulin (IVIg) inhibits the MHC II-restricted CD4⁺ T cell activation induced by the presentation of immune complexes. Because native antigens can also play a role in the induction of several autoimmune diseases, we determined whether IVIg could also affect CD4⁺ T cell activation following presentation of native antigens by APCs. Here we report that IVIg significantly reduces the activation of CD4⁺ T cells by native ovalbumin. The inhibitory effect is FcγR-independent and occurs following internalization of IVIg inside APCs, where it interferes with the intracellular events leading to MHC II-dependent antigen presentation. The effect of IVIg on native antigen presentation could therefore contribute to dampen the autoimmune reaction by reducing CD4⁺ T cell activation and the subsequent inflammatory response induced by these cells.     

Efficiency of peptide presentation by dendritic cells compared with other cell types: implications for cross-priming

Dendritic cells (DCs) play a key role in the induction of cellular immune responses by harvesting antigens from peripheral tissue for cross-priming CD8(+) T cells. It has been demonstrated that apoptotic bodies, whole- or degraded-cell-associated or soluble antigens as well as heat shock protein-bound peptides can be taken up, processed and cross-presented by DCs. Since cells are continuously releasing peptides from their surface MHC molecules, DCs in the tissues are exposed to such peptides and might process and present them to T cells as an additional pathway for cross-priming. To investigate this possibility, we compared and characterized the presentation of exogenous peptides by DCs and other cell types employing novel recombinant antibodies with TCR-like specificities for specific peptide-MHC complexes (pMHCs). These analyses reveal that loading of immature and mature DCs with peptide is far less efficient than it is for monocytes, T and B lymphocytes, B-lymphoblastoid, melanoma and TAP-deficient T2 cells. This inefficiency of peptide transfer to the MHC molecules of DCs makes it unlikely that these cells recycle peptides released from the MHC molecules of other cells and may explain why cross-presentation of such peptides has not yet been observed.     

树突状细胞摄取和提呈颗粒化抗原的能力与颗粒载体的大小相关

目的：研究体外小鼠骨髓树突状细胞对2种不同大小bead-OVA复合物（0.04 μm bead和1.0μm bead）的摄取及class I途径抗原提呈能力。方法：以2h骨髓粘附细胞为前体细胞，用GM－CSF（1000U／ml）和IL－3（10ng/ml）培养5d，观察细胞对FITC标记的2种bead-OVA复合物的摄取，PMA、amiloride、cytochalasin D对摄取的抑制，以及细胞摄取后表达MHC分子和共刺激分子的情况，同时用OVA表位特异性T细胞杂交检测细胞摄取后通过class I途径活化CTL应答的能力。结果：树突状细胞对1．0μm bead-OVA的摄取明显高于对0．04μm bead-OVA，前者被上述3种抑制剂显著抑制，后者仅对amiloride和PMA抑制作用敏感，CCD无明显抑制作用。与摄取结果相反，0．04μm bead－OVA较1．0μm bead－OVA诱导更强的CD8细胞免疫应答，表型分析显示，细胞摄取0．04μm bead后，MHC分子和共刺激分子表达显著高于1．0μm的bead。结论：树突状细胞对2种bead的摄取能力和摄取机制不一样，0．04μm bead尽管摄取效率不如1．0μm bead，但通过class I途径提呈抗原的效率显著高于后者。     

T cell and dendritic cell interactions in lymphoid organs: More than just being in the right place at the right time

The initiation of T lymphocyte responses within secondary lymphoid organs involves interactions with different subsets of dendritic cells (DCs). Recent studies have revealed the complexity of microanatomical organization within lymphoid organs. Exactly how T cells and DCs locate each other and the type of cellular interactions required for optimal priming of effector and memory T cell responses are beginning to be unraveled. Here we review advances in our understanding of how T cell priming is choreographed during infections, highlight the importance of cell positioning in this process and discuss how a spectrum of cellular interactions shapes T cell activation and differentiation.     

Cross presentation of antigen by dendritic cells: mechanisms and implications for immunotherapy

Dendritic cells (DCs) possess the specialized potential to present exogenously derived antigen to cytotoxic T lymphocytes to elicit an immune response. This process, termed cross presentation, is crucial in the generation of immune response to viruses, tumors and in autoimmune disease. The ability of DCs to cross-present exogenous antigen to cytotoxic T lymphocytes makes them an attractive target for exploitation in immunotherapy. In recent years, significant advances have been made in understanding the mechanism of cross-presentation and the DC subsets involved. The recent discovery of the human cross presenting DC has given this field a new lease of life. In this report, the authors provide an overview of cross-presentation of antigen by DCs, focusing on the current understanding of the molecular mechanisms of the process. The authors also discuss the DC subsets involved in cross presentation and its role in health and disease.     

Molecular basis of lipid antigen presentation by CD1d and recognition by natural killer T cells

Together with peptides, T lymphocytes respond to hydrophobic molecules, mostly lipids, presented by the non-classical CD1 family (CD1a–e). These molecules have evolved complex and diverse binding grooves in order to survey different cellular compartments for self and exogenous antigens, which are then presented for recognition to T-cell receptors (TCRs) on the surface of T cells. In particular, most CD1d-presented antigens are recognized by a population of lymphocytes denominated natural killer T (NKT) cells, characterized by a strong immunomodulatory potential. Among NKT cells, two major subsets (type I and type II NKT cells) have been described, based on their TCR repertoire and antigen specificity. Here we review recent structural and biochemical studies that have shed light on the molecular details of CD1d-mediated antigen recognition by type I and II NKT cells, which are in many aspects distinct from what has been observed for peptide major histocompatibility complex-reactive TCRs.     

Antigen presentation by T cells: T cell receptor ligation promotes antigen acquisition from professional antigen-presenting cells

The purpose of this study was to determine whether the clonotypic specificity of the T cell receptor influences the specificity of T cell-mediated antigen presentation. We have previously shown that myelin basic protein (MBP)-specific Lewis rat GP2.E5/R1 (R1) T cells cultured with antigen, irradiated syngeneic splenocytes (IrrSPL) and tolerogenic monoclonal antibody become highly effective antigen-presenting cells (APC). In the current studies, we investigated the transfer of specific (MBP) and unrelated (conalbumin) antigens from antigen-pulsed SPL to R1 T cells. R1 T cells cultured with IrrSPL that were pulsed simultaneously with both MBP and conalbumin acquired and presented both antigens to the appropriate T cell responders in a secondary assay. These results suggested a physical transfer of major histocompatibility complex (MHC)/peptide complexes from professional APC to R1 T cells. Transfer of conalbumin from professional APC to R1 T cells required specific recognition of MBP and was optimal when both conalbumin and MBP were presented on the same group of professional APC. Antigens transfer did not occur when allogeneic SPL were used as APC. The anti-I-A mAb OX6 inhibited antigen transfer but only when added during the initiation of culture. OX6 also inhibited antigen acquisition by R1-trans, a variant of the R1 T cell line which constitutively synthesizes high levels of I-A, from MBP-pulsed IrrSPL but blockade of I-A did not inhibit antigen acquisition when soluble MBP was added directly to the culture. Despite constitutive synthesis of I-A, R1-trans T cells did not acquire guinea pig MBP from pulsed allogeneic APC. These studies demonstrate that although T cells of a particular specificity can present unrelated antigens, the cognate interaction of the T cell antigen receptor with the appropriate antigen/self-MHC complex strongly promotes acquisition of these complexes from professional APC.     

Podocytes present antigen to activate specific T cell immune responses in inflammatory renal disease

Infiltration of activated T cells into renal tissue plays an essential role in inflammatory nephropathy. However, the mechanism enabling the renal recruitment and activation of T cells remains elusive. Here we report that inflammatory cytokine-promoted antigen presentation by podocytes is a key for recruiting and activating specific T cells. Our results showed that diabetes-associated inflammatory cytokines IFNγ and IL-17 all upregulated expression of MHC-I, MHC-II, CD80 and CD86 on the podocyte surface. Both IFNγ and IL-17 stimulated the uptake and processing of ovalbumin (OVA) by mouse podocytes, resulting in presentation of OVA antigen peptide on the cell surface. OVA antigen presentation by podocytes was also validated using human podocytes. Furthermore, OVA antigen-presenting mouse podocytes were able to activate OT-I mouse T cell proliferation and inflammatory cytokine secretion, which in turn caused podocyte injury and apoptosis. Finally, OT-I mice subjected to direct renal injection of OVA plus IFNγ/IL-17 but not OVA alone exhibited OVA antigen presentation by podocytes and developed nephropathy in 4 weeks. In conclusion, antigen presentation by podocytes under inflammatory conditions plays an important role in activating T cell immune responses and facilitating immune-mediated glomerular disease development. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

A role for the Hsp90 molecular chaperone family in antigen presentation to T lymphocytes via major histocompatibility complex class II molecules

The heat shock protein (HSP) Hsp90 is known to chaperone cytosolic peptides for MHC class I (MHCI)-restricted antigen presentation to T lymphocytes. We now demonstrate a role for Hsp90 activity in presentation of antigens on MHCII. Treatment of mouse antigen-presenting cells (APC) with the pharmacological Hsp90 inhibitor, geldanamycin, inhibited MHCII-mediated presentation of endocytosed and cytosolic proteins as well as synthetic peptides to specific T cells. Ectopic expression of human Hsp90 in APC enhanced MHCII-mediated antigen presentation. Further, pharmacological Hsp90 inhibition reduced, while retroviral Hsp90 overexpression enhanced, the levels of stable compact MHCII heterodimers correlating with the antigen presentation phenotype. Pharmacological inhibition of Hsp90 activity in IFN-gamma-treated APC resulted in severe abrogation of MHCII-restricted presentation of cytosolic antigen, but only partially inhibited exogenous antigen presentation. Our data suggest a major role for Hsp90 activity in MHCII-mediated antigen presentation pathways, and implicate IFN-gamma-inducible Hsp90-independent mechanisms.     

An approach to isolating T cell lines that react to antigens presented on the surface of dendritic cells.

We describe an approach that might be useful for identifying antigens on surfaces of antigen presenting cells. It is known that dendritic cells carry antigens in situ and are efficient at clustering antigen-specific T cells. Using the human mixed lymphocyte reaction (MLR) system, we have shown that alloreactive CD4+ T cells can be selected by their capacity to cluster with dendritic cells in the first 2 days of the MLR. Small numbers of clustered cells, 1-10/culture well, could then be expanded as antigen-specific lines in presence of either antigen or mitogen, sodium periodate. Few antigen-specific lines could be isolated from the nonclustered fraction. When T cell lines derived from the dendritic T cell clusters were maintained without antigen, i.e. using second party (syngeneic antigen-presenting cells (APC] or irrelevant antigen bearing APC, i.e. third-party (HLA-mismatched) stimulator cells plus mitogen, the T cells retained their specificity for the original stimulating alloantigen over the time course tested, several weeks to months. These findings show that by using dendritic cells as immunoadsorbents one can prepare antigen-specific cell lines and maintain the specificity of the lines without the need for adding exogeneous antigen during either immunoselection or cloning. We discuss the possible use of dendritic cells as a means for raising T cell lines and clones that recognize antigens being carried by APC and which might be pertinent to protective immunity and autoimmunity.     

Contribution of enhanced engagement of antigen presentation machinery to the clinical immunogenicity of a human interleukin (IL)‐21 receptor‐blocking therapeutic antibody

Reliable risk assessment for biotherapeutics requires accurate evaluation of risk factors associated with immunogenicity. Immunogenicity risk assessment tools were developed and applied to investigate the immunogenicity of a fully human therapeutic monoclonal antibody, ATR‐107 [anti‐interleukin (IL)‐21 receptor] that elicited anti‐drug antibodies (ADA) in 76% of healthy subjects in a Phase 1 study. Because the ATR‐107 target is expressed on dendritic cells (DCs), the immunogenicity risk related to engagement with DC and antigen presentation pathways was studied. Despite the presence of IL‐21R on DCs, ATR‐107 did not bind to the DCs more extensively than the control therapeutic antibody (PF‐1) that had elicited low clinical ADA incidence. However, ATR‐107, but not the control therapeutic antibody, was translocated to the DC late endosomes, co‐localized with intracellular antigen‐D related (HLA‐DR) molecules and presented a dominant T cell epitope overlapping the complementarity determining region 2 (CDR2) of the light chain. ATR‐107 induced increased DC activation exemplified by up‐regulation of DC surface expression of CD86, CD274 (PD‐L1) and CD40, increased expansion of activated DC populations expressing CD86^hi, CD40^hi, CD83^hi, programmed death ligand 1 (PD‐L1)^hi, HLA‐DR^hi or CCR7^hi, as well as elevated secretion of tumour necrosis factor (TNF)‐α by DCs. DCs exposed to ATR‐107 stimulated an autologous T cell proliferative response in human donor cells, in concert with the detection of immunoglobulin (Ig)G‐type anti‐ATR‐107 antibody response in clinical samples. Collectively, the enhanced engagement of antigen presentation machinery by ATR‐107 was suggested. The approaches and findings described in this study may be relevant to identifying lower immunogenicity risk targets and therapeutic molecules.     

Evidence for antigen presentation to sensitized T cells by thyroid peroxidase (TPO)-specific B cells in mice injected with fibroblasts co-expressing TPO and MHC class II

Injection of AKR/N mice with fibroblasts co-expressing MHC class II and TPO in the absence of adjuvant induces IgG-class TPO antibodies that resemble spontaneously arising human thyroid autoantibodies. We have used this model to examine the effect of iodide on TPO antibody induction as well as to analyse the interaction between T and B cells. Despite its importance as a major environmental factor in thyroid autoimmunity, variable iodide intake had no detectable effects on TPO antibody levels, lymphocytic infiltration of the thyroid or thyroid hormone levels. In terms of T cell responsiveness, splenocytes from TPO fibroblast-injected mice, but not from control mice, proliferated in response to TPO. Intriguingly, B cell-depleted splenocytes (mainly T cells without reduction of macrophages) proliferated in response to TPO only when co-cultured with irradiated autologous splenocytes from TPO fibroblast-injected mice but not from control mice. These data suggest that TPO-specific B cells are involved in antigen presentation to sensitized T cells and are supported by the ability of spleen cells from TPO cell-injected (but not control) mice to secrete TPO antibodies spontaneously in culture. In conclusion, we provide the first evidence for the presence of thyroid autoantigen-specific B cells and their ability to present their autoantigen to sensitized T cells in mice induced to develop TPO antibodies resembling autoantibodies in humans.