Antigen-bearing Cells Research Articles

Vaginal type-II mucosa is an inductive site for primary CD8⁺ T-cell mucosal immunity.

The structured lymphoid tissues are considered the only inductive sites where primary T cell immune responses occur. The naïve T cells in structured lymphoid tissues, once being primed by antigen -bearing dendritic cells, differentiate into memory T cells and traffic back to the mucosal sites through the bloodstream. Contrary to this belief, here we show that the vaginal type-II mucosa itself, despite lack of structured lymphoid tissues, can act as an inductive site during primary CD8+ T cell immune responses. We provide evidence that the vaginal mucosa supports both the local immune priming of naïve CD8+ T cells and the local expansion of antigen-specific CD8+ T cells, thereby demonstrating a different paradigm for primary mucosal T cell immune induction.

A novel “priming-boosting” strategy for immune interventions in cervical cancer

Despite the encouraging development of a preventive vaccine for human papillomavirus (HPV), it cannot improve ongoing infections. Therefore, a new vaccine is urgently needed that can prevent and treat cervical cancer, and cure pre-cancerous lesions. In this study, we constructed two peptide-based vaccines. The first was a short-term, long-peptide (ST-LP) vaccine that simultaneously targeted three key carcinogenic epitopes (E5–E6–E7) on HPV16. We tested this vaccine in murine TC-1 cells infected with a recombinant adeno-associated virus (rAAV) fused with HPV16E5 DNA (rTC-1 cells), which served as a cell model; we also tested it in immune-competent mice loaded with rTC-1 cells, which served as an ectopic tumor model. The ST-LP injections resulted in strong, cell-mediated immunity, capable of attacking and eliminating abnormal antigen-bearing cells. Furthermore, to prolong immunogenic capability, we designed a unique rAAV that encoded the three predicted epitopes for a second, long-term, long-peptide (LT-LP) vaccine. Moreover, we used a new immune strategy of continuous re-injections, where three ST-LP injections were performed at one-week intervals (days 0, 7, 14), then one LT-LP injection was performed on day 120. Our in vitro and in vivo studies revealed that this strategy could boost the immune response to produce longer and stronger protection against target cells, and mice were thoroughly protected from tumor growth. Our results showed that priming the immune system with the ST-LP vaccine, followed by boosting the immune system with the LT-LP vaccine could generate a rapid, robust, durable cytotoxic T-lymphocyte response to HPV16-positive tumors.

Strategically Localized Dendritic Cells Promote Rapid T Cell Responses to Lymph-Borne Particulate Antigens

Upon infection, adaptive immune responses play catch-up with rapidly replicating pathogens. While mechanisms for efficient humoral responses to lymph-borne antigens have been characterized, the current paradigm for T cell responses to infections and particulate vaccines involves delayed migration of peripheral antigen-bearing dendritic cells (DCs) to lymph nodes (LNs), where they elicit effector T cell responses. Utilizing whole LN 3D imaging, histo-cytometry, and intravital 2-photon microscopy, we have identified a specialized population of DCs, enriched in the LN-resident CD11b(+) subset, which resides within the lymphatic sinus endothelium and scans lymph with motile dendrites. These DCs capture draining particles and present associated antigens to T lymphocytes, inducing T cell responses much sooner than and independently of migratory DCs. Thus, strategic DC subset positioning in LNs limits a potentially costly delay in generation of T cell responses to lymph-borne antigens, contributing to effective host defense. These findings are also highly relevant to vaccine design.

A GMP-compliant protocol to expand and transfect cancer patient T cells with mRNA encoding a tumor-specific chimeric antigen receptor.

Chimeric antigen receptors (CARs), which combine an antibody-derived binding domain (single chain fragment variable) with T-cell-activating signaling domains, have become a promising tool in the adoptive cellular therapy of cancer. Retro- and lenti-viral transductions are currently the standard methods to equip T cells with a CAR; permanent CAR expression, however, harbors several risks like uncontrolled auto-reactivity. Modification of T cells by electroporation with CAR-encoding RNA to achieve transient expression likely circumvents these difficulties. We here present a GMP-compliant protocol to activate and expand T cells for clinical application. The protocol is optimized in particular to produce CAR-modified T cells in clinically sufficient numbers under full GMP-compliance from late-stage cancer patients. This protocol allows the generation of 6.7 × 10(8) CAR-expressing T cells from one patient leukapheresis. The CAR-engineered T cells produced pro-inflammatory cytokines after stimulation with antigen-bearing tumor cells and lysed tumor cells in an antigen-specific manner. This functional capacity was maintained after cryopreservation. Taken together, we provide a clinically applicable protocol to transiently engineer sufficient numbers of antigen-specific patient T cells for use in adoptive cell therapy of cancer.

Harnessing the heterogeneity of T cell differentiation fate to fine-tune generation of effector and memory T cells (LYM4P.759)

Abstract Recent studies show that naïve T cells bearing identical T cell receptors experience heterogeneous differentiation and clonal expansion processes. The factors controlling this outcome are not well characterized, and their contributions to immune cell dynamics are similarly poorly understood. In this study, we develop a computational model to elaborate mechanisms occurring within and between two important physiological compartments, lymph nodes and blood, to determine how immune cell dynamics are controlled. Our multi-organ (multi-compartment) model integrates cellular and tissue level events and allows us to examine the heterogeneous differentiation of individual precursor cognate naïve T cells to generate both effector and memory T lymphocytes. Using this model, we simulate a hypothetical immune response and reproduce both primary and recall responses to infection. Increased numbers of antigen-bearing dendritic cells are predicted to raise production of both effector and memory T cells, and distinct “sweet spots” of peptide-MHC levels on those dendritic cells exist that favor CD4+ or CD8+ T cell differentiation towards either effector or memory cell phenotypes. This has important implications for vaccine development and immunotherapy.

LFA-1 and CD62L dependent transient surface CCR5 expression in naïve CD8+ T cells enhances T cell immune memory (CCR3P.214)

Abstract The initiation of an effective adaptive immune response requires choreographed interaction among rare antigen-bearing dendritic cells (DCs) with antigen-specific CD4+ and CD8+ T cells in the lymph node (LN). This interaction is especially critical for the magnitude of memory T cell generation. We and others have previously demonstrated that CCL3/CCL4 secretion from productive interaction between antigen-specific T cell and antigen-bearing DCs promote local recruitment of naïve CD8+ T cells in the inflamed LNs, implying that naïve CD8+ T cells can express CCR5 in the inflamed LN microenvironment. Here, we examined the kinetics of CCR5 expression on naïve T cells upon entering an inflamed LN. Maximum surface CCR5 expression occurs in 10-30% of newly arrived naive T cells within the first 6 hours via mobilization of pre-existing CCR5 protein found in intracellular vesicles, and this transient up-regulation of surface CCR5 expression in CD8+ T cells was dependent on direct physical contact with LFA and CD62L, as ligation of CD8+ T cells with anti-CD11a and/or anti-CD62L antibodies enhances surface CCR5 expression within 1 hour in the absence of TCR stimulation. More importantly, CCR5+ fraction of naïve CD8+ T cells exhibit enhanced ability to become memory T cells. Our study implicates an important functional role for CCR5 expression by naïve lymphocytes prior to cognate antigen recognition for enhanced memory T cell development.

Open questions: A rose is a rose is a rose - or not?

Open questions: A rose is a rose is a rose - or not?

Harnessing the Heterogeneity of T Cell Differentiation Fate to Fine-Tune Generation of Effector and Memory T Cells

Recent studies show that naïve T cells bearing identical T cell receptors experience heterogeneous differentiation and clonal expansion processes. The factors controlling this outcome are not well characterized, and their contributions to immune cell dynamics are similarly poorly understood. In this study, we develop a computational model to elaborate mechanisms occurring within and between two important physiological compartments, lymph nodes and blood, to determine how immune cell dynamics are controlled. Our multi-organ (multi-compartment) model integrates cellular and tissue level events and allows us to examine the heterogeneous differentiation of individual precursor cognate naïve T cells to generate both effector and memory T lymphocytes. Using this model, we simulate a hypothetical immune response and reproduce both primary and recall responses to infection. Increased numbers of antigen-bearing dendritic cells (DCs) are predicted to raise production of both effector and memory T cells, and distinct “sweet spots” of peptide-MHC levels on those DCs exist that favor CD4+ or CD8+ T cell differentiation toward either effector or memory cell phenotypes. This has important implications for vaccine development and immunotherapy.

CD8+ T-cell responses rapidly select for antigen-negative tumor cells in the prostate.

Stimulation of patients' immune systems for the treatment of solid tumors is an emerging therapeutic paradigm. The use of enriched autologous T cells for adoptive cell therapy or vaccination with antigen-loaded dendritic cells have shown clinical efficacy in melanoma and prostate cancer, respectively. However, the long-term effects of immune responses on selection and outgrowth of antigen-negative tumor cells in specific tumor types must be determined to understand and achieve long-term therapeutic effects. In this study, we have investigated the expression of a tumor-specific antigen in situ after treatment with tumor-specific CD8(+) T cells in an autochthonous mouse model of prostate cancer. After T-cell treatment, aggregates of dead antigen-positive tumor cells were concentrated in the lumen of the prostate gland and were eventually eliminated from the prostate tissue. Despite the elimination of antigen-positive tumor cells, prostate tumor continued to grow in T-cell-treated mice. Interestingly, the remaining tumor cells were antigen negative and downregulated MHC class I expression. These results show that CD8(+) T cells are effective in eliminating antigen-bearing prostate tumor cells but they also can select for the outgrowth of antigen-negative tumor cells. These findings provide insights into the requirements for an effective cancer immunotherapy within the prostate that not only induces potent immune responses but also avoids selection and outgrowth of antigen-negative tumor cells.

Conjunctival Inflammation in Thrombospondin-1 Deficient Mouse Model of Sjögren’s Syndrome

Lacrimal gland inflammation during autoimmune Sjögren’s syndrome (SS) leads to ocular surface inflammation – Keratoconjunctivitis sicca (KCS). This condition afflicts both the cornea and conjunctiva that form the ocular surface. Thrombospondin-1 (TSP-1) deficiency in mice results in lacrimal gland and corneal inflammation that resembles the human disease. In this study we report conjunctival pathology in this mouse model of SS. We found that TSP-1 null mice develop inflammation in the conjunctiva and associated loss of goblet cell function similar to that seen in patients with SS. Increased expression of Th1 (IFN-γ, TNF-α) and Th17 (IL-6, IL-17A) inflammatory cytokines and related transcription factors (Tbet and RORγt) were detected in TSP-1 null conjunctiva as well as their draining lymph nodes (LNs). The conjunctival inflammation was also accompanied by an increase in local lymphatic vessels. Interestingly, migration of antigen-bearing dendritic cells (DCs) from the ocular surface to the LNs was dependent on the TSP-1 available in the tissue. These results not only reveal potential immunopathogenic mechanisms underlying KCS in SS but also highlight the therapeutic potential of TSP-1.

Fcγ receptors enable anticancer action of proapoptotic and immune-modulatory antibodies

Antibodies have important roles in controlling cellular immunity through interaction with activating or inhibitory Fcγ receptors (FcγRs). FcγR engagement can facilitate receptor cross-linking on target cells, or induce retrograde FcγR signals to stimulate or suppress antibody-dependent, cell-mediated depletion of antigen-bearing target cells. Recent studies uncover unexpectedly important roles for FcγRs in the anticancer action of antibodies designed to trigger tumor cell apoptosis or enhance antitumor immunity. Here, we outline a conceptual framework for understanding these findings and discuss their mechanistic and translational implications.

Antigen-bearing dendritic cells from the sublingual mucosa recirculate to distant systemic lymphoid organs to prime mucosal CD8 T cells.

Effector T cells are described to be primed in the lymph nodes draining the site of immunization and to recirculate to effector sites. Sublingual immunization generates effector T cells able to disseminate to the genital tract. Herein, we report an alternative mechanism that involves the recirculation of antigen-bearing dendritic cells (DCs) in remote lymphoid organs to prime T cells. Sublingual immunization with a muco-adhesive model antigen unable to diffuse through lymphatic or blood vessels induced genital CD8 T cells. The sublingual draining lymph nodes were not mandatory to generate these lymphocytes, and antigen-bearing DCs from distant lymph nodes and spleen were able to prime specific CD8 T cells in a time- and dose-dependent manner. This study demonstrates, for the first time, that antigen-bearing DCs originating from the site of immunization recirculate to distant lymphoid organs and provides insights into the mechanism of distant CD8 T-cell generation by sublingual immunization.

Specialized microenvironment within the lymph node controls antigen-presentation properties of dendritic cells and Th2 differentiation. (P1119)

Abstract T cells need to encounter antigen-bearing dendritic cells (DCs) to undergo clonal expansion and effector development. These interactions, within lymphoid tissues, are orchestrated by the expression of chemokines and chemokine receptors. Indeed, CCR7 expression by both T cells and DCs is thought to facilitate DC/T cell encounters within the T cell area of the lymph node. However, it is unknown whether co-localization of the DCs and T cells within the T cell area is obligate for effector generation. Here, we show that, following infection with an intestinal nematode, antigen-bearing DCs and the responding CD4+ T cells upregulate expression of CXCR5 and migrate in a CXCR5/CXCL-13 dependent fashion to the B cell area of the mesenteric lymph node. We further demonstrate that CXCR5-dependent localization of DCs and T cells near the CXCL13-expressing B cell follicles are required for optimal Th2 responses. Furthermore, we found that disrupting DCs from accumulating in the perifollicular region prevents the DCs from initiating Th2 differentiation. Finally, we demonstrated that DCs receive conditioning signals within the perifollicular microenvironment that change Class II Transactivator (CIITA) expression, maturation and antigen-presenting properties of DCs that allow the DCs to initiate Th2 priming. These results show that specialized microenvironments within the lymph node directly influence dendritic cell antigen presentation and function for effective priming of Th2 responses.

Delivery of viral-vectored vaccines by B cells represents a novel strategy to accelerate CD8+ T-cell recall responses

AbstractRapid boosting of memory CD8+ T cells (TM) is essential in cancer immunotherapy and the control of certain infectious diseases. However, effector T cells (TE) are a barrier to booster vaccination because they can rapidly kill antigen-bearing antigen-presenting cells (APCs) before TM are engaged. We demonstrate that viral-vectored vaccines delivered by B cells elicit robust TM expansion in the presence of TE, enabling booster immunizations to bypass TE-mediated negative feedback regulation. Our data indicate that viral vector–loaded B cells home to the follicular regions in secondary lymphoid organs, which are anatomically separated from TE and in close proximity to TM. The B cells, however, do not serve as APCs in this area. Rather, classic CD11c+ dendritic cells serve to stimulate the secondary CD8+ T-cell response. Our data reveal that B cells represent a novel and readily accessible delivery system that can effectively engage secondary CD8+ T-cell activation for prime-boost strategies.

A novel method of CD34+ cell separation from umbilical cord blood

Umbilical cord blood (UCB) is rich in the heavily glycosylated CD34 antigen-bearing hematopoietic stem cells that are valuable for transplantation therapy of malignant and nonmalignant disease. CD34+ cell yields (0.13%-0.25%-0.3%) of mononuclear cells (UCMCs) isolated by anti-CD34 monoclonal antibody (MoAb) on immunomagnetic particles (e.g., Miltenyi particles) are insufficient to treat adults. We fractionated UCMCs by physicochemical charge-based methods. Avoiding Miltenyi particles and HESPAN yielded 30 x 10(6) to 54 x 10(6) UCMCs/20 mL UCB by Ficoll-Isopaque for serial depletion fractionation, using nylon wool column (NWC) or direct rosetting with sheep red blood cells (SRBCs) without incubation in the cold. CD34+ cell yields (approx. 5.12%) were 39 times greater than 0.13% (Korean study, 11,098 UCB units) and 10 to 20 times greater than 0.25% to 0.3% harvested by anti-CD34 Miltenyi particles. SRBC depletion of most high-specific-gravity T cells achieved considerable enrichment of CD34+ and BY55+ cells. Using NWC achieved 2.5-fold enrichment of CD34+ cells and twofold enrichment of BY55+ cells. Direct SRBC rosetting provided better or higher enrichment of CD34+ cells. Overall CD34+ cell yield in low-density fraction was more than twice after direct rosetting (38% vs. 16%) in contrast to separation by NWC followed by SRBC rosetting. CD3+ cell yields (by three CD markers) were approximately 8.83%, far below approximately 30 x 10(7) /kg considered acceptable to avoid graft-versus-host disease. Natural killer cell yields (CD16+/CD56+ and BY55+/ CD160+) are in perfect agreement. Achieving approximately 5% CD34+ cell yields from single UCB donations, a major advance, holds great promise for CD34+ cell therapy of adults and larger children, and cheaper cultured RBC manufacture.

Current and historical perspectives on methodological flaws in processing umbilical cord blood

Umbilical cord blood (UCB) hematopoietic stem cells (HSC-CD34+) are valuable for treating malignant or nonmalignant disease. Processing UCB by HESPAN-6% and anti-CD34-Miltenyi particles provides insufficient cells for treating adults. Physicochemical-electrokinetic studies on UCB-mononuclear cells (MNCs) under conditions of delayed processing, ice or very low temperatures, and some cell separation media identified artifacts introduced by procedures. Adsorption of biomaterials from cell damage by temperature, degradation products after using enzymes, harsh reagents, dithiothreitol, and HESPAN affect cell properties and distribution. Miltenyi particles internalized by cells could release iron that accumulating in liver or spleen would then risk toxicity. Summary topics included the effects of temperature, HESPAN (fast sedimenting agent), glycoproteases, DNase, and dithiothreitol risk affecting cell receptors in recognition, "homing," leading to possible unintended iatrogenic bioeffects should such cells be transfused into humans. The loss of undetectable and uncaptured low CD34 antigen-bearing cells by Miltenyi particles seems to occur when the current methods of isolation of CD34+ cells and other cells are critically assessed. The purpose here is to highlight and suggest avoiding the procedural flaws involved. Preventing ice temperatures avoids ice-damaged platelets releasing biomaterials that are adsorbed on cells altering UBC-MNCs/HSC properties and cell loss. Omitting the positive selection with antibody-linked Miltenyi particles obviates the use of harsh reagents to release the cells. Internalized Miltenyi particles are a toxicity hazard that needs investigations. Achieving approximately 5% yields of CD34+ cells (153 × 10(5) /110 mL cord-placenta blood) is a major advance holding great promise, for the first time increasing the prospect of stem cell therapy of 70-kg adults, using a single UCB donation (with dose of 1.5 × 10(5) cells/kg) and considerably cheaper cultured red blood cells manufacture (multiple packs/2 × 10(12) ).

Downregulation of Key Early Events in the Mobilization of Antigen-bearing Dendritic Cells by Leukocyte Immunoglobulin-like Receptor B4 in a Mouse Model of Allergic Pulmonary Inflammation

Leukocyte Immunoglobulin-like Receptor B4 (LILRB4) null mice have an exacerbated T helper cell type 2 (Th2) immune response and pulmonary inflammation compared with Lilrb4+/+ animals when sensitized intranasally with ovalbumin (OVA) and low-dose lipopolysaccharide (LPS) followed by challenge with OVA. Moreover, OVA-challenged Lilrb4 −/− mice exhibit greater migration of antigen (Ag)-bearing dendritic cells (DCs) to lymph nodes and accumulation of interleukin 4- and interleukin 5-producing lymph node lymphocytes. The main objective of this study was to determine how the absence of LILRB4 leads to a greater number of DCs in the lymph nodes of Ag-challenged mice and increased lung Th2 inflammation. Mice were sensitized intranasally with PBS alone or containing OVA and LPS; additional cohorts were subsequently challenged with OVA. Expression of chemokine (C-C motif) ligand 21 (CCL21) in the lung was assessed immunohistologically. OVA ingestion and expression of LILRB4 and chemokine (C-C motif) receptor 7 (CCR7) were quantified by flow cytometry. Inhalation of OVA and LPS induced upregulation of LILRB4 selectively on lung Ag-bearing DCs. After sensitization and challenge, the lung lymphatic vessels of Lilrb4 −/− mice expressed more CCL21, a chemokine that directs the migration of DCs from peripheral tissue to draining lymph nodes, compared with Lilrb4+/+ mice. In addition, lung DCs of challenged Lilrb4 −/− mice expressed more CCR7, the CCL21 receptor. The lungs of challenged Lilrb4 −/− mice also contained significantly greater numbers of CD4+ cells expressing interleukin-4 or interleukin-5, consistent with the greater number of Ag-bearing DCs and Th2 cells in lymph nodes and the attendant exacerbated Th2 lung pathology. Our data establish a new mechanism by which LILRB4 can downregulate the development of pathologic allergic inflammation: reduced upregulation of key molecules needed for DC migration leading to decreases in Th2 cells in lymph nodes and their target tissue.

Cytomegalovirus Impairs Antiviral CD8+ T Cell Immunity by Recruiting Inflammatory Monocytes

Inflammatory monocytes are key early responders toinfection that contribute to pathogen-host interactions in diverse ways. Here, we report that the murine cytomegalovirus-encoded CC chemokine, MCK2, enhanced CCR2-dependent recruitment of these cells to modulate antiviral immunity, impairing virus-specific CD8(+) Tcell expansion and differentiation into effector cytotoxic T lymphocytes, thus reducing the capacity to eliminate viral antigen-bearing cells and slowing viral clearance. Adoptive transfer of inflammatory monocytes into Ccr2(-/-)Ccl2(-/-) mice impaired virus antigen-specific clearance. Cytomegalovirus therefore enhancesa natural CCR2-dependent immune regulatory network to modulate adaptive immunity via nitric oxide production, reminiscent of the monocytic subtype of myeloid-derived suppressor cells primarily implicated in cancer immunomodulation.

Regulation of TH2 development by CXCR5+ dendritic cells and lymphotoxin-expressing B cells

Although cognate encounters between CCR7-expressing antigen-bearing dendritic cells (DCs) and CCR7+ naïve T cells take place within the T cell zone of lymph nodes, it is unknown whether co-localization of the DCs and T cells within the T cell area is obligate for effector generation. Here, we show that, following nematode infection, antigen-bearing DCs and CD4+ T cells upregulate CXCR5 and co-localize in a CXCL13, B cell and lymphotoxin-dependent fashion outside of the T zone. Importantly, lymphotoxin-expressing B cells, CXCL13 and CXCR5-expressing DCs and T cells are also necessary for development of interleukin 4 (IL-4) producing TH2 cells, suggesting that TH2 differentiation can initiate outside of the T cell zone.

Leukocyte Ig-like Receptor B4 (LILRB4) Downregulates Key Steps in the Migration of Antigen-bearing Lung Dendritic Cells to Lymph Nodes in Th2 Inflammation (173.30)

Abstract We previously reported that Lilrb4-/- mice have exacerbated Th2 immune responses and pulmonary inflammation compared with Lilrb4+/+ animals when sensitized with OVA and low-dose LPS followed by challenge with OVA. In addition, LILRB4 is selectively upregulated on Ag-bearing dendritic cells (DCs) in the lung and draining lymph nodes (LNs). Ag-challenged Lilrb4-/- mice exhibit increased migration of Ag-bearing DCs to LNs and accumulation of IL-4- and IL-5-producing LN lymphocytes. To determine how the absence of LILRB4 leads to more DCs in the LNs of Ag-challenged mice, animals were sensitized intranasally with PBS alone or OVA/LPS and were challenged with OVA. Four hours later, lungs were evaluated by immunohistology, and lung mononuclear cells were isolated for flow cytometry. The lung lymphatic vessels of Lilrb4-/- mice expressed significantly more CCL21, a chemokine that directs the migration of DCs to draining LNs. In addition, lung DCs of Lilrb4-/- mice expressed more CCR7, the CCL21 receptor. The lungs of Lilrb4-/- mice also contained significantly greater numbers of CD4+ cells expressing IL-4 or IL-5, consistent with the greater number of Ag-bearing DCs and Th2 cells in LNs and the attendant exacerbated Th2 lung pathology. Our data reveal that LILRB4 downregulates expression of two fundamental molecules that induce the migration of Ag-bearing DCs to LNs, thereby decreasing Th2 cell accumulation in LNs and lung and the ensuing pathologic pulmonary inflammation.