John Connolly

Human Tissues Contain CD141hi Cross-Presenting Dendritic Cells with Functional Homology to Mouse CD103+ Nonlymphoid Dendritic Cells

Summary Dendritic cell (DC)-mediated cross-presentation of exogenous antigens acquired in the periphery is critical for the initiation of CD8+ T cell responses. Several DC subsets are described in human tissues but migratory cross-presenting DCs have not been isolated, despite their potential importance in immunity to pathogens, vaccines, and tumors and tolerance to self. Here, we identified a CD141hi DC present in human interstitial dermis, liver, and lung that was distinct from the majority of CD1c+ and CD14+ tissue DCs and superior at cross-presenting soluble antigens. Cutaneous CD141hi DCs were closely related to blood CD141+ DCs, and migratory counterparts were found among skin-draining lymph node DCs. Comparative transcriptomic analysis with mouse showed tissue DC subsets to be conserved between species and permitted close alignment of human and mouse DC subsets. These studies inform the rational design of targeted immunotherapies and facilitate translation of mouse functional DC biology to the human setting.

Dendritic cell subsets in health and disease

Summary:  The dendritic cell (DC) system of antigen‐presenting cells controls immunity and tolerance. DCs initiate and regulate immune responses in a manner that depends on signals they receive from microbes and their cellular environment. They allow the immune system to make qualitatively distinct responses against different microbial infections. DCs are composed of subsets that express different microbial receptors and express different surface molecules and cytokines. Our studies lead us to propose that interstitial (dermal) DCs preferentially activate humoral immunity, whereas Langerhans cells preferentially induce cellular immunity. Alterations of the DC system result in diseases such as autoimmunity, allergy, and cancer. Conversely, DCs can be exploited for vaccination, and novel vaccines that directly target DCs in vivo are being designed.

Sustained expansion of NKT cells and antigen-specific T cells after injection of α-galactosyl-ceramide loaded mature dendritic cells in cancer patients

Natural killer T (NKT) cells are distinct glycolipid reactive innate lymphocytes that are implicated in the resistance to pathogens and tumors. Earlier attempts to mobilize NKT cells, specifically, in vivo in humans met with limited success. Here, we evaluated intravenous injection of monocyte-derived mature DCs that were loaded with a synthetic NKT cell ligand, α-galactosyl-ceramide (α-GalCer; KRN-7000) in five patients who had advanced cancer. Injection of α-GalCer–pulsed, but not unpulsed, dendritic cells (DCs) led to >100-fold expansion of several subsets of NKT cells in all patients; these could be detected for up to 6 mo after vaccination. NKT activation was associated with an increase in serum levels of interleukin-12 p40 and IFN-γ inducible protein-10. In addition, there was an increase in memory CD8+ T cells specific for cytomegalovirus in vivo in response to α-GalCer–loaded DCs, but not unpulsed DCs. These data demonstrate the feasibility of sustained expansion of NKT cells in vivo in humans, including patients who have advanced cancer, and suggest that NKT activation might help to boost adaptive T cell immunity in vivo.

Dendritic cells loaded with killed allogeneic melanoma cells can induce objective clinical responses and MART-1 specific CD8+ T-cell immunity.

Dendritic cells (DCs) loaded with killed allogeneic tumors can cross-prime tumor-specific naive CD8+ T cells in vitro, thereby providing an option to overcome human leukocyte antigen restriction inherent to loading DC vaccines with peptides. We have vaccinated 20 patients with stage IV melanoma with autologous monocyte-derived DCs loaded with killed allogeneic Colo829 melanoma cell line. DCs were generated by culturing monocytes with granulocyte macrophage-colony stimulating factor (granulocyte macrophage-colony stimulating factor) and interleukin (IL-4) and activated by additional culture with tumor necrosis factor and CD40 ligand. A total of 8 vaccines were administered at monthly intervals. The first patient was accrued December 2002 and the last November 2003. Fourteen patients were alive at 12 months, 9 patients were alive at 24 months, and 8 patients are alive as of January 2006. The estimated median overall survival is 22.5 months with a range of 2 to 35.5 months. Vaccinations were safe and tolerable. They induced, in 2 patients who failed previous therapy, durable objective clinical responses, 1 complete regression (CR) and 1 partial regression (PR) lasting 18 and 23 months, respectively. Three out of 13 analyzed patients showed T-cell immunity to melanoma antigen recognized by autologous T cells (MART-1) tissue differentiation antigen. Two of 3 patients showed improved immune function after vaccinations demonstrated by improved secretion of interferon (IFN)-γ or T-cell proliferation in response to MART-1 derived peptides. In one of these patients, vaccination led to elicitation of CD8+ T-cell immunity specific to a novel peptide-derived from MART-1 antigen, suggesting that cross-priming/presentation of melanoma antigens by DC vaccine had occurred. Thus, the present results justify the design of larger follow-up studies to assess the clinical response to DC vaccines loaded with killed allogeneic tumor cells in patients with metastatic melanoma.

IL-15-induced human DC efficiently prime melanoma-specific naive CD8+ T cells to differentiate into CTL.

Monocytes differentiate into dendritic cells (DC) in response to GM‐CSF combined with other cytokines including IL‐4 and IL‐15. Here, we show that IL15‐DC are efficient in priming naive CD8+ T cells to differentiate into melanoma antigen‐specific cytotoxic T lymphocytes (CTL). While both melanoma peptide‐pulsed IL15‐DC and IL4‐DC expand high‐precursor frequency MART‐1‐specific CD8+ T cells after two stimulations in vitro, IL15‐DC require much lower peptide concentration for priming. IL15‐DC are more efficient in expanding gp100‐specific CD8+ T cells and can expand CD8+ T cells specific for Tyrosinase and MAGE‐3. CTL primed by IL15‐DC are superior in their function as demonstrated by (i) higher IFN‐γ secretion, (ii) higher expression of Granzyme B and Perforin, and (iii) higher killing of allogeneic melanoma cell lines, most particularly the HLA‐A*0201+ Sk‐Mel‐24 melanoma cells that are resistant to killing by CD8+ T cells primed with IL4‐DC. Supernatants of the sonicated cells demonstrate unique expression of IL‐1, IL‐8 and IL‐15. Therefore, membrane‐bound IL‐15 might contribute to enhanced priming by IL15‐DC. Thus, IL‐15 induces myeloid DC that are efficient in priming and maturation of melanoma antigen‐specific CTL.

Immune and clinical outcomes in patients with stage IV melanoma vaccinated with peptide-pulsed dendritic cells derived from CD34+ progenitors and activated with type I interferon.

Twenty-two HLA A*0201+ patients with stage IV melanoma were enrolled in a phase 1 safety and feasibility trial using a composite dendritic cell (DC) vaccine generated by culturing CD34+ hematopoietic progenitors and activated with IFN-α. The DC vaccine was loaded with peptides derived from four melanoma tissue differentiation antigens (MART-1, tyrosinase, MAGE-3, and gp100) and influenza matrix peptide (Flu-MP). Twenty patients were evaluable, 14 of whom received vaccination with peptide-pulsed DCs without keyhole limpet hemocyanin (KLH) and 6 of whom received vaccination with KLH-loaded DCs. Patients were vaccinated until disease progression or until they had received eight vaccinations. None of the analyzed patients showed the expansion of melanoma-peptide-specific circulating effector memory T cells that secrete IFN-γ in direct ELISPOT. Melanoma-peptide-specific recall memory CD8+ T cells able to secrete IFN-γ and to proliferate could be detected in six of the seven analyzed patients. There were no objective clinical responses. The estimated median overall survival was 12 months (range 2-38), and the median event-free survival was 4 months (range 1-12). There was no statistically significant survival advantage in patients who received KLH-loaded vaccines. As of March 2005, four patients remained alive, 26+, 28+, 28+, and 36+ months. Three of them had received KLH-loaded vaccines and all of them had had additional therapy. Overall, these results suggest that IFN-α-activated CD34-DCs are safe but elicit only limited immune responses, underscoring the need to test different DC maturation factors.

Citrullinated peptide dendritic cell immunotherapy in HLA risk genotype-positive rheumatoid arthritis patients.

Citrullinated peptide-exposed DCs induced immune regulatory effects in HLA risk genotype–positive RA patients. Immunotherapy out of joint Autoantibodies to anti–citrullinated peptides (ACPA) are found in most patients with rheumatoid arthritis (RA), especially those with HLA-DRB1 risk alleles. Benham et al. report a first-in-human phase 1 trial of a single injection of autologous dendritic cells modified with an NF-κB inhibitor that have been exposed to four citrullinated peptide antigens. They find that HLA risk genotype–positive RA patients had reduced numbers of effector T cells and decreased production of proinflammatory cytokines compared with untreated RA patient controls. The therapy was safe and did not induce disease flares. These data support larger studies of antigen-specific immunotherapy for RA. In animals, immunomodulatory dendritic cells (DCs) exposed to autoantigen can suppress experimental arthritis in an antigen-specific manner. In rheumatoid arthritis (RA), disease-specific anti–citrullinated peptide autoantibodies (ACPA or anti-CCP) are found in the serum of about 70% of RA patients and are strongly associated with HLA-DRB1 risk alleles. This study aimed to explore the safety and biological and clinical effects of autologous DCs modified with a nuclear factor κB (NF-κB) inhibitor exposed to four citrullinated peptide antigens, designated “Rheumavax,” in a single-center, open-labeled, first-in-human phase 1 trial. Rheumavax was administered once intradermally at two progressive dose levels to 18 human leukocyte antigen (HLA) risk genotype–positive RA patients with citrullinated peptide–specific autoimmunity. Sixteen RA patients served as controls. Rheumavax was well tolerated: adverse events were grade 1 (of 4) severity. At 1 month after treatment, we observed a reduction in effector T cells and an increased ratio of regulatory to effector T cells; a reduction in serum interleukin-15 (IL-15), IL-29, CX3CL1, and CXCL11; and reduced T cell IL-6 responses to vimentin447–455–Cit450 relative to controls. Rheumavax did not induce disease flares in patients recruited with minimal disease activity, and DAS28 decreased within 1 month in Rheumavax-treated patients with active disease. This exploratory study demonstrates safety and biological activity of a single intradermal injection of autologous modified DCs exposed to citrullinated peptides, and provides rationale for further studies to assess clinical efficacy and antigen-specific effects of autoantigen immunomodulatory therapy in RA.

Selective blockade of the inhibitory Fcγ receptor (FcγRIIB) in human dendritic cells and monocytes induces a type I interferon response program

The ability of dendritic cells (DCs) to activate immunity is linked to their maturation status. In prior studies, we have shown that selective antibody-mediated blockade of inhibitory FcγRIIB receptor on human DCs in the presence of activating immunoglobulin (Ig) ligands leads to DC maturation and enhanced immunity to antibody-coated tumor cells. We show that Fcγ receptor (FcγR)–mediated activation of human monocytes and monocyte-derived DCs is associated with a distinct gene expression pattern, including several inflammation-associated chemokines, as well as type 1 interferon (IFN) response genes, including the activation of signal transducer and activator of transcription 1 (STAT1). FcγR-mediated STAT1 activation is rapid and requires activating FcγRs. However, this IFN response is observed without a detectable increase in the expression of type I IFNs themselves or the need to add exogenous IFNs. Induction of IFN response genes plays an important role in FcγR-mediated effects on DCs, as suppression of STAT1 by RNA interference inhibited FcγR-mediated DC maturation. These data suggest that the balance of activating/inhibitory FcγRs may regulate IFN signaling in myeloid cells. Manipulation of FcγR balance on DCs and monocytes may provide a novel approach to regulating IFN-mediated pathways in autoimmunity and human cancer.

Yaa autoimmune phenotypes are conferred by overexpression of TLR7

The Y‐linked autoimmune accelerating (Yaa) locus drives the transition to fatal lupus nephritis when combined with B6.Sle1 in our C57BL/6J (B6)‐congenic model of systemic autoimmunity. We and others recently demonstrated that the translocation of a cluster of X‐linked genes onto the Y chromosome is the genetic lesion underlying Yaa (Subramanian, S. et al., Proc. Natl. Acad. Sci. USA 2006. 103: 9970–9975; Pisitkun, P. et al., Science 2006. 312: 1669–1672). In male mice carrying Yaa, the transcription of several genes within the translocated segment is increased roughly twofold. Although the translocated X chromosome segment in Yaa may contain as many as 16 genes, the major candidate gene for causation of the Yaa‐associated autoimmune phenotypes has been TLR7. To confirm the role of TLR7 in Yaa‐mediated autoimmune phenotypes, we introgressed a targeted disruption of TLR7 (TLR7–) onto B6.Sle1Yaa to produce B6.Sle1YaaTLR7– and examined evidence of disease at 6 and 9 months of age. Our results demonstrate that the up‐regulation of TLR7 in the B6.Sle1Yaa strain is responsible for splenomegaly, glomerular nephritis and the majority of the cellular abnormalities of B, T and myeloid cells. The up‐regulation of TLR7 was also responsible for driving the infiltration and activation of leukocytes in the kidney, in which activated T cells were a primary component. However, the resolution of TLR7 up‐regulation did not eliminate the enhanced humoral autoimmunity observed in B6.SleYaa, suggesting that additional elements in the translocation may contribute to the disease phenotype.

The Role of mRNA Turnover in the Regulation of Tristetraprolin Expression: Evidence for an Extracellular Signal-Regulated Kinase-Specific, AU-Rich Element-Dependent, Autoregulatory Pathway

Tristetraprolin (TTP) is a regulator of TNF-α mRNA stability and is the only trans-acting factor shown to be capable of regulating AU-rich element-dependent mRNA turnover at the level of the intact animal. Using the THP-1 myelomonocytic cell line, we demonstrated for the first time that TTP is encoded by an mRNA with a short half-life under resting conditions. Using pharmacologic inhibitors of the mitogen-activated protein kinase pathways, we show that the induction of TTP by LPS activation is mediated through changes in transcription, mRNA stability, and translation. A coordinate increase in both TTP and TNF-α mRNA stability occurs within 15 min of LPS treatment, but is transduced through different mitogen-activated protein kinase pathways. This regulation of TTP and TNF-α mRNA stability is associated with the finding that TTP binds these mRNA under both resting and LPS-activated conditions in vivo. Finally, we demonstrate that TTP can regulate reporter gene expression in a TTP 3′ untranslated region-dependent manner and identify three distinct AU-rich elements necessary to mediate this effect. Thus, TTP regulates its own expression in a manner identical to that seen with the TNF-α 3′ untranslated region, indicating that this autoregulation is mediated at the level of mRNA stability. In this manner, TTP is able to limit the production of its own proteins as well as that of TNF-α and thus limit the response of the cell to LPS.