CD3 limits the efficacy of TCR gene therapy in vivo.

The function of T-cell receptor (TCR) gene modified T cells is dependent on efficient surface expression of the introduced TCR α/ß heterodimer. We tested whether endogenous CD3 chains are rate-limiting for TCR expression and antigen-specific T-cell function. We show that co-transfer of CD3 and TCR genes into primary murine T cells enhanced TCR expression and antigen-specific T-cell function in vitro. Peptide titration experiments showed that T cells expressing introduced CD3 and TCR genes recognized lower concentration of antigen than T cells expressing TCR only. In vivo imaging revealed that TCR+CD3 gene modified T cells infiltrated tumors faster and in larger numbers, which resulted in more rapid tumor elimination compared with T cells modified by TCR only. After tumor clearance, TCR+CD3 engineered T cells persisted in larger numbers than TCR-only T cells and mounted a more effective memory response when rechallenged with antigen. The data demonstrate that provision of additional CD3 molecules is an effective strategy to enhance the avidity, anti-tumor activity and functional memory formation of TCR gene modified T cells in vivo.

Specificity for the tumor-associated self-antigen WT1 drives the development of fully functional memory T cells in the absence of vaccination.

Recently, vaccines against the Wilms Tumor antigen 1 (WT1) have been tested in cancer patients. However, it is currently not known whether physiologic levels of WT1 expression in stem and progenitor cells of normal tissue result in the deletion or tolerance induction of WT1-specific T cells. Here, we used an human leukocyte antigen-transgenic murine model to study the fate of human leukocyte antigen class-I restricted, WT1-specific T cells in the thymus and in the periphery. Thymocytes expressing a WT1-specific T-cell receptor derived from high avidity human CD8 T cells were positively selected into the single-positive CD8 population. In the periphery, T cells specific for the WT1 antigen differentiated into CD44-high memory phenotype cells, whereas T cells specific for a non-self-viral antigen retained a CD44(low) naive phenotype. Only the WT1-specific T cells, but not the virus-specific T cells, displayed rapid antigen-specific effector function without prior vaccination. Despite long-term persistence of WT1-specific memory T cells, the animals did not develop autoimmunity, and the function of hematopoietic stem and progenitor cells was unimpaired. This is the first demonstration that specificity for a tumor-associated self-antigen may drive differentiation of functionally competent memory T cells.

Malaria infection changes the ability of splenic dendritic cell populations to stimulate antigen-specific T cells.

The capacity of splenic CD11c+ dendritic cell (DC) populations to present antigen (Ag) to T cells differs during malarial infection with Plasmodium chabaudi in mice. Both CD11c+ CD8+ and CD8- DCs presented malarial peptides on their surface during infection. However, although both DC subsets expressing malaria peptides could induce interferon-gamma production by CD4 T cells, only CD8- DCs isolated at the acute phase of infection stimulated Ag-specific T cell proliferation and interleukin (IL)-4 and -10 production from MSP1-specific T cell receptor for Ag transgenic T cells coincidental with our reported Th1 to Th2 switch at this stage in response to the pathogen. The timing of these distinct DC responses coincided with increased levels of apoptosis in the CD8+ population and an increase in the numbers of CD8- DCs in the spleen. Our data suggest that the switch in CD4 T cell responses observed in P. chabaudi-infected mice may be the result of the presentation by different DC populations modified by the malaria infection.

Migrating monocytes recruited to the spleen play an important role in control of blood stage malaria.

Host responses controlling blood-stage malaria include both innate and acquired immune effector mechanisms. During Plasmodium chabaudi infection in mice, a population of CD11b(high)Ly6C(+) monocytes are generated in bone marrow, most of which depend on the chemokine receptor CCR2 for migration from bone marrow to the spleen. In the absence of this receptor mice harbor higher parasitemias. Most importantly, splenic CD11b(high)Ly6C(+) cells from P chabaudi-infected wild-type mice significantly reduce acute-stage parasitemia in CCR2(-/-) mice. The CD11b(high)Ly6C(+) cells in this malaria infection display effector functions such as production of inducible nitric oxide synthase and reactive oxygen intermediates, and phagocytose P chabaudi parasites in vitro, and in a proportion of the cells, in vivo in the spleen, suggesting possible mechanisms of parasite killing. In contrast to monocyte-derived dendritic cells, CD11b(high)Ly6C(+) cells isolated from malaria-infected mice express low levels of major histocompatibility complex II and have limited ability to present the P chabaudi antigen, merozoite surface protein-1, to specific T-cell receptor transgenic CD4 T cells and fail to activate these T cells. We propose that these monocytes, which are rapidly produced in the bone marrow as part of the early defense mechanism against invading pathogens, are important for controlling blood-stage malaria parasites.

Alterations of splenic architecture in malaria are induced independently of Toll-like receptors 2, 4, and 9 or MyD88 and may affect antibody affinity.

Splenic microarchitecture is substantially altered during acute malaria infections, which may affect the development and regulation of immune responses. Here we investigated whether engagement of host Toll-like receptor 2 (TLR2), TLR4, TLR9, and the adaptor protein MyD88 is required for induction of the changes and whether antibody responses are modified when immunization takes place during the period of splenic disruption. The alterations in splenic microarchitecture were maximal shortly after the peak of parasitemia and were not dependent on engagement of TLR2, TLR4, or TLR9, and they were only minimally affected by the absence of the MyD88 adaptor molecule. Although germinal centers were formed in infected mice, they did not contain the usual light and dark zones. Immunization of mice with chicken gamma globulin 2 weeks prior to acute Plasmodium chabaudi infection did not affect the quantity or avidity of the immunoglobulin G antibody response to this antigen. However, immunization at the same time as the primary P. chabaudi infection resulted in a clear transient reduction in antibody avidity in the month following immunization. These data suggest that the alterations in splenic structure, particularly the germinal centers, may affect the quality of an antibody response during a malaria infection and could impact the development of immunity to malaria or to other infections or immunizations given during a malaria infection.

Nitric oxide and indoleamine 2,3-dioxygenase mediate CTLA4Ig-induced survival in heart allografts in rats.

Cytotoxic T lymphocyte-associated antigen 4 immunoglobulin (CTLA4Ig) leads to transplantation tolerance in mice depending on indoleamine 2,3-dioxygenase (IDO). We have shown that CTLA4Ig induces indefinite heart allograft survival in rats and that nitric oxide (NO) was implicated in the in vitro active tolerogenic mechanisms mediated by dendritic cells (DCs). Here we studied the in vivo tolerogenic mechanisms by which CTLA4Ig induces graft survival in rats receiving a cardiac allograft. Treatment of recipients with the IDO inhibitor 1-methyltryptophan (1-MT) did not abrogate the indefinite graft survival observed with CTLA4Ig alone. This was also the case after administration of the inducible nitric oxide synthase inhibitor aminoguanidine when again, indefinite allograft survival was maintained. However, administration of both inhibitors led to acute rejection. We show that IDO and NO are responsible for the impaired capacity of DCs from CTLA4Ig-treated rats to stimulate allogeneic T cells. In conclusion, we show that NO and IDO mediate CTLA4Ig-induced tolerance in rat allograft recipients.

Increased vaccination efficiency with apoptotic cells by silica-induced, dendritic-like cells.

We have demonstrated previously the ability of apoptotic cells to prime a functional immune response using an i.p. vaccination protocol with apoptotic cells and interleukin 2, before injecting a lethal dose of tumor cells into syngeneic rats. This protocol resulted in a survival rate of 33%. To elucidate the nature and the activity of the phagocytes involved in the clearance of apoptotic cells in vivo, we modulated the peritoneal cavity environment by administrating either thioglycollate or silica i.p. before injecting the apoptotic cells. Our results showed that thioglycollate abrogated vaccination efficiency, because none of the rats survived under these conditions. In fact, thioglycollate treatment induced a massive recruitment and activation of inflammatory macrophages that efficiently engulfed apoptotic cells, bypassing induction of specific immune responses. In contrast, silica treatment enhanced the vaccination efficiency of apoptotic cells plus interleukin 2 up to 66%. We distinguished a population of dendrite-like cells among the cells derived from the silica-treated peritoneal cavity both by their phenotype (MHC II(+)/CD80(+)/CD86(+)) and by their ability to induce the proliferation of allogeneic T cells in a mixed leukocyte reaction. Our results demonstrate the different roles of macrophages and dendritic-like cells in the physiological clearance of dead tumor cells and their implication in the design of immunomodulating vaccines.

Classical CD11c+ dendritic cells, not plasmacytoid dendritic cells, induce T cell responses to Plasmodium chabaudi malaria.

Dendritic cells play an important role in the development of immune responses in malaria, but the contribution of plasmacytoid dendritic cells (pDC) to CD4 T cell activation and immunopathology is unknown. We have investigated pDC in a Plasmodium chabaudi infection in mice. During infection, pDC increased in number and transiently up-regulated expression of Major Histocompatibility Complex class II and co-stimulatory molecules. However, in contrast to classical CD11c(high) DC, pDC could not phagocytose parasites or process parasite proteins, to activate CD4 T cells. Activation of naïve pDC, but not CD11c(high) DC, by infected red blood cells induced IFN alpha in vitro, which was dependent on the Toll-like receptor, TLR9. However, inactivation of TLR9 in knock-out mice had no effect on a P. chabaudi infection suggesting that TLR9 was not crucial for parasite elimination or pathology. Neither pDC nor IFN alpha beta were essential for parasite clearance as mice depleted of pDC or IFN alpha beta Receptor-knock-out mice could control infection. However, these mice lost significantly more weight than untreated or wild-type mice. We conclude that classical DC are the major antigen-presenting cells for CD4 T cells in this infection, but that pDC and IFN alpha beta may play minor roles in controlling the magnitude of acute stage pathology.

Differential pattern recognition receptor expression but stereotyped responsiveness in rat spleen dendritic cell subsets.

Dendritic cells (DC) are a heterogeneous population of APC endowed with specific functions. The nature of the DC subset involved in the course of an immune response to a specific pathogen might be important for inducing the appropriate effectors. In addition, each DC subset might also exhibit intrinsic functional plasticity. In the rat, spleen DC can be separated into three morphological and phenotypical distinct subsets, namely CD4+, CD4-, and plasmacytoid DC (pDC), whose frequencies are strain dependent. We correlated the expression of TLR and nucleotide-binding oligomerization domain 2 (NOD2) in these DC subsets to their in vitro responsiveness to specific ligands. CD4- DC expressed high levels of TLR1, 2, 3, and 10 mRNA, low TLR4, 5, 6, 7, and 9, and very low, if any, TLR8. pDC had a restricted repertoire characterized by high TLR7 and 9. CD4+ DC expressed all TLR and 10-fold higher levels of NOD2 mRNA than CD4- and pDC. Upon stimulation by TLR and NOD2 ligands, each DC subset responded in quite a stereotyped fashion. TLR2/6, 3, 4, 5, 9, and NOD2 triggering induced CD4- DC to mature and produce high IL-12p40, low IL-10, and TNF-alpha. TLR7/8 and 9 triggering induced pDC to mature and produce copious amounts of IL-6, IL-12p40, and TNF-alpha and low IFN-alpha. CD4+ DC were very poor producers of inflammatory cytokines. This study suggests that the nature of spleen DC responses to pathogens is dependent on subset specific-stimulation rather than intrinsic plasticity.

Immature CD4- CD103+ rat dendritic cells induce rapid caspase-independent apoptosis-like cell death in various tumor and nontumor cells and phagocytose their victims.

We previously reported the characterization of a MHC class II(low) CD4- CD103+ (CD4-) subset of dendritic cells (DC) in rat spleen that exhibit a Ca2+-, Fas ligand-, TRAIL- and TNF-alpha-independent cytotoxic activity against specific targets in vitro. In this study, we demonstrate that this DC subset was also found in lymph nodes. Freshly extracted and, therefore, immature CD4- DC exhibited a potent cytotoxic activity against a large panel of tumor cell lines as well as primary endothelial cells. The cytotoxic activity of immature CD4- DC required cell-to-cell contact and de novo protein expression. CD4- DC-mediated cell death resembled apoptosis, as evidenced by outer membrane phosphatidylserine exposure and nuclear fragmentation in target cells, but was caspase as well as Fas-associated death domain and receptor-interacting protein independent. Bcl-2 overexpression in target cells did not protect them against DC-mediated cell death. Immature CD4- DC phagocytosed efficiently apoptotic cells in vitro and, therefore, rapidly and specifically engulfed their victims following death induction. Maturation induced a dramatic down-regulation of the killing and phagocytic activities of CD4- DC. In contrast, CD4+ DC were both unable to kill target cells and to phagocytose apoptotic cells in vitro. Taken together, these data indicate that rat immature CD4- CD103+ DC mediate an unusual cytotoxic activity and can use this function to efficiently acquire Ag from live cells.

Rat plasmacytoid dendritic cells are an abundant subset of MHC class II+ CD4+CD11b-OX62- and type I IFN-producing cells that exhibit selective expression of Toll-like receptors 7 and 9 and strong responsiveness to CpG.

We have identified in the rat a new subset of MHC class II(+) CD4(+)CD3(-)CD11b(-) leukocytes that produce high amounts of type I IFN upon viral stimulation and that appeared homologous to plasmacytoid DC (pDC) previously described in humans and mice. These cells exhibited the following phenotype: CD5(+),CD90(+),CD45R(+),CD45RC(+),CD11c(-),CD161a(+),CD200(+),CD172a(+),CD32(+),CD86(+). Rat pDC did not express the DC-specific marker OX62 and were more abundant in the spleen than the classical CD4(+) and CD4(-) subsets of OX62(+)CD11b(+) DC we previously described that produced very little, if any, type I IFN. Spleen pDC exhibited an undifferentiated morphology and rapidly died in vitro, but showed extensive dendrite formation, survival, maturation, and moderate type I IFN production upon stimulation by oligonucleotides containing type B CpG motifs (CpG ODN). Type A CpG ODN and CD40 ligand induced pDC to produce large amounts of type I IFN, but did not promote maturation. CpG ODN and CD40 ligand, but not influenza virus, induced IL-12p40 and IL-6 secretion. Spleen pDC did not produce IL-12p70, TNF-alpha, IL-1beta, or IL-10 using these stimulation conditions. Correlating with their strong responsiveness to virus and CpG ODN, rat pDC specifically expressed Toll-like receptor 7 and 9 mRNA. Fresh spleen pDC were poor stimulators of allogenic CD4(+) and CD8(+) T cells, but became potent inducers of allogenic T cell proliferation as well as Th1 differentiation after stimulation by type B CpG. Therefore, rat pDC appear very similar to human pDC, indicating that the specific phenotype and functions of pDC have been highly conserved between species.

Two phenotypically distinct subsets of spleen dendritic cells in rats exhibit different cytokine production and T cell stimulatory activity.

We recently reported that splenic dendritic cells (DC) in rats can be separated into CD4(+) and CD4(-) subsets and that the CD4(-) subset exhibited a natural cytotoxic activity in vitro against tumor cells. Moreover, a recent report suggests that CD4(-) DC could have tolerogenic properties in vivo. In this study, we have analyzed the phenotype and in vitro T cell stimulatory activity of freshly isolated splenic DC subsets. Unlike the CD4(-) subset, CD4(+) splenic DC expressed CD5, CD90, and signal regulatory protein alpha molecules. Both fresh CD4(-) and CD4(+) DC displayed an immature phenotype, although CD4(+) cells constitutively expressed moderate levels of CD80. The half-life of the CD4(-), but not CD4(+) DC in vitro was extremely short but cells could be rescued from death by CD40 ligand, IL-3, or GM-CSF. The CD4(-) DC produced large amounts of the proinflammatory cytokines IL-12 and TNF-alpha and induced Th1 responses in allogeneic CD4(+) T cells, whereas the CD4(+) DC produced low amounts of IL-12 and no TNF-alpha, but induced Th1 and Th2 responses. As compared with the CD4(+) DC that strongly stimulated the proliferation of purified CD8(+) T cells, the CD4(-) DC exhibited a poor CD8(+) T cell stimulatory capacity that was substantially increased by CD40 stimulation. Therefore, as previously shown in mice and humans, we have identified the existence of a high IL-12-producing DC subset in the rat that induces Th1 responses. The fact that both the CD4(+) and CD4(-) DC subsets produced low amounts of IFN-alpha upon viral infection suggests that they are not related to plasmacytoid DC.

Dendritic cells, pro-inflammatory responses, and antigen presentation in a rodent malaria infection.

An infection of mice with Plasmodium chabaudi is characterized by a rapid and marked inflammatory response with a rapid but regulated production of interleukin-12 (IL-12), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma). Recent studies have shown that dendritic cells (DCs) are activated in vivo in the spleen, are able to process and present malaria antigens during infection, and may provide a source of cytokines that contribute to polarization of the CD4 T-cell response. P. chabaudi-infected erythrocytes are phagocytosed by DCs, and peptides of malaria proteins are presented on major histocompatibility complex (MHC) class II. The complex disulfide-bonded structure of some malaria proteins can impede their processing in DCs, which may affect the magnitude of the CD4 T-cell response and influence T-helper 1 (Th1) or Th2 polarization. DCs exhibit a wide range of responses to parasite-infected erythrocytes depending on their source, their maturational state, and the Plasmodium species or strain. P. chabaudi-infected erythrocytes stimulate an increase in the expression of costimulatory molecules and MHC class II on mouse bone marrow-derived DCs, and they are able to induce the production of pro-inflammatory cytokines such as IL-12, TNF-alpha, and IL-6, thus enhancing the Th1 response of naïve T cells. IFN-gamma and TNF-alpha play a role in both protective immunity and the pathology of the infection, and the inflammatory disease may be regulated by IL-10 and transforming growth factor-beta. It will therefore be important to elucidate the host and parasite molecules that are involved in activation or suppression of the DCs and to understand the interplay between these opposing forces on the host response in vivo during a malaria infection.