Mahyar Nouri-Shirazi

Dendritic cells capture killed tumor cells and present their antigens to elicit tumor-specific immune responses.

Due to their capacity to induce primary immune responses, dendritic cells (DC) are attractive vectors for immunotherapy of cancer. Yet the targeting of tumor Ags to DC remains a challenge. Here we show that immature human monocyte-derived DC capture various killed tumor cells, including Jurkat T cell lymphoma, malignant melanoma, and prostate carcinoma. DC loaded with killed tumor cells induce MHC class I- and class II-restricted proliferation of autologous CD8+ and CD4+ T cells, demonstrating cross-presentation of tumor cell-derived Ags. Furthermore, tumor-loaded DC elicit expansion of CTL with cytotoxic activity against the tumor cells used for immunization. CTL elicited by DC loaded with the PC3 prostate carcinoma cell bodies kill another prostate carcinoma cell line, DU145, suggesting recognition of shared Ags. Finally, CTL elicited by DC loaded with killed LNCap prostate carcinoma cells, which express prostate specific Ag (PSA), are able to kill PSA peptide-pulsed T2 cells. This demonstrates that induced CTL activity is not only due to alloantigens, and that alloantigens do not prevent the activation of T cells specific for tumor-associated Ags. This approach opens the possibility of using allogeneic tumor cells as a source of tumor Ag for antitumor therapies.

Evidence for the immunosuppressive role of nicotine on human dendritic cell functions

Nicotine alters a wide range of immunological functions, including innate and adaptive immune responses. To date, no studies have been reported showing the immunoregulatory effects of nicotine on dendritic cells (DCs), which are critical cells for initiation of cell‐mediated immunity against infection and neoplastic diseases. In this work, we report that, in a nicotinic environment, monocyte‐derived DCs manifest lower endocytic and phagocytic activities. Interestingly, although immature DCs undergo maturation in response to bacterial antigen lipopolysaccharide, they produce decreased levels of pro‐inflammatory cytokines, notably interleukin‐12, and reveal a reduced ability to stimulate antigen‐presenting cell‐dependent T‐cell responses. Importantly, the reduction in T‐cell responses is associated with a diminished ability of DCs to induce differentiation and expansion of type 1 T cells, as evidenced by a decreased frequency of interferon‐γ‐producing effector cells. These results strongly suggest that nicotine can exert its immunosuppressive effects on immune surveillance through functional impairment of the DC system.

Chitin particles induce size-dependent but carbohydrate-independent innate eosinophilia.

Murine Mφ that phagocytose CMP develop into M1; this response depends on the size and the chemical composition of the particles. In contrast, recent studies concluded that chitin particles induce M2 and eosinophil migration, promoting acquired Th2 immune responses against chitin‐containing microbes or allergens. This study examined whether these apparently inconsistent responses to chitin could be induced by variation in the size and chemical composition of the chitin particles. We compared the responses of Mφ with CMP, LCB, and Sephadex G‐100 beads (>40 μm). Beads were given i.p. to WT mice and to mice deficient in a CRTH2, a receptor for the eosinophil chemoattractant PGD2. In contrast to the M1 activation induced by CMP, i.p. administration of LCB or Sephadex beads induced within 24 h a CRTH2‐dependent peritoneal eosinophilia, as well as CRTH2‐independent activation of peritoneal Mφ that expressed Arg I, an M2 phenotype. LCB‐induced Mφ exhibited elevated Arg I and a surface MR, reduced surface TLR2 levels, and no change in the levels of CHI3L1 or IL‐10 production. Our results indicate that the effects of chitin in vivo are highly dependent on particle size and that large, nonphagocytosable beads, independent of their chemical composition, induce innate eosinophilia and activate Mφ expressing several M2, but not M1, phenotypes.

Dendritic cells as promoters of transplant tolerance

Dendritic cells (DCs) play a crucial role during the initiation of immune responses against non-self antigens. Following organ transplantation, activated donor- and recipient-derived DCs participate actively in graft rejection by sensitising recipient T cells via the direct or indirect pathways of allorecognition, respectively. There is increasing evidence that immature/semi-mature DCs induce antigen-specific unresponsiveness or tolerance to self antigens, both in central lymphoid tissue and in the periphery, through a variety of mechanisms (deletion, anergy and regulation). In the past few years, DC-based therapy of experimental allograft rejection has focused on ex vivo biological, pharmacological and genetic engineering of DCs to mimic/enhance their natural tolerogenicity. Successful outcomes in rodent models have built the case that DC-based therapy may provide a novel approach to transplant tolerance. Ongoing research into the role that DCs play in the induction of tolerance should allow for its clinical application in the near future.

B7-1 and B7-2 differentially control peripheral homeostasis of CD4(+)CD25(+)Foxp3(+) regulatory T cells.

Targeting the CD28/B7 interaction remains among the most promising approaches to treat transplant rejection. A drawback to this approach is however the observations of decreased numbers of naturally occurring regulatory T cells (Tregs) in CD28(-) or B7-deficient non-obese diabetic (NOD) mice, cells that maintain immunological self tolerance, prevent autoimmunity and control immune responses to transplants. In this study, therefore, we investigated the relative contributions of B7-1 and B7-2, the only known ligands of CD28, on the thymic development and peripheral homeostasis of Tregs. Our data indicates that the absence of both B7-1 and B7-2 result in a dramatic reduction in the frequencies of Tregs in thymus and peripheral tissues of B7-1/B7-2-deficient mice with no apparent changes in the percentage and distribution of conventional T-cell subsets. In addition, neither B7-1 nor B7-2 expression alone is sufficient for the development and peripheral homeostasis of Tregs. Interestingly, while B7-1 and B7-2 equally contribute to thymic development of Tregs, the significant loss in peripheral homeostasis of Tregs is more evident in the absence of B7-2 as compared to B7-1. Consistent with these results we found that B7-2-deficient DCs are less effective than B7-1-deficient DCs in maintaining Tregs in vitro due to their inability to induce IL-2 production by conventional T cells and sustain Tregs expression of CD25. This study provides the first demonstration of relative roles of B7-1 and B7-2 in Tregs homeostasis and suggests that therapeutic approaches designed to selectively interrupt CD28/B7-2 interaction could indeed have measurable impact on sustaining Tregs homeostasis.

Exposure to Nicotine Adversely Affects the Dendritic Cell System and Compromises Host Response to Vaccination

The magnitude of Th1 cells response to vaccination is a critical factor in determining protection from clinical disease. Our previous in vitro studies suggested that exposure to the nicotine component of cigarette smoke skews the differentiation of both human and mouse dendritic cell (DC) precursors into atypical DCs (DCs differentiated ex vivo in the presence of nicotine) lacking parameters essential for the development of Th1-mediated immunity. In this study, we determined the causal relationship between nicotine-induced DC alterations and host response to vaccines. We show that animals exposed to nicotine failed to develop and maintain Ag-specific effector memory Th1 cells and Ab production to protein-based vaccine formulated with Th1 adjuvants. Accordingly, both prophylactic and therapeutic vaccines failed to protect and cure the nicotine-exposed mice from disease. More importantly, we demonstrate the nicotine-induced defects in the biological activities of in vivo DCs as an underlying mechanism. Indeed, i.v. administration of DCs differentiated in the presence of nicotine preferentially promoted the development of Ag-specific IL-4–producing effector cells in the challenged mice. In addition, DC subsets isolated from mice exposed to nicotine produced significantly less cytokines in response to Th1 adjuvants and inadequately supported the development of Ag-specific Th1 cells. Collectively, our studies suggest that nicotine-induced defects in the DC system compromises vaccine efficacy in smokers.

Comparative analysis of dendritic cells and anti-CD3/CD28 expanded regulatory T cells for application in transplantation.

The containment of direct and indirect recognition of donor alloantigens by enhancement of the number and activity of regulatory T cells (Tregs) has been one of the promising approaches to achieve transplant tolerance. Two major methods, dendritic cell (DC) and anti-CD3/CD28 antibodies (Abs) have been introduced for ex vivo expansion of Tregs prior to their adoptive transfer. Here we compared the clinical advantage of using these methods of Tregs expansion by evaluating the nature and ability of expanded Tregs to abolish recipient alloreactive T-cell responses in vitro and in vivo. The ovalbumin (OVA)-specific Tregs isolated from DO11.10 mice were exposed to either Abs or syngeneic DC loaded with OVA peptide in the presence of exogenous IL-2 for a week. Using BABL/c as recipient and C57BL/6 as donor, the suppressive activity of these cells was examined. We found that DCs were much more efficient than Abs in expanding (9-fold versus 4-fold) and maintaining the viability (90% versus 35%) of purified Tregs. Interestingly, the Abs-expanded Tregs superbly contained the alloreactive T-cell proliferation and both Tregs were more suppressive when the Tregs cognate antigen and alloantigens were separately expressed on recipient and donor DCs (HVGD) rather than on recipient DC alone (GVHD). Importantly, however, DC-expanded Tregs maintained stable expression of Foxp3, survived longer and effectively contained the differentiation of alloreactive T cells into IFN-gamma-producing effector cells both in vitro and in vivo. Our data suggests that DC-expanded Tregs provides a clinically advantageous means of preventing unwanted immune reactions to allografts.

Nicotine exposure alters the mRNA expression of Notch ligands in dendritic cells and their response to Th1-/Th2-promoting stimuli.

Dendritic cells (DCs) utilize polarizing signals to instruct the differentiation of T helper (Th) cells into Th1 and Th2 effector cells: antigen‐specific ‘signal 1’, costimulatory ‘signal 2’ and polarizing cytokines ‘signal 3’. Accumulating evidence suggests the involvement of an additional signal, the Notch signalling pathway. We reported that in response to Th1‐promoting stimuli, both mouse and human DCs generated in the presence of the immune modulator nicotine (nicDCs) fail to support the development of effector memory Th1 cells. However, in response to Th2‐promoting stimuli, these nicDCs preferentially support the differentiation of antigen‐specific IL‐4‐producing Th2 effector cells. Here, we show that when compared to their control counterparts, immature mouse and human nicDCs display higher levels of the Notch ligands D1, D4 and J2 mRNA expression. In response to Th1‐ and Th2‐promoting stimuli, mouse nicDCs display higher levels of the Notch ligands D1, D4 and J2, while human nicDCs show higher levels of D1, D4 and J1 mRNA expression. Furthermore, both stimulated mouse and human nicDCs express higher CD86 to CD80 ratio and produce lower amount of IL‐12. Collectively, our data suggest that these changes in addition to an increase in Jagged expression correlate with the ability of nicDCs to modulate the Th1/Th2 balance in favour of Th2 generation.

The genetic background influences the cellular and humoral immune responses to vaccines

The assessment of Toll‐like receptor (TLR) agonists as candidate adjuvants for induction of effective T helper type 1 (Th1) immunity continues to rely on the use of mice. However, the genetic variation among inbred mice may influence the efficacy of adjuvants and bias a studys conclusions. Here, we evaluated the differences in cellular and humoral responses of genetically non‐identical mouse strains immunized with ovalbumin (OVA) plus alum, TLR‐3, TLR‐4, TLR‐7/8 or TLR‐9 agonists. We found that all the tested TLR agonists recruited dendritic cells (DCs) and natural killer (NK) cells significantly into the lymph nodes, promoted DC–NK cross‐talk and enhanced the cellular responses in B6 strain. In contrast, TLR‐3 and TLR‐7/8 were the only two agonists that showed the cellular adjuvanticity in the BALB/c strain. Compared with other TLR agonists, TLR‐3 and TLR‐7/8 were demonstrated to be the most effective adjuvants to generate interferon (IFN)‐γ‐producing effector NK, CD4, and CD8 T cells in B6 and BALB/c strains, respectively. We also found that compared with alum, all adjuvants induced the recruitment of B cells and production of OVA‐specific immunoglobulin (Ig)G2a more effectively in both strains. In addition, the B6 strain recruited more B cells, but surprisingly produced significantly lower amounts of OVA‐specific IgG2a in response to all adjuvants. However, consistent with the frequency of IFN‐γ‐producing effector cells observed in individual strains following immunizations, we detected more OVA‐specific IgG2a in serum of B6 and BALB/c strains in response to TLR‐3 and TLR‐7/8, respectively. Our data suggest that genetic background should be taken into consideration when evaluating the activities of TLR agonists for the development of prophylactic and therapeutic vaccines.

TLR8 combined withTLR3 or TLR4 agonists enhances DC-NK driven effector Tc1 cells

BACKGROUND Most current prophylactic vaccines confer protection primarily through humoral immunity. Indeed, aluminum salts which have been widely used as adjuvants in vaccines primarily enhance Th2-driven antibody responses. Therefore, new vaccines formulation is moving toward a careful selection of adjuvants that also elicit significant Th1 or Tc1 responses. Several TLR agonists have been tested as potential new adjuvants in clinical and preclinical studies with some efficacy. These studies suggest that combining more than one of TLR ligands enhances the magnitude of immune responses to cancer and infectious disease. OBJECTIVES In order to evaluate the synergistic effect of TLR agonists for effective induction of cellular immunity, we investigated the effects of single and/or combined TLR agonists on monocyte-derived DC maturation, DC-NK crosstalk and ultimately naïve T cells polarization into effector T cells. RESULTS Among the adjuvants tested, we found that TLR3, TLR4, TLR7/8 and TLR8 agonists were the most effective adjuvants to increase the expression levels of antigen-presenting, co-stimulatory molecules and production of cytokines by maturing DCs. When combined, TLR3+8 and TLR4+8 synergistically optimized DC maturation and IFN-γ secretion from NK cells co-cultured with DCs. Interestingly, co-culture of DC-NK-T treated with aluminum salt produced the highest percentage of effector memory CFSE-CCR7- Th1 cells whereas TLR3+8 and TLR4+8 treated co-cultures produced the highest percentage of effector memory CFSE-CCR7- Tc1 cells producing IFN-γ. Finally, while both TLR3+8 or TLR4+8 treated co-cultures generated similar frequency of Th1 and Tc1 effector cells, the effector cells from the latter co-culture produced quantitatively more IFN-γ in the supernatant. CONCLUSION Our data indicate that if in need of an enhanced DC-NK mediated cellular immunity one may select TLR agonists with defined synergistic effects.