Unconventional cytokine profiles and development of T cell memory in long-term survivors after cancer vaccination

Cancer vaccine trials frequently report on immunological responses, without any clinical benefit. This paradox may reflect the challenge of discriminating between effective and pointless immune responses and sparse knowledge on their long-term development. Here, we have analyzed T cell responses in long-term survivors after peptide vaccination. There were three main study aims: (1) to characterize the immune response in patients with a possible clinical benefit. (2) To analyze the long-term development of responses and effects of booster vaccination. (3) To investigate whether the Th1/Th2-delineation applies to cancer vaccine responses. T cell clones were generated from all nine patients studied. We find that surviving patients harbor durable tumor-specific responses against vaccine antigens from telomerase, RAS or TGFβ receptor II. Analyses of consecutive samples suggest that booster vaccination is required to induce robust T cell memory. The responses exhibit several features of possible clinical advantage, including combined T-helper and cytotoxic functionality, recognition of naturally processed antigens and diverse HLA-restriction and fine-specificity. CD4⁻CD8⁻ T cell clones display unconventional cytotoxicity and specifically kill tumor cells expressing mutated TGFβ receptor II. Cytokine profiling on the long-term survivors demonstrates high IFNγ/IL10-ratios, favoring immunity over tolerance, and secretion of multiple chemokines likely to mobilize the innate and adaptive immune system. Interestingly, these pro-inflammatory cytokine profiles do not follow a Th1/Th2-delineation. Most IFNγ^high/IL4^low/IL10^low cultures include high concentrations of hallmark Th2-cytokines IL-5 and IL-13. This does not reflect a mixture of Th1- and Th2-clones, but applies to 19/20 T cell clones confirmed to be monoclonal through TCR clonotype mapping. The present study identifies several factors that may promote clinical efficacy and suggests that cytokine profiling should not rely on the Th1/Th2-paradigm, but assess the overall inflammatory milieu and the balance between key cytokines. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

CpG-ODN but not other TLR-ligands restore the antitumor responses in old mice: the implications for vaccinations in the aged

Aim There is accumulative evidence indicating that targeting antigen presenting cells (APCs) with different types of adjuvants could result in the induction of antitumor immune responses. It has been hypothesized that APCs function may be altered in the elderly contributing to a decline in the immune function. We evaluated whether targeting APCs following injection with Poly I:C, LPS, flagellin, imiquimod and CpG-ODN would induce an antitumor response in the old. Materials and methods The immune and antitumor responses induce Poly I:C, LPS, flagellin, imiquimod and CpG-ODN were compared in young (2 month old) and old (18 months) mice. Results Our results indicated that only intratumoral (i.t.) injections of CpG-ODN completely rejected the tumor in both young and old mice. Injections of Poly I:C also induced the rejection of tumors in the young but not in the old. Furthermore, i.t. injections of CpG-ODN promoted the development of protective memory responses in the young and the old. Analysis of the immune responses in the old indicated that CpG-ODN but not Poly-I:C induces: a pro-inflammatory Th1 type response; accumulation and activation of CD4⁺, CD8⁺ T and, NK cell responses; activation of APCs; and reduction in the number of T_regs. The activation of these immune-parameters positively correlates with the induction of an antitumor response. Conclusions These studies indicate that there are differences in the level of stimulation with TLR-ligands between young and old APCs and that the aged immune responses can be rescued and exploited for the induction of tumor immunity by targeting APCs with specific TLR-ligands. These results have important clinical implications for developing immunization strategies containing TLR-ligands that will be effective in both the young and old.     

DNA fusion vaccine designed to induce cytotoxic T cell responses against defined peptide motifs: implications for cancer vaccines

DNA vaccination offers a strategy to induce immune attack on cancer cells, but tumor Ags are often weak. Inclusion of a "foreign" protein increases immunogenicity, and we found previously that fusion of the fragment C (FrC) of tetanus toxin to the tumor Ag sequence promotes Ab and CD4(+) responses against B cell tumors. For CTL responses, use of the full two-domain FrC may be less helpful, because known immunogenic MHC class I-binding peptides in the second domain could compete with attached tumor-derived epitopes. Therefore, we removed the second domain, retaining the N-terminal domain, which contains a "universal" helper epitope. We investigated the ability to induce CTL responses of candidate peptides placed at the C terminus of this domain. As test peptides, we repositioned the two known CTL motifs from the second domain to this site. Strong CTL responses to each peptide were induced by the engineered construct, as compared with the native FrC construct. Induced CTLs were able to specifically kill tumor cells transfected with FrC as a surrogate tumor Ag both in vitro and in vivo. Further reduction of the domain to a short helper epitope generated only weak CTL responses against fused peptides, and synthetic peptides mixed with the plasmid containing the first domain were ineffective. The single FrC domain-peptide vaccine design also was able to induce high levels of CTLs against a known epitope from carcinoembryonic Ag. Response to peptide was suppressed if two FrC domains were present, consistent with immunodominance. These principles and designs may have relevance for cancer vaccines delivered via DNA.     

The murine model of infection with Leishmania major and its importance for the deciphering of mechanisms underlying differences in Th cell differentiation in mice from different genetic backgrounds

Mice from the majority of inbred strains are resistant to infection by Leishmania major, an obligate intracellular protozoan parasite of macrophages in the mammalian host. In contrast, mice from BALB strains are unable to control infection and develop progressive disease. In this model of infection, genetically determined resistance and susceptibility have been clearly shown to result from the appearance of parasite-specific CD4+ T helper 1 or T helper 2 cells, respectively. This murine model of infection is considered as one of the best experimental systems for the study of the mechanisms operating in vivo at the initiation of polarised T helper 1 and T helper 2 cell maturation. Among the several factors influencing Th cell development, cytokines themselves critically regulate this process. The results accumulated during the last years have clarified some aspects of the role played by cytokines in Th cell differentiation. They are providing critical information that may ultimately lead to the rational devise of means by which to tailor immune responses to the effector functions that are most efficient in preventing and/or controlling infections with pathogens.     

Altered antibody production and helper T cell function in mice lacking chemokines CCL19 and CCL21-Ser

The roles of chemokines CCL19 and CCL21 in Ab production were investigated using plt mutant mice, which lack expression of CCL19 and CCL21-ser in their lymphoid organs. In these mice, the Th response has been shown to tend towards the Th1 type because of accumulation of inflammatory dendritic cells. When plt mice were immunized with 100 μg OVA in CFA, the number of Ab-forming cells in the draining LN, and serum concentrations of OVA-specific IgM and IgG Ab, were very close to those of the control, yet IgG2a Ab in plt mice was increased. In vitro IFN-γ production by the draining LN cells of plt mice was increased. In addition, the ability of helper T cells from plt mice to stimulate Ab production in vitro was prolonged. Also, in the plt mice, in vivo challenge with OVA in incomplete Freund's adjuvant elicited a stronger IgG2a response and a weaker IgG1 response, which is suggestive of a Th1-dominant response. Similar findings were obtained when mice were immunized with 100 μg OVA in alum, except that with alum the increases observed in plt mice were IgG1 produced in vivo and IL-4 produced in vitro by draining LN cells. Furthermore, immunization with alum adjuvant also induced a prolonged in vitro recall response of IFN-γ and IL-4. These findings indicate that plt mice mount an anti-OVA Ab response, and suggest that CCL19 and CCL21 induce prompt Ab responses to antigen, and negatively regulate helper T cell responses in vivo.     

Antigenicity studies in humans and immunogenicity studies in mice: an MSP1P subdomain as a candidate for malaria vaccine development

The newly identified GPI-anchored Plasmodium vivax merozoite surface protein 1 paralog (MSP1P) has a highly antigenic C-terminus that binds erythrocytes. To characterize the antigenicity and immunogenicity of two regions (PvMSP1P-19 and -33) of the highly conserved C-terminus of MSP1P relative to PvMSP1-19, 30 P. vivax malaria-infected patients and two groups of mice (immunized with PvMSP1P-19 or -33) were tested for IgG subclass antibodies against PvMSP1P-19 and -33 antigens. In the patients infected with P. vivax, IgG1 and IgG3 levels were significantly higher than those levels in healthy individuals, and were the predominant response to the two C-terminal fragments of PvMSP1P (p < 0.05). In mice immunized with PvMSP1P-19, IgG1 levels were the highest while IgG2b levels were similar to IgG1 levels. The levels of Th1 cytokines in mice immunized with PvMSP1P-19 or -33 were significantly higher than those in mice immunized with PvMSP1-19 (p < 0.05). Our results indicate that: (i) IgG1 and IgG3 (IgG2b in mice) are predominant IgG subclasses in both patients infected with P. vivax and mice immunized with PvMSP1P-19 or -33; (ii) the C-terminus of MSP1P induces a Th1-cytokine response. This immune profiling study provides evidence that MSP1P may be a potential candidate for vivax vaccine.     

Glucuronoxylomannan of Cryptococcus neoformans exacerbates in vitro yeast cell growth by interleukin 10-dependent inhibition of CD4+ T lymphocyte responses

Glucuronoxylomannan (GXM), the major capsular polysaccharide of Cryptococcus neoformans, is the most important virulence factor of this fungus. We analyzed the molecular events related to protective immune responses against a non-encapsulated strain of C. neoformans, mediated by murine splenic CD4(+) T lymphocytes in vitro, and the impact of GXM addition upon these events. Both the lymphoproliferation of CD4(+) T cells and the control of fungus growth were dependent on B7 co-stimulation. Addition of GXM did not modify CD4(+) T cell proliferation, but exacerbated infection in cultures obtained from normal and infected hosts. GXM enhanced the secretion of IL-10 and IL-4, while it reduced the production of pro-inflammatory cytokines TNF-alpha and IFN-gamma. The blockade of IL-10 activity with neutralizing antibodies increased TNF-alpha production and reduced yeast cell growth. The findings suggest that GXM exacerbates infection by down-regulating cell-mediated protective immune response and that IL-10 is implicated in yeast evasion.     

Regulation of immunity to Plasmodium: Implications from mouse models for blood stage malaria vaccine design

Malaria, a disease caused by the protozoan parasite Plasmodium, remains a serious healthcare problem in developing countries worldwide. While the host-parasite relationship in humans has been difficult to determine, the pliability of murine malaria models has enabled valuable contributions to the understanding of the pathogenesis of disease. Although no single model reflects precisely malaria infection of the human, different models collectively provide important information on the mechanisms of protective immunity and immunopathogenesis. This review summarizes progress towards understanding the broad spectrum of immune responsiveness to the blood stages of the malaria parasite during experimental infections in mice and highlights how examination of murine malarias sheds light on the factors involved in the modulation of vaccine-potentiated immunity.     

CD4+ T cell responses to HLA-DP5-restricted wild-type sequence p53 peptides in patients with head and neck cancer

Wild-type sequence (wt) p53 peptides are attractive candidates for broadly applicable cancer vaccines. Evidence has been accumulating which indicates that CD4+ Th cells have an important role in generating and maintaining antitumor immune responses. To elucidate the nature of CD4+ Th responses to wt p53 epitopes in patients with squamous cell carcinoma of the head and neck (SCCHN), peripheral blood mononuclear cells (PBMCs) from HLA-DP5+ patients were stimulated with HLA-DP5-restricted wt p53 peptides, p53_108–122 or p53_153–166, and tested for the release of IFN-γ and IL-5 in ELISPOT assays. Immunohistochemistry for p53 accumulation in tumors, and ELISA for serum antibodies to p53 were also performed. Eleven (57.9%) of 19 HLA-DP5+ patients but none of 5 healthy donors had detectable Th1 and/or Th2 responses to wt p53 peptides by ELISPOT assay. Among these 11 responding patients, 9 (81.8%) and all 11 (100%) patients had a tumor burden and p53 accumulation, respectively. On the other hand, two responding patients were in post-operative condition. Interestingly, among nine patients with a tumor burden, four patients with early disease showed either Th1-polarized or mixed Th1/Th2 responses, while five patients with advanced disease showed either Th2-polarized or mixed Th1/Th2 responses. Our results suggest that wt p53_108–122 and p53_153–166 peptides stimulate both Th1- and Th2-type CD4+ T cell responses in patients with SCCHN, and anti-p53 Th responses may persist even after surgical resection of the tumor; however, the presence of a tumor and its progression may affect the nature of immune responses to wt p53 peptides.     

Altered T helper responses in CD40 and interleukin-12 deficient mice reveal a critical role for Th1 responses in eliminating the helminth parasite Taenia crassiceps

A key feature of helminth infections is the induction of strong Th2-biased immune responses in their hosts. We have previously found that Th2-like responses mediate susceptibility to the helminth parasite Taenia crassiceps, probably by inhibiting Th1 responses required for the development of protective immunity against this parasite. Here we show that mice lacking interleukin-12p35 (IL-12p35-/-) following T. crassiceps infection, failed to mount a Th1 response, but developed a strong Th2-type response, produced higher levels of IgG1, IgE, interleukin-4, interleukin-5 as well as interleukin-13 than wild-type mice, and became highly susceptible to the larval stage of this cestode. In contrast, similarly-infected CD40 deficient BALB/c mice (CD40-/-) displayed impairment of both Th1 and Th2-type responses associated with low levels of interferon-gamma as well as IgE, interleukin-4, interleukin-5 and interleukin-13, but efficiently controlled T. crassiceps infection. Together, these findings suggest a detrimental role for Th2-biased responses during the larval stage of T. crassiceps infection. Furthermore, they also suggest a pivotal role for CD40 in developing Th2-type responses.     

Subcutaneous injection of interleukin 12 induces systemic inflammatory responses in humans: implications for the use of IL-12 as vaccine adjuvant

Interleukin 12 (IL-12) is a cytokine with important regulatory functions bridging innate and adaptive immunity. It has been proposed as an immune adjuvant for vaccination therapy of infectious diseases and malignancies. The inflammatory properties of IL-12 play an important role in the adjuvant effect. We studied the effect of s.c. injections of recombinant human IL-12 (rHuIL-12) in 26 patients with renal cell cancer and demonstrated dose-dependent systemic activation of multiple inflammatory mediator systems in humans. rHuIL-12 at a dose of 0.5 g/kg induced degranulation of neutrophils with a significant increase in the plasma levels of elastase (p<0.05) and lactoferrin (p=0.01) at 24 h. Additionally, rHuIL-12 injection mediated the release of lipid mediators, as demonstrated by a sharp increase in the plasma secretory phospholipase A₂ (sPLA₂) level (p=0.003). rHuIL-12, when administered at a dose of 0.1 g/kg, showed minimal systemic effects. In conclusion, when IL-12 is used as an adjuvant, doses should not exceed 0.1 g/kg, in order to avoid severe systemic inflammatory responses.     

Murine T cell clones specific for Hymenolepis nana: generation and functional analysis in vivo and in vitro.

To examine the role of the T cell in protective immunity to Hymenolepis nana, H. nana-specific clonal lymphocytes were generated from mesenteric lymph nodes of BALB/c mice infected with H. nana, and some of their functions were analyzed in vitro and in vivo. Following limiting dilution techniques, five clones were generated from mesenteric lymph node cell populations. All of these clones expressed the L3T4⁺, Lyt-2.2⁻ phenotype and proliferated in vitro in response to soluble egg antigen of H. nana. Of five clones, three secreted interleukin 2 (IL-2) and interferon-γ (IFN-γ) after stimulation with egg antigen. Furthermore, these three clones conferred local delayed-type hypersensitivity to egg antigen. The remaining two clones produced interleukin 4 (IL-4) in response to egg antigen, and could not mediate local delayed-type hypersensitivity. Adoptive transfer experiments using clonal lymphocytes were also undertaken in an attempt to define cell types involved in protective immunity. Clonal lymphocytes secreting both IL-2 and IFN-γ transferred protective immunity, equivalent to that obtained by non-cultured-sensitized mesenteric lymph node cells. They were effective in very small numbers. However, clonal lymphocytes that secreted IL-4 did not transfer protective immunity. These results suggest that helper T lymphocytes, especially the Th1 subtype, are involved in protective immunity against H. nana.     

Efnb1 and Efnb2 proteins regulate thymocyte development, peripheral T cell differentiation, and antiviral immune responses and are essential for interleukin-6 (IL-6) signaling

Erythropoietin-producing hepatocellular kinases (Eph kinases) constitute the largest family of cell membrane receptor tyrosine kinases, and their ligand ephrins are also cell surface molecules. Because of promiscuous interaction between Ephs and ephrins, there is considerable redundancy in this system, reflecting the essential roles of these molecules in the biological system through evolution. In this study, both Efnb1 and Efnb2 were null-mutated in the T cell compartment of mice through loxP-mediated gene deletion. Mice with this double conditional mutation (double KO mice) showed reduced thymus and spleen size and cellularity. There was a significant decrease in the DN4, double positive, and single positive thymocyte subpopulations and mature CD4 and CD8 cells in the periphery. dKO thymocytes and peripheral T cells failed to compete with their WT counterparts in irradiated recipients, and the T cells showed compromised ability of homeostatic expansion. dKO naive T cells were inferior in differentiating into Th1 and Th17 effectors in vitro. The dKO mice showed diminished immune response against LCMV infection. Mechanistic studies revealed that IL-6 signaling in dKO T cells was compromised, in terms of abated induction of STAT3 phosphorylation upon IL-6 stimulation. This defect likely contributed to the observed in vitro and in vivo phenotype in dKO mice. This study revealed novel roles of Efnb1 and Efnb2 in T cell development and function.     

Cytokine cascade in dengue hemorrhagic fever: implications for pathogenesis

Dengue virus produces a mild acute febrile illness, dengue fever (DF) and a severe illness, dengue hemorrhagic fever (DHF). The characteristic feature of DHF is increased capillary permeability leading to extensive plasma leakage in serous cavities resulting in shock. The pathogenesis of DHF is not fully understood. This paper presents a cascade of cytokines, that in our view, may lead to DHF. The main feature is the early generation of a unique cytokine, human cytotoxic factor (hCF) that initiates a series of events leading to a shift from Th1-type response in mild illness to a Th2-type response resulting in severe DHF. The shift from Th1 to Th2 is regulated by the relative levels of interferon-gamma and interleukin (IL)-10 and between IL-12 and transforming growth factor-beta, which showed an inverse relationship in patients with DF.     

Sialic acids in T cell development and function

Virtually all cell surface proteins and many cell membrane lipids are glycosylated, creating a cell surface glycocalyx. The glycan chains attached to cell surface glycoproteins and glycolipids are complex structures with specific additions that determine functions of the glycans in cell–cell communication and cell sensing of the environment. One type of specific modification of cell surface glycans is decoration of glycan termini by sialic acids. On T cells, these terminal sialic acid residues are involved in almost every aspect of T cell fate and function, from cell maturation, differentiation, and migration to cell survival and cell death. The roles that sialylated glycans play in T cell development and function, including binding to specific sialic acid-binding lectins, are reviewed here.     

Recombinant salmonella-based 4-1BBL vaccine enhances T cell immunity and inhibits the development of colorectal cancer in rats: in vivo effects of vaccine containing 4-1BBL

Background

Immunotherapy with vaccines is attractive for the treatment of cancer. This study is aimed at determining the effect of recombinant Salmonella (SL3261)-based 4-1BB ligand (4-1BBL) vaccine on the development of colorectal cancers and the potential immune mechanisms in rats.

Results

In comparison with that in the PBS group, similar levels of 4-1BBL expression, the frequency of T cells, IFN-γ responses, and comparable numbers of tumors were detected in the SL3261 and SL3261C groups of rats. In contrast, significantly fewer numbers of tumors, increased levels of 4-1BBL expression in the spleens and colorectal tissues, higher frequency of peripheral blood and splenic CD3⁺CD25⁺ T cells, and stronger splenic T cell IFN-γ responses were detected in the SL3261R group of rats.

Conclusion

Our results indicated that vaccination with recombinant attenuated Salmonella harboring the 4-1BBL gene efficiently enhanced T cell immunity and inhibited the development of carcinogen-induced colorectal cancers in rats.     

Loss of T cell antigen recognition arising from changes in peptide and major histocompatibility complex protein flexibility: implications for vaccine design

Modification of the primary anchor positions of antigenic peptides to improve binding to major histocompatibility complex (MHC) proteins is a commonly used strategy for engineering peptide-based vaccine candidates. However, such peptide modifications do not always improve antigenicity, complicating efforts to design effective vaccines for cancer and infectious disease. Here we investigated the MART-1(27-35) tumor antigen, for which anchor modification (replacement of the position two alanine with leucine) dramatically reduces or ablates antigenicity with a wide range of T cell clones despite significantly improving peptide binding to MHC. We found that anchor modification in the MART-1(27-35) antigen enhances the flexibility of both the peptide and the HLA-A*0201 molecule. Although the resulting entropic effects contribute to the improved binding of the peptide to MHC, they also negatively impact T cell receptor binding to the peptide·MHC complex. These results help explain how the "anchor-fixing" strategy fails to improve antigenicity in this case, and more generally, may be relevant for understanding the high specificity characteristic of the T cell repertoire. In addition to impacting vaccine design, modulation of peptide and MHC flexibility through changes to antigenic peptides may present an evolutionary strategy for the escape of pathogens from immune destruction.