Supplementary Data from Clinical Benefit of Allogeneic Melanoma Cell Lysate–Pulsed Autologous Dendritic Cell Vaccine in MAGE-Positive Colorectal Cancer Patients

Background: Cytokines have been found to play a role in the disease activity of multiple sclerosis (MS). Previous studies indicate that acupuncture can affect cytokine levels in persons with other inflammatory diseases. Objectives: The aim of this study is to investigate the effect of acupuncture on cytokine levels and health-related quality of life (HRQoL) in persons with MS. Materials and Methods: A single-blind, randomized controlled trial was performed. Participants (n = 66) were randomized into three groups (real acupuncture, sham acupuncture, and reference). Participants in the real acupuncture and sham groups received six treatments during a period of 4 weeks. The serum levels of 11 pro- and anti-inflammatory cytokines (IFNγ, IL-1β, IL-6, IL-8, IL-12p70, IL-13, TNFα, IL-10, IL-4, IL-2, and IL-17A) were assessed at baseline, after 2 and 4 weeks of treatment, and 4 weeks after the final treatment. Changes in HRQoL were assessed using the Functional Assessment of Multiple Sclerosis questionnaire. Results: No statistically significant differences in plasma levels between the three groups were seen for either of the cytokines, nor were there any differences between the groups for HRQoL. Conclusions: In this study, the authors could not demonstrate that a 4-week acupuncture treatment had a measurable effect on the plasma levels of seven selected cytokines or on HRQoL among people with MS. The trial was registered with the ISRCTN registry as ISRCTN34352011.

Enteroantigen specific B cells have previously been demonstrated to play an immunomodulatory role in experimental colitis such as the T cell transfer model of colitis and regulatory B cells are known to be important for suppressing immunopathology in human IBD. Establishing immortalized B cell hybridomas with enteroantigen specificity would be an important tool to investigate further the role of B cells in the immunopathology of experimental colitis and inflammatory bowel disease. Here, based on polyethylene glycol fusion of X63 cells and enteroantigen reactive murine CD19+ B cells, we generated enteroantigen specific B cell hybridomas with proliferative capacity and IL-10 secretion against enteroantigen. The establishment of such hybridomas might be the basis for further assessing the enteroantigen specificity of gut-derived B cells, enteroantigens relevant for experimental colitis as well as human IBD including the immunoregulatory role of such enteroantigen specific B cells.

The aim of the present study was to identify influenza A-derived peptides which bind to both HLA class I and -II molecules and by immunization lead to both HLA class I and class II restricted immune responses. Eight influenza A-derived 9-11 mer peptides with simultaneous binding to both HLA-A*02:01 and HLA-DRB1*01:01 molecules were identified by bioinformatics and biochemical technology. Immunization of transgenic HLA-A*02:01/HLA-DRB1* 01: 01 mice with four of these double binding peptides gave rise to both HLA class I and class II restricted responses by CD8 and CD4 T cells, respectively, whereas four of the double binding peptides did result in HLA-A*02:01 restricted responses only. According to their cytokine profile, the CD4 T cell responses were of the Th2 type. In influenza infected mice, we were unable to detect natural processing in vivo of the double restricted peptides and in line with this, peptide vaccination did not decrease virus titres in the lungs of intranasally influenza challenged mice. Our data show that HLA class I and class II double binding peptides can be identified by bioinformatics and biochemical technology. By immunization, double binding peptides can give rise to both HLA class I and class I restricted responses, a quality which might be of potential interest for peptide-based vaccine development.

Melatonin has been shown to play a fundamental part in neuroimmunomodulation. Besides regulating the circadian rhythm it works as a natural antioxidant with immune stimulatory and anti-cancer properties. Melatonin is a regulator of haemopoiesis and modifies various cells and cytokines of the immune system. Also, melatonin elicits oncostatic properties in a variety of different tumour cell lines. A number of studies have documented that when given in combination with chemo-therapy to patients with disseminated disease, melatonin increases the overall one-year survival and reduces toxic side effects.

Naive CD4(+) T cells depleted of natural T-reg (CD25(+)) cells proliferate extensively when exposed to a fecal extract [enteroantigen (eAg)] pulsed on antigen-presenting cells (APC). When transplanted into SCID recipient mice, the CD25-depleted T cells induce a chronic colitis with a lethal course. We observed here that if T cells, pre-activated for 48h by eAg from BALB/c or SCID mice, are removed and then reexposed to either of the two sources of antigen, these T cells have completely lost their anti-eAg proliferative capacity in vitro. This observation indicates that eAgs derived from Balb/c and SCID mice are recognized by similar subsets of T cells. However, when transferred into SCID mice, eAg-activation-depleted T cells are still capable of inducing a severe colitis fully comparable with the disease induced by naive CD4(+) T cells.

Identification of new antigenic peptides, derived from infectious agents or cancer cells, which bind to human leukocyte antigen (HLA) class I and II molecules, is of importance for the development of new effective vaccines capable of activating the cellular arm of the immune response. However, the barrier to the development of peptide-based vaccines with maximum population coverage is that the restricting HLA genes are extremely polymorphic resulting in a vast diversity of peptide-binding HLA specificities and a low population coverage for any given peptide-HLA specificity. One way to reduce this complexity is to group thousands of different HLA molecules into several so-called HLA supertypes: a classification that refers to a group of HLA alleles with largely overlapping peptide binding specificities. In this chapter, we focus on the state-of-the-art classification of HLA supertypes including HLA-I supertypes and HLA-II supertypes and their application in development of peptide-based vaccines.

Studies reporting beneficial effects of B lymphocytes in autoimmune diseases have been accumulating and a regulatory role for certain B cell subsets is hence getting more and more recognition. Recently, B cells were shown to exhibit a regulatory effect in a T cell transfer model of colitis. Here, B cells exposed to enteroantigen (eAg) ex vivo abrogated the colitogenic effect if co-transplanted with Treg-depleted (CD4+CD25-) T cells into severe combined immune deficiency (SCID) mice. These data may imply a role for B cells that bind eAg (eAg+ B cells) in the immunopathology of colitis. Here, we report the detection of a subset of eAg+ B cells, including both B2 and B1 lineages, and show that these cells are present in all peripheral lymphoid organs of the mouse including the peritoneal cavity. eAg+ B cells are far more efficient as eAg-presenting cells than unfractionated splenocytes or eAg- B cells in causing proliferation of eAg-specific T cells. In comparison with eAg- B cells, eAg+ B cells secrete a significant amount of IL-10 in vitro, suggesting an anti-inflammatory potential. Compared with wild-type B cells, B cell receptor (BCR) transgenic, hen egg lysozyme-specific B cells show inferior eAg binding and T cell stimulatory activity suggesting involvement of the BCR in eAg binding and processing. After activation of CD19+ B cells by eAg and hybridization with hypoxanthine-aminopterin-thymidine (HAT) sensitive 963 lymphoma cells followed by cloning at limiting dilution conditions, around 10% of the hybridoma cells secrete eAg-specific antibodies.

Background:Treatment with the TRPV1 agonist, capsaicin, was previously shown to protect against experimental colitis in the severe combined immunodeficiency (SCID) T-cell transfer model. Here, we investigate trpv1 gene expression in lymphoid organs and cells from SCID and BALB/c mice to identify a potential target for the anti-inflammatory effect of capsaicin. Methods:The trpv1 expression was studied by real-time PCR in lymphoid tissues and gut of untreated and capsaicin-treated colitic SCID mice. Effects of capsaicin and a TRPV1 antagonist on T cells were studied in vitro. Results:In contrast to BALB/c mice, spleen, lymph nodes, and rectum of colitic and noncolitic SCID mice express trpv1 mRNA. Capsaicin treatment in vivo attenuated T-cell transfer colitis and capsaicin in vitro also attenuated T-cell proliferation induced by enteroantigen, mitogen, and anti-CD3/CD28 beads in BALB/c, C57BL/6 mice, and B6.129X1-trpv1tm1Jul/J trpv1 knockout mice. Proliferation and cytokine secretion were fully comparable in mice with and without trpv1 expression. Likewise, enteroantigen- and mitogen-stimulated T cells from wild-type and trpv1 knockout mice were equally inhibited by capsaicin. Surprisingly, the TRPV1 antagonist BCTC also inhibited enteroantigen- and mitogen-induced T-cell proliferation. Conclusions:The trpv1 mRNA expression in lymphoid organs and the rectum of SCID mice suggests that the TRPV1 signaling in these organs could play a role in capsaicin-mediated attenuation of colitis. In addition, capsaicin-induced inhibition of T-cell proliferation of wild-type T cells lacking trpv1 expression suggests that capsaicin inhibits colitogenic T cells in a TRPV1 receptor-independent way, which might be linked to its anti-inflammatory effect.

Background. Measles vaccine (MV) has a greater effect on child survival when administered in early infancy, when maternal antibody may still be present.Methods. To test whether MV has a greater effect on overall survival if given in the presence of maternal measles antibody, we reanalyzed data from 2 previously published randomized trials of a 2-dose schedule with MV given at 4-6 months and at 9 months of age. In both trials antibody levels had been measured before early measles vaccination.Results. In trial I (1993-1995), the mortality rate was 0.0 per 1000 person-years among children vaccinated with MV in the presence of maternal antibody and 32.3 per 1000 person-years without maternal antibody (mortality rate ratio [MRR], 0.0; 95% confidence interval [CI], 0-. 52). In trial II (2003-2007), the mortality rate was 4.2 per 1000 per-son-years among children vaccinated in presence of maternal measles antibody and 14.5 per 1000 person-years without measles antibody (MRR, 0.29; 95% CI,.09-. 91). Possible confounding factors did not explain the difference. In a combined analysis, children who had measles antibody detected when they received their first dose of MV at 4-6 months of age had lower mortality than children with no maternal antibody, the MRR being 0.22 (95% CI,.07-. 64) between 4-6 months and 5 years.Conclusions. Child mortality in low-income countries may be reduced by vaccinating against measles in the presence of maternal antibody, using a 2-dose schedule with the first dose at 4-6 months (earlier than currently recommended) and a booster dose at 9-12 months of age.

Enteroantigens (eAgs) drive tolerogenic and inflammatory immune responses in the gut and are of importance for sustained immune homeostasis in colonic mucosa. Decline of regulatory activity in the gut mucosa might result in chronic colitis. B7-H4 is a co-inhibitory receptor expressed by professional antigen-presenting cells. By delivering signal 2 during T cell activation, it inhibits T cell proliferation and inflammation. In this study, we have used a newly developed B7-H4-Ig fusion protein and evaluated its effect on eAg-activated effector and regulatory T cells (Treg) in vitro and in vivo. T cells were recovered from the mesenteric lymph nodes (MLNs) of untreated or B7-H4-Ig-treated BALB/c mice. Treatment of cells in vitro did neither affect the proliferation of effector T cells nor the function of Tregs. In vivo, B7-H4 treatment increased the total number of MLN-derived CD4(+) and CD8(+) T cell subsets as well as the functional activity of MLN-derived Tregs, whereas the proliferative activity of eAg or alloantigen specific effector T cells was not influenced, although treatment resulted in less secretion of inflammatory cytokines and chemokines from these cells. B7-H4-Ig treatment of severe combined immune-deficient (SCID) mice undergoing T cell transfer colitis did not influence the course of disease probably reflecting the lack of Tregs in this model of chronic colitis. In conclusion, we show that treatment with B7-H4-Ig in vivo changes lymphocyte homeostasis and increases the regulatory potential in normal mice, but does not affect the course of disease development in SCID mice undergoing T cell transfer colitis.

Immune cells often infiltrate testicular germ cell neoplasms, including pre-invasive carcinoma in situ (CIS), but the significance of this phenomenon remains unknown. The composition and distribution of infiltrating immune cells were examined by immunohistochemistry in testis samples with CIS and overt seminoma, in comparison to biopsies from infertile men without neoplasia. The composition of immune cells was similar across all the groups studied. Macrophages, CD8+ and CD45R0+ T lymphocytes constituted the majority of infiltrates, B lymphocytes were present in an intermediate proportion and very few CD4+ and FoxP3+ T cells were detected. HLA-I antigen was more abundant in Sertoli cells in tubules containing CIS than in those with normal spermatogenesis. This study showed a phenotypically comparable composition of infiltrating immune cells independently of the presence of neoplasia, suggesting the absence of active immune surveillance in testicular germ cell cancer.

Background: We evaluated the clinical and immunological effects of dendritic cell (DC) vaccination of patients with NSCLC. Autologous DCs were pulsed with a MAGE containing allogeneic melanoma cell lysate (MelCancerVac?, Dandrit Biotech,Copenhagen,Denmark). Imiquimod cream, proleukin and celecoxib were used as adjuvants to the vaccines. The objective of the study was to evaluate specific T cell response in vitro by IFNg EliSpot. Secondary objectives were overall survival, response and quality of life (QoL). Results: Twenty-two patients initiated the vaccination program consisting of ten vaccinations. Seven patients remained in stable disease (SD) three months after the first vaccination. After ten vaccinations (six months), four patients still showed SD and continued vaccinations on a monthly basis. These four patients received a total of 12, 16, 26 and 35 vaccinations, respectively. Five patients showed an unexpectedly prolonged survival. The treatment was well tolerated and only minor adverse events were reported. Quality of life did not change during the study period. In four of the seven patients with SD, vaccine-specific T cells were detected by IFNγ EliSpot assays, whereas only one patient with progressive disease (PD) showed vaccine-specific responses. Conclusion: This DC-based vaccine trial has indicated a correlation between vaccine-specific immunity and sustained SD. Furthermore, we observed an unexpectedly prolonged survival in some patients, which may indicate delayed effect of DC vaccination after completion of the treatment. A prospective randomized phase-IIb or -III is needed to further evaluate the use of MelCancerVac? vaccine treatment in patients with progressive NSCLC.

B7-H4 has been shown to inhibit T cell proliferation, cytokine production and cell cycle in vitro. B7-H4 deficient mice develop exacerbated disease in the mouse models of Rheumatoid Arthritis (RA), Type 1 Diabetes (T1D) and Experimental Autoimmune Encephalomyelitis (EAE). On the other hand, B7-H4-Ig fusion protein has been documented to assuage the symptoms in mouse models of RA, T1D, and multiple sclerosis in vivo. In the present study, B7-H4-Ig bound to the majority of human peripheral blood monocytes and NK cells, but not to either normal or activated T cells. B7-H4-Ig fusion protein was assayed for its effects in allogeneic mixed lymphocyte culture (MLC) systems. Soluble B7- H4-Ig had no significant effect in the MLC, but with a tendency to promote allogeneic response. Immobilized, but not soluble B7-H4-Ig inhibited plastic bound anti-CD3 mediated activation of T cells. This inhibition however was largely due to B7-H4-Ig mediated displacement of anti-CD3 antibody from the plastic plate. Finally, B7-H4-Ig had no effect on the cytotoxicity mediated by NK and LAK cells in PBMC. Our findings thus caution against the interpretation of suppressive effect observed solely in plate-bound anti-CD3 mediated T cell co-stimulation in vitro.

Background: T helper (T-H) 17 cells are believed to play a pivotal role in development of inflammatory bowel disease, and their contribution to intestinal inflammation has been studied in various models of colitis. T(H)17 cells produce a range of cytokines, some of which are potential targets for immunotherapy. However, blockade of IL-17A alone with secukinumab was not effective in Crohn's disease. In this regard, the pathogenic impact of IL-17A versus IL-17F during intestinal inflammation is still unresolved.Methods:Development of IFN--producing, IL-17A-producing, and IL-17F-producing CD4(+) T cells was analyzed in the CD4(+)CD25(-) T-cell transfer model of colitis at varying degrees of colitis. The pathogenic roles of IL-17A and IL-17F were investigated by treating colitic mice with neutralizing antibodies against these 2 cytokines.Results:We found that colitis development was associated with an increase in IL-17A-producing T(H)17 cells in spleen, mesenteric lymph nodes, and lamina propria. In contrast, the relative abundance of IFN--producing T(H)1 cell was stable in all 3 organs during progression of colitis, and the frequency of IFN-+IL-17A(+) double-positive cells declined in spleen and mesenteric lymph node but not in lamina propria. IL-17F was coexpressed in T(H)17 cells and IFN-+IL-17A(+) double positive but not in T(H)1 cells and its expression inversely correlated with colitis development. In vivo neutralization of both IL-17A and IL-17F ameliorated colitis in particular at early administration, whereas neutralization of IL-17A or IL-17F alone was inefficient.Conclusions:T(H)17 cell development correlates with colitis progression, and concurrent neutralization of their cytokine products IL-17A and IL-17F ameliorates intestinal inflammation. These findings suggest combined IL-17A and IL-17F blockade as a potential strategy in inflammatory bowel disease therapy.

The majority of clear-cell renal cell carcinomas (ccRCC) show high and homogeneous expression levels of the tumor associated antigen (TAA) carbonic anhydrase IX (CAIX), and treatment with interleukin-2 (IL-2) based immunotherapy can lead to cure in patients with metastatic renal cell carcinoma (mRCC). However, the involvement of CAIX specific CD8+ T cells and/or NK cells in the tumor eradication is unknown. We investigated T cell and antibody reactivity against overlapping 15-mer CAIX-peptides as well as HLA haplotype frequency and NK cell cytotoxicity in 11 patients with no evidence of disease (NED) following treatment with IL-2 based immunotherapy, and thus potentially cured. Immune reactivity in these patients was compared with samples from patients with dramatic tumor response obtained immediately at the cessation of therapy, samples from patients that experienced progressive disease during treatment and samples from healthy controls. We observed more focused but only weak and not consistent CAIX specific T-cells in the late observation and early observation response groups compared with the healthy control group. An increased frequency of the class II alleles HLA-DRB4 01:01, HLA-DPB 01:01 and HLA-DPB 03:01 was noted in the NED patients. In contrast, NK cytotoxicity was low even in the late observation response group as compared with controls. In particular, a HLA-B*40:01 restricted CD8+ T cell response recognizing the CAIX- derived peptide SEEEGSLKL was identified. This may have interest in future cancer vaccines, but more studies are needed to elucidate the immunological mechanisms of action in potentially cured patients treated with an immunotherapeutic agent.

Mice were immunized twice with a pool of five peptides selected among twenty 8–9-mer peptides for their ability to form stable complexes at 37°C with recombinant H-2Kb (half-lives 10–15h). Vaccine-induced immunity of splenic CD8+ T cells was studied in a 24h IFNγ Elispot assay. Surprisingly, IFNγ spot-formation was observed without addition of peptide to the assay culture at 3 weeks and 3 months after immunization. To clarify if IFNγ spot formation in the absence of peptide exposure ex vivo is caused by the peptide-pool per se, mice were immunized with single peptides. Three of the five peptides induced normal peptide immunity i.e. the specific T cell reactivity in the Elispot culture was strictly dependent on exposure to the immunizing peptide ex vivo. However, immunization with two of the peptides, a VSV- and a Mycobacterium-derived peptide, resulted in IFNγ spot formation without peptide in the Elispot culture. Immunization with a mixture of the VSV-peptide and a “normal” peptide also resulted in IFNγ spot formation without addition of peptide to the assay culture. Peptide-tetramer staining of CD8+ T cells from mice immunized with a mixture of VSV-peptide and “normal” peptide showed peptide specific binding by CD8+ T cells for both of the peptides. Thus, although immunization with certain peptides alone or in a mixture of peptides may result in IFNγ spot formation without peptide in the assay culture, specific immunity against the individual immunizing peptide in the mixture remains intact. Our data suggest that certain peptides exhibit sustained immunogenicity in vivo for prolonged periods of time.

Within a neonatal vitamin A supplementation (VAS) trial, we investigated the effect of VAS on TNF-α, IL-10, IL-5 and IL-13 production after lipopolysaccharide, purified protein derivative (PPD) of Mycobacterium tuberculosis and phytohaemagglutinin stimulation using a whole blood culture protocol. We found that VAS recipients had lower unstimulated TNF-α concentrations than placebo recipients. In the present paper, we investigated whether the SNP TNF-α − 308, TNF-α − 238, IL-10 − 592, IL-10 − 1082 and toll-like receptor 4 ( TLR4 )+896 modified the effect of VAS on cytokine production. DNA and cytokine concentrations were available from 291 children. We found a significant interaction between TNF-α − 308 genotype and VAS for the unstimulated TNF-α production ( P interaction = 0·04); among G homozygotes, TNF-α concentrations were significantly lower after VAS compared with placebo, whereas for A carriers, VAS did not appear to have any effect. For TNF-α − 238, there was a tendency towards an increase in PPD-stimulated TNF-α production after VAS for the G homozygotes, but the opposite tendency for A allele carriers ( P interaction = 0·07). Stratification by sex revealed a significant VAS–genotype interaction for boys for TNF-α − 238. There was a borderline-significant three-way interaction ( P = 0·05) between sex, VAS and TLR4 +896 genotype. Although the present study had very limited representation of the genetic variation with potential for modification of the response to VAS, it adds to the efforts of untangling the diverse effects and impact of VAS.

A number of mainly observational studies suggest that many African females below the age of one year die each year from the nonspecific effects of vaccination with diphtheria-tetanus toxoids and killed (whole-cell) Bordetella pertussis (DTwP). In contrast, similar studies suggest that many African females and males may have their lives saved each year by the nonspecific immunological benefits of Bacillus Calmette-Guerin (BCG) vaccination. From an immunological point of view, we hypothesise that the adverse effects of DTwP vaccine may occur because of the Th2-polarising effect of the aluminium phosphate adjuvant in the vaccine and because intramuscular administration of the vaccine may cause chronic inflammation at the site of injection. However, the Th1-polarising effect of BCG is likely to be beneficial. Sexual dimorphism affecting immune functions and vitamin A supplementation may influence both the deleterious and beneficial nonspecific effects of immunisation.