Simona Rolla

Concordant morphologic and gene expression data show that a vaccine halts HER-2/neu preneoplastic lesions

While much experimental data shows that vaccination efficiently inhibits a subsequent challenge by a transplantable tumor, its ability to inhibit the progress of autochthonous preneoplastic lesions is virtually unknown. In this article, we show that a combined DNA and cell vaccine persistently inhibits such lesions in a murine HER-2/neu mammary carcinogenesis model. At 10 weeks of age, all of the ten mammary gland samples from HER-2/neu-transgenic mice displayed foci of hyperplasia that progressed to invasive tumors. Vaccination with plasmids coding for the transmembrane and extracellular domain of rat p185neu followed by a boost with rp185neu+ allogeneic cells secreting IFN-gamma kept 48% of mice tumor free. At 22 weeks, their mammary glands were indistinguishable from those of 10-week-old untreated mice. Furthermore, the transcription patterns of the two sets of glands coincided. Of the 12,000 genes analyzed, 17 were differentially expressed and related to the antibody response. The use of B cell knockout mice as well as the concordance of morphologic and gene expression data demonstrated that the Ab response is the main mechanism facilitating tumor growth arrest. This finding suggests that a new way can be found to secure the immunologic control of the progression of HER-2/neu preneoplastic lesions.

Distinct and Non-Overlapping T Cell Receptor Repertoires Expanded by DNA Vaccination in Wild-Type and HER-2 Transgenic BALB/c Mice

Central tolerance to tumor-associated Ags is an immune-escape mechanism that significantly limits the TCR repertoires available for tumor eradication. The repertoires expanded in wild-type BALB/c and rat-HER-2/neu (rHER-2) transgenic BALB-neuT mice following DNA immunization against rHER-2 were compared by spectratyping the variable (V)β and the joining (J)β CDR 3. Following immunization, BALB/c mice raised a strong response. Every mouse used one or more CD8+ T cell rearrangements of the Vβ9-Jβ1.2 segments characterized by distinct length of the CDR3 and specific for 63-71 or 1206-1214 rHER-2 peptides. In addition, two CD4+ T cell rearrangements recurred in >50% of mice. Instead, BALB-neuT mice displayed a limited response to rHER-2. Their repertoire is smaller and uses different rearrangements confined to CD4+ T cells. Thus, central tolerance in BALB-neuT mice acts by silencing the BALB/c mice self-reactive repertoire and reducing the size of the CD8+ T cell component. CD8+ and CD4+ T cells from both wild-type and transgenic mice home to tumors. This definition of the T cell repertoires available is critical to the designing of immunological maneuvers able to elicit an effective immune reaction against HER-2-driven carcinogenesis.

Vaccination With ENO1 DNA Prolongs Survival of Genetically Engineered Mice With Pancreatic Cancer

BACKGROUND & AIMS Pancreatic ductal adenocarcinoma (PDA) is an aggressive tumor, and patients typically present with late-stage disease; rates of 5-year survival after pancreaticoduodenectomy are low. Antibodies against α-enolase (ENO1), a glycolytic enzyme, are detected in more than 60% of patients with PDA, and ENO1-specific T cells inhibit the growth of human pancreatic xenograft tumors in mice. We investigated whether an ENO1 DNA vaccine elicits antitumor immune responses and prolongs survival of mice that spontaneously develop autochthonous, lethal pancreatic carcinomas. METHODS We injected and electroporated a plasmid encoding ENO1 (or a control plasmid) into Kras(G12D)/Cre (KC) mice and Kras(G12D)/Trp53(R172H)/Cre (KPC) mice at 4 weeks of age (when pancreatic intraepithelial lesions are histologically evident). Antitumor humoral and cellular responses were analyzed by histology, immunohistochemistry, enzyme-linked immunosorbent assays, flow cytometry, and enzyme-linked immunosorbent spot and cytotoxicity assays. Survival was analyzed by Kaplan-Meier analysis. RESULTS The ENO1 vaccine induced antibody and a cellular response and increased survival times by a median of 138 days in KC mice and 42 days in KPC mice compared with mice given the control vector. On histologic analysis, the vaccine appeared to slow tumor progression. The vaccinated mice had increased serum levels of anti-ENO1 immunoglobulin G, which bound the surface of carcinoma cells and induced complement-dependent cytotoxicity. ENO1 vaccination reduced numbers of myeloid-derived suppressor cells and T-regulatory cells and increased T-helper 1 and 17 responses. CONCLUSIONS In a genetic model of pancreatic carcinoma, vaccination with ENO1 DNA elicits humoral and cellular immune responses against tumors, delays tumor progression, and significantly extends survival. This vaccination strategy might be developed as a neoadjuvant therapy for patients with PDA.

Th22 cells are expanded in multiple sclerosis and are resistant to IFN-β

Th1 and Th17 cells have been considered as effectors in mouse EAE and in the human counterpart, MS. Recently, IL‐22, a Th17‐related, proinflammatory cytokine, has been associated with a new Th cell subset, defined as Th22, involved in chronic inflammatory conditions, such as psoriasis; the role of IL‐22 in MS has not yet been elucidated. Here, we report that similar to Th17 cells, the number of Th22 cells increased in the PB and the CSF of RR MS patients, especially during the active phases of the disease. However, as opposed to Th17 cells, the expansion of Th22 cells occurred before the active phases of the disease. Th22 cells were found to be specific for the autoantigen MBP and also expressed high levels of CCR6 and T‐bet, as for Th17 cells, indicating that Th22 self‐reactive cells could have CNS‐homing properties and be pathogenic in active RRMS patients. Conversely to Th17 cells, Th22 cells displayed lower levels of IFNAR1 and were insensitive to IFN‐β inhibition. These data suggest that expansion of Th22 cells in MS could be one of the factors that critically influence resistance to IFN‐β therapy.

Erbb2 DNA Vaccine Combined with Regulatory T Cell Deletion Enhances Antibody Response and Reveals Latent Low-Avidity T Cells: Potential and Limits of Its Therapeutic Efficacy

Rat (r)Erbb2 transgenic BALB-neuT mice genetically predestined to develop multiple invasive carcinomas allow an assessment of the potential of a vaccine against the stages of cancer progression. Because of rErbb2 expression in the thymus and its overexpression in the mammary gland, CD8+ T cell clones reacting at high avidity with dominant rErbb2 epitopes are deleted in these mice. In BALB-neuT mice with diffuse and invasive in situ lesions and almost palpable carcinomas, a temporary regulatory T cells depletion combined with anti-rErbb2 vaccine markedly enhanced the anti-rErbb2 Ab response and allowed the expansion of latent pools of low-avidity CD8+ T cells bearing TCRs repertoire reacting with the rErbb2 dominant peptide. This combination of a higher Ab response and activation of a low-avidity cytotoxic response persistently blocked tumor progression at stages in which the vaccine alone was ineffective. However, when diffuse and invasive microscopic cancers become almost palpable, this combination was no longer able to secure a significant extension of mice survival.

Protective Immunity Against neu-Positive Carcinomas Elicited by Electroporation of Plasmids Encoding Decreasing Fragments of Rat Neu Extracellular Domain

We have shown that electroporation of plasmid carrying extracellular and transmembrane domains (EC-TM plasmid) encoded by the rat neu oncogene triggers a protective immune response toward rat p185(neu)-positive tumors in both wild-type BALB/c mice and cancer-prone rat neu-transgenic BALB-neuT mice. To identify the critical fragments that confer this protective immunity, mice were electroporated with plasmids encoding the TM domain associated with decreasing fragments of the EC domain and the antitumor protection afforded, the titer of antibody, and cytotoxic T lymphocyte (CTL) activity elicited to Neu protein were evaluated. Plasmids encoding EC fragments shortened by 70 (EC1-TM plasmid), 150 (EC2-TM), 230 (EC3-TM), 310 (EC4-TM), and 390 (EC5-TM) NH(2)-terminal residues afforded effective protection. Plasmids encoding shorter truncated proteins were ineffective. When the immunogenic protein was retained in the cytoplasm (EC1-TM, EC2-TM, and EC5-TM), only a CTL response was elicited, whereas when it was also expressed on the membrane (EC4-TM) both CTLs and antibodies were induced. EC4-TM encoding a truncated protein with an EC portion of only 344 amino acids conferred protection on both BALB/c and BALB-neuT mice comparable to that of EC-TM.

Acute-Phase Protein Hemopexin Is a Negative Regulator of Th17 Response and Experimental Autoimmune Encephalomyelitis Development

Hemopexin (Hx) is an acute-phase protein synthesized by hepatocytes in response to the proinflammatory cytokines IL-6, IL-1β, and TNF-α. Hx is the plasma protein with the highest binding affinity to heme and controls heme-iron availability in tissues and also in T lymphocytes, where it modulates their responsiveness to IFN-γ. Recent data have questioned regarding an anti-inflammatory role of Hx, a role that may be both heme-binding dependent and independent. The aim of this study was to investigate the role of Hx in the development of a T cell–mediated inflammatory autoimmune response. During experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis, Hx content in serum increased and remained high. When EAE was induced in Hx knockout (Hx−/−) mice, they developed a clinically earlier and exacerbated EAE compared with wild-type mice, associated to a higher amount of CD4+-infiltrating T cells. The severe EAE developed by Hx−/− mice could be ascribed to an enhanced expansion of Th17 cells accounting for both a higher disposition of naive T cells to differentiate toward the Th17 lineage and a higher production of Th17 differentiating cytokines IL-6 and IL-23 by APCs. When purified human Hx was injected in Hx−/− mice before EAE induction, Th17 expansion, as well as disease severity, were comparable with those of wild-type mice. Taken together, these data indicate that Hx has a negative regulatory role in Th17-mediated inflammation and prospect its pharmacological use to limit the expansion of this cell subset in inflammatory and autoimmune disease.

Alemtuzumab long-term immunologic effect: Treg suppressor function increases up to 24 months

Objective: To analyze changes in T-helper (Th) subsets, T-regulatory (Treg) cell percentages and function, and mRNA levels of immunologically relevant molecules during a 24-month follow-up after alemtuzumab treatment in patients with relapsing-remitting multiple sclerosis (RRMS). Methods: Multicenter follow-up of 29 alemtuzumab-treated patients with RRMS in the Comparison of Alemtuzumab and Rebif Efficacy in Multiple Sclerosis (CARE-MS) I and CARE-MS II trials. Peripheral blood (PB) samples were obtained at months 0, 6, 12, 18, and 24. We evaluated (1) mRNA levels of 26 immunologic molecules (cytokines, chemokines, chemokine receptors, and transcriptional factors); (2) Th1, Th17, and Treg cell percentages; and (3) myelin basic protein (MBP)–specific Treg suppressor activity. Results: We observed 12 relapses in 9 patients. mRNA levels of the anti-inflammatory cytokines interleukin (IL)–10, IL-27, and transforming growth factor–β persistently increased whereas those of proinflammatory molecules related to the Th1 or Th17 subsets persistently decreased after alemtuzumab administration throughout the follow-up period. PB CD4+ cell percentage remained significantly lower than baseline while that of Th1 and Th17 cells did not significantly change. A significant increase in Treg cell percentage was observed at month 24 and was accompanied by an increase in Treg cell suppressive activity against MBP-specific Th1 and Th17 cells. Conclusions: The long-lasting therapeutic benefit of alemtuzumab in RRMS may involve a shift in the cytokine balance towards inhibition of inflammation associated with a reconstitution of the PB CD4+ T-cell subsets that includes expansion of Treg cells with increased suppressive function.

Immune prevention of mammary carcinogenesis in HER-2/neu transgenic mice: a microarray scenario

Neoplastic transformation is a multistep process in which gene products of specific regulatory pathways are involved at each stage. Identification of these overexpressed or mutated gene products provides an unprecedented opportunity to address the immune system against defined antigens and eliminate transformed cells. Mice transgenic for these oncogenes (e.g. HER-2/neu, a prototype of deregulated oncogenic protein kinase membrane receptors) are ideal experimental models for assessing the potential of active immunization. The demonstration that vaccines can cure HER-2/neu transplantable tumors, prevent their onset and delay the progression of preneoplastic lesions in mice at risk suggests that efficient immunological inhibition of HER-2/neu carcinogenesis can be achieved by specific vaccination. To further explore this issue, halting of tumor progression in the mammary glands of BALB-neuT mice with two immunization protocols in two laboratories has been studied independently by DNA microarray analysis. Combination of the two sets of results revealed a clear correlation between them when the tumor mass was titrated by transcription profiling. It was also clear that both protocols induced a strong, polyclonal antibody response and halted tumor growth at a condition very similar to that at which the vaccination began. Differences in the expression profiles were mainly related to the expression levels of a few chemokines and T-cell-specific genes that may be in some way correlated with the efficacy of the vaccination. Last, combination of the expression data with the protection results indicated that chronic vaccination is needed to maintain an active IFN-γ-mediated response in the mammary gland.

Th17 Cells in Multiple Sclerosis Express Higher Levels of JAK2, Which Increases Their Surface Expression of IFN-γR2

IFN-β inhibits the expansion of Th17 cells in active multiple sclerosis (AMS), and this might contribute to improve the clinical symptoms. The effectiveness of this inhibition, however, requires intact IFN-γ signaling in T cells. In this study, we report that both mRNA and cell surface expression of the signaling chain of the IFN-γ receptor (IFN-γR2) and its cognate tyrosine kinase JAK2 are enhanced in peripheral blood Th17 cells and clones from patients with AMS compared with those with inactive multiple sclerosis (IMS) or healthy subjects (HS). IFN-γ decreased the frequency of Th17 peripheral cells and proliferation of Th17 clones from AMS patients. Stimulation of PBMCs from HS in Th17-polarizing conditions resulted in the enhancement of JAK2 expression and accumulation of cell surface IFN-γR2. The role of JAK2 in the modulation of IFN-γR2 was demonstrated as its transduction prevented rapid internalization and degradation of IFN-γR2 in JAK2-deficient γ2A cells. In conclusion, these data identify JAK2 as a critical factor that stabilizes IFN-γR2 surface expression in Th17 cells from AMS patients, making them sensitive to IFN-γ. These data may have clinical implications for a better use of IFNs in multiple sclerosis and possibly other inflammatory diseases.