New tools are needed to assess at a single cell level allergen-specific T cell responses in allergic or healthy individuals, as well as in the course of immunotherapy. We developed MHC-class II peptide tetramers to monitor T cell responses against Bet v 1, Der p 1, Der p 2 in individuals with either an HLA-DRB1∗0101, ∗0301, ∗0401, ∗1501 or HLA-DPB1∗0401 background. Expanded cells following in vitro allergen stimulation were stained with tetramers and subsequently assessed for IL-5, IL-10 or IFN-γ production using a cytokine surface capture. Samples were analysed by cytofluorometry. High affinity epitopes for multiple HLA-class II haplotypes have been identified for Bet v 1141-155, Der p 116-30, Der p 226-40, leading to the development of corresponding tetramers detecting allergen specific T cells with a high specificity and sensitivity. MHC class II Bet v 1141-155 tetramer+ T cells produce IFN-γ and IL-10 in response to Bet v 1 in healthy individuals, whereas IL-5 secreting cells are mostly detected in allergic patients. Frequencies of Bet v 1-specific CD4+ T cells circulating in the blood of allergic or non allergic individuals, range from 10-6 to 10-3 CD4+ T cells, outside or within the pollen season, respectively. MHC-class II peptide tetramers are valuable tools to assess allergen-specific T cell responses, both qualitatively and quantitatively. Peripheral IFNγ/IL10 producing CD4+ T cells undergoing a three-log expansion during allergen exposure play a key role in establishing tolerance.

Background: We report herein critical methodological principles for assessing, at a single cell level, allergen-specific T cell responses using MHC class II peptide tetramers. Methods: We developed MHC class II peptide tetramers to monitor T cell responses against the immunodominant Bet v 1141–155 peptide in individuals with either an HLA-DRB1*0101, DRB1*0401 or DRB1*1501 background. In vitro stimulation was performed with serially truncated versions of the Bet v 1141–155 epitope chemically conjugated to the Ii-Key peptide. Results: Identification of Bet v 1141–155 as a high-affinity epitope for multiple HLA-DRB1 allotypes led to the development of corresponding tetramers detecting Bet v 1141–155-specific T cells with a high specificity and sensitivity. Stimulation with Bet v 1141–155 Ii-Key conjugate peptides is the most efficient procedure to expand Bet v 1141–155-specific CD4+ T cells, allowing to detect such cells in both allergic and healthy individuals. MHC class II Bet v 1141–155 tetramer-positive T cells produce IFN-γ and IL-10 in healthy individuals, and IL-5 in allergic patients. Frequencies of Bet v 1-specific CD4+ T cells circulating in the blood of allergic or nonallergic individuals range from approximately 10–5 to 10–3 CD4+ T cells, outside or within the pollen season, respectively. Conclusions: MHC class II peptide tetramers are valuable tools to assess allergen-specific T cell responses, both qualitatively and quantitatively. Selection of a high-affinity T cell epitope, as well as optimization of in vitro stimulation conditions to expand rare T cell progenitors are critical success factors in those analyses.

We have demonstrated that coupling an immunoregulatory segment of the MHC class II-associated invariant chain (Ii), the Ii-Key peptide, to a promiscuous MHC class II epitope significantly enhances its presentation to CD4+ T cells. Here, a series of homologous Ii-Key/HER-2/neu(776-90) hybrid peptides, varying systematically in the length of the epitope(s)-containing segment, are significantly more potent than the native peptide in assays using T cells from patients with various types of tumors overexpressing HER-2/neu. In particular, priming normal donor and patient PBMCs with Ii-Key hybrid peptides enhances recognition of the native peptide either pulsed onto autologous dendritic cells (DCs) or naturally presented by IFN-γ-treated autologous tumor cells. Moreover, patient-derived CD4+ T cells primed with the hybrid peptides provide a significantly stronger helper effect to autologous CD8+ T cells specific for the HER-2/neu(435-43) CTL epitope, as illustrated by either IFN-γ ELISPOT assays or specific autologous tumor cell lysis. Hybrid peptide-specific CD4+ T cells strongly enhanced the antitumor efficacy of HER-2/neu(435-43) peptide-specific CTL in the therapy of xenografted SCID mice inoculated with HER-2/neu overexpressing human tumor cell lines. Our data indicate that the promiscuously presented vaccine peptide HER-2/neu(776-90) is amenable to Ii-Key-enhancing effects and supports the therapeutic potential of vaccinating patients with HER-2/neu+ tumors with such Ii-Key/HER-2/neu(776-90) hybrid peptides.

We tested the hypothesis that Epstein-Barr virus (EBV) might actually infect leukemic hairy cells in vivo by examining those cells for the EBV-receptor, EBV nuclear antigen (EBNA) and membrane antigen (MA), for spontaneous transformation and rescue of infectious virus and for presence of EBV genome. EBV-receptors were found on subpopulations of leukemic cells from each of 7 patients with hairy cell leukemia (HCL) tested. MA was present on low numbers (1-5 per cent) of fresh leukemic cells of 7 patients and in some instances occurred with a greater frequency after 3 to 5 days in culture, with or without 12-O-tetradecanoylphorbol-13-acetate. In 11 fresh leukemic cell preparations from 8 HCL patients, no EBNA was demonstrated. However, 2 samples after 4 days in culture expressed low frequencies of EBNA-positive cells. Spontaneous, EBV-positive cell lines were established with a high transformation efficiency from 3 HCL blood samples but not from 8 other specimens. Infectious EBV could be rescued from some hairy leukemic cell preparations by co-cultivation with cord blood lymphocytes. These results demonstrated that leukemic cell populations harbored infectious EBV, that the leukemic cells expressed virus receptors and suggested that a small subpopulation of leukemic cells might become infected in vivo at least transiently and possibly transformed in vitro by EBV. To test for the extent of occult in vivo infection of leukemic cells with EBV, Southern type hybridization studies were performed with a probe for EBV genome (Bam HI W). At a sensitivity level of 0.1 genome per cell, EBV genome was not detected in the leukemic cell populations of 7 patients. We conclude that host defence mechanisms protecting these individuals from EBV also prevent infections of the leukemic cell and/or most hairy leukemic cells are not suitable targets for both infection and transformation.

SummaryOne function of the major histocompatibility complex (MHC) class II‐associated invariant chain (Ii) is to prevent MHC class II molecules from binding endogenously generated antigenic epitopes. Ii inhibition leads to MHC class II presentation of endogenous antigens by APC without interrupting MHC class I presentation. We present data that in vivo immunization of BALB/c mice with HIV gp120 cDNA plus an Ii suppressive construct significantly enhances the activation of both gp120‐specific T helper (Th) cells and cytotoxic T lymphocytes (CTL). Our results support the concept that MHC class II‐positive/Ii‐negative (class II+/Ii–) antigen‐presenting cells (APC) present endogenously synthesized vaccine antigens simultaneously by MHC class II and class I molecules, activating both CD4+ and CD8+ T cells. Activated CD4+ T cells locally strengthen the response of CD8+ CTL, thus enhancing the potency of a DNA vaccine.

Life-threatening diseases, such as cancer and pandemic influenza, demand new efforts towards effective vaccine design. Peptides represent a simple, safe and adaptable basis for vaccine development; however, the potency of peptide vaccines is insufficient in most cases for significant therapeutic efficacy. Several methods, such as Ligand Epitope Antigen Presentation System and ISCOMATRIX®, have been developed to enhance the potency of peptide vaccines. One way of increasing the loading of MHC class II peptides occurs through the use of Ii-Key technology. Ii-Key (LRMK), a portion of the MHC class II-associated invariant chain (Ii), facilitates the direct loading of epitopes to the MHC class II molecule groove. Linking the Ii-Key moiety via a simple polymethylene bridge to an MHC class II epitope, to generate an Ii-Key/MHC class II epitope hybrid, greatly enhances the vaccine potency of the tethered epitope. The combination of such Ii-Key/MHC class II epitope hybrids with MHC class I epitope-containing peptides might generate a potent peptide vaccine for malignancies and infectious diseases. The Ii-Key hybrid technology is compared with other methods that enhance the potency of a peptide vaccine.

: Substantial progress has been made in creating a simple, feasible method to induce an anti-cancer immune response in prostate cancers or metastases by manipulating the regulation of the immune response. Cancer cells are transformed into antigen presenting cells by inducing of the MHC class II molecules and suppressing the co-induced immunoregulatory Ii protein with antisense or siRNA methods. The resulting anticancer immune response is profound, curing up to 80% of mice with established prostate tumors, transplanted into their normal prostates. The important achievement of this past year has been to design, synthesize and validate two second generation reagents, for mice and humans, that are more potent, simpler to use, and set the stage for definite preclinical validation in mice, and with human tumors in tissue culture. Substantial improvement on the reagent of the original grant proposal was achieved by a) tripling the expression of the reverse gene construct which suppresses Ii protein expression, and b) by both chemical and plasmid genetic forms of small inhibitory RNA, which achieve the same end, but with surprising potency. These substantial technical advances (under Task 1 and 2) set the stage for advancement toward clinical trails (when justified by data from next year's effort).