Unique and cross-reactive T cell epitope peptides of the major Bahia grass pollen allergen, Pas n 1.

BACKGROUND: Bahia grass pollen (BaGP) is a major cause of allergic rhinitis. Subcutaneous allergen-specific immunotherapy is effective for grass pollen allergy, but is unsuitable for patients with moderate to severe asthma due to the risk of anaphylaxis. T cell-reactive but IgE nonreactive peptides provide a safer treatment option. This study aimed to identify and characterize dominant CD4(+) T cell epitope peptides of the major BaGP allergen, Pas n 1. METHODS: Pas n 1-specific T cell lines generated from the peripheral blood of BaGP-allergic subjects were tested for proliferative and cytokine response to overlapping 20-mer Pas n 1 peptides. Cross-reactivity to homologous peptides from Lol p 1 and Cyn d 1 of Ryegrass and Bermuda grass pollen, respectively, was assessed using Pas n 1 peptide-specific T cell clones. MHC class II restriction of Pas n 1 peptide T cell recognition was determined by HLA blocking assays and peptide IgE reactivity tested by dot blotting. RESULTS: Three Pas n 1 peptides showed dominant T cell reactivity; 15 of 18 (83%) patients responded to one or more of these peptides. T cell clones specific for dominant Pas n 1 peptides showed evidence of species-specific T cell reactivity as well as cross-reactivity with other group 1 grass pollen allergens. The dominant Pas n 1 T cell epitope peptides showed HLA binding diversity and were non-IgE reactive. CONCLUSIONS: The immunodominant T cell-reactive Pas n 1 peptides are candidates for safe immunotherapy for individuals, including those with asthma, who are allergic to Bahia and possibly other grass pollens.

Immunological heterogeneity of the RCC microenvironment: do targeted therapies influence immune response?

The introduction of targeted agents has substantially improved treatment of metastatic clear-cell renal cell carcinoma (RCC). However, complete responses are rare and therapy is not curative. Moreover, information on the latest generation of potent and selective vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors (TKI) suggests that a plateau has been reached in terms of efficacy. Recent data reveal that targeted agents are involved in modulating immune responses in RCC. In addition, current research adds to our understanding of how RCC escapes an effective anti-tumor response with the potential to modulate these processes by drug development. This review provides specific insight into targeted therapy induced changes in the immunological microenvironment of RCC, summarizes the available evidence, and discusses potential therapeutic implications.

Visualizing fewer than 10 mouse T cells with an enhanced firefly luciferase in immunocompetent mouse models of cancer.

Antigen specific T cell migration to sites of infection or cancer is critical for an effective immune response. In mouse models of cancer, the number of lymphocytes reaching the tumor is typically only a few hundred, yet technology capable of imaging these cells using bioluminescence has yet to be achieved. A combination of codon optimization, removal of cryptic splice sites and retroviral modification was used to engineer an enhanced firefly luciferase (ffLuc) vector. Compared with ffLuc, T cells expressing our construct generated >100 times more light, permitting detection of as few as three cells implanted s.c. while maintaining long term coexpression of a reporter gene (Thy1.1). Expression of enhanced ffLuc in mouse T cells permitted the tracking of <3 x 10(4) adoptively transferred T cells infiltrating sites of vaccination and preestablished tumors. Penetration of light through deep tissues, including the liver and spleen, was also observed. Finally, we were able to enumerate infiltrating mouse lymphocytes constituting <0.3% of total tumor cellularity, representing a significant improvement over standard methods of quantitation including flow cytometry.

Refinement in the production and purification of recombinant HCMV IE1-pp65 protein for the generation of epitope-specific T cell immunity.

Human cytomegalovirus (HCMV) remains one of the most common opportunistic infections causing disease following stem cell transplantation, despite the availability of anti-viral therapies. Adoptive immunotherapy has the potential to further aid in counteracting chronic viral reactivation and subsequent disease by restoring viral immunity through the transfer of virus-specific T cells from transplant donors to their recipients. Our study refines the production and purification of a recombinant HCMV protein containing two of the most immunodominant antigens (IE1 and pp65) for the generation of polyclonal HCMV-specific T cells. In doing so, a 6x His-tagged IE1-pp65 protein was generated using a serum-free baculovirus/insect cell expression system and soluble IE1-pp65 protein was subsequently purified using Ni-NTA affinity chromatography under stringent conditions to obtain a highly pure product. The ability of the recombinant IE1-pp65 protein to elicit a functional T cell mediated immune response was demonstrated by the vigorous reactivation and expansion of HLA-A2-restricted pp65(495-503)-specific CD8+ T cells. This recombinant IE1-pp65 protein can potentially generate a multitude of HLA-restricted HCMV-specific T cells, providing a better alternative to using costly overlapping peptides or HCMV lysates for expansion of T cells for use in adoptive immunotherapy strategies.

TGF-beta1 induces preferential rapid expansion and persistence of tumor antigen-specific CD8+ T cells for adoptive immunotherapy.

Adoptive cell transfer of expanded, autologous tumor-infiltrating lymphocytes (TIL) into lymphodepleted melanoma patients can induce the regression of bulky, metastatic disease. To generate the large numbers of T cells needed for infusion, TIL undergo a rapid expansion protocol (REP) in vitro using anti-CD3 antibody, interleukin-2, and irradiated peripheral blood feeder cells that typically results in an approximately 1000-fold expansion over 14 days. However, we have found that the conventional REP (C-REP) often favors the expansion of CD4+ T cells at the expense of tumor antigen-specific CD8+ T cells, which are the most potent cytolytic effector cells. In this study, we demonstrate that addition of transforming growth factor (TGF)-beta1 to the TIL culture at the onset of rapid expansion (T-REP ) maintained the percentage of CD8+ T cells while not inhibiting overall T-cell expansion. Of T cells expanded from different melanoma patient tumors, 13 of 15 TIL demonstrated improved yields and percentages of both CD8+ and MART-1 melanoma antigen-specific T cells after 14 days of expansion in TGF-beta1 compared with the C-REP. This was associated with a marked improvement in the antitumor activity of the resulting bulk TIL culture in terms of interferon-gamma production and melanoma tumor-specific cytotoxic T-lymphocyte activity. In addition, T-REP T cells demonstrated a higher potential for continued expansion in vitro for up to 3 weeks after the expansion compared with C-REP T cells, suggesting that they may also be capable of increased persistence after adoptive cell transfer. Our results suggest that TGF-beta1-expanded TIL have attributes that might predict efficacy superior to that of conventional TIL.

Expression and purification of the minor histocompatibility antigen, HA-1H generated in Escherichia coli.

The minor histocompatibility antigen HA-1H is a potential immunotherapeutic molecule. It can be used as a target for graft versus leukaemia reactions to eliminate residual HA-1H expressing leukaemic cells in patients following haemopoietic stem cell transplantation, whereby HA-1H primed donor cells can be transferred into a patient via adoptive immunotherapy. However, thus far only synthetic peptides corresponding to a HLA-A *0201 restricted HA-1H epitope have been used to generate HA-1H specific T cells. We are the first laboratory to clone, express and purify a region of HA-1H using an Escherichia coli expression system. The recombinant HA-1H protein was purified under denaturing conditions and the affinity purification tag removed using thrombin to remove non-specific amino acids. The 92 amino acid recombinant protein was characterised by mass spectrometry. Our rationale is that by using a recombinant HA-1H protein rather than peptide, HA-1H specific T cells may be generated from presentation of multiple HA-1H epitopes complexed in different HLA molecules. A multi-epitope approach may have wider applicability and maybe more effective at leukaemia control. The recombinant HA-1H protein may also be used as a research tool to identify novel CD4(+) helper T cell and CD8(+) cytotoxic T cell epitopes.