Surveillance Study of Influenza Occurrence and Immunity in a Wisconsin Cohort During the 2009 Pandemic.

BACKGROUND: Antibody and T-cell immunity to conserved influenza virus antigens can protect animals against infection with diverse influenza strains. Although immunity against conserved antigens occurs in humans, whether such responses provide cross-protection in humans and could be harnessed as the basis for universal influenza vaccines is controversial. The 2009 pandemic provided an opportunity to investigate whether pre-existing cross-reactive immunity affected susceptibility to infection. METHODS: In 2009, we banked sera and peripheral blood mononuclear cells (PBMC) from blood donors, then monitored them for pandemic influenza infection (pH1N1) by polymerase chain reaction or seroconversion. Antibodies to hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix 2 (M2), and HA-pseudotypes were measured in sera. T-cell inteferon-γ enzyme-linked immunospot responses were measured in PBMC. RESULTS: There were 13 infections in 117 evaluable donors. Pre-existing T-cell reactivity to pH1N1 was substantial (of 153 donors tested, 146 had >100 spot-forming cells/106 cells). Antibodies reactive with pH1N1 were common: anti-NP (all donors) and anti-M2 (44% of donors). Pseudotype-neutralizing antibodies to H1 were detected, but not to highly conserved HA epitopes. Unexpectedly, donors with symptomatic pH1N1 infection had sharp rises in HA pseudotype-neutralizing antibodies, not only pH1N1 but also against multiple seasonal H1s. In addition, an exploratory study of a T-cell marker (response to NP418-426) identified probable infection missed by standard criteria. CONCLUSIONS: Although the number of infections was inadequate for conclusions about mechanisms of protection, this study documents the wide variety of pre-existing, cross-reactive, humoral and cellular immune responses to pandemic influenza virus antigens in humans. These responses can be compared with results of other studies and explored in universal influenza vaccine studies.

Adoptive immunotherapy with CMV-specific cytotoxic T lymphocytes for stem cell transplant patients with refractory CMV infections.

Adoptive immunotherapy with cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTL) is an effective strategy for preventing and treating viral reactivation after allogeneic stem cell transplantation (SCT). We have shown previously that CMV CTL can be generated in 1 to 2 weeks by stimulating donor lymphocytes with peptide mixes derived from full-length pp65 and IE1. We conducted a multi-institutional study of CMV-specific CTL for patients with persistent or anti-viral-resistant CMV infections after allogeneic SCT, to determine the safety, feasibility, and immunologic effects of this approach. We were successful in stimulating CTL from 10/10 donors with pooled CMV overlapping peptide mixes. Five of the 7 subjects who met infusion criteria had new onset CMV-specific CTL activity detected within 4 to 6 weeks after infusion. Of the 2 subjects who did not have immunologic responses after infusion, 1 received CTL with a low viability after thawing, and the other patient received cyclosporine A and systemic corticosteroids at the time of the infusion, achieving only a low, transient increase (10%) in pp65-specific activity. There was no graft-versus-host disease attributable to these infusions. These findings indicate that the infusion of CTL stimulated over 1 to 2 weeks with overlapping CMV peptides can result in virus-specific immune reconstitution in SCT recipients, without exacerbations of graft-versus-host disease.

ELISPOT Assay for Monitoring Cytotoxic T Lymphocytes (CTL) Activity in Cancer Vaccine Clinical Trials.

The profiling and monitoring of immune responses are key elements in the evaluation of the efficacy and development of new biotherapies, and a number of assays have been introduced for analyzing various immune parameters before, during, and after immunotherapy. The choice of immune assays for a given clinical trial depends on the known or suggested immunomodulating mechanisms associated with the tested therapeutic modality. Cell-mediated cytotoxicity represents a key mechanism in the immune response to various pathogens and tumors. Therefore, the selection of monitoring methods for the appropriate assessment of cell-mediated cytotoxicity is thought to be crucial. Assays that can detect both cytotoxic T lymphocytes (CTL) frequency and function, such as the IFN-γ enzyme-linked immunospot assay (ELISPOT) have gained increasing popularity for monitoring clinical trials and in basic research. Results from various clinical trials, including peptide and whole tumor cell vaccination and cytokine treatment, have shown the suitability of the IFN-γ ELISPOT assay for monitoring T cell responses. However, the Granzyme B ELISPOT assay and Perforin ELISPOT assay may represent a more direct analysis of cell-mediated cytotoxicity as compared to the IFN-γ ELISPOT, since Granzyme B and perforin are the key mediators of target cell death via the granule-mediated pathway. In this review we analyze our own data and the data reported by others with regard to the application of various modifications of ELISPOT assays for monitoring CTL activity in clinical vaccine trials.

Wilms' tumor 1-specific cytotoxic T lymphocytes can be expanded from adult donors and cord blood.

The use of WT1-specific CTL is one potential strategy to treat leukemic relapse following allogeneic stem cell transplant (SCT). Previous studies have largely focused on generating WT1-CTL from adult donors by cloning. We demonstrate that WT1-CTL can be generated from healthy adult donors and from cord blood by stimulating with an overlapping pool of peptides derived from full length WT1 and selecting antigen-specific cells based on the expression of CD137. The rapid expansion with anti-CD3 and IL-2 resulted in a 100-200-fold expansion. These CTL lysed WT1 expressing targets, including leukemia lines, in a HLA restricted manner.

MAGE-A1, MAGE-A3, and NY-ESO-1 can be upregulated on neuroblastoma cells to facilitate cytotoxic T lymphocyte-mediated tumor cell killing.

Approximately half of patients with stage IV neuroblastoma are expected to relapse despite current therapy, and when this occurs, there is little likelihood of achieving a cure. Very few clinical trials have been conducted to determine whether cellular immune responses could be harnessed to fight this tumor, largely because potential tumor antigens for cytotoxic T lymphocytes (CTL) are limited. MAGE-A1, MAGE-A3, and NY-ESO-1 are cancer-testis (CT) antigens expressed on a number of malignant solid tumors, including neuroblastoma, but many tumor cell lines down-regulate the expression of CT antigens as well as major histocompatibility (MHC) antigens, precluding recognition by antigen-specific T cells. If expression of cancer antigens on neuroblastoma could be enhanced pharmacologically, CT antigen-specific immunotherapy could be considered for this tumor. We have demonstrated that the expression of MAGE-A1, MAGE-A3, and NY-ESO-1 can be upregulated on neuroblastoma cells following exposure to pharmacologic levels of the demethylating agent 5-aza-2'-deoxycytidine (decitabine, DAC). Expression of NY-ESO-1, MAGE-A1, or MAGE-A3 was induced in 10/10 neuroblastoma cell lines after 5 days of exposure to DAC. Culture of neuroblastoma cell lines with IFN-γ was also associated with an increased expression of either MHC Class I or II by cytofluorometry, as reported by other groups. MAGE-A1, MAGE-A3, and NY-ESO-1-specific CTL were cultured from volunteer donors by stimulating peripheral blood mononuclear cells with dendritic cells pulsed with overlapping peptide mixes derived from full-length proteins, and these CTL preferentially lysed HLA partially matched, DAC-treated neuroblastoma and glioblastoma cell lines. These studies show that demethylating chemotherapy can be combined with IFN-γ to increase the expression of CT antigens and MHC molecules on neuroblastoma cells, and pre-treatment with these agents makes tumor cell lines more susceptible to CTL-mediated killing. These data provide a basis to consider the use of demethylating chemotherapy in neuroblastoma patients, in conjunction with immune therapies that facilitate the expansion of CT antigen-specific CTL.

The detection of CMV pp65 and IE1 in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a highly lethal brain tumor affecting children and adults, with the majority of affected individuals dying from their disease by 2 years following diagnosis. Other groups have reported the association of cytomegalovirus (CMV) with GBM, and we sought to confirm these findings in a large series of patients with primary GBM from our institution. Immunohistochemical analysis of paraffin embedded tissue sections was performed on 49 newly diagnosed GBM tumors, the largest series reported to date. We confirmed the presence of CMV pp65 on 25/49 (51%) and of IE1 on 8/49 (16%) of these tumors. While pp65 and IE1 are generally found in the nucleus of cells that are permissibly infected by CMV, GBM in this series had mostly cytoplasmic staining, with only 16% having nuclear staining for one or both of these antigens. We infected GBM cell lines with a laboratory strain of CMV, and found that most of the staining was cytoplasmic, with some perinuclear localization of IE1. To test the potential for CMV infected GBM cells to be recognized by CMV pp65 and IE1 specific cytotoxic T lymphocytes (CTL), we used CMV infected GBM cell lines in cytotoxicity assays with human leukocyte antigen partially matched CMV CTL. Lysis of CMV infected GBM tumor cells was accentuated by pre-treating these cell lines with either the demethylating agent decitabine or interferon-γ, both of which were shown to increase MHC Class I and II expression on tumor cells in vitro. These studies confirm the presence of CMV pp65 or IE1 on approximately half of GBM, with the possibility that CMV positive tumor cells can be recognized by CMV pp65/IE1 specific T cells.

Infusion of cytomegalovirus specific cytotoxic T lymphocytes from a sero-negative donor can facilitate resolution of infection and immune reconstitution.

We report a stem cell transplant patient with a therapy-refractory cytomegalovirus (CMV) infection who received CMV-specific T cells from his sero-negative stem cell donor. This donor received the Towne strain CMV vaccine, and T cells were expanded using monocytes pulsed with pp65 overlapping peptides. CMV DNA decreased after the CTL infusion, and CMV-specific cytotoxicity increased. This strategy could be implemented in similar situations or with persistent viremia post-transplant.

Expansion of cytomegalovirus pp65 and IE-1 specific cytotoxic T lymphocytes for cytomegalovirus-specific immunotherapy following allogeneic stem cell transplantation.

Adoptive immunotherapy with antigen-specific cytotoxic T lymphocytes (CTLs) has proven effective in restoring cellular immunity to cytomegalovirus (CMV) and preventing viral reactivation after allogeneic stem cell transplantation (SCT). In an effort to develop a cost-effective, relatively rapid method of CMV CTL expansion, we investigated the use of a pool of overlapping CMV peptides. Because the possibility exists of vaccinating CMV-seronegative donors, and these individuals may have T cell responses predominantly against IE-1, commercially available peptide mixes for pp65 as well as IE-1 were used to stimulate CTLs from 10 seropositive donors. Of these 10 donors, 4 responded to pp65 only, 1 did not respond to either pp65 or IE-1, 4 responded to both pp65 and IE-1, and 1 responded to IE-1 only. These CMV- specific T cells included a mixture of CD4(+) and CD8(+) effectors, and specific cytotoxicity correlated with interferon-gamma production. The costs associated with a 28-day maintenance course of intravenous ganciclovir, cidofovir, foscarnet, and valganciclovir, as well as the preparation and shipping a single dose of CTLs, were determined. The price of generating CMV CTLs using this method was comparable to or less expensive than a 28-day maintenance course for these agents, not including the costs associated with drug administration, supportive care, and the treatment of drug-related complications. Considering the relative ease, low cost, and the fact that CTL administration can result in CMV-specific immune reconstitution, this option should be considered for patients with CMV reactivation or for prophylaxis in patients at high risk for infection.

Interferon-gamma expressing EBV LMP2A-specific T cells for cellular immunotherapy.

To generate therapeutic T cells for adoptive immunotherapy, T cells specific to Epstein-Barr virus LMP2A were enriched on the basis of antigen-specific production of interferon-gamma (IFNgamma). The enriched T cells, contained over 60% LMP2A-specific effectors, were polyclonal and targeted multiple LMP2A epitopes. A high proportion of the enriched T cells produced the Th1 cytokines interleukin (IL)-2 and granulocyte monocyte colony stimulating factor, while few cells expressed the Th2 cytokines IL4 and IL10. The enriched T cells specifically lysed LMP2A-expressing target cells, with concomitant production of IFNgamma and surface expression of CD107, suggesting the involvement of the granule exocytosis-mediated cytolytic pathway. In addition, the enriched T cells expressed CD45RO, CD28 and CD27, but not CD45RA, consistent with a differentiation stage capable of self-renewal for long-term persistence. LMP2A-specific T cells enriched based on IFNgamma-production may provide improved efficacy for the treatment of Epstein-Barr virus related malignancy.