Ainhoa Perez-Diez

Status of Activation of Circulating Vaccine-Elicited CD8+ T Cells

Selective blunting of the status of activation of circulating tumor-specific T cells was invoked to explain their paradoxical coexistence with unhampered tumor growth. By analogy, lack of tumor regression in the face of observable melanoma vaccine-induced T cell responses might be attributed to their status of activation. We enumerated with HLA-A*0201/peptide tetramers (tHLA) vaccine-elicited T cell precursor frequency directly in PBMC of patients with melanoma undergoing vaccination with the HLA-A*0201-associated gp100:209–217(210 M) epitope (g209-2 M). Furthermore, we tested by intracellular (IC)-FACS analysis and quantitative real-time PCR (qRT-PCR) the ability of postvaccination PBMC to produce cytokine in response to challenge with vaccine-related epitopes or vaccine-matched (HLA-A*0201) melanoma cells. Vaccine-induced enhancement of T cell precursor frequency could be detected with tHLA in PBMC from six of eight patients studied at frequencies ranging between 0.3 and 2.3% of the total CD8+ population. Stimulation with vaccine-related epitopes induced IFN-γ expression detectable by IC-FACS or qRT-PCR, respectively, in five and six of these patients. Furthermore, down-regulation of tHLA staining was noted upon cognate stimulation that could be utilized as an additional marker of T cell responsiveness. Finally, we observed in six patients an enhancement of reactivity against vaccine-matched tumor targets that was partly independent of documented vaccine-specific immune responses. A strong correlation was noted between tHLA staining of postvaccination PBMC and IFN-γ expression by the same samples upon vaccine-relevant stimulation and assessed either by IC-FACS or qRT-PCR. Thus, blunting of the status of T cell activation on itself cannot easily explain the lack of clinical responses observed with vaccination.

Intensity of the vaccine-elicited immune response determines tumor clearance

Tumor Ag-specific vaccines used for cancer immunotherapy can generate specific CD8 responses detectable in PBMCs and in tumor-infiltrating lymphocytes. However, human studies have shown that detection of a systemic vaccine-induced response does not necessarily correlate with the occasional instances of tumor rejection. Because this discrepancy might partially be attributable to the genetic heterogeneity of human cancers, as well as to the immunosuppressive effects of previous treatments, we turned to a mouse model in which these variables could be controlled to determine whether a relationship exists between the strength of vaccine-induced immune responses and tumor rejection. We challenged mice with the β-galactosidase (β-gal)-expressing tumor cells, C25.F6, vaccinated them with β-gal-carrying viral vectors, and used quantitative RT-PCR to measure the vaccine-induced immune response of splenocytes directly ex vivo. We found that the strength of the response increased with increasing doses of β-gal-carrying vector and/or upon boosting with a heterologous β-gal-carrying virus. Most importantly, we found that the strength of the detected immune response against this foreign Ag strongly correlated with reduction in the number of lung metastases. The results from this mouse model have major implications for the implementation of tumor vaccines in humans.

Kinetics of TCR Use in Response to Repeated Epitope-Specific Immunization

Selection of T cell-directed immunization strategies is based extensively on discordant information derived from preclinical models. We characterized the kinetics of T cell selection in response to repeated antigenic challenge. By enumerating with epitope/HLA tetrameric complexes (tHLA) vaccine-elicited T cell precursor frequencies (Tc-pf) in melanoma patients exposed to the modified gp100 epitope gp100:209–217 (g209-2M) we observed in most patients that the Tc-pf increased with number of immunizations. One patient’s kinetics were further characterized. Dissociation kinetics of g209-2M/tHLA suggested enrichment of T cell effector populations expressing TCR with progressively higher affinity. Furthermore, vaccine-elicited T cells maintained the ability to express IFN-γ ex vivo and proliferate in vitro. Thus, repeated exposure to immunogenic peptides benefited immune competence. These results provide a rationale for immunization strategies.

Microarrays for Cancer Diagnosis and Classification

Microarray analysis has yet to be widely accepted for diagnosis and classification of human cancers, despite the exponential increase in microarray studies reported in the literature. Among several methods available, a few refined approaches have evolved for the analysis of microarray data for cancer diagnosis. These include class comparison, class prediction and class discovery. Using as examples some of the major experimental contributions recently provided in the field of both hematological and solid tumors, we discuss the steps required to utilize microarray data to obtain general and reliable gene profiles that could be universally used in clinical laboratories. As we show, microarray technology is not only a new tool for the clinical lab but it can also improve the accuracy of the classical diagnostic techniques by suggesting novel tumor-specific markers. We then highlight the importance of publicly available microarray data and the development of their integrated analysis that may fulfill the promise that this new technology holds for cancer diagnosis and classification.

Molecular Profiling Improves Diagnoses of Rejection and Infection in Transplanted Organs

The monitoring of transplanted hearts is currently based on histological evaluation of endomyocardial biopsies, a method that is fairly insensitive and that does not always accurately discriminate between rejection and infection in the heart. Accurate diagnosis of rejection and infection is absolutely crucial, however, as the respective treatments are completely different. Using microarrays, we analyzed gene expression in 76 cardiac biopsies from 40 heart recipients undergoing rejection, no rejection, or Trypanosoma cruzi infection. We found a set of genes whose expression patterns were typical of acute rejection, and another set of genes that discriminated between rejection and T cruzi infection. These sets revealed acute rejection episodes up to 2 weeks earlier, and trypanosome infection up to 2 months earlier than did histological evaluation. When applied to raw data from other institutions, the 2 sets of predictive genes were also able to accurately pinpoint acute rejection of lung and kidney transplants, as well as bacterial infections in kidneys. In addition to their usefulness as diagnostic tools, the data suggest that there are similarities in the biology of the processes involved in rejection of different grafts and also in the tissue responses to pathogens as diverse as bacteria and protozoa.

Resident Peritoneal NK Cells

In this study, we describe a new population of NK cells that reside in the normal, uninflamed peritoneal cavity. Phenotypically, they share some similarities with the small population of CD49b−, CD27+ immature splenic NK cells, as well as liver NK cells, but they differ in their expression of CD62L, TRAIL, and EOMES. Functionally, the peritoneal NK cells resemble the immature splenic NK cells in their production of IFN-γ, GM-CSF, and TNF-α and in the killing of YAC-1 target cells. We also found that the peritoneum induces different behavior in mature and immature splenic NK cells. When transferred i.v. into RAGγc knockout mice, both populations undergo homeostatic proliferation in the spleen, but only the immature splenic NK cells are able to reach the peritoneum. When transferred directly into the peritoneum, the mature NK cells survive but do not divide, whereas the immature NK cells proliferate profusely. These data suggest that the peritoneum is not only home to a new subset of tissue-resident NK cells, but that it differentially regulates the migration and homeostatic proliferation of immature versus mature NK cells.

Administration of interleukin-7 increases CD4 T cells in idiopathic CD4 lymphocytopenia.

Idiopathic CD4 lymphopenia (ICL) is a rare syndrome defined by low CD4 T-cell counts (<300/µL) without evidence of HIV infection or other known cause of immunodeficiency. ICL confers an increased risk of opportunistic infections and has no established treatment. Interleukin-7 (IL-7) is fundamental for thymopoiesis, T-cell homeostasis, and survival of mature T cells, which provides a rationale for its potential use as an immunotherapeutic agent for ICL. We performed an open-label phase 1/2A dose-escalation trial of 3 subcutaneous doses of recombinant human IL-7 (rhIL-7) per week in patients with ICL who were at risk of disease progression. The primary objectives of the study were to assess safety and the immunomodulatory effects of rhIL-7 in ICL patients. Injection site reactions were the most frequently reported adverse events. One patient experienced a hypersensitivity reaction and developed non-neutralizing anti-IL-7 antibodies. Patients with autoimmune diseases that required systemic therapy at screening were excluded from the study; however, 1 participant developed systemic lupus erythematosus while on study and was excluded from further rhIL-7 dosing. Quantitatively, rhIL-7 led to an increase in the number of circulating CD4 and CD8 T cells and tissue-resident CD3 T cells in the gut mucosa and bone marrow. Functionally, these T cells were capable of producing cytokines after mitogenic stimulation. rhIL-7 was well tolerated at biologically active doses and may represent a promising therapeutic intervention in ICL. This trial was registered at www.clinicaltrials.gov as #NCT00839436.

T-Cell Depletion in the Colonic Mucosa of Patients With Idiopathic CD4+ Lymphopenia

Idiopathic CD4(+) lymphopenia (ICL) is a rare syndrome characterized by low peripheral CD4(+) T-cell counts that can lead to serious opportunistic infections. The pathogenesis of ICL remains unclear, and whether effector sites are also lymphopenic is unknown. In this study, rectosigmoid mucosal biopsy specimens from patients with ICL and healthy controls were evaluated. Significant T-cell lymphopenia was observed in the mucosal tissue of patients with ICL by flow cytometry and immunohistochemistry, compared with healthy controls. Functional capacity of T cells, assessed by production of interferon γ and interleukin 17, was preserved in the mucosa of patients with ICL. In contrast to T lymphocytes, the frequency of myeloid cells (neutrophils and macrophages) was elevated in the colonic mucosa of patients with ICL. Despite the observed mucosal abnormalities, plasma levels of intestinal fatty acid binding protein, a marker of enterocyte turnover and other inflammatory biomarkers, including interleukin 6, C-reactive protein, and tumor necrosis factor, were not elevated in patients with ICL, compared with healthy controls, whereas soluble CD14 levels were minimally elevated. These data suggest that patients with ICL, despite gut mucosal lymphopenia and local tissue inflammation, have preserved enterocyte turnover and T-helper type 17 cells with minimal systemic inflammation. These observations highlight differences from patients with human immunodeficiency virus infection, with or without AIDS, and may partially explain their distinct clinical prognosis.