Cécile Gouttefangeas

Cytomegalovirus infection: A driving force in human T cell immunosenescence

Abstract:  The human immune system evolved to defend the organism against pathogens, but is clearly less well able to do so in the elderly, resulting in greater morbidity and mortality due to infectious disease in old people, and higher healthcare costs. Many age‐associated immune alterations have been reported over the years, of which probably the changes in T cell immunity, often manifested dramatically as large clonal expansions of cells of limited antigen specificity together with a marked shrinkage of the T cell antigen receptor repertoire, are the most notable. It has recently emerged that the common herpesvirus, cytomegalovirus (CMV), which establishes persistent, life‐long infection, usually asymptomatically, may well be the driving force behind clonal expansions and altered phenotypes and functions of CD8 cells seen in most old people. In those few who are not CMV‐infected, another even more common herpesvirus, the Epstein‐Barr virus, appears to have the same effect. These virus‐driven changes are less marked in “successfully aged” centenarians, but most marked in people whom longitudinal studies have shown to be at higher risk of death, that is, those possessing an “immune risk profile” (IRP) characterized by an inverted CD4:8 ratio (caused by the accumulation primarily of CD8+ CD28− cells). These findings support the hypothesis that persistent herpesviruses, especially CMV, act as chronic antigenic stressors and play a major causative role in immunosenescence and associated mortality.

Identification of tumor-associated MHC class I ligands by a novel T cell-independent approach.

Specific immunotherapy of cancer utilizes tumor‐directed cytotoxic T lymphocytes (CTL) that lyse tumor cells presenting MHC class I‐associated peptides derived from tumor‐associated proteins. Many tumor‐associated gene products are known, but corresponding T cell epitopes are only known for relatively few of these. The most commonly used approaches to identify such antigens require pre‐existing CTL lines or clones. By using a CTL‐independent high performance liquid chromatography mass spectrometry (HPLC MS)–based approach we identified HLA‐A2‐presented peptides from carcinoembryonic antigen and wild‐type p53 with a copy number as low as eight molecules per cell. Potential epitopes were predicted from the sequences of known tumor antigens and the corresponding synthetic peptides were analyzed by nanocapillary HPLC MS. In parallel, peptides were extracted from fresh, solid tumor tissue or tumor cell lines and analyzed in the same way. Upon co‐elution of a natural peptide with a predicted peptide of the same mass, the peptide sequence was confirmed by on‐line tandem MS. This approach allows rapid screening of large numbers of tumor‐associated gene products for naturally processed peptides presented by different MHC class I molecules as a prerequisite for efficient epitope identification and rapid transfer to therapeutic vaccine trials.

The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8+ T lymphocytes by structural and functional assays

The interpretation of the results obtained from immunomonitoring of clinical trials is a difficult task due to the variety of methods and protocols available to detect vaccine-specific T-cell responses. This heterogeneity as well as the lack of standards has led to significant scepticism towards published results. In February 2005, a working group was therefore founded under the aegis of the Association for Immunotherapy of Cancer (“CIMT”) in order to compare techniques and protocols applied for the enumeration of antigen-specific T-cell responses. Here we present the results from two consecutive phases of an international inter-laboratory testing project referred to as the “CIMT monitoring panel”. A total of 13 centers from six European countries participated in the study in which pre-tested PBMC samples, synthetic peptides and PE-conjugated HLA-tetramers were prepared centrally and distributed to participants. All were asked to determine the number of antigen-specific T-cells in each sample using tetramer staining and one functional assay. The results of the first testing round revealed that the total number of cells analyzed was the most important determinant for the sensitive detection of antigen-specific CD8+ T-cells by tetramer staining. Analysis by ELISPOT was influenced by a combination of cell number and a resting phase after thawing of peripheral blood mononuclear cells. Therefore, the experiments were repeated in a second phase but now the participants were asked to change their protocols according to the new guidelines distilled from the results of the first phase. The recommendations improved the number of antigen-specific T-cell responses that were detected and decreased the variability between the laboratories. We conclude that a two-step approach in inter-laboratory testing allows the identification of distinct variables that influence the sensitivity of different T-cell assays and to formally show that a defined correction to the protocols successfully increases the sensitivity and reduces the inter-center variability. Such “two-step” inter-laboratory projects could define rational bases for accepted international guidelines and thereby lead to the harmonization of the techniques used for immune monitoring.

Response definition criteria for ELISPOT assays revisited

No consensus has been reached on how to determine if an immune response has been detected based on raw data from an ELISPOT assay. The goal of this paper is to enable investigators to understand and readily implement currently available methods for response determination. We describe empirical and statistical approaches, identifying the strengths and limitations of each approach to allow readers to rationally select and apply a scientifically sound method appropriate to their specific laboratory setting. Five representative approaches were applied to data sets from the CIMT Immunoguiding Program and the response detection and false positive rates were compared. Simulation studies were also performed to compare empirical and statistical approaches. Based on these, we recommend the use of a non-parametric statistical test. Further, we recommend that six medium control wells or four wells each for both medium control and experimental conditions be performed to increase the sensitivity in detecting a response, that replicates with large variation in spot counts be filtered out, and that positive responses arising from experimental spot counts below the estimated limit of detection be interpreted with caution. Moreover, a web-based user interface was developed to allow easy access to the recommended statistical methods. This interface allows the user to upload data from an ELISPOT assay and obtain an output file of the binary responses.

T cell assays and MIATA: the essential minimum for maximum impact.

The Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA*Correspondence: cedrik.britten@tron-mainz.dehttp://dx.doi.org/10.1016/j.immuni.2012.07.010The field of immunology has recentlyexperienced enormous advances fromwhich most have so far not been incorpo-rated into standard medical practice (Da-vis, 2008). One approach to fully exploitthe existing wealth of knowledge is toimplement a systematic strategy to eval-uate the immune system. The potentialbenefit of such an approach is that itmay lead to results that can be translatedinto the rational development of diagnos-tics and therapeutics (Hoos et al., 2011).Two prerequisites for its application arethat (1) applied immune assays lead toreproducible results (van der Burg et al.,2011), and (2) sets of experimental resultswill be reported and deposited in a waythat supports maximum use of data (Ja-netzki et al., 2009; Sansone et al., 2012).The task of establishing robust assayshas been shown to be particularly chal-lenging for cellular assays. This hasled to multiple independent activities toharmonize and standardize immuneassays(vanderBurgetal.,2011;Maeckeret al., 2010; Roep et al., 2012). Experi-ments conducted by more than 100 labsshowed that (1) a plethora of differentprotocols exists, (2) results generatedacrossinstitutionsvarygreatly,(3)multipleprocess steps contribute to test variation,(4) many labs performing T cell assays donot control critical process steps, and (5)the majority of reports comprising resultsfrom T cell assay experiments lack criticalinformation. At first these findings weremet with skepticism, given that theyseemed to contradict promising resultsshowing that cellular assays can consti-tute robust tools to quantify antigen-specific T cell responses. However, boththe optimistic and the pessimistic viewhave validity. T cell assays can delivercompelling performance in the hands ofskilled researchers. Nonetheless, pullingdata from heterogeneous groups of labsdemonstrates that controlling the perfor-mance of cellular assays requires explicitknowledgeoftheircriticalprotocoldetails.Notably,publicationslackingthosecriticalprotocol details may be difficult to inter-pret and to reproduce. Such reportingissues have the potential to hamper prog-ress in the field.To provide a solution, the Minimal Infor-mationaboutTCellAssays(MIATA)projectwas initiated in 2009 (Janetzki et al.,2009) as a field-spanning initiative whosemain objective is to generate a broadlyacceptable framework for the reporting ofresultsfromcommonlyusedTcellassays,which is envisioned to be expandable tonew, complex assays (Sharma et al.,2011; for a full list of MIATA contributors,see http://www.miataproject.org/index.php?option=com_content&view=article&id=4&Itemid=6).The generated MIATA guidelines pre-sented here are the outcome of one ofthe most intensive community-widevetting processes in the field of immu-nology so far (Britten et al., 2011). Thisprocess and all its steps are transparentlydisplayed at the project’s website (www.miataproject.org). Importantly, MIATA isImmunity 37, July 27, 2012 a2012 Elsevier Inc. 1

Novel Multi-Peptide Vaccination In Hla-A2+ Hormone Sensitive Patients With Biochemical Relapse of Prostate Cancer

A phase I/II trial was conducted to assess feasibility and tolerability of tumor associated antigen peptide vaccination in hormone sensitive prostate carcinoma (PC) patients with biochemical recurrence after primary surgical treatment.

Towards patient‐specific tumor antigen selection for vaccination

Summary: In this review, we discuss the possibilities for combining the power of molecular analysis of the antigens expressed in a given individual tumor with the design of a tailored vaccine containing defined antigens. Step 1 is a differential gene expression analysis of tumor and corresponding normal tissue. Step 2 is the analysis of human leukocyte antigen (HLA) ligands on tumor cells. Step 3 is data mining with the aim to select those antigens that might be suitable for tumor attack by the adaptive immune system. Step 4 is the on‐the‐spot clinical grade production of the constituents of the patient tailored vaccine, e.g. peptides. Step 5 is then vaccination and monitoring. Although it will not be possible to cover all relevant antigens expressed in a tumor, the antigens that can be identified with our present technical possibilities might be enough for improved immunotherapy. The scope of the present review is to explore the possibilities and the formidable technical and logistical challenge for such individual patient‐oriented antigen definition to be used for therapeutic immunization.

Unexpected Abundance of HLA Class II Presented Peptides in Primary Renal Cell Carcinomas

Purpose: To elicit a long-lasting antitumor immune response, CD8+ and CD4+ T cells should be activated. We attempted to isolate HLA-DR–presented peptides directly from dissected solid tumors, in particular from renal cell carcinoma, to identify MHC class II ligands from tumor-associated antigens (TAA) for their use in peptide-based immunotherapy. Experimental Design: Tumor specimens were analyzed by immunohistochemical staining for their HLA class II expression. HLA class II peptides were subsequently isolated and identified by mass spectrometry. Gene expression analysis was done to detect genes overexpressed in tumor tissue. Peptides from identified TAAs were used to induce peptide-specific CD4+ T-cell responses in healthy donors and in tumor patients. Results: In the absence of inflammation, expression of MHC class II molecules is mainly restricted to cells of the immune system. To our surprise, we were able to isolate and characterize hundreds of class II peptides directly from primary dissected solid tumors, especially from renal cell carcinomas, and from colorectal carcinomas and transitional cell carcinomas. Infiltrating leukocytes expressed MHC class II molecules and tumor cells, very likely under the influence of IFNγ. Our list of identified peptides contains ligands from several TAAs, including insulin-like growth factor binding protein 3 and matrix metalloproteinase 7. The latter bound promiscuously to HLA-DR molecules and were able to elicit CD4+ T-cell responses. Conclusions: Thus, our direct approach will rapidly expand the limited number of T-helper epitopes from TAAs for their use in clinical vaccination protocols.

Simultaneous Infiltration of Polyfunctional Effector and Suppressor T Cells into Renal Cell Carcinomas

Renal cell carcinoma is frequently infiltrated by cells of the immune system. This makes it important to understand interactions between cancer cells and immune cells so they can be manipulated to bring clinical benefit. Here, we analyze subsets and functions of T lymphocytes infiltrating renal cell tumors directly ex vivo following mechanical disaggregation and without any culture step. Subpopulations of memory and effector CD4(+) Th1, Th2, and Th17 and CD8(+) Tc1 cells were identified based on surface phenotype, activation potential, and multicytokine production. Compared with the same patients peripheral blood, T lymphocytes present inside tumors were found to be enriched in functional CD4(+) cells of the Th1 lineage and in effector memory CD8(+) cells. Additionally, several populations of CD4(+) and CD8(+) regulatory T cells were identified that may synergize to locally dampen antitumor T-cell responses.

Toward the harmonization of immune monitoring in clinical trials: Quo vadis?

A constantly increasing number of cancer immunotherapies are being investigated in clinical trials but no reliable biomarkers to predict clinical benefit currently exist. For some cancer types, biomarkers have proven to be meaningful predictors of patient outcomes (e.g., BCR-ABL in Chronic Myeloid Leukemia or PSA in prostate cancer) and were established as routine tools. As effects of cancer immunotherapy are mediated through the immune system, immune responses may act as natural biomarkers for clinical efficiency if the right factors can be reliably measured. Following this concept, cancer immunotherapy trials over the last decade have often included measures of tumor-specific cellular immune responses as endpoints to identify reliable surrogates for clinical benefit. Substantial efforts were invested throughout the immunotherapy field in setting up suitable cellular immune assays. However, to date, data from clinical trials do not consistently show that immune responses are correlated with clinical endpoints [1]. This lack of correlation is partially interpreted as a consequence of the high variability in assay results, due to the lack of assay standardization, validation and harmonization across laboratories. If harmonization of immune assays can be achieved, assays can be tested as surrogate endpoints in clinical trials, substantially accelerate the development of immunotherapeutic agents and, in addition, offer a rationale to pre-select groups of patients with high probability to benefit from subsequent immunotherapy. A plethora of different immunological assays to monitor antigen-specific T cell responses are available, and some are used by a majority of laboratories. Prime examples are the ELISPOT assay, intracellular cytokine staining (ICS), MHC-peptide multimer staining for detection of antigen-specific CD8+ and increasingly for CD4+ T cells and proliferation assays based on carboxyfluorescein succinimidyl ester (CFSE)-labelling. Additional functional assays that have been introduced more recently are based on CD154 up-regulation on activated antigen-specific CD4+ T cells or the detection of CD107a or cytotoxicity related molecules like perforin or granzyme in CD8+ T cells. For each of these assays, the use of different protocols that have evolved over time translates into a wide range of performances. Differences in the interpretation of the results obtained make comparisons of published data even more complex. This has led to lack of comparability as well as doubts about the integrity of the results that were obtained and published worldwide [2]. Recent developments clearly suggest that “proper” immunomonitoring will have to encompass the parallel use of several tests, which together assess the frequency, function and homing capacity of induced/stimulated T cells present in the circulation and preferentially also locally within the targeted tissue. In view of the influence of both natural and adaptive regulatory T cells on clinical outcome, immunomonitoring should also include screening for these cells. Only the combined use of several techniques may lead to a valid set of surrogate markers for specific immune interventions that allows clinical decisions or optimization of immunotherapeutic strategies. A related proposal was made by a community-wide consensus workshop on clinical trials with cancer vaccines, in which criteria for the use of immune assays were recommended including at least two validated assays to be used in parallel [3]. There is also a surprising disparity between the substantial financial and logistic efforts invested in reagents and man hours to develop immune monitoring techniques and the comparatively minor investment made to date in actually validating and standardizing the available techniques within and between laboratories. This has to be critically re-evaluated as efforts put in the adequate harmonization of assays will support the investments already made. They are also a pre-requisite to meet regulatory requirements on assay standards prior to them being considered as surrogate endpoints for clinical trials. This affects academic institutions and industry partners alike and may reach beyond cancer into the fields of autoimmune and infectious diseases.