Prognostic Indicators after Musculoskeletal Injuries in Standardbred Racehorses in Italy.

The objective of this study was to report recovery time, retirement rate, racing performance, and presence of compensation pattern after musculoskeletal injuries (MSIs) in Standardbred racehorses (STBRs). This is a retrospective single open-cohort study, enrolling 356 STBRs in training in a single racetrack. Musculoskeletal injury was defined as any training-related injury after which the horse did not train for at least 15 days. The first and second MSIs encountered during the horse's racing career were considered. Medical records, training logbooks, and racing data were reviewed to determine recovery time, retirement rate, racing performance, and compensation pattern after MSIs. Kaplan-Meier estimators considered the return to activity as primary endpoint. The hazard ratio (HR) for return to activity was determined using Cox proportional hazard models, after classifying horses based on the injury type. A total of 543 MSIs were considered. The 33.6% (95% confidence limits [CL]: 29.5, 37.8) of STBRs suffering from MSI had to be retired as consequence of that. The median recovery time after MSI was 119 days (95% CL: 47, 179). Horses with traumatic osteoarthritis had a greater likelihood of returning to athletic activity compared with horses with stress fractures (HR = 4.8, 95% CL: 3.5, 6.7) and tendon/ligament strains (HR = 4.1, 95% CL: 3.1, 5.4). Increased racing speed was recorded after injuries. The second MSI was more often localized to the contralateral limb compared with the first injury (odds ratio (OR) 6.35, 95% CL: 4.29, 8.51), diagonal (OR 4.59, 95% CL: 3.05, 6.27) and to the same limb (OR 3.17, 95% CL: 2.03, 4.47) than to the ipsilateral limb.

Specific removal of alloreactive T-cells to prevent GvHD in hemopoietic stem cell transplantation: rationale, strategies and perspectives.

Hemopoietic stem cell transplantation (HSCT) is a standard procedure for treatment of malignant and non-malignant hematological diseases. HSCT donors include HLA-identical siblings, matched or mismatched unrelated donors and haploidentical related donors. Graft-versus-host disease (GvHD), mediated by donor alloreactive T-cells in the graft, can be triggered by minor histocompatibility antigens in HLA-identical pairs, by alleles at loci not considered for MUD-matching or by the mismatched haplotype in haplo-HSCT. Therefore, removal of donor T-cells, that contain the alloreactive precursors, is required, but T-cell depletion associates with opportunistic infections and with reduced graft-versus-leukemia effect. Selective T-cell depletion strategies have been introduced, like removal of αß T-lymphocytes and of naive T-cells, two subsets including the alloreactive precursors, but the ultimate goal is specific removal of alloreactive T-cells. Here we review the different approaches to deplete alloreactive T-cells only and discuss pros and cons, specificity, efficiency and efficacy. Combinations of different methods and innovative approaches are also proposed for depleting specific alloreactive T-cells with high efficiency.

A registry of HLA-typed donors for production of virus-specific CD4 and CD8 T lymphocytes for adoptive reconstitution of immune-compromised patients.

BACKGROUND: Virus-specific CD4 and CD8 T lymphocytes from HLA-matched donors are effective for treatment and prophylaxis of viral infections in immune-compromised recipients of hematopoietic stem cell transplant recipients. Adoptive immune reconstitution is based on selection of specific T cells or on generation of specific T-cell lines from the graft donor. Unfortunately, the graft donor is not always immune to the relevant pathogen or the graft donor may not be available (registry-derived or cord blood donors). STUDY DESIGN AND METHODS: Since the possibility of using T cells from a third-party subject is now established, we screened potential donors for T-cell responses against cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus, the viruses most frequently targeted by adoptive immune reconstitution. Specific T-cell responses against viral antigens were analyzed in 111 donors using a miniaturized interferon-γ release assay. RESULTS: Responders to CMV were 64%, to EBV 40%, and to adenovirus 51%. Simultaneous responders to the three viruses were 49%. CMV-specific CD4 and CD8 T-cell lines could be generated from 11 of 12 donors defined as positive responders according to the T-cell assay. CONCLUSIONS: These data demonstrate that a large fraction of volunteers can be recruited in a donor registry for selection or expansion of virus specific T cells and that our T-cell assay predicts the donors' ability to give rise to established T-cell lines endowed with proliferative potential and effector function for adoptive immune reconstitution.

Miniaturized and high-throughput assays for analysis of T-cell immunity specific for opportunistic pathogens and HIV.

Monitoring of antigen-specific T-cell responses is valuable in numerous conditions that include infectious diseases, vaccinations, and opportunistic infections associated with acquired or congenital immune defects. A variety of assays that make use of peripheral lymphocytes to test activation markers, T-cell receptor expression, or functional responses are currently available. The last group of assays calls for large numbers of functional lymphocytes. The number of cells increases with the number of antigens to be tested. Consequently, cells may be the limiting factor, particularly in lymphopenic subjects and in children, the groups that more often require immune monitoring. We have developed immunochemical assays that measure secreted cytokines in the same wells in which peripheral blood mononuclear cells (PBMC) are cultured. This procedure lent itself to miniaturization and automation. Lymphoproliferation and the enzyme-linked immunosorbent spot (ELISPOT) assay have been adapted to a miniaturized format. Here we provide examples of immune profiles and describe a comparison between miniaturized assays based on cytokine secretion or proliferation. We also demonstrate that these assays are convenient for use in testing antigen specificity in established T-cell lines, in addition to analysis of PBMC. In summary, the applicabilities of miniaturization to save cells and reagents and of automation to save time and increase accuracy were demonstrated in this study using different methodological approaches valuable in the clinical immunology laboratory.

Lymphocyte proliferation specific for recall, CMV and HIV antigens in miniaturized and automated format.

Lymphoproliferation assay (LPA) is used to test specific T-cell responses. LPA is performed in 96-well plates with 2-5×105 PBMC/well. In order to test numerous antigens, as in the case of epitope mapping or screening of antigenic panels from relevant pathogens, PBMC numbers may not be sufficient. We developed a miniaturized and automated procedure to perform LPA in 384- and 1536-well plates with one fourth to one twentieth of PBMC numbers used for standard assays. Here, we demonstrate that the procedure is reliable and robust using recall antigens and protein and peptide antigens from CMV and HIV. By using HIV specific T-cell lines, we also demonstrate that sensitivity ranges overlap with those of standard LPA and that as few as 3 specific cells/well provide a positive signal. This procedure is consistent with our policy to miniaturize assays for specific T-cell immunity, as we have already established for cytokine secretion assays.

Selective binding of CD4 and CD8 T-cells to antigen presenting cells for enrichment of CMV and HIV specific T-lymphocytes.

Adherent antigen presenting cells (APC) pulsed with protein or peptide antigens were used to capture specific CD4 or CD8 T-cells derived from established T-cell lines or from PBMC of immune subjects based on physiological interaction between TCR and MHC-peptide complex. This method could be applied independently of epitope specificity, HLA restriction alleles, activation markers and secreted cytokines, parameters required by other methods for selection of specific T cells. Non specific T-cells were removed by applying a 1g force that did not affect binding of specific T-lymphocytes. Lymphocyte selection was specific and the average recovery was 36% for CD4 T-cells. CD8 T-cells proved trickier to purify, since solid phase APC were recognized as targets for cytotoxicity. Specificity was comparable to CD4 cells, but the average recovery for CD8 cells was 26%. No residual alloreactivity was detected in expanded T-cells. Frequency and recovery of specific T-cells were comparable to other current technologies, such as generation of T-cell lines and cytokine capture method. Since antigen and IL2 are the only reagents added to the cultures, this physiological procedure can be proposed for selection and expansion of pathogen specific T-cells not only for research purposes, but also for adoptive reconstitution of immunocompromised subjects if performed under GMP conditions.

The PEDVAC trial: preliminary data from the first therapeutic DNA vaccination in HIV-infected children.

The PEDVAC study is the first trial designed to analyze safety and immunogenicity of a therapeutic vaccination with a multiclade multigene HIV DNA vaccine (HIVIS) in infected children. Twenty HIV-1 vertically infected children (6-16 years of age), on stable antiretroviral treatment for at least 6 months with HIV-1 RNA<50 copies/ml and stable CD4 counts (> 400 cells/mm³ or 25%) over 12 months of follow-up, were recruited into the study. Enrolled patients have been randomized into two arms: a control group of 10 children who continued previous antiretroviral treatment (HAART) (arm A) and a group of 10 children immunized intramuscularly with the HIVIS DNA vaccine in addition to previous HAART (arm B). Immunizations took place at week 0, 4, 12 and the boosting dose is planned at week 36. The 10 children in the vaccine group have received the first 3 priming doses of the HIVIS vaccine. Safety data showed good tolerance to the vaccination schedule. Mild cutaneous self-limeted reactions consisted of local irritation, usually itching or erythema +/- swelling at the injection site, were reported. No severe systemic adverse events have been observed. No vaccinated children had a decrease of CD4 T-cell counts from baseline. None experienced virological failure. Analysis of cellular immune responses was scheduled at week 0, 4, 12, 16, 20, 40, 60, 72 and 96 by standard lymphoproliferation assay, intracellular cytokine staining and cell-ELISA, a miniaturized assay to measure antigen-induced IFNγ secretion. Evaluation of these results is in progress and will provide key information on the status and changes of antigen specific immunity during HIV DNA immunization.

High throughput T epitope mapping and vaccine development.

Mapping of antigenic peptide sequences from proteins of relevant pathogens recognized by T helper (Th) and by cytolytic T lymphocytes (CTL) is crucial for vaccine development. In fact, mapping of T-cell epitopes provides useful information for the design of peptide-based vaccines and of peptide libraries to monitor specific cellular immunity in protected individuals, patients and vaccinees. Nevertheless, epitope mapping is a challenging task. In fact, large panels of overlapping peptides need to be tested with lymphocytes to identify the sequences that induce a T-cell response. Since numerous peptide panels from antigenic proteins are to be screened, lymphocytes available from human subjects are a limiting factor. To overcome this limitation, high throughput (HTP) approaches based on miniaturization and automation of T-cell assays are needed. Here we consider the most recent applications of the HTP approach to T epitope mapping. The alternative or complementary use of in silico prediction and experimental epitope definition is discussed in the context of the recent literature. The currently used methods are described with special reference to the possibility of applying the HTP concept to make epitope mapping an easier procedure in terms of time, workload, reagents, cells and overall cost.

Validation of a miniaturized assay based on IFNg secretion for assessment of specific T cell immunity.

A miniaturized method for detection of antigen induced secretion of IFNg by specific T cells cultured in 384 well plates has been recently reported. In order to confidently apply this assay to clinical investigations for monitoring of specific T cell immunity, an intralaboratory validation study has been undertaken. High reproducibility and linearity of reference curves was demonstrated. Consecutive replicate experiments handled by different operators using broad panels of recall antigens were reproducible when tested on individual biological samples. Kinetics of IFNg secretion with different antigens showed a plateau after 24h culture. Similar trends were observed with secretion of TNFa, GM-CSF and IL17, suggesting that the same kinetics can be applied if other cytokines are tested with this assay. It was demonstrated that frozen-thawed cells can be tested by cell-ELISA and that when PBMC are replaced by whole blood similar reactivity profiles were observed even though cytokine concentration was lower. T cell responses were higher in round bottom than in flat bottom wells, but these plates could not be applied to cell-ELISA as clear plates are not available for scanning. In conclusion, the assay proved flexible, since plates can be frozen at different times during the process, fresh or frozen PBMC and PBMC or whole blood could be used, and robust, since reproducibility was remarkable even when different operators performed the procedures.

Pathogen specific T-lymphocytes for the reconstitution of the immunocompromised host.

Cellular immune functions are impaired in hemopoietic stem cell and solid organ transplantation or in cancer and autoimmune diseases treated with intensified immunosuppression. Thus, control of opportunistic pathogens is lost and severe infections break out. Defective cellular immunity can be restored upon endogenous immunoreconstitution or, if delayed, exogenous immunoreconstitution with pathogen specific T-lymphocytes selected or expanded from appropriate donors can be applied. Here we describe how recent developments in basic immunology knowledge and techniques have accelerated progresses of clinical trials in this attractive field. In particular, methods for the identification of appropriate antigens, for selection and expansion of specific T-cells and for safer manipulation of cellular products have been applied with promising advances. Finally, the development of biobanks of specific T-cells is described as an attractive perspective to reconstruct pathogen specific cellular immunity.

Evaluation of antigen-specific T-cell responses with a miniaturized and automated method.

The evaluation of antigen-specific T-cell responses is helpful for both research and clinical settings. Several techniques can enumerate antigen-responsive T cells or measure their products, but they require remarkable amounts of peripheral blood mononuclear cells (PBMCs). Since screening numerous antigens or testing samples from pediatric or lymphopenic patients is hampered in clinical practice, we refined a miniaturized, high-throughput assay for T-cell immunity. Antigens and cells in 10-microl volumes were dispensed into 1,536-well culture plates precoated with anti-gamma interferon (anti-IFN-gamma) antibodies. After being cultured, the wells were developed by enzyme-linked immunosorbent assay for bound cytokine. Miniaturization and automation allowed quantitation of antigen-specific responses on 10(4) PBMCs. This method was applied for epitope mapping of mycobacterial antigens and was used in the clinic to evaluate T-cell immunity to relevant opportunistic pathogens by using small blood samples. A comparison with conventional methods showed similar sensitivity. Therefore, current flow cytometric methods that provide information on frequency and phenotype of specific T cells can be complemented by this assay that provides extensive information on cytokine concentrations and profiles and requires 20- to 50-fold fewer PBMCs than other analytical methods.

Positive selection and expansion of cytomegalovirus-specific CD4 and CD8 T cells in sealed systems: potential applications for adoptive cellular immunoreconstitution.

Administration of pathogen-specific T-cell lines can reconstitute the cellular immune function of immunocompromised patients. Selection and expansion of specific T cells for reinfusion pose unique challenges owing to the fact that good manufacturing procedures must be implemented. Cytokine secretion-based methods can identify and select specific T cells. We showed here that it is possible to combine this method with procedures for cell handling performed in a sealed, unbreached system from start to end. Peripheral blood mononuclear cells, obtained from blood samples and processed in a sealed system, were stimulated in Teflon bags with a library of selected CD4 and CD8 peptides derived from the immunodominant cytomegalovirus protein pp65. The stimulated T cells were labeled with reagents for interferon-gamma surface capture and selected on a magnetic column using a sealed system connected to the Teflon bags. Elution and final expansion were also performed with an unbreached protocol with preservation of sterility even if the steps were run on the bench top. Expanded cells exhibited the appropriate functions. The use of this unbreached procedure proves that safety of cellular products generated in a good manufacturing procedures facility can be further improved. Similar sealed protocols can also be applied for T-cell therapies directed against tumor antigens.

Antigenic properties of HCMV peptides displayed by filamentous bacteriophages vs. synthetic peptides.

Several efforts have been invested in the identification of CTL and Th epitopes, as well as in the characterization of their immunodominance and MHC restriction, for the generation of a peptide-based HCMV vaccine. Small synthetic peptides are, however, poor antigens and carrier proteins are important for improving the efficacy of synthetic peptide vaccines. Recombinant bacteriophages appear as promising tools in the design of subunit vaccines. To investigate the antigenicity of peptides carried by recombinant bacteriophages we displayed different HCMV MHCII restricted peptides on the capsid of filamentous bacteriophage (fd) and found that hybrid bacteriophages are processed by human APC and activate HCMV-specific CD4 T-cells. Furthermore we constructed a reporter T-cell hybridoma expressing a chimeric TCR comprising murine alphabeta constant regions and human variable regions specific for the HLA-A2 restricted immunodominant NLV peptide of HCMV. Using the filamentous bacteriophage as an epitope carrier, we detected a more robust and long lasting response of the reporter T-cell hybridoma compared to peptide stimulation. Our results show a general enhancement of T-cell responses when antigenic peptides are carried by phages.

Characterization of migratory activity and cytokine profile of helper and cytotoxic CMV-specific T-cell lines expanded by a selective peptide library.

OBJECTIVE: Reconstitution of cellular immunity by infusion of cytomegalovirus (CMV)-specific T lymphocytes is an attractive alternative to drugs currently used to control CMV reactivation in immunocompromised patients. For this purpose, we established a method for generating both anti-CMV CD4 and CD8 T cells following Good Manufacturing Practice indications, and we extensively characterized their immune functions. MATERIALS AND METHODS: For generating CD4 and CD8 CMV-specific lymphocytes, T cells from 11 CMV-seropositive donors were stimulated three times with dendritic cells (DC) pulsed with a library of selected CMV peptides, recognized by >85% of the Caucasian population. At the end of the culture, T cells were analyzed for their specificity, cytotoxicity, chemotactic migration, proliferation, and cytokine production. RESULTS: T cells were successfully expanded and enriched in CMV-specific subsets with an effector memory or an effector memory CD45 RA(+) phenotype. CMV-specific T-cell lines showed specific cytotoxicity (average lysis: 47%) against CMV peptides-pulsed DCs, and were depleted of auto- and alloreactivity. Moreover, the ability to proliferate following antigenic stimulation and the presence of functional CD4 lymphocytes producing Th1 and Th2 cytokines can ensure long-term antiviral immunity after in vivo injection. CMV-specific T lymphocytes also proved to be fully equipped to reach CMV-infected tissues, because they expressed CD49d and CCR1, CXCR3, CXCR4, necessary to recruit effector cells to inflamed sites. In accordance with this profile, they significantly migrated towards inflammatory chemokines and towards the supernatant collected from inflamed lung fibroblasts, frequently involved in CMV pathology. CONCLUSION: This strategy allows expansion of effector T cells capable to exert CD8 and CD4-mediated immune functions and, thus, is suitable for clinical use.

High throughput functional microdissection of pathogen-specific T-cell immunity using antigen and lymphocyte arrays.

The analysis of the human T-cell response specific for relevant pathogens is useful for diagnostic purposes and for research. Several methods enumerate antigen specific T-cells and measure their functions. Since screening of numerous antigens from pathogens is often needed to evaluate immunocompetence, lymphocytes, labor and cost are limiting factors. To examine pathogen-specific T-cell immunity, we have miniaturized the analysis of T-cell responses using an array approach in 384- and 1536-well plates with as few as 10 x 10(3) PBMC per well instead of the 500 x 10(3) PBMC used for current assays. Secreted cytokines were detected in the same wells used for lymphocyte cultures. The method can detect about ten CMV specific T-cells diluted into 50 x 10(3) PBMC (0.02%), and can quantify secreted cytokines. The microarray approach allowed evaluation of T-cell immunity in children with a sensitivity higher than current methods. When applied to CMV epitope mapping, the data obtained with conventional methods were confirmed. The assay could be automated, allowing high throughput processing. The assay provides quantitative information on cytokines induced by antigen stimulation and can be applied in a simplified format as a field test to monitor T-cell immunity in vaccine trials or in veterinary medicine.

Measurement of antigen specific immune responses: 2006 update.

Measuring antigen-specific immune responses (MASIR) is essential for basic immunological research and in the clinical setting. Numerous techniques have been used and the recent years have witnessed a flourishing of flow cytometry based methods for the identification of antigen specific T cells, in addition to other methodologies. The second MASIR conference held in Santorini, Greece, from 14 to 18 June 2006 has been a forum for the discussion of methodological issues and for research or clinical applications of these techniques, as reviewed here. In addition to flow cytometry based techniques, other emerging techniques with different degrees of complexity can be applied. These novel methods are highly promising in numerous conditions to look for correlates of protection, to test responses to natural infections or to vaccination trials, to evaluate the immune status of immunocompromised patients and to monitor persistence and function of specific T cells administered as adoptive therapy.

Comparative analysis of new innovative vaccine formulations based on the use of procaryotic display systems.

A T helper epitope was expressed in three innovative delivery vehicles recently developed in our laboratories and based respectively, on the filamentous bacteriophage fd, the E2 protein from the PDH complex of Bacillus stearothermophilus and the protein CotC of Bacillus subtilis spores. Studies of antigenicity and immunogenicity were performed by using a specific T cell hybridoma and by priming mononuclear cells isolated from the venous blood of human donors. The results indicate that the E2 system is the best suited for inducing a specific immune response towards a CD4 T cell epitope. Importantly, TCR clonal analysis demonstrated the persistence over years of a previously described antigen specific clonotype and its presence correlates with the immunogenic strength of the antigen delivery system.

Helper function of cytolytic lymphocytes: switching roles in the immune response.

T helper (Th) cells and cytolytic T lymphocytes (CTL) play defined roles in the cellular immune response. This distinction wavered when Th lymphocytes were shown to kill antigen-presenting cells displaying the relevant antigen. Here we demonstrate that also the opposite can be true: CTL can exert helper functions. We noticed that certain CMV-specific CTL lines grew after antigen activation also without exogenous IL-2. These lines produced their own IL-2, which supported the expansion of other CTL and Th cell lines. High levels of helper cytokines like IL-4, IL-5 and IL-6 were detected in the culture supernatants. Thus, we set up a helper assay to study the functional interactions between T cells (or their supernatants) and B cells. Conditioned media from helper CTL lines induced secretion of antigen-specific antibodies by B cells pulsed with antigen as first signal. We conclude that it is possible to isolate CTL lines that exhibit helper functions for T cells and B cells. If this possibility is proven also in vivo, we should revise some of our views on the pathogenesis of diseases in which CD8 cells are key players, such as in viral infections, graft rejection and GVHD.

Human bone marrow stromal cells hamper specific interactions of CD4 and CD8 T lymphocytes with antigen-presenting cells.

Bone marrow stromal cells (BMSCs) may inhibit T-cell functions in vitro and thus have been proposed as immunoregulators to control in vivo graft-versus-host disease (GVHD) in haploidentical hemopoietic stem cell transplants. To better investigate this phenomenon, we used a defined experimental system in which responding T cells are antigen-specific and devoid of alloreactivity against BMSC from a different subject. Thus, we established antigen-specific human CD4 and CD8 T-cell lines as the readout system. Antigen-dependent proliferation was reduced with both T-cell subsets cultured on confluent BMSCs, and also on confluent human skin fibroblasts (HSF) inhibited T-cell proliferation with similar efficiency. Morphological observations of the cocultures showed impairment of physical interactions between T-cell and antigen-presenting cells in the presence of BMSC, with lack of formation of antigen-dependent clusters of T cells and antigen-presenting cells (APCs). In contrast, no effects were seen with BMSC-conditioned medium. Since suppression was seen only with confluent mesenchymal cells, this phenomenon may not be relevant in vivo, where BMSCs are at low frequency. In addition, if the reported suppressive effect of BMSCs on GVHD in vivo is confirmed, a different in vitro system should be envisaged to better understand and exploit the underlying mechanism.