A model for harmonizing flow cytometry in clinical trials.

Complexities in sample handling, instrument setup and data analysis are barriers to the effective use of flow cytometry to monitor immunological parameters in clinical trials. The novel use of a central laboratory may help mitigate these issues.

The mechanism of action of tofacitinib - an oral Janus kinase inhibitor for the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterised by infiltration of immune cells into the affected synovium, release of inflammatory cytokines and degradative mediators, and subsequent joint damage. Both innate and adaptive arms of the immune response play a role, with activation of immune cells leading to dysregulated expression of inflammatory cytokines. Cytokines work within a complex regulatory network in RA, signalling through different intracellular kinase pathways to modulate recruitment, activation and function of immune cells and other leukocytes. As our understanding of RA has advanced, intracellular signalling pathways such as Janus kinase (JAK) pathways have emerged as key hubs in the cytokine network and, therefore, important as therapeutic targets. Tofacitinib is an oral JAK inhibitor for the treatment of RA. Tofacitinib is a targeted small molecule, and an innovative advance in RA therapy, which modulates cytokines critical to the progression of immune and inflammatory responses. Herein we describe the mechanism of action of tofacitinib and the impact of JAK inhibition on the immune and inflammatory responses in RA.

Development of immunotoxicity testing strategies for immunomodulatory drugs.

The ICH S8 immunotoxicity testing guideline for human pharmaceuticals was published in 2006 and was intended to provide guidance for assessing the immunotoxicity potential of low-molecular-weight drugs that are not intended to alter the immune system. For drugs intended to modulate the immune system, immunotoxicity testing strategies are generally developed on a case-by-case approach since the targets, intended patient population, and mechanisms of action of the test compound will determine the type of testing needed. Some of the general principles of ICH S8, however, may be applied to immunotoxicity testing strategies for immunomodulatory drugs. A weight-of-evidence approach using factors discussed in ICH S8 in concert with an assessment of the potential value of additional immunotoxicity testing should be considered. For most situations, immunotoxicity studies with immunomodulatory compounds evaluate off-target effects on the immune system and exaggerated pharmacology. The potential use of data from these studies and considerations such as translatability to humans are discussed.

Immunological responses to exogenous insulin.

Regardless of purity and origin, therapeutic insulins continue to be immunogenic in humans. However, severe immunological complications occur rarely, and less severe events affect a small minority of patients. Insulin autoantibodies (IAAs) may be detectable in insulin-naive individuals who have a high likelihood of developing type 1 diabetes or in patients who have had viral disorders, have been treated with various drugs, or have autoimmune disorders or paraneoplastic syndromes. This suggests that under certain circumstances, immune tolerance to insulin can be overcome. Factors that can lead to more or less susceptibility to humoral responses to exogenous insulin include the recipient's immune response genes, age, the presence of sufficient circulating autologous insulin, and the site of insulin delivery. Little proof exists, however, that the development of insulin antibodies (IAs) to exogenous insulin therapy affects integrated glucose control, insulin dose requirements, and incidence of hypoglycemia, or contributes to beta-cell failure or to long-term complications of diabetes. Studies in which pregnant women with diabetes were monitored for glycemic control argue against a connection between IAs and fetal risk. Although studies have shown increased levels of immune complexes in patients with diabetic microangiopathic complications, these immune complexes often do not contain insulin or IAs, and insulin administration does not contribute to their formation. The majority of studies have shown no relationship between IAs and diabetic angiopathic complications, including nephropathy, retinopathy, and neuropathy. With the advent of novel insulin formulations and delivery systems, such as insulin pumps and inhaled insulin, examination of these issues is increasingly relevant.

Immunogenicity of biologically-derived therapeutics: assessment and interpretation of nonclinical safety studies.

An evaluation of potential antibody formation to biologic therapeutics during the course of nonclinical safety studies and its impact on the toxicity profile is expected under current regulatory guidance and is accepted standard practice. However, approaches for incorporating this information in the interpretation of nonclinical safety studies are not clearly established. Described here are the immunological basis of anti-drug antibody formation to biopharmaceuticals (immunogenicity) in laboratory animals, and approaches for generating and interpreting immunogenicity data from nonclinical safety studies of biotechnology-derived therapeutics to support their progression to clinical evaluation. We subscribe that immunogenicity testing strategies should be adapted to the specific needs of each therapeutic development program, and data generated from such analyses should be integrated with available clinical and anatomic pathology, pharmacokinetic, and pharmacodynamic data to properly interpret nonclinical studies.

Covalent binding of the nitroso metabolite of sulfamethoxazole is important in induction of drug-specific T-cell responses in vivo.

Immune-mediated drug hypersensitivity reactions (IDHRs) represent a significant problem due to their unpredictable and severe nature, as well as the lack of understanding of the pathogenesis. Sulfamethoxazole (SMX), a widely used antibiotic, has been used as a model compound to investigate the underlying mechanism of IDHRs because it has been associated with a relatively high incidence of hypersensitivity. Previous studies by others showed that administration of 4-(nitroso)-N-(5-methyl-1,2-oxazol-3-yl)benzenesulfonamide (SMX-NO), the reactive metabolite of SMX, to rats resulted in the generation of SMX-specific antibodies and ex vivo splenocyte proliferative responses, as well as haptenation of skin keratinocytes, circulating peripheral blood mononuclear cells, and splenocytes. The objective of the present study was to further investigate SMX-NO-protein binding in relationship to its immunogenicity. In female DBA/1 mice treated with SMX-NO, varying degrees of SMX-NO-dependent T-cell responses and SMX-NO-protein adduct formation were observed in the spleen and in inguinal, brachial, and axillary lymph nodes. The data suggested a tissue-specific threshold of SMX-NO dosage that triggers the detection of adducts and immune response. Furthermore, serum albumin and immunoglobulin were identified as protein targets for SMX-NO modification. It seemed that these adducts were formed in the blood, circulated to lymphoid tissues, and initiated SMX-NO-dependent immune responses. Collectively, these data revealed a causal link between the deposition of SMX-NO-protein adducts in a lymphoid tissue and the induction of immune response in that tissue. Our findings also suggest that the immunogenicity of SMX-NO is determined by the immunogenic nature of the hapten, rather than special characteristics of the adducted protein.

Optimization and validation of a flow cytometric method for immunophenotyping peripheral blood lymphocytes from cynomolgus monkeys (Macaca fascicularis).

BACKGROUND: Guidelines published by the Food and Drug Administration and Center for Human Medicinal Products describe the need to assess immunotoxic effects in nonclinical studies that evaluate drug toxicity, including the use of immunophenotyping to measure immunotoxicity. We are not aware of previous studies, however, that have validated methods for immunophenotyping peripheral blood lymphocyte subsets in whole blood samples from cynomolgus monkeys. OBJECTIVE: The purpose of this study was to optimize and validate a flow cytometric assay for immunophenotyping lymphocytes in the peripheral blood of cynomolgus monkeys. METHODS: A series of prevalidation experiments were done to determine optimal reagents, volumes, timing, and other procedural details of the flow cytometric assay. Using the optimized method, we then determined precision, interindividual variation, laboratory-to-laboratory variability, and sample stability. Stabilized human blood was used as a positive control for staining, processing, and analysis. The percentage and number of pan-T cells (CD3+), T-helper cells (CD3+4+), T cytotoxic/suppressor cells (CD3+8+), natural killer cells (CD3-16+), and B-cells (CD3-20+) were determined in 146 male and 140 female, clinically healthy monkeys and reference intervals were calculated. RESULTS: By doing 4-color staining with a lyse-wash method, intra- and interassay precision were <5% for all lymphocyte subsets. Variability between technicians and laboratories was minimal (CVs<3%). Samples were stable for up to 24 hours after staining and fixing. CONCLUSIONS: The validated method is extremely robust and can be performed under good laboratory practice conditions to support nonclinical studies. Reference intervals for lymphocyte subsets were similar to those previously reported.

Recommendations for the design, optimization, and qualification of cell-based assays used for the detection of neutralizing antibody responses elicited to biological therapeutics.

The administration of biological therapeutics can evoke some level of immune response to the drug product in the receiving subjects. An immune response comprised of neutralizing antibodies can lead to loss of efficacy or potentially more serious clinical sequelae. Therefore, it is important to monitor the immunogenicity of biological therapeutics throughout the drug product development cycle. Immunoassays are typically used to screen for the presence and development of anti-drug product antibodies. However, in-vitro cell-based assays prove extremely useful for the characterization of immunoassay-positive samples to determine if the detected antibodies have neutralizing properties. This document provides scientific recommendations based on the experience of the authors for the development of cell-based assays for the detection of neutralizing antibodies in non-clinical and clinical studies.

Insulin antibodies with pulmonary delivery of insulin.

BACKGROUND AND METHODS: Delivery of insulin to the deep lung presents unique challenges to the body's mucosal defense system. Pulmonary mucosal defense has the ability to discriminate between self and non-self antigens and has the potential for induction of immunologic tolerance. Published data concerning the immunogenicity of inhaled human insulin in drug trials will be reviewed, and data regarding the possible adverse effects of anti-insulin antibody development will be presented. Examination of the immunologic safety of inhaled human insulin will include discussion of comparator studies, factors affecting immunogenicity, the effects of insulin immunity on glycemic control and pulmonary function, and the relationship of insulin antibodies to dose requirements, pharmacodynamics, and hypoglycemia. CONCLUSIONS: Inhaled human insulin, whether formulated as a powder or liquid, has been shown to be more immunogenic than comparator insulins given by subcutaneous routes; however, adverse effects of antibody formation have not been demonstrated.

Synthesis and in vitro evaluation of 18F- and 19F-labeled insulin: a new radiotracer for PET-based molecular imaging studies.

A new and regioselective strategy was developed for the preparation of fluorine-18-labeled insulin as a novel positron emission tomography (PET) tracer. [18F]-4-Fluorobenzoic acid (4-18FBA), which was produced in 83 +/- 8% yield (n = 10), through the use of succinimidyl [18F]-4-fluorobenzoate (4-(18)FSB), was conjugated through a short spacer (6-aminohexanoic acid, AHx) to the PheB1 residue of a protected form of insulin. 18FB-AHx-insulin (8b) was repeatedly prepared in practical quantities (10-20 mCi, 370-740 MBq) in good radiochemical yield (9 +/- 5%, n = 9) and in a specific activity of 7.8 mCi/micromol. The final product was characterized by comparing the radioHPLC and radioTLC of 8b with that of the 19F-analogue (19FB-AHx-insulin, 8a) and by analyzing a carrier-added synthesis by mass spectrometry. Dithiothreitol and endoproteinase Glu-C digestion experiments on 8a confirmed that the prosthetic group was in fact conjugated to the PheB1 residue. An insulin receptor (IR) phosphorylation assay using CHO-hIR cells overexpressing recombinant human insulin receptors indicated no statistical difference in the extent of autophosphorylation stimulated by 8a as compared to that for human insulin (EC50 values of 0.82 nM and 1.0 nM, respectively). The stimulation of 2-deoxyglucose uptake in 3T3-L1 mouse adipocytes utilizing 8a versus unmodified human insulin gave similar EC50 values of 0.68 nM and 0.41 nM, respectively. The IC50 values for 8a versus native insulin for the displacement of 125I-insulin from HEK-293 cells were also the same within experimental error (2.6 nM for 8a versus 2.4 nM for unmodified human insulin). These results support the use of the 18F-insulin analogue as a PET tracer for imaging the distribution of insulin in vivo.

Assessing the potential to induce respiratory hypersensitivity.

Acute and repeat dose inhalation studies have been an important part of the safety assessment of drugs, chemicals, and other products throughout the world for many years. It is known that damage to the respiratory tract can be triggered either by nonspecific irritation or by specific immune-mediated pathogenesis, and it is acknowledged that traditional inhalation studies are not designed to address fully the impact of the latter. It is also recognized that different types of immune-mediated responses can be triggered by different classes of compounds and that some immune reactions in the lung are life threatening. As such, it is important to understand as fully as possible the basis for the immune-mediated damage to the lung in order to characterize adequately the risks of individual chemicals or proteins. It is against this background that a review of the methods used to assess the potential for immune-mediated respiratory hypersensitivity was conducted. The primary objectives of this review are to discuss appropriate methods for identifying and characterizing respiratory hypersensitivity hazards and risks; and to identify key data gaps and related research needs with respect to respiratory hypersensitivity testing. The following working definition of respiratory hypersensitivity was formulated: a hypersensitivity response in the respiratory tract precipitated by a specific immune response, mediated by multiple mechanisms, including IgE antibody. Because of the importance played by various classes of compounds, the subsequent sections of this review will consider protein-specific, chemical-specific, and drug-specific aspects of respiratory hypersensitivity.

Characterization of the draining lymph node response in the mouse drug allergy model: A model for drug hypersensitivity reactions.

The mouse drug allergy model (MDAM) was developed as a tool to predict the potential of systemically administered drugs to produce hypersensitivity reactions (HR). Drugs associated with HR in the clinic produce a marked increase in the cellularity of the draining lymph nodes (DLN) in the MDAM. The objective of this study was to characterize the changes in the DLN following exposure to drugs associated with HR and to investigate whether lymphocyte migration and/or proliferation play a role in the response. These endpoints were also investigated in the local lymph node assay (LLNA) to determine whether responses between the two assays occur via similar mechanisms. Results demonstrated that total numbers of T- and B-cells were proportionally increased in the DLN of mice treated with positive control drugs (i.e. abacavir, amoxicillin, ofloxacin, and sulfamethoxazole) compared to animals administered the vehicle or negative control drugs (metformin and cimetidine). In contrast, a significant increase in the B-cell population of the DLN was observed for 2,4-dinitrofluorobenzene (DNFB) following the LLNA protocol. Down-regulation of CD62L and up-regulation of CCR7 were observed for T-cells from the DLN of the positive control treated mice in the MDAM, but not with DNFB in the LLNA. A mild increase in T-cell proliferation was observed in the MDAM with positive control drugs, while DNFB in the LLNA induced proliferation within the B-cell population only. Anti-CD40L antibody administration inhibited MDAM responses to positive control drugs, but did not affect DNFB-induced increases in total cell number in the LLNA. These results suggest that the increased cellularity of the DLN in the MDAM may be the result of drug-induced alterations in lymphocyte migration and/or effects on lymphocyte proliferation. Moreover, it appears that different mechanisms may be involved in driving the MDAM and LLNA responses.

Development of a fluorescence-based in vivo phagocytosis assay to measure mononuclear phagocyte system function in the rat.

The mononuclear phagocyte system (MPS) which provides protection against infection is made up of phagocytic cells that engulf and digest bacteria or other foreign substances. Suppression of the MPS may lead to decreased clearance of pathogenic microbes. Drug delivery systems and immunomodulatory therapeutics that target phagocytes have a potential to inhibit MPS function. Available methods to measure inhibition of MPS function use uptake of radioactively-labeled cells or labor-intensive semi-quantitative histologic techniques. The objective of this work was to develop a non-radioactive quantitative method to measure MPS function in vivo by administering heat-killed E. coli conjugated to a pH-sensitive fluorescent dye (Bioparticles(®)). Fluorescence of the Bioparticles(®) is increased at low pH when they are in phagocytic lysosomes. The amount of Bioparticles(®) phagocytosed by MPS organs in rats was determined by measuring fluorescence intensity in livers and spleens ex vivo using an IVIS(®) Spectrum Pre-clinical In Vivo Imaging System. Phagocytosis of the particles by peripheral blood neutrophils was measured by flow cytometry. To assess method sensitivity, compounds likely to suppress the MPS [clodronate-containing liposomes, carboxylate-modified latex particles, maleic vinyl ether (MVE) polymer] were administered to rats prior to injection of the Bioparticles(®). The E. coli particles consistently co-localized with macrophage markers in the liver but not in the spleen. All of the compounds tested decreased phagocytosis in the liver, but had no consistent effects on phagocytic activity in the spleen. In addition, administration of clodronate liposomes and MVE polymer increased the percentage of peripheral blood neutrophils that phagocytosed the Bioparticles(®). In conclusion, an in vivo rat model was developed that measures phagocytosis of E. coli particles in the liver and may be used to assess the impact of test compounds on MPS function. Still, the detection of inhibition of splenic macrophage function will require further assay development.

Development and validation of radioligand binding assays to measure total, IgA, IgE, IgG, and IgM insulin antibodies in human serum.

Radioligand binding assays for total and Ig classes of insulin antibodies (IAB) were developed and validated. For each assay, insulin-extracted serum samples were incubated with radiolabeled insulin in the presence and absence of high levels of unlabeled insulin to determine nonspecific binding and total binding, respectively. To measure total IAB, antibody-bound insulin was precipitated with a polyethylene glycol solution, washed, and counted in a gamma-counter. To measure IgG IAB, samples were treated with protein G-Sepharose beads, centrifuged, washed, and counted. For the measurement of IgA, IgE, and IgM IAB, IgG was removed from the samples and treated with anti-IgA, -IgE, or -IgM conjugated to Sepharose beads, centrifuged, washed, and counted. The acid/charcoal extraction of bound and unbound insulin from serum samples was optimized. Specificity and binding capacity of the protein G and antibody-bound beads were evaluated and optimized. The linear region of the total and IgG IAB assays was determined using serum samples containing high levels of insulin antibodies. The limit of quantitation, limit of detection, and precision for all the assays were also determined.

Development and partial validation of a mouse model for predicting drug hypersensitivity reactions.

Animal models that can be used to predict the allergenic potential of drug candidates have not been adequately optimized, validated, or characterized. While initial validation data from an inter-laboratory study of the mouse lymph node proliferation assay (LNPA) appeared promising, no additional investigations in this model have been reported. The objectives of this study were to use positive and negative control drugs to further optimize and validate the LNPA utilizing a non-radioactive endpoint and determine the sensitivity, specificity, and predictivity of the model. Drugs associated with hypersensitivity reactions in the literature were chosen to test in the model in addition to drugs with few or no reports of hypersensitivity. Mice received a subcutaneous injection of drug or vehicle into the scruff of the neck once daily for a period of 3 days. On Day 6, draining lymph nodes were harvested, single cell suspensions prepared, and total cell numbers determined for each animal by flow cytometry. A stimulation index was calculated by dividing the mean total cell number for the drug-treated group by the mean total cell number for the vehicle-treated animals. Based on statistical analysis of the data, animals with a total cell number ≥2.5× the mean of the vehicle group were classified as 'responders'. Based on data generated to date with 12 positive control and six negative control drugs, the model had a sensitivity of 75%, a specificity of 74%, and a relatively good predictive value (measured by the Receiver Operating Characteristic AUC of 0.80). The data here suggest that this model may be a useful tool for identifying drug candidates with the potential to produce allergic responses in the clinic. Future studies will investigate the mechanism(s) for the lymph node responses in order to develop additional endpoints that may increase the sensitivity and specificity of the model.

Development of a screening assay to evaluate the potential of drugs to cause immune-mediated hypersensitivity reactions.

Evidence suggests that bio-activation of drugs to generate chemically reactive metabolites (RM) that act as haptens to form immunogenic protein conjugates may be an important cause of immune-mediated drug hypersensitivity reactions (IDHR). Although many drugs that form RMs raise concerns about producing IDHR, standard non-clinical testing methods are rarely able to identify compounds with the potential to produce IDHR in humans. The objective of this study was to develop a predictive assay for IDHR that involves: (1) the use of an in vitro drug-metabolizing system to generate the RM that is captured by GSH, (2) conjugating the RM-GSH conjugate to mouse serum albumin (MSA) by using a chemical cross-linker, (3) immunization of mice with RM-GSH-MSA adducts, and (4) ex vivo challenge with RM-GSH-MSA adduct and measurement of lymphocyte proliferation to determine if the RM is immunogenic. The predictivity of the assay was evaluated by using drugs that produce RM and have been strongly, weakly, or not associated with IDHRs in the clinic. While this method requires additional validation with more drugs, the results demonstrate the feasibility of identifying drugs strongly associated with IDHR and the utility of the assay for rank ordering drugs with respect to their potential to cause IDHR.

Measuring T-cell responses against LCV and CMV in cynomolgus macaques using ELISPOT: potential application to non-clinical testing of immunomodulatory therapeutics.

A number of immunomodulatory therapeutics increase the risk of disease associated with latent herpesviruses such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV), a member of the lymphocryptovirus (LCV) family that infects humans. The diseases associated with loss of immunity to these viruses can have major impacts on patients as well as on the commercial viability of the immunomodulatory therapeutics. In an effort to develop non-clinical methods for measuring effects on anti-viral immunity, we have developed an interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISPOT) assay to quantify the number of CMV or LCV-reactive T-cells in peripheral blood of cynomolgus macaques. After optimization of various parameters, the IFN-γ ELISPOT assay was characterized for specificity, intra-assay, monkey-to-monkey, and longitudinal variability and sensitivity to immunosuppression. The results show that nearly all animals have detectable responses against both CMV and LCV and responses were derived from T-cells specific to the virus of interest. Analyses of variability show assay reproducibility (≤23% CV), and that variability over time in anti-viral responses in individual animals (larger for LCV than for CMV) was ∼2-fold in most animals over a 3-month time period, which is predicted to allow for detection of drug-induced changes when using group sizes typical of non-clinical studies. In addition, the IFN-γ ELISPOT assay was capable of detecting decreases in the numbers of CMV and LCV reactive T-cells induced by immunosuppressive drugs in vitro. This assay may allow for non-clinical assessment of the effects of immunomodulatory therapeutics on anti-viral T-cell immunity in monkeys, and may help determine if therapeutics increase the risk of reactivating latent viral infections.

Herpes zoster and tofacitinib therapy in patients with rheumatoid arthritis.

OBJECTIVE: Patients with rheumatoid arthritis (RA) are at increased risk for herpes zoster (HZ) (i.e., shingles). The aim of this study was to determine whether treatment with tofacitinib increases the risk of HZ in patients with RA. METHODS: HZ cases were identified as those reported by trial investigators from the databases of the phase II, phase III, and long-term extension (LTE) clinical trials in the Tofacitinib RA Development Program. Crude incidence rates (IRs) of HZ per 100 patient-years (with 95% confidence intervals [95% CIs]) were calculated by exposure group. Logistic regression analyses were performed to evaluate potential risk factors for HZ (e.g., age, prednisone use). RESULTS: Among 4,789 participants, 239 were identified as having tofacitinib-associated HZ during the phase II, phase III, and LTE trials, of whom 208 (87%) were female and whose median age was 57 years (range 21-75 years). One HZ case (0.4%) was multidermatomal; none of the cases involved visceral dissemination or death. Twenty-four patients with HZ (10%) permanently discontinued treatment with tofacitinib, and 16 (7%) were either hospitalized or received intravenous antiviral drugs. The crude HZ IR across the development program was 4.4 per 100 patient-years (95% CI 3.8-4.9), but the IR was substantially higher within Asia (7.7 per 100 patient-years, 95% CI 6.4-9.3). Older age was associated with HZ (odds ratio 1.9, 95% CI 1.5-2.6), and IRs for HZ were similar between patients receiving 5 mg tofacitinib twice daily (4.4 per 100 patient-years, 95% CI 3.2-6.0) and those receiving 10 mg twice daily (4.2 per 100 patient-years, 95% CI 3.1-5.8). In the phase III trials among placebo recipients, the incidence of HZ was 1.5 per 100 patient-years (95% CI 0.5-4.6). CONCLUSION: In the Tofacitinib RA Development Program, increased rates of HZ were observed in patients treated with tofacitinib compared with those receiving placebo, particularly among patients within Asia. Complicated HZ among tofacitinib-treated patients was rare.

Summary of roundtable discussion meeting: non-human primates to assess risk for EBV-related lymphomas in humans.

Epstein-Barr virus (EBV)-associated lymphomas are a known risk for immunosuppressed individuals. Non-clinical methods to determine the potential of new immunomodulatory compounds to produce EBV-associated lymphomas (hazard identification) have not been developed. Since lymphocryptovirus (LCV) in non-human primates (NHP) has similar characteristics to EBV in humans, a Roundtable meeting was held in October 2010 to explore how the potential for EBV-related lymphomas in humans can be assessed by using surrogate biomarkers for lymphoma risk in NHP toxicity studies. Stakeholders from regulatory agencies, academia, and industry came together to determine the research gaps and potential benefits and considerations of such an approach given the current state-of-the-science. Key conclusions from the discussion included considerations raised about the potential usefulness of LCV-related biomarkers from NHP studies since there is significant controversy over the reliability of using EBV viral load or EBV-specific T-lymphocytes to predict for lymphoproliferative disorders in transplant patients. In addition, there are technical challenges that need to be further addressed in order to develop methods to measure LCV viral load and LCV-specific T-lymphocytes from cynomolgus monkeys.