Monitoring Antigen-specific T Cell Immunity by ELISPOT

By Cellular Technology Limited (CTL)

T and B lymphocytes can each generate immune responses to specific antigens. The resulting immunity helps in the prevention of infectious diseases and in rejection of cancer cells. Immune responses are not always beneficial. Allergies, including adverse drug reactions, autoimmune diseases, and transplant rejection are examples of unwanted immune responses. Activating or inactivating/deviating antigen-specific immunity is therefore a major goal in various therapeutic strategies. Clearly, measuring the impact of such treatments on the immune system is a primary concern for the developers of such vaccines/treatments. Until recently, assessments of induced immunity have been confined to detection of antigen-specific antibodies in serum. However, the presence of antibodies does not always necessarily translate into specific immunity with certain pathogens. For example, antibodies do not provide protection against many infections, including HIV and M. tuberculosis. Additionally, there is little to no involvement of antibodies in the rejection of cancer cells or transplants; likewise, antibodies do not play a significant role in many autoimmune diseases, nor in adverse drug reactions. Whereas T cells play the major role in mediating the immune responses in these conditions, reliable measurements of T cell immunity have, until recently, been next to impossible to produce. In the following, we will briefly outline the challenges that researchers in this field have faced and how we have overcome these difficulties at CTL.

Challenge 1: Working with live cell material
CTL’s novel cryopreservation strategies provide a reliable source of primary cells for use in T cell assays.
Serum antibodies are stable proteins that can be easily collected, shipped and stored until they are measured as proxies for B cell immunity. In contrast, measuring T cell immunity invariably involves testing live cells. As with all primary cell types, T cells are highly sensitive and need to be tested within hours after isolation from whole blood. Until recently, T cells could not be cryopreserved without major losses in recovery and functionality; this necessitated working with perishable fresh cells. For clinical trials, this was a logistical nightmare. It was necessary to process and test blood samples one-by-one at or in the vicinity of the collection site. This fact necessitated the set-up, training and maintenance of a laboratory for each clinical site participating in a study. While all scientists would agree that it is most relevant in immune-monitoring studies to measure T cell immunity, a large majority of trials have not done so due to the cost and effort involved in working with fresh cells. CTL has developed reagents, protocols and logistics for loss-free cryopreservation of peripheral blood mononuclear cells (PBMC). Our technique allows for the storage and eventual thawing of the cryopreserved PBMC for use in T cell assays; these thawed T cells display full functionality relative to the freshly isolated cells. Using CTL’s cryopreservation techniques, PBMC can be collected at any clinical site, frozen, and shipped for cost-efficient batch testing in CTL’s central laboratory.

Challenge 2: Detection of rare antigen-specific T cells
The ELISPOT assay can detect antigen-specific T cell responses at extremely low frequency with single-cell resolution.
With very few exceptions (e.g. acute infections), antigen-specific T cells occur in very low frequencies in the blood. For example, shortly after immunization with a protein such as tetanus toxoid (TT), the TT-specific T cells are typically < 1/10,000 in PBMC. As most flow cytometry-based assays reach their detection limits at 1/1,000 (with even the most sensitive assay systems reaching their limit at 1/10,000), such rare cells can not be reliably detected by tetramer/pentamer or intracytoplasmic cytokine staining. In contrast, ELISPOT assays provide reliable detection of T cells in frequencies as low as 1/1,000,000, thus reliably covering the range in which antigen-specific T cells occur in PBMC.

In ELISPOT assays, the test cell population rests in a monolayer on a membrane coated with the analyte-specific antibody. Thus, when an antigen-stimulated T cell secretes the analyte, it is captured directly around the secreting cell, producing a “spot”, which is visualized by subsequent secondary antibody staining. The proximity of the source of secretion (the T cell) to the capture and detection means (the membrane) results in the unprecedented sensitivity of ELISPOT. ELISA or cytokine bead arrays (CBA), in contrast, detect the analyte secreted into cell culture supernatant after the analyte has been greatly diluted. Detection in supernatant is further complicated due to the capture of the analyte by high-affinity receptors on bystander cells present in the assay, as well as cleavage of the analyte by proteases. For these reasons, ELISPOT assays are typically 500-fold more sensitive in detecting low frequency T cells than ELISA or CBA. Since CTL’s founding scientist first pioneered ELISPOT assays, ELISPOT has become the gold standard for detecting low frequency antigen-specific T cells in PBMC. CTL offers GLP-compliant, high or low throughput ELISPOT testing for humans, non-human primates, mice, pigs, and other species.

Challenge 3: T cell assay development, qualification and validation 
CTL has standardized the ELISPOT for regulated T cell assays in clinical trials.
Until recently, it was generally believed that T cell assays were too difficult to reproduce, and therefore could not be validated. This still holds true if one works solely with fresh cell material. The cells from each blood draw rapidly perish – each test is thus a “one shot” determination which can not be reproduced. Due to environmental stimuli, the composition of white blood cells can change at any time – even in healthy individuals. Re-testing by re-bleeding thus involves different cell material. The capability of cryopreserving PBMC from single blood draws, in multiple identical aliquots, allows for the reproducibility of assay results as well as refinement of test conditions. Furthermore, in addition to re-testing of samples, one can perform additional follow-up experiments to test the same cells under different assay conditions, to measure different analytes, or for use in parallel experiments. Reference PBMC can be generated which permit rapid assay development and qualification while working with aliquots of the identical cell material. Assays that have been validated in the past primarily included measurements of soluble molecules. T cell assays are an entirely different matter. Both the viability and functionality of the T cells need to be verified as well as that of the antigen presenting cells (APC). Further, both cell types must have optimal access to each other, and their functionality must be maintained during cell culture. Using ELISPOT, CTL was the first to validate a T cell assay, and is now doing so routinely for its clients.

Challenge 4: The multiplicity of T cell effector lineages
ELISPOT is capable of reliably detecting the many various T cell effector classes by measuring each specific effector molecule.
Naïve T cells differentiate into distinct effector classes. Interferon gamma (IFN-γ)-producing “Th1” T cells play a role in inhibiting viral replication. Interleukin 17 (IL-17) producing “Th17” cells mediate delayed type hypersensitivity (DTH), and as such are critical for controlling bacterial and fungal infections. Via IL-5 production, “Th5” cells recruit and activate eosinophils, thereby protecting against helminthes or mediating allergies. Cytolytic T cells secrete granzyme B and perforin to kill their target cells.

T cells occur in many distinct effector lineages – each releasing different molecules through which they carry out their specific functions. Each of these secreted molecules can be detected, at single cell resolution, by ELISPOT, to enumerate the T cells in each lineage. In this way, the “relevant” effector cell class can be monitored. A vaccine directed against tuberculosis would specifically trigger IL-17 producing T cells which mediate DTH. Likewise, an effective cancer vaccine would specifically induce cytolytic CD8 cells which secret perforin and granzyme B. CTL offers ELISPOT testing of all the above analytes, and more.

Recently, multi-cytokine expressing T cells have sparked interest as they seem to be associated with protective immunity in infections such as HIV. Cytokine co-expression can easily be studied by using double color ELISPOT assays, of which a representative image is shown above.

Challenge 5: Measurement of several analytes and their co-expression
CTL’s cryopreservation and ELISPOT assays are capable of testing multiple analytes from a uniform cell sample.
Often it is not known, and can not be predicted, which type of effector T cell lineage will be induced by a specific vaccine, nor is it known how the lineages are altered by therapeutic interventions. In such cases, it is wise to screen for analytes secreted by all classes of effector T cells. ELISPOT assays are highly efficient in cell utilization for such multi-plexed screening. Two analytes can be readily measured in as few as 100,000 PBMC (corresponding to 0.1 ml of whole blood) via double color analysis. Thus, a typical blood draw (e.g. 10 ml) can provide sufficient PBMC for repeated screening for multiple analytes and their co-expression. CTL can provide the experience and expertise in assisting the customizing of such assays for different needs.

Since T cells are involved in all aspects of immunity, scientists have known for decades that it is important to monitor T cell immunity. Recently, CTL has shown that this has become possible, and has developed simple, reliable and affordable techniques for use in clinical trials.