Immune Functional Assays, From Custom to Standardized Tests for Precision Medicine

Abstract

The immune response is a dynamic system that maintains the integrity of the body, and more specifically fight against infections. However, an unbalanced host immune response is highlighted in many diseases. Exacerbated responses lead to autoimmune and allergic diseases, whereas, low or inefficient responses favor opportunistic infections and viral reactivations. Conflicting situations may also occur, such as in sepsis where inflammation and compensatory immunosuppression make it difficult to deploy the appropriate drug treatment. Until the current day, assessing the immune profile of patients remains a challenge. This is especially due to the inter-individual variability-a key feature of the immune system-which hinders precise diagnosis, prognosis, and therapeutic stratification. Our incapacity to practically interpret the host response may contribute to a high morbidity and mortality, such as the annual 6 million worldwide deaths in sepsis alone. Therefore, there is a high and increasing demand to assess patient immune function in routine clinical practice, currently met by Immune Functional Assays. Immune Functional Assays (IFA) hold a plethora of potentials that include the precise diagnosis of infections, as well as prediction of secondary and latent infections. Current available products are devoted to indirect pathogen detection such as Mycobacteria tuberculosis interferon gamma release assays (IGRA). In addition, identifying the status and the underlying factors of immune dysfunction (e.g., in septic patients) may guide immune targeted therapies. Tools to monitor and stratify the immune status are currently being studied but they still have many limitations such as technical standardization, biomarkers relevance, systematic interpretation and need to be simplified, in order to set the boundaries of "healthy," "ill," and "critically ill" responses. Thus, the design of new tools that give a comprehensive insight into the immune functionality, at the bedside, and in a timely manner represents a leap toward immunoprofiling of patients.

Keywords: IGRA; critically-ill patients; host response; immune functional assay; immune monitoring; immunoprofiling; sepsis; stimulation.

Figure 1

Interferon-gamma-based in vitro assays (IGRA). (A) Mycobacterium tuberculosis IFN-γ-release assay. In the ELISA method (QuantiFERON®-TB Gold In-Tube Test; Quest Diagnostics, USA), whole blood is stimulated with M. tuberculosis antigens, and the amount of IFN-γ secreted into the supernatant is quantified by ELISA. (B) In the ELISPOT method (T-SPOT.TB; Oxford Immunotec, UK), PBMCs are prepared by density gradient centrifugation (Ficoll method). A defined number of cells is then stimulated with M. tuberculosis antigens for 24 h on plates coated with anti-IFN-γ antibodies. Antigen-responsive cells secrete IFN-γ, which binds to these antibodies. After removal of the cells, antigens are detected by a second labeled anti-IFN-γ antibody. The number of spots on the plate corresponds to the number of IFN-γ+ cells in the sample.

Figure 2

“Ideal” Immune Function Assay. Many intrinsic factors contribute to the increased variability among subjects, such as genetic factors or vaccination record. The ideal IFA should be minimally impacted by those factors that create diversity in the physiological context. Taking into consideration the challenge to interpret results due to inter-individual diversity, technical factors, as minimal sample handling or robust platforms, can be optimized to allow reproducibility between tests and decrease the bias accumulated during the manipulation workflow.