It is essential to identify donors who have not been infected with human cytomegalovirus (HCMV) in order to avoid transmission of HCMV to recipients of blood transfusions or organ transplants. In the present study, we tested the reliability of seronegativity as an indicator for the lack of HCMV exposure in healthy human blood donors. Eighty-two HCMV seronegative individuals were identified, and their peripheral blood mononuclear cells (PBMC) were tested in ImmunoSpot® assays for the presence of HCMV-specific T- and B-memory lymphocytes. Eighty-two percent (67 of 82) of these HCMV seronegative individuals featured at least one memory cell that was lineage specific for HCMV, with the majority of these subjects possessing CD4+ and CD8+ T cells, as well as B cells, providing three independent lines of evidence for having developed immunity to HCMV. Only 15 of these 82 donors (18%) showed neither T- nor B-cell memory to HCMV, consistent with immunological naïveté to the virus. The data suggest that measurements of serum antibodies frequently fail to reveal HCMV exposure in humans, which may be better identified by direct detection of HCMV-specific memory lymphocytes.

Healthy human subjects develop spontaneous CD8+ T cell responses to melanoma associated antigens (MA) expressed by normal melanocytes, such as Tyrosinase, MAGE-A3, Melan/Mart-1, gp100, and NY-ESO-1. This natural autoimmunity directed against melanocytes might confer protection against the development of malignant melanoma (MM), where MA are present as overexpressed tumor-associated antigens. Consistent with this notion we report here that functional T cell reactivity to MA was found to be significantly diminished to MAGE-A3, Melan-A/Mart-1, and gp100 in untreated MM patients. Three lines of evidence suggest that the MA-reactive T cells present in healthy subjects undergo exhaustion once MM establishes itself. First, only the MA-specific T cell reactivity was affected in the MM patients; that to third party recall antigens was not. Second, in these patients, the residual MA-specific T cells, unlike third party antigen reactive T cells, were functionally impaired, showing a diminished per cell IFN-γ productivity. Third, we show that immunization with MA restored natural CD8+ T cell autoimmunity to MA in 85% of the MM patients. The role of natural T cell autoimmunity to tumor-associated MA is discussed based on discrete levels of T cell activation thresholds.

Testing of peripheral blood mononuclear cells (PBMC) for immune monitoring purposes requires verification of their functionality. This is of particular concern when the PBMC have been shipped or stored for prolonged periods of time. While the CEF (Cytomegalo-, Epstein-Barr and Flu-virus) peptide pool has become the gold standard for testing CD8 cell functionality, a positive control for CD4 cells is so far lacking. The latter ideally consists of proteins so as to control for the functionality of the antigen processing and presentation compartments, as well. Aiming to generate a positive control for CD4 cells, we first selected 12 protein antigens from infectious/environmental organisms that are ubiquitous: Varicella, Influenza, Parainfluenza, Mumps, Cytomegalovirus, Streptococcus, Mycoplasma, Lactobacillus, Neisseria, Candida, Rubella, and Measles. Of these antigens, three were found to elicited interferon (IFN)-γ-producing CD4 cells in the majority of human test subjects: inactivated cytomegalo-, parainfluenza-, and influenza virions (CPI). While individually none of these three antigens triggered a recall response in all donors, the pool of the three (the ‘CPI pool’), did. One hundred percent of 245 human donors tested were found to be CPI positive, including Caucasians, Asians, and African-Americans. Therefore, the CPI pool appears to be suitable to serve as universal positive control for verifying the functionality of CD4 and of antigen presenting cells.

Monitoring antigen-specific T cell immunity relies on functional tests that require T cells and antigen presenting cells to be uncompromised. Drawing of blood, its storage and shipment from the clinical site to the test laboratory, and the subsequent isolation, cryopreservation and thawing of peripheral blood mononuclear cells (PBMCs) before the actual test is performed can introduce numerous variables that may jeopardize the results. Therefore, no T cell test is valid without assessing the functional fitness of the PBMC being utilized. This can only be accomplished through the inclusion of positive controls that actually evaluate the performance of the antigen-specific T cell and antigen presenting cell (APC) compartments. For Caucasians, CEF peptides have been commonly used to this extent. Moreover, CEF peptides only measure CD8 cell functionality. We introduce here universal CD8+ T cell positive controls without any racial bias, as well as positive controls for the CD4+ T cell and APC compartments. In summary, we offer new tools and strategies for the assessment of PBMC functional fitness required for reliable T cell immune monitoring.

One of the primary effector functions of immune cells is the killing of virus-infected or malignant cells in the body. Natural killer (NK) and CD8 effector T cells are specialized for this function. The gold standard for measuring such cell-mediated cytolysis has been the chromium release assay, in which the leakage of the radioactive isotope from damaged target cells is being detected. Flow cytometry-based single cell analysis of target cells has recently been established as a non-radioactive alternative. Here we introduce a target cell visualization assay (TVA) that applies similar target cell staining approaches as used in flow cytometry but based on single cell computer image analysis. Two versions of TVA are described here. In one, the decrease in numbers of calcein-stained, i.e., viable, target cells is assessed. In the other, the CFSE/PI TVA, the increase in numbers of dead target cells is established in addition. TVA assays are shown to operate with the same sensitivity as standard chromium release assays, and, leaving data audit trails in form of scanned (raw), analyzed, and quality-controlled images, thus meeting requirements for measuring cell-mediated cytolysis in a regulated environment.