T-cell Immune Monitoring Research Articles

Skewed Cellular Distribution and Low Activation of Functional T-Cell Responses in SARS-CoV-2 Non-Seroconvertors.

The role of T cells in the control of SARS-CoV-2 infection has been underestimated in favor of neutralizing antibodies. However, cellular immunity is essential for long-term viral control and protection from disease severity. To understand T-cell immunity in the absence of antibody generation we focused on a group of SARS-CoV-2 Non-Seroconvertors (NSC) recovered from infection. We performed an immune comparative analysis of SARS-CoV-2 infected individuals stratified by the absence or presence of seroconversion and disease severity. We report high levels of total naïve and low effector CD8+ T cells in NSC. Moreover, reduced levels of T-cell activation monitored by PD-1 and activation-induced markers were observed in the context of functional SARS-CoV-2 T-cell responses. Longitudinal data indicate the stability of the NSC phenotype over three months of follow-up after infection. Together, these data characterized distinctive immunological traits in NSC including skewed cellular distribution, low activation and functional SARS-CoV-2 T-cell responses. This data highlights the value of T-cell immune monitoring in populations with low seroconversion rates in response to SARS-CoV-2 infection and vaccination.

Evaluation of suitable target antigens and immunoassays for high-accuracy immune monitoring of cytomegalovirus and Epstein–Barr virus-specific T cells as targets of interest in immunotherapeutic approaches

Adoptive immunotherapy with donor-derived antiviral T cells can prevent viral complications such as with cytomegalovirus (CMV) and Epstein–Barr virus (EBV). In this context accurate monitoring of cellular immunity is essential and requires suitable quantitative and qualitative assays for high-throughput screening.We comparatively analyzed 57 HLA-typed healthy donors for memory T-cell responses to CMV- and EBV-derived proteins, peptide pools and single HLA-restricted peptides by five commonly used immunoassays in parallel: enzyme-linked immunospot (ELISPOT), cytokine secretion assay (CSA), intracellular cytokine staining (ICS), enzyme-linked immunosorbent assay (ELISA) and pMHC multimer staining. T-cell responses varied greatly between the different target antigens in the investigated assays. IFN-γ ELISPOT consistently detected the highest T-cell response levels against CMV and EBV. CMV-specific T cells were detected in 100% of CMV-seropositive donors tested using CMVpp65 protein and/or overlapping CMVpp65 peptide pool. CMV-specific T cells in HLA-A*02:01-positive/CMV-seropositive donors were identified directly by HLA-A02/CMVpp65 (A02pp65) multimer staining and, after short in vitro stimulation with HLA-A*02:01-restricted pp65 peptide, by ELISPOT, ELISA, ICS and CSA. A peptide-specific T-cell response was detected in only 4 HLA-A*02:01-positive donors (50%). Despite A02pp65 peptide negativity, T-cell responses to CMVpp65 protein and/or overlapping peptide pool were detected.Comparing the specific immune response against EBV antigens in healthy donors overall, BZLF1-specific T cells (<92.9% peptides, <56.3% peptide pool) were more frequent than EBNA-specific T cells (<64.3% peptides, <46.9% peptide pool) with higher percentage of positive findings for single HLA-restricted EBV peptides. T-cell response against HLA-B*08 peptide epitopes was predominant (multimer staining: EBNA3A: 9/14 and BZLF1: 7/14, IFN-γ ELISPOT: EBNA3A: 13/14 and BZLF1: 11/14). The fact that responses to EBV-specific antigens were not detected in every single EBV-seropositive donor as well as that the T-cell frequencies in response to the investigated EBV antigens differed strongly in the donor cohort indicates that these epitopes are less immunodominant than CMVpp65.Taken together, precise monitoring of T-cell immunity against infectious agents in potential T-cell donors and post-transplant recipients requires individual selection of antigens and immunoassays for the efficient detection and generation of clinically relevant T cells. Due to its lower detection limit and direct visualization of each IFN-γ-secreting cell we identified ELISPOT analysis to be preferable for high-throughput pre-screening. CSA was found to be advantageous for a more detailed analysis of antigen-specific T-cell subsets.

T-Cell Immune Monitoring in Organ Transplantation

Chronic allograft injury remains the leading cause of graft loss despite improvements in immunosuppression, clinical risk stratification, and state-of-the-art antibody testing. Emerging results indicate that T-cell immune monitoring by cytokine enzyme-linked immunosorbent spot, as part of a comprehensive risk assessment platform, has the potential to guide decision making and improve outcomes after transplantation.

ELISPOT assays provide reproducible results among different laboratories for T-cell immune monitoring—even in hands of ELISPOT-inexperienced investigators

Measurements of antibodies in bodily fluids (e.g., by ELISA) have provided robust and reproducible results for decades and such assays have been validated for monitoring of B-cell immunity. In contrast, measuring T-cell immunity has proven to be a challenge due to the need to test live cells in functional assays ex vivo. Several previous efforts looking into the reproducibility of ex vivo T-cell assays between different laboratories, or even within the same laboratory, have provided rather discouraging results. The hypothesis we tested in this study is that those poor results are due to the lack of assay and data analysis standardization, rather than the inherent complexity of T-cell assays. In this study, 11 laboratories across Europe and the United States were provided identical reagents and were asked to follow the same protocol while testing aliquots of the same three cryopreserved peripheral blood mononuclear cells (PBMC) in an interferon-γ (IFNγ) ELISPOT assay measuring the antigen-specific T-cell response to a CMV peptide. All individuals performing the assays were ELISPOT novices. At their first attempt, while three of these individuals failed with the basic logistics of the trial, eight detected the peptide-specific CD8+ T-cells in frequencies approximating the values established by the Reference Laboratory. The data show that ELISPOT assays provide reproducible results among different laboratories when the assay procedure and data analysis is standardized. Since ELISPOT assays have been qualified and validated for regulated studies, they are ideal candidates for robust and reproducible monitoring of T-cell activity in vivo.

ELISPOT assays provide reproducible results among different laboratories for T-cell immune monitoring—even in hands of ELISPOT-inexperienced investigators

ELISPOT assays provide reproducible results among different laboratories for T-cell immune monitoring—even in hands of ELISPOT-inexperienced investigators

Diagnostic value of T-cell monitoring assays in kidney transplantation

Personalized immunosuppressive therapy on the basis of the recognition of individual alloreactive and anti-infectious immune responses is a major goal in clinical transplantation. It requires the development of reliable assays for quantification of T-cell responses. Here, we review recent findings in the field of T-cell immune monitoring focusing on candidate assays with clinical utility for predicting outcome in kidney transplantation. Promising assays for routine monitoring of T-cell reactivity in transplant patients include IFNgamma Elispot, multiparameter flow cytometry and intracellular ATP assay. Although large randomized multicenter studies are still lacking, first clinical applications have demonstrated remarkable differences in alloreactive and anti-infectious T-cell reactivity of transplant patients with potential for predicting outcome. Currently, standardization of the tests is a major challenge for translation into multicenter trials. A definitive picture of the (allo)immune response would require the assessment of multiple biological and genetic markers. However, frequent and reliable T-cell monitoring is feasible by simple cellular assays such as IFNgamma Elispot and intracellular ATP determination. International efforts are warranted for further standardization of these assays. Furthermore, the implementation of T-cell monitoring assays in large randomized clinical studies assessing different immunosuppressive regimens and weaning procedures is clearly required.

T-cell immune monitoring in organ transplantation

Chronic injury and late allograft loss remain major causes of morbidity in clinical transplantation. Biomarkers that can reliably assess the risk of posttransplant complications are required to direct and individualize therapy aimed at prolonging graft survival and improving patient health. The purpose of this review is to provide a framework for understanding how to use biomarkers in the context of clinical transplantation and to summarize current data on available noninvasive cellular-based immune monitoring methods to predict transplant outcome. New microarray and gene profiling data reveal peripheral blood cell gene expression patterns that identify operational tolerance, raising the possibility that the measurements can be used to direct immunosuppression withdrawal. Additional data support the use of selective urine gene products and soluble CD30 measurements in serum as reliable biomarkers of acute graft injury. Finally, recent studies demonstrate that measurement of T-cell alloimmunity by cytokine enzyme-linked immunospot is a promising, supplementary pretransplant risk assessment tool. Recently published studies in organ transplantation suggest that results derived from assays focused on markers of T-cell immunity can segregate transplant candidates or recipients into high and low-risk subgroups for posttransplant graft injury. Larger prospective studies are needed, however, before any proposed biomarker can be incorporated into the transplant physicians' armamentarium to guide individualized therapeutic decision-making.

Early determinants of long-term T-cell reconstitution after hematopoietic stem cell transplantation for severe combined immunodeficiency

The immune system of patients with severe combined immunodeficiency (SCID) reconstitutes to a large extent during the first years after hematopoietic stem cell transplantation (HSCT). It was suggested, however, that accelerated loss of thymus output may cause impaired immune function at the long term. To address this issue, we studied patients with SCID who underwent allogeneic HSCT 5 to 32 years earlier and identified early determinants of long-term T-cell reconstitution. A variety of immune parameters were analyzed both early (1-4 years) and late (5-32 years) after HSCT. Late after HSCT, a clear distinction could be made between a group of 8 patients with impaired T-cell reconstitution and 11 patients with good immune reconstitution. Importantly, in patients with decreased long-term T-cell reconstitution, T-cell recovery was already poor early after HSCT, demonstrating that long-term immune failure was not caused by accelerated loss of thymus output or long-term graft failure, but resulted from poor early grafting. The number of T-cell receptor excision circles (TRECs) early after HSCT was most predictive for long-term T-cell reconstitution. Frequent monitoring of T-cell immunity and TREC numbers early after HSCT may thus serve to timely identify patients who will fail to reconstitute properly and who may need additional treatment.

Pro- and anti-inflammatory cytokine production by autoimmune T cells against preproinsulin in HLA-DRB1*04, DQ8 Type 1 diabetes.

Preproinsulin is a target T cell autoantigen in human Type 1 diabetes. This study analyses the phenotype and epitope recognition of preproinsulin reactive T cells in subjects with a high genetic risk of diabetes [HLA-DRB1*04, DQ8 with Ab+ (autoantibody-positive) or without islet autoantibodies (control subjects)], and in HLA-matched diabetic patients. A preproinsulin peptide library approach was used to screen for cytokine profiles and epitope specificities in human peripheral blood lymphocytes, and CD4(+)CD45RA(-) and CD4(+)CD45RA(+) T cell subfractions, representing memory and naive and recently primed T cells respectively. In CD4(+) T cell subsets we identified immunodominant epitopes and cytokine production patterns that differed profoundly between patients, Ab+ subjects and non-diabetic HLA-matched control subjects. In Ab+ subjects, a C-peptide epitope C13-29 and insulin B-chain epitope B11-27 were preferentially recognised, whereas insulin-treated Type 1 diabetic patients reacted to native insulin and B-chain epitope B1-16. In peripheral blood lymphocytes of Ab+ subjects, an increase in T helper (Th) 1 (IFNgamma, IL-2) and Th2 (IL-4) cytokines was detectable, wheras in CD45RA(+) and CD45RA(-) subsets, IL-4 and IL-10 phenotypes dominated, compatible with the contribution of non-CD4 cells to IFNgamma content. In insulin-treated Type 1 diabetic patients, naive and recently primed CD4(+) cells were characterised by increasd IFNgamma, TNFalpha, and IL-5. Our data show that T cell reactivity to preproinsulin in CD45RA subsets is Th2-dominant in Ab+ subjects, challenging the Th1 paradigm in Type 1 diabetes. Characteristic immunodominant epitopes and cytokine patterns distinguish diabetic patients and Ab+ subjects from HLA-matched healthy individuals. This could prove useful in monitoring of T-cell immunity in clinical diabetes intervention trials.