Rapid culling of the CD4+ T cell repertoire in the transition from effector to memory.

Requirements for CD4+ T cell memory differentiation were analyzed with adoptively transferred SMARTA T cell receptor (TCR) transgenic cells specific for alymphocytic choriomeningitis virus (LCMV) epitope. LCMV-induced effector and memory differentiation of SMARTA cells mimicked the endogenous CD4+ T cell response. In contrast, infection with a recombinant Listeria expressing the LCMV epitope, although resulting initially in massive SMARTA expansion, led to loss of effector function and rapid cell death characterized by high expression of the apoptosis regulator Bim. Defective memory differentiation was seen after stimulation of naive but not memory SMARTA cells, was independent of precursor frequency, and correlated with a lower TCR avidity compared to endogenous responders. In addition, long-lived endogenous CD4+ memory T cells skewed to a higher functional avidity over time. These results support a model in which CD4+ T cell memory differentiation and longevity depend on the strength of the TCR signal during the primary response.

Converting an HLA-B27 flow assay from the BD FACSCanto to the BD FACSLyric.

HLA-B27 is a major histocompatibility complex (MHC) class I antigen which exhibits strong association (90%) with ankylosing spondylitis. HLA-B27 detection in patients by flow cytometry is a widely used clinical test, performed on many different flow cytometer models. We sought to develop and validate a test conversion protocol for the HLA-B27 test performed on the BD FACSCanto to BD's newer FACSLyric flow cytometers. The development and validation experiments were performed using anti-HLA-B27*FITC/CD3*PE antibody-stained whole blood patient specimens. The anti-HLA-B27*FITC logarithmic median fluorescence (LMF) results on the BD FACSCanto were converted to median fluorescence intensity (MFI) values on the BD FACSLyric. Clustering of the HLA-B27 positive and negative values, using a 3rd order polynomial equation, resulted in a conversion of the BD FACSCanto cutoff values, negative (<150 LMF) and positive (≥160 LMF), to negative (<4530 MFI) and positive (≥6950 MFI) on the BD FACSLyric. Accuracy was assessed by comparing the flow results obtained on the BD FACSCanto and BD FACSLyric to a molecular PCR based assay. Additional validation parameters (compensation verification, intra- and inter-assay precision, and instrument comparison) were performed per the recommendations outlined in the Clinical and Laboratory Standards Institute (CLSI) H62 guidelines for validation of flow cytometry assays.

Reduced Monocyte Proportions and Responsiveness in Convalescent COVID-19 Patients.

The clinical manifestations of acute severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection and COVID-19 suggest a dysregulation of the host immune response that leads to inflammation, thrombosis, and organ dysfunction. It is less clear whether these dysregulated processes persist during the convalescent phase of disease or during long COVID. We investigated the effects of SARS-CoV-2 infection on the proportions of classical, intermediate, and non-classical monocytes, their activation status, and their functional properties in convalescent COVID-19 patients and uninfected control subjects. We found that the percentage of total monocytes was decreased in convalescent COVID-19 patients compared to uninfected controls. This was due to decreased intermediate and non-classical monocytes. Classical monocytes from convalescent COVID-19 patients demonstrated a decrease in activation markers, such as CD56, in response to stimulation with bacterial lipopolysaccharide (LPS). In addition, classical monocytes from convalescent COVID-19 patients showed decreased expression of CD142 (tissue factor), which can initiate the extrinsic coagulation cascade, in response to LPS stimulation. Finally, we found that monocytes from convalescent COVID-19 patients produced less TNF-α and IL-6 in response to LPS stimulation, than those from uninfected controls. In conclusion, SARS-CoV-2 infection exhibits a clear effect on the relative proportions of monocyte subsets, the activation status of classical monocytes, and proinflammatory cytokine production that persists during the convalescent phase of disease.

Reduced monocyte proportions and responsiveness in convalescent COVID-19 patients.

Introduction: The clinical manifestations of acute severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection and coronavirus disease 2019 (COVID-19) suggest a dysregulation of the host immune response that leads to inflammation, thrombosis, and organ dysfunction. It is less clear whether these dysregulated processes persist during the convalescent phase of disease or during long COVID. We sought to examine the effects of SARS-CoV-2 infection on the proportions of classical, intermediate, and nonclassical monocytes, their activation status, and their functional properties in convalescent COVID-19 patients. Methods: Peripheral blood mononuclear cells (PBMCs) from convalescent COVID-19 patients and uninfected controls were analyzed by multiparameter flow cytometry to determine relative percentages of total monocytes and monocyte subsets. The expression of activation markers and proinflammatory cytokines in response to LPS treatment were measured by flow cytometry and ELISA, respectively. Results: We found that the percentage of total monocytes was decreased in convalescent COVID-19 patients compared to uninfected controls. This was due to decreased intermediate and non-classical monocytes. Classical monocytes from convalescent COVID-19 patients demonstrated a decrease in activation markers, such as CD56, in response to stimulation with bacterial lipopolysaccharide (LPS). In addition, classical monocytes from convalescent COVID-19 patients showed decreased expression of CD142 (tissue factor), which can initiate the extrinsic coagulation cascade, in response to LPS stimulation. Finally, we found that monocytes from convalescent COVID-19 patients produced less TNF-α and IL-6 in response to LPS stimulation, than those from uninfected controls. Conclusion: SARS-CoV-2 infection exhibits a clear effect on the relative proportions of monocyte subsets, the activation status of classical monocytes, and proinflammatory cytokine production that persists during the convalescent phase of disease.

Distinct roles for IL-2 and IL-15 in the differentiation and survival of CD8+ effector and memory T cells.

IL-2 provides a memory differentiation signal to CD8+ T cells during the primary response that impacts the ability of the subsequent memory pool to mount a successful recall response. In this study, we find that although primary effector CTL development is modestly decreased in the absence of IL-2, the persistence of short-term and long-term effector memory CD8+ T cells on pathogen clearance is greatly diminished. Furthermore, secondary challenge of CD8+ memory T cells lacking the high-avidity IL-2R results in a failure to repopulate the effector pool. The role of IL-2 in promoting effector differentiation is not shared with the highly related cytokine, IL-15. Although IL-15 supports the survival of effector CD8+ T cells after pathogen clearance, its absence does not impair either primary or secondary effector CTL differentiation, nor does it impact the differentiation of long-term effector memory CD8+ T cells. These findings indicate a unique role for IL-2, but not IL-15, in promoting the differentiation not only of primary effector CD8+ T cells, but also of CD8+ memory T cells capable of secondary effector differentiation.

Validation of cytomegalovirus immune competence assays for the characterization of CD8(+) T cell responses posttransplant.

Cytomegalovirus (CMV) infection is one of the most important infectious complications of transplantation. Monitoring CMV-specific CD8 T cell immunity is useful for predicting active CMV infection and for directing targeted antiviral therapy. In this study, we examined four basic parameters for validation of CMV-specific tetramer staining and peptide stimulation assays that cover five most frequent HLA class I alleles. We also examined the potential use of CMV-specific CD8(+) T cell numbers and functional and cytolytic responses in two autologous HSCT recipients treated for multiple myeloma. The coefficient of variation (CV %) of the precision within assays was 3.1-24% for HLA-tetramer staining, 2.5-47% for IFN-γ, and 3.4-59.7% for CD107a/b production upon peptide stimulation. The precision between assays was 5-26% for tetramer staining, 4-24% for IFN-γ, and 5-48% for CD107a/b. The limit of detection was 0.1-0.23 cells/µL of blood for tetramer staining, 0-0.23 cell/µL for IFN-γ, and 0.11-0.98 cells/µL for CD107a/b. The assays were linear and specific. The reference interval with 95% confidence level was 0-18 cells/µL for tetramer staining, 0-2 cells/µL for IFN-γ, and 0-3 cells/µL for CD107a/b. Our results provide acceptable measures of test performance for CMV immune competence assays for the characterization of CD8(+) T cell responses posttransplant measured in the absolute cell count per µL of blood.

The magnitude of CD4+ T cell recall responses is controlled by the duration of the secondary stimulus.

The parameters controlling the generation of robust CD4(+) T cell recall responses remain poorly defined. In this study, we compare recall responses by CD4(+) and CD8(+) memory T cells following rechallenge. Homologous rechallenge of mice immune to either lymphocytic choriomeningitis virus or Listeria monocytogenes results in robust CD8(+) T cell recall responses but poor boosting of CD4(+) T cell recall responses in the same host. In contrast, heterologous rechallenge with a pathogen sharing only a CD4(+) T cell epitope results in robust boosting of CD4(+) T cell recall responses. The disparity in CD4(+) and CD8(+) T cell recall responses cannot be attributed to competition for growth factors or APCs, as robust CD4(+) and CD8(+) T cell recall responses can be simultaneously induced following rechallenge with peptide-pulsed dendritic cells. Instead, CD4(+) T cell recall responses are dependent on the duration of the secondary challenge. Increasing the rechallenge dose results in more potent boosting of CD4(+) T cell recall responses and artificially limiting the duration of secondary infection following heterologous rechallenge adversely impacts the magnitude of CD4(+) T cell, but not CD8(+) T cell, recall responses. These findings suggest that rapid pathogen clearance by secondary CTL following homologous rechallenge prevents optimal boosting of CD4(+) T cell responses and therefore have important practical implications in the design of vaccination and boosting strategies aimed at promoting CD4(+) T cell-mediated protection.

Evaluation of Mass Cytometry in the Clinical Laboratory.

BACKGROUND: Mass cytometry can differentiate more channels than conventional flow cytometry. However, for clinical use, standardization and agreement with well-established methods is paramount. We compared mass cytometry to standard clinical flow cytometry. METHODS: Mass and flow cytometry were performed in parallel on peripheral blood samples from 25 healthy individuals. Antibody staining was performed on the same samples at the same time, and analyzed for granulocyte, monocyte, lymphocyte, T, B, NK, CD4 and CD8 percentages. Validation parameters included comparison to flow cytometry, inter- and intra-assay precision and establishment of reference intervals. RESULTS: There was a positive correlation between mass and flow cytometry for the eight populations studied (R2 between 0.26 and 0.97). Slopes of the best-fit lines varied from 0.50 to 1.21 (fluorescence/mass). No significant differences in variance were found (F-test, P > 0.05). However, paired t-tests were significantly different for four of the eight markers (granulocytes, NK cells, T cells and CD4 cells), resulting in different reference intervals. Signal intensities were correlated for monocytes, lymphocytes, T, CD4 and CD8 cells (R2 = 0.41-0.57). The mass cytometry intra-assay precisions were 0.7-8.5% and inter-assay precisions 1.5-13.8%. CONCLUSION: Mass and flow cytometry evaluations of whole blood for major cell populations correlate with similar precision and signal intensity. However, for clinical use, separate reference interval studies are required. Cell population identification should rely on gating strategies that take advantage of the characteristics offered by each method. © 2019 International Clinical Cytometry Society.

A modified alpha-galactosyl ceramide for staining and stimulating natural killer T cells.

CD1d presentation of alpha-galactosyl ceramides to natural killer T cells has been a focal point of the study of regulatory T cells. KRN7000, an alpha-galactosyl ceramide originally generated from structure activity studies of antitumor properties of marine sponge glycolipids, is currently the most commonly used agonist ligand and is used to stain NKT cells. However, this glycolipid suffers from poor solubility and availability. We have developed an alpha-galactosyl ceramide with improved solubility over KRN7000 that effectively stains NKT cells, both mouse and human, and stimulates cytokine release at low concentrations.

Identification and validation of shrimp-tropomyosin specific CD4 T cell epitopes.

BACKGROUND: Shellfish allergy is an immune-mediated adverse reaction to allergenic shellfish and is responsible for significant morbidity and mortality. CD4 T cell responses play an important role in the pathophysiological mechanisms of sensitization and in production of IgE. OBJECTIVE: We sought to identify and validate CD4 T cell shrimp tropomyosin-derived epitopes and characterize CD4 T cell responses in subjects with a clinical history of shellfish allergy. METHOD: Using an in vitro MHC-peptide binding assay, we screened 91 overlapping peptides and identified 28 epitopes with moderate and strong binding capacities; 3 additional peptides were included based on MHC binding prediction score. These peptides were then examined in proliferation and cytokine release assays with T cells from allergic subjects. RESULT: 17 epitopes restricted to DRB(∗)01:01, DRB1(∗)03:01, DRB1(∗)04:01, DRB1(∗)09:01, DQB1(∗)02:01, DQB1(∗)03:02 and DQB1(∗)05:01 alleles were identified and validated by both the MHC binding and the functional assays. Two peptides showed specificities to more than one MHC class II allele. We demonstrated that these peptides exert functional responses in an epitope specific manner, eliciting predominantly IL-6 and IL-13. CONCLUSION: The identified epitopes are specific to common MHC class II alleles in the general population. Our study provides important data for the design of peptide-based immunotherapy of shrimp-allergic patients.

Immediate early effector functions of virus-specific CD8+CCR7+ memory cells in humans defined by HLA and CC chemokine ligand 19 tetramers.

Memory T cells exhibit a high degree of heterogeneity in terms of their phenotype and functional characteristics. It has been proposed that the CCR7 chemokine receptor divides memory T cell populations into central memory T cells and effector memory T cells with distinct functions in secondary immune responses. We were interested whether this hypothesis holds true in experiments performed on Ag-specific CD8(+) T cells. To identify CCR7(+) cells, we engineered a fluorescent ligand for CCR7; results with the new CC chemokine ligand 19 chemotetramer were verified by staining with a CCR7 mAb. Staining with the CC chemokine ligand 19 chemotetramer reveals two subsets within CCR7(+) cells: a CCR7(int) population containing memory cells and a CCR7(high) population containing naive T cells. Phenotypic analysis of MHC class I/peptide tetramer-positive cells revealed that HLA-A2-restricted CMV-specific CD8 T cells exhibit the lowest percentage of CCR7(+) cells (0.5-5%), while HLA-A2-restricted flu- and HLA-B8-restricted EBV-specific CD8 T cells showed the highest (45-70%). Intracellular staining of unstimulated cells revealed that both CCR7(int)- and CCR7(-)-specific CD8 T cells exhibit a detectable level of perforin. Both CCR7(int) and CCR7(-) Ag-specific CD8(+) T cells produced IFN-gamma and TNF-alpha following short-term peptide stimulation. Therefore, our finding that CCR7(+)CD8(+) T cells are able to exert immediate effector functions requires a substantial revision to the central and effector memory hypothesis.