scholarly journals Proliferation of PD-1+ CD8 T cells in peripheral blood after PD-1–targeted therapy in lung cancer patients

2017 ◽  
Vol 114 (19) ◽  
pp. 4993-4998 ◽  
Author(s):  
Alice O. Kamphorst ◽  
Rathi N. Pillai ◽  
Shu Yang ◽  
Tahseen H. Nasti ◽  
Rama S. Akondy ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cancer Patients ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd8 T Cell ◽  
Blood Analysis ◽  
Advanced Stage ◽  
Nsclc Patients ◽  
Peripheral Blood Analysis

Exhausted T cells in chronic infections and cancer have sustained expression of the inhibitory receptor programmed cell death 1 (PD-1). Therapies that block the PD-1 pathway have shown promising clinical results in a significant number of advanced-stage cancer patients. Nonetheless, a better understanding of the immunological responses induced by PD-1 blockade in cancer patients is lacking. Identification of predictive biomarkers is a priority in the field, but whether peripheral blood analysis can provide biomarkers to monitor or predict patients’ responses to treatment remains to be resolved. In this study, we analyzed longitudinal blood samples from advanced stage non–small cell lung cancer (NSCLC) patients (n = 29) receiving PD-1–targeted therapies. We detected an increase in Ki-67+ PD-1+ CD8 T cells following therapy in ∼70% of patients, and most responses were induced after the first or second treatment cycle. This T-cell activation was not indiscriminate because we observed only minimal effects on EBV-specific CD8 T cells, suggesting that responding cells may be tumor specific. These proliferating CD8 T cells had an effector-like phenotype (HLA-DR+, CD38+, Bcl-2lo), expressed costimulatory molecules (CD28, CD27, ICOS), and had high levels of PD-1 and coexpression of CTLA-4. We found that 70% of patients with disease progression had either a delayed or absent PD-1+ CD8 T-cell response, whereas 80% of patients with clinical benefit exhibited PD-1+ CD8 T-cell responses within 4 wk of treatment initiation. Our results suggest that peripheral blood analysis may provide valuable insights into NSCLC patients’ responses to PD-1–targeted therapies.

T Cell Recognition of HCMV: Pan-Genome Analysis of Immunogenic Open-Reading Frames.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 606-606 ◽  
Author(s):  
Louis J. Picker ◽  
Andrew W. Sylwester ◽  
Bridget L. Mitchell ◽  
Cara Taormina ◽  
Christian Pelte ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cell Response ◽  
Cd8 T Cell ◽  
Cross Reactivity ◽  
T Cell Response ◽  
Cd4 T Cell ◽  
Cell Responses

Abstract Human Cytomegalovirus (HCMV) is among the largest and most complex of known viruses with 150–200nm virions enclosing a double stranded 230kb DNA genome capable of coding for >200 proteins. HCMV infection is life-long, and for the vast majority of immune competent individuals clinically benign. Disease occurs almost exclusively in the setting of immune deficiency, suggesting that the stable host-parasite relationship that characterizes these infections is the result of an evolutionarily “negotiated” balance between viral mechanisms of pathogenesis and the host immune response. In keeping with, and perhaps because of this balance, the human CD4+ T cell response to whole HCMV viral lysates is enormous, with median peripheral blood frequencies of HCMV-specific cells ~5–10 fold higher than for analogous preparations of other common viruses. Although certain HCMV ORFs have been identified as targets of either the CD4+ or CD8+ T cell response, the specificities comprising the CD4+ T cell response, and both the total frequencies and component parts of the CD8+ T cell response are unknown. Here, we used cytokine flow cytometry and ~14,000 overlapping 15mer peptides comprising all 213 HCMV ORFs encoding proteins >100 amino acids in length to precisely define the total CD4+ and CD8+ HCMV-specific T cell responses and the HCMV ORFs responsible for these responses in 33 HCMV-seropositive, HLA-disparate donors. An additional 9 HCMV seronegative donors were similarly examined to define the extent to which non-HCMV responses cross-react with HCMV-encoded epitopes. We found that when totaled, the median frequencies of HCMV-specific CD4+ and CD8+ T cells in the peripheral blood of the seropositive subjects were 4.0% and 4.5% for the total CD4+ or CD8+ T cell populations, respectively (which corresponds to 9.1% and 10.5% of the memory populations, respectively). The HCMV-specific CD4+ and CD8+ T cell responses included a median 12 and 7 different ORFs, respectively, and all told, 73 HCMV ORFs were identified as targets for both CD4+ and CD8+ T cells, 26 ORFs as targets for CD8+ T cells alone, and 43 ORFS as targets for CD4+ T cells alone. UL55, UL83, UL86, UL99, and UL122 were the HCMV ORFs most commonly recognized by CD4+ T cells; UL123, UL83, UL48, UL122 and UL28 were the HCMV ORFs most commonly recognized by CD8+ T cells. The relationship between immunogenicity and 1) HLA haplotype and 2) ORF expression and function will be discussed. HCMV-seronegative individuals were non-reactive with the vast majority of HCMV peptides. Only 7 potentially cross-reactive responses were identified (all by CD8+ T cells) to 3 ORFs (US32, US29 and UL116) out of a total of almost 4,000 potential responses, suggesting fortuitous cross-reactivity with HCMV epitopes is uncommon. These data provide the first glimpse of the total human T cell response to a complex infectious agent, and will provide insight into the rules governing immunodominance and cross-reactivity in complex viral infections of humans.

AAV-2 Capsid-Specific CD8+ T Cells Limit the Duration of Gene Therapy in Humans and Cross-React with AAV-8 Capsid.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 455-455 ◽  
Author(s):  
Federico Mingozzi ◽  
Marcela V. Maus ◽  
Denise E. Sabatino ◽  
Daniel J. Hui ◽  
John E.J. Rasko ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Cell Population ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Transgene Expression ◽  
Cd8 T Cell ◽  
Target Cells

Abstract Efforts to establish an adeno-associated viral (AAV) vector-mediated gene therapy for the treatment of hemophilia B have been hindered by an immune response to the viral capsid antigen. Preclinical studies in small and large animal models of the disease showed long-term factor IX (F.IX) transgene expression and correction of the phenotype. However, in a recent phase I/II clinical trial in humans (Manno et al., Nat. Med. 2006), after hepatic gene transfer with an AAV-2 vector expressing human F.IX transgene, expression lasted for only a few weeks, declining to baseline concurrently with a peak in liver enzymes. We hypothesized that T cells directed towards AAV capsid antigens displayed by transduced hepatocytes were activated and these mediated destruction of the transduced hepatocytes, thereby causing loss of transgene expression and a transient transaminitis. Peripheral blood mononuclear cells isolated from AAV-infused subjects were stained with an AAV capsid-specific MHC class I pentamer either directly or after in vitro expansion. Two weeks after vector infusion 0.14% of circulating CD8+ T cells were capsid-specific on direct staining, and five weeks after infusion the capsid-specific population had expanded to 0.5% of the circulating CD8+ T cells, indicating proliferation of this T cell subset. By 20 weeks after vector infusion, the capsid-specific CD8+ T cell population had contracted to the level seen at 2 weeks. The expansion and contraction of this capsid-specific CD8+ T cell population paralleled the rise and fall of serum transaminases in the subject observed. Subsequent ex vivo studies of PBMC showed the presence of a readily expandable pool of capsid-specific CD8+ T cells up to 2.5 years post vector-infusion. Similarly, we were able to expand AAV-specific CD8+ T cells from peripheral blood of normal donors, suggesting the existence of a T cell memory pool. Expanded CD8+ T cells were functional as evidenced by specific lysis of HLA-matched target cells and by IFN-γsecretion in response to AAV epitopes. It has been argued that potentially harmful immune responses could be avoided by switching AAV serotypes, however, capsid protein sequences are highly conserved among different serotypes, as are some immunodominant epitopes that we identified. Indeed, we demonstrated that capsid-specific CD8+ T cells from AAV-infused hemophilic subjects functionally cross-react with AAV-8. Moreover, cells expanded from normal donors with AAV-2 vector capsids proliferated upon culture with AAV-8 capsids, demonstrating that both vectors could be processed appropriately in vitro to present the epitopic peptide to capsid-specific T cells. This suggests that AAV-2-specific memory CD8+ T cells normally present in humans likely would expand upon exposure to AAV-8 capsid epitopes. We conclude that the use of immunomodulatory therapy may be a better approach to achieving durable transgene expression in the setting of AAV-mediated gene therapy.

Autoproliferation Contributes to Clonal Expansion and Changes In Cellular Topography In T Cell Large Granular Lymphocyte (LGL) Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1373-1373
Author(s):  
JianXiang Zou ◽  
Jeffrey S Painter ◽  
Fanqi Bai ◽  
Lubomir Sokol ◽  
Thomas P. Loughran ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd8 T Cell ◽  
Effector Memory ◽  
Ki67 Expression ◽  
Terminal Effector ◽  
Lgl Leukemia

Abstract Abstract 1373 Introduction: LGL leukemia is associated with cytopenias and expansion of clonally-derived mature cytotoxic CD8+ lymphocytes. The etiology of LGL leukemia is currently unknown, however, T cell activation, loss of lymph node homing receptor L-selectin (CD62L), and increased accumulation of T cells in the bone marrow may lead to suppressed blood cell production. The broad resistance to Fas (CD95) apoptotic signals has lead to the hypothesis that amplification of clonal cells occurs through apoptosis resistance. However, the proliferative history has not been carefully studied. To define possible mechanism of LGL leukemia expansion, T cell phenotype, proliferative history, and functional-related surface marker expression were analyzed. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from 16 LGL leukemia patients that met diagnostic criteria based on the presence of clonal aβ T cells and >300 cells/ml CD3+/CD57+ T cells in the peripheral blood. Samples were obtained from 10 age-matched healthy individuals from the Southwest Florida Blood Services for comparisons. Multi-analyte flow cytometry was conducted for expression of CD3, CD4/8, CD45RA, CD62L, CD27, CD28, CD25, CD127, IL15Ra, IL21a, CCR7 (all antibodies from BD Biosciences). The proliferative index was determined by Ki67 expression in fixed and permeabilized cells (BD Biosciences) and the proliferative history in vivo was assessed by T-cell-receptor excision circle (TREC) measurement using real-time quantitative PCR (qRT-PCR) in sorted CD4+ and CD8+ T cells. TRECs are episomal fragments generated during TCR gene rearrangements that fail to transfer to daughter cells and thus diminish with each population doubling that reflects the in vivo proliferative history. Results: Compared to healthy controls, significantly fewer CD8+ naïve cells (CD45RA+/CD62L+, 8.4 ± 10.8 vs 24.48 ± 11.99, p=0.003) and higher CD8+ terminal effector memory (TEM) T cells (CD45RA+/CD62L-, 67.74 ± 28.75 vs 39.33 ± 11.32, p=0.007) were observed in the peripheral blood. In contrast, the percentage of CD4+ naïve and memory cells (naïve, central memory, effector memory, and terminal effector memory based on CD45RA and CD62L expression) was similar in patients as compared to controls. The expression of CD27 (31.32 ± 34.64 vs 71.73 ± 20.63, p=0.003) and CD28 (31.38 ± 31.91 vs 70.02 ± 22.93, p=0.002) were lower in CD8+ T cell from patients with LGL leukemia and this reduction predominated within the TEM population (17.63±24.5 vs 70.98±22.5 for CD27, p<0.0001 and 13±20.5 vs 69.43± 21.59 for CD28, p<0.0001). Loss of these markers is consistent with prior antigen activation. There was no difference in CD25 (IL2Ra, p=0.2) expression on CD4+ or CD8+ T cells, but CD127 (IL7Ra, p=0.001), IL15Ra, and IL21Ra (p=0.15) were overexpressed in TEM CD8+ T cell in patients vs controls. All of these cytokine receptors belong to the IL2Rβg-common cytokine receptor superfamily that mediates homeostatic proliferation. In CD8+ T cells in patients, the IL-21Ra was also overexpressed in naïve, central and effector memory T cells. The topography of the expanded CD8+ T cell population was therefore consistent with overexpression of activation markers and proliferation-associated cytokine receptors. Therefore, we next analyzed Ki67 expression and TREC DNA copy number to quantify actively dividing cells and determine the proliferative history, respectively. We found that LGL leukemia patients have more actively dividing CD8+ TEM T cells compared to controls (3.2 ± 3.12 in patients vs 0.44 ± 0.44 in controls, p=0.001). Moreover, the TREC copy number in CD8+ T cells was statistically higher in healthy individuals after adjusting for age (177.54 ± 232 in patients vs 1015 ± 951 in controls, p=0.019). These results show that CD8+ cells in the peripheral compartment have undergone more population doublings in vivo compared to healthy donors. In contrast, the TREC copies in CD4+ T-cells were similar between LGL patients and controls (534.4 ± 644 in patients vs 348.78 ± 248.16 in controls, p>0.05) demonstrating selective cellular proliferation within the CD8 compartment. Conclusions: CD8+ T- cells are undergoing robust cellular activation, contraction in repertoire diversity, and enhanced endogenous proliferation in patients with LGL leukemia. Collectively, these results suggest that clonal expansion is at least partially mediated through autoproliferation in T-LGL leukemia. Disclosures: No relevant conflicts of interest to declare.

Peripheral blood CD4+CRTH2+ cells in advanced stage non small cell lung cancer

Open Medicine ◽  
2010 ◽  
Vol 5 (4) ◽  
pp. 431-436
Author(s):  
Bülent Karagöz ◽  
Oğuz Bilgi ◽  
Emin Kandemir ◽  
Alev Erikçi ◽  
Özkan Sayan ◽  
...  
Keyword(s):  
Lung Cancer ◽  
T Cells ◽  
T Cell ◽  
Patient Group ◽  
Peripheral Blood ◽  
Control Group ◽  
Advanced Stage ◽  
Cell Percentage ◽  
Nsclc Patients ◽  
Cd4 Cell

AbstractTo investigate CD4+CRTH2+ cells in peripheral blood in advanced stage non small cell lung cancer (NSCLC) patients. Forty-six patients with advanced stage NSCLC, who are chemotherapy or radiotherapy naïve, and 17 healthy volunteers, were enrolled in this study. The study was performed using flow cytometry and a complete blood cell counter analyser. CD4+ T cell percentage, CD4/CD8 ratio, CRTH2+CD4+ cell percentages, counts, and mean fluorescein intensity (MFI) and hematological parameters were evaluated in both groups. A survival analysis was performed to compare the patients with high CD4+CRTH2+ cell percentage and those with low CD4+CRTH2+ percentage. CD4+ T cell percentage in total lymphocytes and the CD4/CD8 ratio were lower in the patient group than in the control group. The absolute CD8 T cell count was higher in the patient group than in the control group, whereas the total T cells was not different. The CRTH2+ cell percentage in CD4+ T cells (7.96% ± 6.21% vs 3.37% ± 3.55%; respectively; p: 0,001) and the absolute count of CRTH2+CD4+ cells ( 97 mm-3 ± 109 mm-3 vs 37 mm-3 ± 38 mm-3, respectively; p: 0,033) in the patient group were higher than in the control group, but CRTH2-PE MFI values were not different between groups. Cox regression analysis did not show that CRTH2+CD4+ cell count or percentage is an independent prognostic factor. The study found that CRTH2 expression of CD4+ T cells and CRTH2+CD4+ cell number are higher in the peripheral blood of NSCLC patients than in that of healthy subjects. Further studies that explore the biological significance of high CD4+CRTH2+ cells in lung cancer patients, should be pursued.

scholarly journals Differentiation of a CD4/CD8αβ Double Positive T Cell Population from the CD8 Pool Is Both Predictive and Sufficient to Mediate Graft-Vs-Host Disease

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2761-2761
Author(s):  
Nicholas J. Hess ◽  
David Turicek ◽  
Amy Hudson ◽  
Peiman Hematti ◽  
Jenny Gumperz ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Cell Population ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd8 T Cell ◽  
T Cell Differentiation ◽  
Cell Populations ◽  
Human T Cell

Abstract Acute graft-vs-host disease (aGVHD) and cancer relapse remain the primary complications following an allogeneic hematopoietic stem cell transplantation (allo-HSCT) for malignant blood disorders. While post-transplant cyclophosphamide combined with standard GVHD prophylaxis has greatly reduced the overall prevalence and severity of aGVHD, relapse rates remain a concern. There is thus a need to identify the specific human T cell populations mediating GVHD vs GVL activity as a means to develop targeted therapeutics capable of controlling aGVHD without inhibiting GVL activity. In this study, we identify a novel human T cell population that develops after transplant that is predictive and sufficient for GVHD pathology. To determine the role of human T cell populations in aGVHD, we performed xenogeneic transplantation studies using primary human graft tissue from a variety of sources (peripheral blood, G-CSF mobilized peripheral blood, bone marrow and umbilical cord blood) in addition to collecting primary human aGVHD blood samples from our clinic. Using the LD50 dose of human graft tissue, we identified a novel mature CD4 +/CD8αβ + double positive (DP) T cell population that only developed after transplantation. The development of this population was further confirmed in aGVHD patients from our clinic. The presence of DP T cells, irrespective of graft source, was also predictive of lethal GVHD in as early as one week after xenogeneic transplantation. To identify the origin of DP T cells, we transplanted isolated human CD4 or CD8 T cells into mice which showed that DP T cells only arise from the CD8 pool. Furthermore, re-transplantation of flow-sorted CD8 T cells from GVHD mice did not reveal a 2nd wave of DP T cell differentiation. This data, in addition to their highly proliferative state, suggests that DP T cells represent highly activated CD8 T cell clones. The ability of these CD8-derived DP T cells to gain CD4 expression coincides with their co-expression of both RUNX3 and THPOK, the master transcription factors of the CD8 and CD4 lineages respectively, that classically repress each other. Intracellular cytokine staining also revealed that DP T cells are the primary activated T cell population in xenogeneic GVHD, secreting both modulatory and cytotoxic cytokines (e.g. IFNγ, IL-17A, IL-22, perforin and granzyme). Ex vivo re-stimulation or re-transplantation of flow-sorted DP T cells showed that this T cell population is capable of dividing and expanding independent of CD4 and CD8 single positive T cells with the majority of the isolated DP T cells retaining their co-expression of CD4 and CD8. Finally, transplantation of either isolated human peripheral blood CD4 or CD8 T cell populations were capable of causing lethal GVHD. Conversely, re-transplantation of flow-sorted DP, CD8 or CD4 T cells from GVHD mice revealed that DP and CD4 T cells are sufficient to mediate GVHD pathology but re-transplanted CD8 T cell are not. This correlates with the absence of DP T cell differentiation in that re-transplanted CD8 population. The differentiation of DP T cells from chronically activated CD8 T cells represents a novel mechanism of GVHD pathology not previously described. The presence of DP T cells in other chronic inflammatory human diseases also suggests a broader pathology mediated by DP T cells. Further understanding of DP T cell differentiation and pathology may lead to targeted prophylaxis and/or treatment regimens for aGVHD and other human chronic inflammatory diseases. Figure 1 Figure 1. Disclosures Capitini: Nektar Therapeutics: Honoraria; Novartis: Honoraria.

scholarly journals DOP70 High-dimensional analysis reveals a novel signature for IL-17 producing CD8 T cells in inflammatory bowel disease

2020 ◽  
Vol 14 (Supplement_1) ◽  
pp. S106-S107
Author(s):  
A M Globig ◽  
P Otto-Mora ◽  
A Hipp ◽  
M Heeg ◽  
H Schwacha ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Population ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Intestinal Tissue ◽  
Cell Responses ◽  
Distinct Cell

Abstract Background The pathogenesis of IBD has previously mainly been associated with a dysregulation of CD4 T-cell responses, and active disease is linked to induction of pathogenic IL-17 producing T helper cells. Importantly, CD8 T cells can also produce IL-17 and demonstrate a significant increase in active IBD. However, it remains unclear whether this is due to conventional or unconventional T-cell responses and whether these CD8 responses could play a role as novel biomarkers or therapeutic targets of active IBD. Methods We isolated lymphocytes from the peripheral blood and intestinal tissue of IBD patients (n = 56) and performed a detailed analysis of CD8 T-cell phenotype and function using high-parametric flow cytometry and mass cytometry in combination with algorithm-aided bioinformatic analysis. Results We observe a significant increase in IL-17 production by CD8 T cells in active IBD, primarily produced by conventional CD8 T cells. Unconventional T-cell subsets (e.g. MAIT cells, γδ T cells and NKT cells) represented only ~30% (peripheral blood) or ~25% (intestinal tissue) of IL-17 producing CD8 T cells (Tc17). The mass cytometric analysis identified Tc17 cells as a distinct cell population within the intestinal CD8 T-cell compartment that can be further subdivided into 3 subsets which share expression of phenotypic markers such as CD6, CD39, CD69 and PD1 and a low expression of CD27. This novel signature was validated in a separate cohort of IBD patients. Moreover, at initial IBD diagnosis, the IL-17 signature is associated with flare-free survival in a retrospective cohort analysis based on published transcriptome data. Conclusion Our data indicate that conventional IL-17 producing CD8 T cells are a very distinct cell population that is linked to IBD activity. The identification of a novel IL-17 CD8 signature may help guide treatment decisions as a biomarker and for immunotherapeutic approaches.

scholarly journals CD8 T cells compensate for impaired humoral immunity in COVID-19 patients with hematologic cancer

2021 ◽  
Author(s):  
Erin Bange ◽  
Nicholas Han ◽  
E. Paul Wileyto ◽  
Justin Kim ◽  
Sigrid Gouma ◽  
...  
Keyword(s):  
T Cells ◽  
Viral Load ◽  
T Cell ◽  
B Cells ◽  
Cancer Patients ◽  
Humoral Immunity ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Immune Phenotype ◽  
Flow Cytometric

Abstract Cancer patients have increased morbidity and mortality from Coronavirus Disease 2019 (COVID-19), but the underlying immune mechanisms are unknown. In a cohort of 100 cancer patients hospitalized for COVID-19 at the University of Pennsylvania Health System, we found that patients with hematologic cancers had a significantly higher mortality relative to patients with solid cancers after accounting for confounders including ECOG performance status and active cancer status. We performed flow cytometric and serologic analyses of 106 cancer patients and 113 non-cancer controls from two additional cohorts at Penn and Memorial Sloan Kettering Cancer Center. Patients with solid cancers exhibited an immune phenotype similar to non-cancer patients during acute COVID-19 whereas patients with hematologic cancers had significant impairment of B cells and SARS-CoV-2-specific antibody responses. High dimensional analysis of flow cytometric data revealed 5 distinct immune phenotypes. An immune phenotype characterized by CD8 T cell depletion was associated with a high viral load and the highest mortality of 71%, among all cancer patients. In contrast, despite impaired B cell responses, patients with hematologic cancers and preserved CD8 T cells had a lower viral load and mortality. These data highlight the importance of CD8 T cells in acute COVID-19, particularly in the setting of impaired humoral immunity. Further, depletion of B cells with anti-CD20 therapy resulted in almost complete abrogation of SARS-CoV-2-specific IgG and IgM antibodies, but was not associated with increased mortality compared to other hematologic cancers, when adequate CD8 T cells were present. Finally, higher CD8 T cell counts were associated with improved overall survival in patients with hematologic cancers. Thus, CD8 T cells likely compensate for deficient humoral immunity and influence clinical recovery of COVID-19. These observations have important implications for cancer and COVID-19-directed treatments, immunosuppressive therapies, and for understanding the role of B and T cells in acute COVID-19.

Efficient identification of HLA-A*2402–restricted cytomegalovirus-specific CD8+ T-cell epitopes by a computer algorithm and an enzyme-linked immunospot assay

Blood ◽  
2001 ◽  
Vol 98 (6) ◽  
pp. 1872-1881 ◽  
Author(s):  
Kiyotaka Kuzushima ◽  
Naomi Hayashi ◽  
Hiroshi Kimura ◽  
Tatsuya Tsurumi
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cell Clone ◽  
Cd8 T Cell ◽  
Computer Assisted ◽  
Efficient Strategy ◽  
T Cell Clone ◽  
Ctl Responses

Abstract Antigenic peptides recognized by virus-specific cytotoxic T lymphocytes (CTLs) are useful tools for studying the CTL responses exclusively among those who own the major histocompatibility complex (MHC) class I molecules that present the peptides. For widening the application, an efficient strategy to determine such epitopes in the context of a given MHC is highly desirable. A rapid and efficient strategy is presented for the determination of CTL epitopes in the context of given MHC molecules of interest through multiple screenings consisting of a computer-assisted algorithm and MHC stabilization and enzyme-linked immunospot assays. A major cytomegalovirus (CMV)–specific CTL epitope, QYDPVAALF, in the amino acid sequence of its lower matrix 65 kd phosphoprotein (pp65) presented by HLA-A*2402 molecules was identified from 83 candidate peptides. The results indicate that the CMV-specific CTL response is highly focused to pp65 in the context of HLA-A*2402. Endogenous processing and presentation was confirmed using a peptide-specific CD8+ T-cell clone as the effectors and autologous fibroblast cells infected with recombinant vaccinia virus expressing pp65 gene or CMV as antigen-presenting cells. Flow cytometric analysis of intracellular interferon-γ production revealed 0.04% to 0.27% of CD8+ T cells in peripheral blood of HLA-A24+ and CMV-seropositive donors to be specific for the peptide. The tetrameric MHC-peptide complexes specifically bound to the reactive T-cell clone and 0.79% of CD8+ T cells in peripheral blood from a seropositive donor. The peptide could be a useful reagent to study CTL responses to CMV among populations positive for HLA-A*2402.

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