High frequency of circulating HBcAg-specific CD8 T cells in hepatitis B infection: a flow cytometric analysis

Abstract Chronic GVHD (cGVHD) is a major risk factor in patients receiving allogeneic hematopoietic cell transplantation (HCT), and is a complicated syndrome with a combination of autoimmune-like features and a range of multiorgan manifestations. Currently, efforts are being made to standardize the criteria for diagnosis and staging of cGVHD, but there is little understanding of the pathogenesis of the disease, associated biomarkers, and the immune perturbations that may result. Reconstitution of the T cell repertoire after allo-HCT often takes several months to a year, and may be significantly impaired or skewed in patients who develop cGVHD. We thus sought to assess the immune T cell status of cGVHD patients by TCR Vβ CDR3-size spectratype analysis. A cohort of 9 patients who underwent allo-HCT (PBMC n=7; BM n=2) were enrolled in the study. The underlying diseases in these patients were CML (n=1), AML (n=4), ALL (n=1), CLL (n=1), and MM (n=2). Patients received either reduced intensity or myeloablative conditioning before transplantation, and 8 of the 9 had a previous history of acute GVHD. Furthermore, the patients did not have evidence of infectious disease. PBMC was collected from each patient at one time point ranging from 2 wk to 3 yr from the time they were diagnosed with cGVHD. The onset of cGVHD ranged from 100 d to 3 yr post-HCT (median of 5 mo). Flow cytometric analysis was performed on peripheral blood lymphocytes from 7 of the 9 patients to analyze recovery of different subpopulations. PCR amplification of the CDR3 region of 21 TCR Vβ genes was used to analyze the diversity of the T cell repertoire. The PCR products were run on a sizing gel to separate the CDR3-lengths, and further analyzed by ABI GeneMapper software. Flow cytometric analysis revealed diverse percentages of CD4+ and CD8+ T cells among the 7 patients tested, which were correlated with the post-HCT period. Two patients who received HCT, 4 and 9 months before blood sampling, had only 3% and 4% CD4+ and 3% and 9% CD8+ T cells in their PBMC sample, respectively. On the other hand, the remaining 5 patients, who were all at later time points post-HCT, had CD4+ and CD8+ T cell percentages within normal range. One patient had a ratio close to the normal 2:1 CD4/CD8 ratio, two patients had a 1:1 ratio, and four had inverse CD4/CD8 ratios. Based on CDR3-size spectratype analysis, we determined the recipient TCR-Vβ complexity index within each resoluble family, which represented the percentage of the number of peaks found for each Vβ relative to that found in the average corresponding Vβ family of 10 healthy donors. We considered Vβ to be fully complex if the complexity index exceeded 85%. The results indicated that 41 to 88% of resolved Vβ in all 9 patients were fully complex, with the lower range corresponding to those patients sampled early post-HCT. Vβ 1, 2, 4, 6, 8, 12, and 13 families revealed the best recovery in all patients, even in patients after 4-mo post-HCT. Importantly, extensive skewing of the repertoire within most of the TCR Vβ families were found in all 9 recipients, suggesting that there were active heterogenous T cell responses in those patients with cGVHD. As to what these T cell responses were directed to remains to be seen, and could theoretically involve autoantigens, alloantigens, tumor antigens, or sub-detectable infectious agents. In any case, the presence of a wide-ranging T cell response in these patients may serve as an important new diagnostic indicator for cGVHD.

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Prevention of Acute GvHD During MHC Haploidentical BMT: Evaluating the Efficacy of T-Cell Costimulation Blockade Using a Novel Rhesus Macaque Transplant Model.

Blood ◽

10.1182/blood.v114.22.2455.2455 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2455-2455

Author(s):

Weston Miller ◽

Caleb E. Wheeler ◽

Angela Panoskaltsis-Mortari ◽

Allan D Kirk ◽

Christian P Larsen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Rhesus Macaque ◽

Cd8 T Cells ◽

Flow Cytometric Analysis ◽

Costimulation Blockade ◽

Flow Cytometric ◽

Gvhd Prophylaxis ◽

High Level ◽

Cd127 Expression

Abstract Abstract 2455 Poster Board II-432 Introduction: While hematopoietic stem cell transplantation (HSCT) offers a cure for many hematologic diseases, it remains plagued by often fatal graft-versus-host disease (GvHD). Despite the inadequacy of current GvHD prevention strategies, especially for MHC-mismatched HSCT, the pace of the clinical introduction of novel therapeutics has been slow, likely due to the lack of a suitable translational model to rigorously test the immunologic and clinical impact of novel biologic therapies. Among the most promising of these therapies include those that block T cell costimulation blockade. While they have been used for both autoimmune disease and to prevent rejection of solid organ transplants, costimulation blockade reagents have not yet been evaluated for efficacy in preventing clinical GvHD. Here we describe a novel primate model of MHC-mismatched GvHD, that has allowed us, for the first time, to evaluate the mechanisms controlling GvHD in a primate translational system, and to evaluate the efficacy of costimulation blockade for the prevention of primate GvHD, even across haplo-MHC barriers. Methods: Using DNA microsatellite-based pedigree analysis and MHC haplotype determination, we have developed the first MHC-defined Rhesus macaque HSCT system. MHC haplo-identical transplant pairs were chosen, and recipients prepared for transplant with TBI (8 Gy, as a single dose, with lung shielding to 6 Gy). Animals were either treated with no immunosuppression post-transplant (controls) or with a costimulation blockade-based regimen which included CD28/B7 blockade with abatacept (20mg/kg every 7 days), CD40/CD154 blockade with the 3A8 anti-CD40 monoclonal antibody (maintenance dosing at 5mg/kg twice weekly) as well as sirolimus to maintain serum trough levels between 5-10 ng/mL. Either leukopheresis-derived peripheral blood stem cells or bone marrow was used for transplant (average total nucleated cell dose = 9.3 +/-2.7×108/kg; average CD3+ cell dose = 1.1 +/- 0.88 ×108/kg) Donor engraftment was measured by microsatellite analysis, and GvHD was graded clinically using standard scales. The immune phenotype after transplant was determined by multicolor cell- and serum-based flow cytometric analyses. Results: Seven haploidentical transplants have been completed. Three controls received no immunosuppression. These animals demonstrated rapid and complete donor engraftment, with donor T cell activation and proliferation occurring within one week of transplant, coincident with the onset of severe clinical GvHD, which predominantly targeted the GI tract. Flow cytometric analysis showed loss of CD127 expression on both CD4+ and CD8+ T cells, consistent with their rapid clonal expansion and differentiation. Multiplexed luminex cytokine analysis demonstrated high-level secretion of the inflammatory cytokines IFNγ, and IL18, as well as the counter-regulatory cytokine IL-1RA. Importantly, no rise in TNF, IL-1b, nor IL17 was measured despite severe GvHD. In contrast, four treated animals received a haplo-identical BMT in the setting of abatacept/anti-CD40 and sirolimus for GvHD prophylaxis. All of these recipients demonstrated rapid donor engraftment, but, unlike the controls, they were protected against clinical GvHD—they displayed neither the skin rash nor the profuse diarrhea noted in the control animals. Flow cytometric analysis demonstrated maintenance of CD127 expression on both CD4+ and CD8+ T cells. Furthermore, luminex analysis revealed that expression of IFNγ, IL18 and IL-1RA were all normal in the setting of GvHD prophylaxis with costimulation blockade and sirolimus. Conclusions: We have established a robust model of haplo-identical HSCT and GvHD using an MHC-defined Rhesus macaque colony. This model has allowed us to begin to determine the mechanisms underlying GvHD during primate haplo-identical BMT and to assess the efficacy of novel regimens to prevent this disease. We find that unprotected primate GvHD is characterized by rapid T cell proliferation, with concomitant loss of expression of CD127 on both CD4+ and CD8+ T cells. In addition, it is associated with a cytokine storm, including high level secretion of IFNγ, IL18 and IL-1RA into the serum. Finally, we find that CD28/CD40-directed costimulation blockade in combination with sirolimus can effectively inhibit both the clinical and cellular hallmarks of GvHD during haplo-identical BMT, and thus may deserve close clinical scrutiny as a possible prophylaxis strategy during these high risk transplants. Disclosures: No relevant conflicts of interest to declare.

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Expression of two alpha chains on the surface of T cells in T cell receptor transgenic mice.

Journal of Experimental Medicine ◽

10.1084/jem.178.5.1807 ◽

1993 ◽

Vol 178 (5) ◽

pp. 1807-1811 ◽

Cited By ~ 90

Author(s):

W R Heath ◽

J F Miller

Keyword(s):

T Cells ◽

T Cell ◽

Transgenic Mice ◽

T Cell Receptor ◽

Cd8 T Cells ◽

Flow Cytometric Analysis ◽

Cell Receptor ◽

Specific Monoclonal Antibody ◽

Beta Chain ◽

Flow Cytometric

CD8+ T cells taken directly from mice expressing a Kb-specific T cell receptor (TCR) transgene expressed the transgenic TCR in a bimodal profile as detected by flow cytometric analysis using a clonotype-specific monoclonal antibody. Those cells expressing the lower density of the transgenic TCR expressed the transgenic beta chain and two different alpha chains on their surface. One alpha chain was the product of the alpha transgene, whereas the other was derived by endogenous rearrangement. This report provides the first demonstration that T cells isolated directly from mice may express two different TCR clonotypes on their surface. The potential consequences of this finding for studies using TCR transgenic mice and for the induction of autoimmunity are discussed.

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CD4+ T Cell Fate in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-117674 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4494-4494

Author(s):

Rachel Elizabeth Cooke ◽

Jessica Chung ◽

Sarah Gabriel ◽

Hang Quach ◽

Simon J. Harrison ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Research Funding ◽

Flow Cytometric Analysis ◽

Advisory Committees ◽

Mitochondrial Mass ◽

Flow Cytometric

Abstract The average incidence of multiple myeloma (MM) is in the 7th decade that coincides with the development of immunosenescence and thymic atrophy, meaning that lymphocyte recovery after lymphopenia-inducing therapies (most notably autologous stem cell transplant, ASCT) is largely reliant on homeostatic proliferation of peripheral T cells rather than replenishing the T cell pool with new thymic emigrants. We have previously shown that there is a significant reduction in circulating naïve T cells with a reciprocal expansion of antigen-experienced cells from newly diagnosed MM (NDMM) to relapsed/refractory disease (RRMM). This results in a reduced TCR repertoire and the accumulation of senescence-associated secretory phenotype cytotoxic T cells, which maintain the ability to produce IFNγ but lose proliferative potential. A reduction in CD4:8 ratio is also a characteristic finding in MM with disease progression, which can be explained by high IL-15 levels in lymphopenic states that preferentially drive expansion of CD8+ memory T cells. We wanted to further evaluate what changes were occurring in the CD4+ T cell population with disease progression in MM. We analyzed paired peripheral blood (PB) samples from patients with NDMM and RRMM, and compared with age-matched normal donors (ND). In the NDMM cohort, we examined T cells from PB samples at baseline, after 4 cycles of lenalidomide and dexamethasone (len/dex), and after ASCT; and in the RRMM cohort samples from baseline and after 6 cycles of len/dex. We firstly confirmed in flow cytometric analysis of T cells at serial intervals in NDMM patients that the reduction in circulating naïve T cells and in CD4:8 ratio occurs post ASCT and does not recover by time of last follow-up. We next utilised RNA-seq to analyse differences in CD4+ T cells from NDMM, RRMM and ND. CD4+ T cells from RRMM showed downregulation of cytosolic ribosomal activity but maintenance of mitochondrial ribosomal activity and significant upregulation of pathways involved with calcium signalling. To this end, we evaluated mitochondrial biogenesis and metabolic pathways involved with mitochondrial respiration. Flow cytometric analysis of mitochondrial mass showed a marked increase in RRMM compared with ND, in keeping with a shift towards memory phenotype. Key rate-limiting enzymes in fatty acid β-oxidation (CPT1-A, ACAA2 and ACADVL) were all significantly increased in RRMM compared with ND. To analyse whether these cells were metabolically active, we also measured mitochondrial membrane potential and reactive oxygen species (ROS), gating on cells with high mitochondrial mass. Mitochondrial membrane potential was significantly increased in RRMM compared with ND, although ROS was reduced. The significance of this is not clear, as ROS are not only implicated in cell senescence and activation-induced cell death, but are also positively involved in tyrosine kinase and PI3K-signalling pathways. PD-1 has been shown to play a role in transitioning activated CD4+ T cells from glycolysis to FAO metabolism, and elevating ROS in activated CD8+ T cells. We analysed PD-1 expression on T cells in RRMM and at treatment intervals in NDMM (as described earlier). The proportion of CD4+ and CD8+ T cells expressing PD-1 was increased 4-6 months post-ASCT and remained elevated in CD4+ T cells 9-12 months post-ASCT, but normalised to baseline levels in CD8+ T cells. Increased PD-1 expressing CD4+ T cells was also evident in RRMM patient samples. This may suggest that in the lymphopenic state, PD-1 expression enhances longevity in a subset of CD4+ T cells by promoting reliance on mitochondrial respiration; however, their ability to undergo homeostatic proliferation is impaired. In CD8+ T cells, high PD-1 expression may lead to cell death via ROS accumulation, and these cells do not persist. ASCT remains a backbone of myeloma treatment in medically fit patients. However, this leads to significant permanent defects in the T cell repertoire, which may have unintended adverse outcomes. Additionally, T cells post-ASCT may not be metabolically adequate for the production of CAR-T cells, nor respond to checkpoint blockade therapies. Disclosures Quach: Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Sanofi Genzyme: Research Funding; Janssen Cilag: Consultancy. Harrison:Janssen-Cilag: Other: Scientific advisory board. Prince:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Response to Zhang et al. re:“CXCR5+ CD8+ T Cells Indirectly Offer B Cell Help and Are Inversely Correlated with Viral Load in Chronic Hepatitis B Infection”

DNA and Cell Biology ◽

10.1089/dna.2018.4538 ◽

2019 ◽

Vol 38 (1) ◽

pp. 113-113

Author(s):

Yun Jin

Keyword(s):

T Cells ◽

Chronic Hepatitis ◽

Viral Load ◽

Hepatitis B ◽

B Cell ◽

Chronic Hepatitis B ◽

Cd8 T Cells ◽

Hepatitis B Infection ◽

Chronic Hepatitis B Infection

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Correlation of CpG methylation of the Pdcd1 gene with PD‐1 expression on CD8 + T cells and medical laboratory indicators in chronic hepatitis B infection

The Journal of Gene Medicine ◽

10.1002/jgm.3148 ◽

2020 ◽

Vol 22 (2) ◽

Author(s):

Lin Jiao ◽

Jie Chen ◽

Xiaojuan Wu ◽

Bei Cai ◽

Zhenzhen Su ◽

...

Keyword(s):

T Cells ◽

Chronic Hepatitis ◽

Hepatitis B ◽

Chronic Hepatitis B ◽

Cd8 T Cells ◽

Cpg Methylation ◽

Medical Laboratory ◽

Hepatitis B Infection ◽

Chronic Hepatitis B Infection

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Donor CD8+ T Cells Facilitate Graft-Versus-Tumor Effect Via Alloantigen Rather Than Tumor-Specific Antigen Recognition Following Murine Myeloablative BMT.

Blood ◽

10.1182/blood.v104.11.3051.3051 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3051-3051

Author(s):

Asha B. Pillai ◽

Pearline A. Teo ◽

Aditi Mukhopadhyay ◽

Samuel Strober

Keyword(s):

Bone Marrow ◽

T Cells ◽

Tumor Cells ◽

Cd8 T Cells ◽

Specific Antigen ◽

Flow Cytometric Analysis ◽

Antigen Recognition ◽

Wild Type ◽

Flow Cytometric ◽

Tumor Specific Antigen

Abstract Previous data from our group and others has shown that donor CD8+ Tcells mediate graft-versus-tumor (GVT) function in murine myeloablative bone marrow transplantation (BMT) via Perforin/Fas ligand-dependent mechanisms. However, there has to date been no analysis of the mechanism of tumor recognition (i.e allo- versus tumor-specific antigen recognition) by donor CD8+ T cells following myeloablative MHC-mismatched BMT. In order to test the hypothesis that donor CD8 T-cells require allo-antigen recognition to maintain graft-versus-tumor effect, we developed stable full chimeras by transplanting T-cell depleted bone marrow (TCD BM) from C57BL/6 (H-2b) donor mice into myeloablated BALB/c (H-2d) hosts given 800 cGy total body irradiation (TBI) and evaluated the in vivo ability of CD8+ TCR+ splenocytes from these donors to kill the BCL1 tumor (a BALB/c-derived B-cell lymphoma carrying a detectable tumor-specific idiotype which can be monitored via peripheral blood flow cytometric analysis). These chimeras showed complete donor chimerism in myeloid, B- and T-lymphocytic lineages by day +100 following transplantation, and splenocytes from these chimeras exhibited tolerance to host-type but not third-party alloantigens. BALB/c hosts were given 800 cGy TBI on day -1, followed on day 0 by intravenous administration of 500 BCL1 tumor cells and infusion of 0.3x 106 CD8+ T cells of C57 origin (H-2b+) sorted by flow cytometric analysis from the either spleens of the chimeric mice or from the spleens of untreated wild-type C57BL/6 mice. All hosts were given 5 x 106 T cell-depleted wild-type C57 bone marrow cells. All mice were observed for clinical signs of graft-versus-host disease (GVHD) and mortality through day +100. Autopsy was performed at death to assess for sub-clinical target organ involvement with GVHD or tumor. Donor chimerism and BCL1 status was assessed at day +28 and day +100 by 3-color flow cytometric analysis for donor-specific MHC versus T-, B- and myeloid lineage markers as well as tumor-specific idiotype in the peripheral blood (or in the spleen at time of death for animals dying prior to day +100). All animals receiving BCL1 tumor cells and sorted CD8+T cells from wild-type untreated C57 donors cleared tumor idiotype but succumbed to GVHD. All animals receiving tumor cells and sorted chimeric C57 CD8+ T cells remained free of clinical or pathologic evidence of GVHD, but died with tumor progression. Control myeloablated animals given C57 TCD BM alone with BCL1 tumor cells all succumbed to tumor, whereas those receiving C57 whole bone marrow with tumor demonstrated tumor survival without GVHD. The data indicate that chimeric donor peripheral CD8+ T cells, which lose their capacity to induce lethal GVHD in BALB/c hosts, also lose the capacity to eradicate BALB/c-type lymphoma cells. We conclude that CD8+ T cell-induced GVT effect in this model is dependent upon alloantigen rather than tumor-specific antigen recognition.

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