Macrophage-derived CXCL9 and CXCL11, T-cell skin homing and disease control in mogamulizumab-treated CTCL patients

Blood ◽  
2021 ◽  
Author(s):  
Adèle de Masson ◽  
Delphine Darbord ◽  
Gabor Dobos ◽  
Marie Boisson ◽  
Marie Roelens ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Disease Control ◽  
High Throughput Sequencing ◽  
Sequencing Analysis ◽  
Cell Clones ◽  
Skin Homing ◽  
T Cell Homing ◽  
T Cell Immune Responses

Cutaneous T-cell lymphoma (CTCL) is a malignancy of skin-homing T-cells. Long-term remissions are rare in CTCL, and the pathophysiology of long-lasting disease control is unknown. Mogamulizumab is a defucosylated anti-human CCR4 antibody that depletes CCR4-expressing CTCL tumor cells and peripheral blood memory regulatory T cells. Prolonged remissions and immune side effects have been observed in mogamulizumab-treated CTCL patients. We report that mogamulizumab induced skin rashes in 32% of 44 CTCL patients. These rashes were associated with long-term CTCL remission, even in the absence of specific CTCL treatment. CTCL patients with mogamulizumab-induced rash had significantly higher overall survival (hazard ratio, 0.16 (0.04-0.73, p=0.01)). Histopathology and immunohistochemistry of the rashes revealed granulomatous and lichenoid patterns with CD163 macrophagic and CD8 T-cell infiltrates. Depletion of skin CTCL cells was confirmed by high-throughput sequencing analysis of TCRβ genes and in blood by flow cytometry. New reactive T-cell clones were recruited in skin. Gene expression analysis showed overexpression of CXCL9 and CXCL11, two chemokines involved in CXCR3-expressing T-cell homing to skin. Single-cell RNA sequencing analysis in skin of CTCL patients confirmed that CXCL9 and CXCL11 were primarily macrophage-derived and that skin T-cells expressed CXCR3. Finally, patients with rashes had a significantly higher proportion of exhausted reactive blood T-cells expressing TIGIT and PD1 at baseline compared to patients without rash, which decreased under mogamulizumab treatment, consistent with an activation of the antitumor immunity. Together, these data suggest that mogamulizumab may induce long-term immune control in CTCL patients by activation of the macrophagic and T-cell immune responses.

Ten Year Survivors of Multiple Myeloma Demonstrate a Differential Expression of Immunological Biomarkers Including a High Incidence of Cytotoxic T-Cell Clones Which Have Not Acquired Myeloma-Associated Anergy,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3924-3924
Author(s):  
Christian E Bryant ◽  
Ross D Brown ◽  
Shihong Yang ◽  
Hayley Suen ◽  
Esther Aklilu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Disease Control ◽  
Th17 Cells ◽  
Cytotoxic T Cell ◽  
Cd4 Cells ◽  
T Cell Clones ◽  
Cell Clones

Abstract Abstract 3924 Prior to the introduction of novel therapies for patients with multiple myeloma (MM) few patients survived for more than 10 years. Several reports have suggested that 10 year survival is associated with a younger age at diagnosis and the duration of exposure to effective agents. Although the number of patients surviving 10 years is increasing, there have been no significant reports of immunological biomarkers in these patients. This is especially true in those for whom the prolonged survival has occurred without novel drugs. Previous studies have shown that the presence of expanded peripheral blood CD8 T-cell clones in MM patients is associated with a better prognosis; raising the possibility that these T-cells, confirmed as clones by TCR sequencing, mediate an anti-tumor effect. However microarray gene set enrichment analysis and proliferation tracking studies demonstrated that these cells are in an anergic state. In addition, there is evidence that Tregs inhibit and Th17 cells enhance autologous immune response in malignancy and that an imbalance in MM may impair disease control. Slan-DCs are a subset of myeloid dendritic cells and are of interest in MM because of their ability to stimulate cytotoxic T-cell responses and reverse anergy in tumor infiltrating lymphocytes. We have investigated the immune mechanisms of disease control which may contribute to long-term survival by analyzing Tregs, Th17 cells, Slan-DCs and the incidence and relative degree of anergy of T-cell clones in all current >10 year survivors. Peripheral blood samples were analyzed for the presence of CD3+ T-cell receptor Vβ restricted T-cell clones (BetaMark Kit), the number of CD3+CD4+CD25h+CD127- Tregs, CD4+IL-17+ Th17 cells and CD16+CD14low M-DC8+ Slan-DCs. Proliferation of T-cells was analyzed using CFSE tracked 4 day cultures stimulated with anti-CD3 and anti-CD28 beads. Results were compared with local data collected from 2 large MM cohorts and age-matched controls. Of 26 patients who had survived for >10 years, 22 were available for testing. At diagnosis the median age was 59y (median=57y in comparison cohort); 54% were female; median β2M was 2.2mg/L (range 1.1–7.3); 92% were ISS I; 73% of paraproteins were IgG, 23% IgA and 4% light chain only. 73% had end-organ effects with; 65% bone disease; 15% anemia and 8% renal impairment. 58% had received cytotoxic chemotherapy; 27% novel agents; 38% autologous transplants; 8% allogeneic transplants and 31% were untreated. Expanded T-cell clones were documented in all (100%) of the 10 year survivors, a significant increase compared with 54% (n=144) and 48% (n=120) in the previous MM cohorts (χ2=43.6;p<0.001). Tregs accounted for 5.7% of CD4+ cells in the 10 year survivors which was significantly less than the 8.9% in the all-MM cohort (t=3.1; p< 0.005) but similar to 6.5% in age-matched controls. Proportions of Th17 cells were significantly greater in the 10 year survivor group (3.3% of CD4+ cells) than in the previous MM cohort (0.72%; U= 78; p< 0.005). Both absolute and percent mean Slan-DC numbers were significantly higher in the 10 year survivors than the all-MM cohort (0.17% vs 0.1%; t=2.5, p<0.02). Proliferation of clonal T cells was markedly greater in the 10-year survivors than the all-MM cohort (median 84% and 4% respectively; p<0.001) suggesting that they are more responsive. Analysis of immunological biomarkers in >10 year survivors of MM was compared to a large MM cohort and demonstrated a statistically significant decrease in T-regs with an increase in Th17 cells, absolute Slan-DCs and incidence of T-cell clones. These T-cell clones are more responsive than those in an all-MM cohort suggesting they have acquired less MM-associated anergy. This demonstrates that immunological mechanisms are active in long term disease control in MM and suggests that overcoming anergy of cytotoxic T-cells should be a focus of future studies. Disclosures: No relevant conflicts of interest to declare.

Stable Gene Transfer and Expression in Human Primary T-Cells by the Sleeping Beauty Transposon System.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5539-5539
Author(s):  
Xianzheng Zhou ◽  
Xin Huang ◽  
Andrew C. Wilber ◽  
Lei Bao ◽  
Dong Tuong ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Transgene Expression ◽  
Sleeping Beauty ◽  
Stable Gene ◽  
Cell Clones ◽  
Stable Gene Expression

Abstract The Sleeping Beauty (SB) transposon system is a non-viral DNA delivery system in which a transposase directs integration of an SB transposon into TA-dinucleotide sites in the genome. To determine whether the SB transposon system can mediate integration and long-term transgene expression in human primary T-cells, freshly isolated peripheral blood lymphocytes (PBLs) without prior activation were nucleofected with SB vectors carrying a DsRed reporter gene. Plasmids containing the SB transposase on the same (cis) (n=10) or separate molecule (trans) (n=8) as the SB transposon mediated long-term and stable reporter gene expression in human primary T-cells. We observed that delivery of SB transposase-encoding plasmid in trans effectively mediated stable gene expression in primary T-cells, exhibiting about a 3-fold increase (11% vs. 3% with 10 microgram plasmid on day 21) in potency in comparison with the cis vector (p&lt;0.0001). In addition, a transposase mutant construct was incapable of mediating stable gene expression in human PBLs (n=6, p&lt;0.0001), confirming that catalytic DDE domain is necessary for transposition in human primary T-cells. Immunophenotyping analysis in transposed T-cells showed that both CD4 and CD8 T-cells were transgene positive. SB-mediated high level of transgene expression in human T-cells was maintained in culture for at least 4 months without losing observable expression. Southern hybridization analysis showed a variety of transposon integrants among the 6 DsRed positive T-cell clones and no transposon sequences identifiable in the 2 DsRed negative clones. Sequencing of transposon:chromosome junctions in 5 out of 6 transposed T-cell clones confirmed that stable gene expression was due to SB-mediated transposition. In other studies, PBLs were successfully transfected using the SB transposon system and shown to stably and functionally express a fusion protein consisting of a surface receptor useful for positive T-cell selection and a “suicide” gene useful for elimination of transfected T-cells after chemotherapy. This study is the first report demonstrating that the SB transposon system can mediate stable gene transfer in human primary PBLs, which may be more advantageous for T-cell based gene therapies over widely used virus-based or conventional mammalian DNA vectors in terms of simplicity, stability, efficiency and safety.

Establishing T Cell Memory by Adoptive Transfer of T Cell Clones.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 866-866
Author(s):  
Carolina Berger ◽  
Michael C. Jensen ◽  
Stanley R. Riddell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Annexin V ◽  
T Cell Memory ◽  
T Cell Clones ◽  
Cell Clones

Abstract Adoptive transfer of T cells has been employed to reconstitute T cell immunity to viruses such as cytomegalovirus (CMV) in immunodeficient allogeneic stem cell transplant (SCT) patients and is being investigated to treat malignancies. In the allogeneic SCT setting, the T cells are derived from the donor and need to be isolated as clones or highly pure populations to avoid graft-versus-host disease. CD8+ T cells can be divided into defined subsets including CD62L− effector memory (TEM) and central memory T cells (TCM) expressing the CD62L lymph node homing molecule. Both TCM and TEM can give rise to cytolytic effector T cells (TE) after antigen stimulation and can be expanded in vitro for immunotherapy. However, the potential of T cells derived from either the TEM or TCM subset to persist in vivo has not been investigated. We used a macaque model to determine whether reconstitution of T cell memory to CMV by adoptive transfer of CD8+ T cell clones depended on their origin from either the CD62L+ TCM or CD62L− TEM subset. T cell clones were retrovirally transduced to express the macaque CD19 or CD20 surface marker to allow tracking of T cells in vivo. Clones derived from both TCM and TEM had similar avidity and proliferative capacity in vitro, and had a TE phenotype (CD62L−CCR7−CD28−CD127−, granzyme B+). TCM and TEM-derived T cell clones were transferred to macaques at doses of 3–6×108/kg and were both detected in the blood one day after transfer at 1.2–2.7% (low dose) to 20–25% (high dose) of CD8+ T cells. However, the frequency of TEM-derived T cells was undetectable after 3–5 days, and the cells were not present in lymph node or bone marrow obtained at day 14. By contrast, TCM-derived clones persisted in peripheral blood, migrated to tissue sites, and were detectable long-term at significant levels. A distinguishing feature of TCM-derived cells was their responsiveness to homeostatic cytokines. Only TCM-derived clones were rescued from apoptotic cell death by low-dose IL15 for &gt;30 days in vitro and this correlated with higher levels of IL15Rα, IL2Rβ, and IL2Rγ, and of Bcl-xL and Bcl-2, which promote cell survival. To determine if the inability of TEM-derived clones to survive in vitro correlated with an increased susceptibility of cell death in vivo, we measured the proportion of infused cells that were positive for propidium iodide (PI) and Annexin V during the short period of in vivo persistence. One day after transfer, 41–45% of TEM-derived T cells were Annexin V+/PI+, analyzed directly in the blood or after 24 hours of culture. By contrast, only a minor fraction of an adoptively transferred TCM-derived T cell clone was Annexin V+/PI+ and the infused cells survived in vivo. A subset of the persisting T cells reacquired TCM marker (CD62L+CCR7+CD127+CD28+) in vivo and regained functional properties of TCM (direct lytic activity; rapid proliferation to antigen). These T cells produced IFN-γ and TNF-α after peptide stimulation, and studies are in progress to assess their in vivo response to antigen by delivery of T cells expressing CMV proteins. Our studies in a large animal model show for the first time that CD8+ TE derived from TCM but not TEM can persist long-term, occupy memory T cell niches, and restore TCM subsets of CMV-specific immunity. Thus, taking advantage of the genetic programming of cells that have become TCM might yield T cells with greater therapeutic activity and could be targeted for human studies of T cell therapy for both viral and malignant disease.

scholarly journals Dynamics of the Cytotoxic T Cell Response to a Model of Acute Viral Infection

2015 ◽  
Vol 89 (8) ◽  
pp. 4517-4526 ◽  
Author(s):  
William S. DeWitt ◽  
Ryan O. Emerson ◽  
Paul Lindau ◽  
Marissa Vignali ◽  
Thomas M. Snyder ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cell ◽  
Viral Infection ◽  
High Throughput Sequencing ◽  
Cell Receptor ◽  
Cell Repertoire ◽  
T Cell Clones ◽  
Cell Clones ◽  
Acute Viral Infection

ABSTRACTA detailed characterization of the dynamics and breadth of the immune response to an acute viral infection, as well as the determinants of recruitment to immunological memory, can greatly contribute to our basic understanding of the mechanics of the human immune system and can ultimately guide the design of effective vaccines. In addition to neutralizing antibodies, T cells have been shown to be critical for the effective resolution of acute viral infections. We report the first in-depth analysis of the dynamics of the CD8+T cell repertoire at the level of individual T cell clonal lineages upon vaccination of human volunteers with a single dose of YF-17D. This live attenuated yellow fever virus vaccine yields sterile, long-term immunity and has been previously used as a model to understand the immune response to a controlled acute viral infection. We identified and enumerated unique CD8+T cell clones specifically induced by this vaccine through a combined experimental and statistical approach that included high-throughput sequencing of the CDR3 variable region of the T cell receptor β-chain and an algorithm that detected significantly expanded T cell clones. This allowed us to establish that (i) on average, ∼2,000 CD8+T cell clones were induced by YF-17D, (ii) 5 to 6% of the responding clones were recruited to long-term memory 3 months postvaccination, (iii) the most highly expanded effector clones were preferentially recruited to the memory compartment, and (iv) a fraction of the YF-17D-induced clones could be identified from peripheral blood lymphocytes solely by measuring clonal expansion.IMPORTANCEThe exhaustive investigation of pathogen-induced effector T cells is essential to accurately quantify the dynamics of the human immune response. The yellow fever vaccine (YFV) has been broadly used as a model to understand how a controlled, self-resolving acute viral infection induces an effective and long-term protective immune response. Here, we extend this previous work by reporting the identity of activated effector T cell clones that expand in response to the YFV 2 weeks postvaccination (as defined by their unique T cell receptor gene sequence) and by tracking clones that enter the memory compartment 3 months postvaccination. This is the first study to use high-throughput sequencing of immune cells to characterize the breadth of the antiviral effector cell response and to determine the contribution of unique virus-induced clones to the long-lived memory T cell repertoire. Thus, this study establishes a benchmark against which future vaccines can be compared to predict their efficacy.

scholarly journals Cytotoxic CD4+ T-cells specific for EBV capsid antigen BORF1 are maintained in long-term latently infected healthy donors

2021 ◽  
Vol 17 (12) ◽  
pp. e1010137
Author(s):  
Alexander C. Dowell ◽  
Tracey A. Haigh ◽  
Gordon B. Ryan ◽  
James E. Turner ◽  
Heather M. Long ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Granzyme B ◽  
T Cell Responses ◽  
Cd4 T Cell ◽  
Structural Proteins ◽  
Cell Responses ◽  
Ebv Infection ◽  
Cell Clones

Epstein Barr Virus (EBV) infects more than 95% of the population whereupon it establishes a latent infection of B-cells that persists for life under immune control. Primary EBV infection can cause infectious mononucleosis (IM) and long-term viral carriage is associated with several malignancies and certain autoimmune diseases. Current efforts developing EBV prophylactic vaccination have focussed on neutralising antibodies. An alternative strategy, that could enhance the efficacy of such vaccines or be used alone, is to generate T-cell responses capable of recognising and eliminating newly EBV-infected cells before the virus initiates its growth transformation program. T-cell responses against the EBV structural proteins, brought into the newly infected cell by the incoming virion, are prime candidates for such responses. Here we show the structural EBV capsid proteins BcLF1, BDLF1 and BORF1 are frequent targets of T-cell responses in EBV infected people, identify new CD8+ and CD4+ T-cell epitopes and map their HLA restricting alleles. Using T-cell clones we demonstrate that CD4+ but not CD8+ T-cell clones specific for the capsid proteins can recognise newly EBV-infected B-cells and control B-cell outgrowth via cytotoxicity. Using MHC-II tetramers we show a CD4+ T-cell response to an epitope within the BORF1 capsid protein epitope is present during acute EBV infection and in long-term viral carriage. In common with other EBV-specific CD4+ T-cell responses the BORF1-specific CD4+ T-cells in IM patients expressed perforin and granzyme-B. Unexpectedly, perforin and granzyme-B expression was sustained over time even when the donor had entered the long-term infected state. These data further our understanding of EBV structural proteins as targets of T-cell responses and how CD4+ T-cell responses to EBV change from acute disease into convalescence. They also identify new targets for prophylactic EBV vaccine development.

scholarly journals Cytolytic function of clonable T cells after human bone marrow transplantation

Blood ◽  
1990 ◽  
Vol 75 (6) ◽  
pp. 1364-1369 ◽  
Author(s):  
A Velardi ◽  
P Varese ◽  
CE Grossi ◽  
N Albi ◽  
C Dembech ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
T Cell ◽  
Marrow Transplantation ◽  
Allogeneic Bone Marrow Transplantation ◽  
Allogeneic Bone ◽  
T Cell Clones ◽  
Cell Clones

Abstract We evaluated T-cell mediated lymphokine activated killer (LAK) function during the late (greater than 5 months) reconstitution phase after T cell-depleted allogeneic bone marrow transplantation (BMT) for hematologic malignancy. Since LAK cells are sustained by interleukin-2 (IL-2), we also investigated the ability of post-BMT T cells to produce IL-2. These functions were investigated at the clonal level. More than 200 T-cell clones from six long-term BMT recipients were generated and compared with 60 T-cell clones derived from two normal controls. Almost all the CD8+ clonal cultures from BMT recipients expressed cytolytic activity in a lectin-dependent cellular cytoxicity assay. Interestingly, a higher proportion of BMT recipient-derived cytolytic clones were able to mediate LAK activity in comparison with control clones (28% versus 4%, P less than .05). However, T-cell clones from BMT recipients, as opposed to control clones, were largely incapable of producing IL-2. Given the high proportions of post-BMT circulating CD8+ T cells, it appears that, in long-term BMT recipients, the precursors of nonspecific LAK effectors are present at above normal levels. However, their function may be defective in vivo due to poor IL-2 production.

scholarly journals Integration features of intact latent HIV-1 in CD4+ T cell clones contribute to viral persistence

2021 ◽  
Vol 218 (12) ◽  
Author(s):  
Amy S. Huang ◽  
Victor Ramos ◽  
Thiago Y. Oliveira ◽  
Christian Gaebler ◽  
Mila Jankovic ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cd4 T Cell ◽  
Small Subset ◽  
Major Barrier ◽  
T Cell Clones ◽  
Cell Clones ◽  
Hiv 1

Latent intact HIV-1 proviruses persist in a small subset of long-lived CD4+ T cells that can undergo clonal expansion in vivo. Expanded clones of CD4+ T cells dominate latent reservoirs in individuals on long-term antiretroviral therapy (ART) and represent a major barrier to HIV-1 cure. To determine how integration landscape might contribute to latency, we analyzed integration sites of near full length HIV-1 genomes from individuals on long-term ART, focusing on individuals whose reservoirs are highly clonal. We find that intact proviruses in expanded CD4+ T cell clones are preferentially integrated within Krüppel-associated box (KRAB) domain–containing zinc finger (ZNF) genes. ZNF genes are associated with heterochromatin in memory CD4+ T cells; nevertheless, they are expressed in these cells under steady-state conditions. In contrast to genes carrying unique integrations, ZNF genes carrying clonal intact integrations are down-regulated upon cellular activation. Together, the data suggest selected genomic sites, including ZNF genes, can be especially permissive for maintaining HIV-1 latency during memory CD4+ T cell expansion.

scholarly journals SARS-CoV-2 escape from cytotoxic T cells during long-term COVID-19

2021 ◽  
Author(s):  
Evgeniia Alekseeva ◽  
Oksana Stanevich ◽  
Maria Sergeeva ◽  
Artem Fadeev ◽  
Kseniya Komissarova ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Neutralizing Antibodies ◽  
Immune Escape ◽  
Cytotoxic T Cells ◽  
Hla Class I ◽  
Cd8 T Cell ◽  
Hla Class I Antigens ◽  
Cell Clones

Abstract Evolution of SARS-CoV-2 in immunocompromised hosts may result in novel variants with changed properties, but the mode of selection underlying this process remains unclear. While escape from humoral immunity certainly plays a role in intra-host evolution, escape from cellular immunity is poorly understood. Here, we report a case of long-term COVID-19 in an immunocompromised patient with non-Hodgkin’s lymphoma who received treatment with rituximab and lacked neutralizing antibodies. Over the 318 days of the disease, the SARS-CoV-2 genome gained a total of 40 changes, 34 of which were present by the end of the study period. Among the acquired mutations, 12 reduced or prevented binding of known immunogenic SARS-CoV-2 HLA class I antigens, suggesting that virus immunoediting is largely driven by cytotoxic CD8 T cell clones. The two changes with the strongest effect, nsp3:T504A and nsp3:T504P, were experimentally assessed in a cytotoxic assay of the patient's CD8 T cells. Both these changes were associated with immune escape, with a stronger effect observed for nsp3:T504P, the change which ultimately got fixed. Together, these results suggest that CD8 T cell escape may be an underappreciated contributor to SARS-CoV-2 evolution in humans.

scholarly journals Cytolytic function of clonable T cells after human bone marrow transplantation

Blood ◽  
1990 ◽  
Vol 75 (6) ◽  
pp. 1364-1369
Author(s):  
A Velardi ◽  
P Varese ◽  
CE Grossi ◽  
N Albi ◽  
C Dembech ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
T Cell ◽  
Marrow Transplantation ◽  
Allogeneic Bone Marrow Transplantation ◽  
Allogeneic Bone ◽  
T Cell Clones ◽  
Cell Clones

We evaluated T-cell mediated lymphokine activated killer (LAK) function during the late (greater than 5 months) reconstitution phase after T cell-depleted allogeneic bone marrow transplantation (BMT) for hematologic malignancy. Since LAK cells are sustained by interleukin-2 (IL-2), we also investigated the ability of post-BMT T cells to produce IL-2. These functions were investigated at the clonal level. More than 200 T-cell clones from six long-term BMT recipients were generated and compared with 60 T-cell clones derived from two normal controls. Almost all the CD8+ clonal cultures from BMT recipients expressed cytolytic activity in a lectin-dependent cellular cytoxicity assay. Interestingly, a higher proportion of BMT recipient-derived cytolytic clones were able to mediate LAK activity in comparison with control clones (28% versus 4%, P less than .05). However, T-cell clones from BMT recipients, as opposed to control clones, were largely incapable of producing IL-2. Given the high proportions of post-BMT circulating CD8+ T cells, it appears that, in long-term BMT recipients, the precursors of nonspecific LAK effectors are present at above normal levels. However, their function may be defective in vivo due to poor IL-2 production.

scholarly journals High Throughput TCR Sequencing Provides Added Value in the Diagnosis of Cutaneous T-Cell Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1683-1683 ◽  
Author(s):  
Ilan Kirsch ◽  
David W Williamson ◽  
James Krueger ◽  
Jessica Teague ◽  
Christopher Elco ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
High Throughput ◽  
Skin Disease ◽  
Malignant Disease ◽  
High Throughput Sequencing ◽  
Employment Equity ◽  
Cell Clones ◽  
Skin Biopsies ◽  
Equity Ownership

Abstract Context Diagnosis of early-stage CTCL can be challenging because skin lesions contain a mixture of both diverse benign and clonal malignant T cells. Currently, clonality is assessed by TCR PCR but this method is qualitative, often fails to detect clones present at low numbers, and cannot be used to identify malignant T cells for subsequent study by flow cytometry or immunohistochemistry. High throughput sequencing (HTS) of the TCR gamma (TCRG) and TCR beta (TCRB) CDR3 regions provide comprehensive analysis of the total repertoire of T cell clones in a specimen, the breadth of repertoire diversity, and an exact quantitation of individual T cell clones. Objective In this study, we investigated whether the Adaptive Biotechnologies immunoSEQ assay of TCRG and TCRB could discriminate between benign and malignant T-cell skin dyscrasias. Patients This study involved a total of 132 patients. We analyzed skin biopsies from 39 patients with histopathologically and clinically suspicious or diagnostic CTCL (over 80% Stage I or Stage II), 9 patients with Lymphomatoid Papulosis, 4 treated CTCL patients in partial or complete remission, non-lesional biopsies from 3 CTCL patients, 6 biopsies from individuals without skin disease, 9 patients with atopic dermatitis, 12 patients with eczematous dermatitis, 24 patients with psoriasis, 15 patients with psoriasis after treatment with etanercept and clinical clearance, and non-lesional biopsies from 11 patients with psoriasis. Methods Genomic DNA was extracted from FFPE or OCT sections of skin biopsy specimens of patients. T cell receptor gamma (TCRG) and beta (TCRB) chain sequences were then independently amplified using multiplex PCR with optimized primer sets. Following HTS, a bioinformatics pipeline clusters the sequences into distinct clonotypes to determine overall frequencies and to identify diagnostic clones. V, (D), and J genes are also identified for each clonotype. Results The assays defined the dominant clonal sequences in every case and distinguished likely gamma/delta from alpha/beta T cell lymphoma. Using the fraction of the top two TCRG sequences as a fraction of the total nucleated cell population defined a cut off of approximately 1/500 above which the biopsy approached 100% specificity for malignant disease and below which the assay approached 100% specificity for non-malignant or treated malignant disease. TCR PCR was negative in 10 out of 34 biopsies suspicious of CTCL—all of these were among those determined to be clonal by HTS. While there was a strong correlation between analyses using the top two TCRG sequences (because both TCRG alleles are VJ rearranged in most T cells) versus the top one TCRB sequence (because only one TCRB allele is V(D)J rearranged in most T cells), the TCRG analysis was more powerful in terms of discrimination and separation of disease categories (see figure; P<0.00001 between CTCL or LyP and any other category) Figure Legend: 10 categories of histopathologically and clinically defined skin disease were studied by high-throughput sequencing of the TCRB and TCRG loci. “Box and whisker” plots of the TCRG analyses are shown. The boxed areas represent the second and third quartiles of the values of the top two TCRG sequences identified as a log fraction of the total nucleated cells in the study sample. Solid dots represent the median values. There is a clear demarcation between CTCL and LyP and the other categories of skin disease. Figure Legend:. 10 categories of histopathologically and clinically defined skin disease were studied by high-throughput sequencing of the TCRB and TCRG loci. “Box and whisker” plots of the TCRG analyses are shown. The boxed areas represent the second and third quartiles of the values of the top two TCRG sequences identified as a log fraction of the total nucleated cells in the study sample. Solid dots represent the median values. There is a clear demarcation between CTCL and LyP and the other categories of skin disease. Conclusions HTS analyses of DNA extracted from skin biopsies of patients with skin disorders can provide important quantitative data of T cell number, clonality, repertoire, and frequency and in so doing provide a useful discriminator of benign versus malignant disease. This same methodology may prove useful for other lymphoid malignancies in which the diagnosis is based on identification of clonal populations within a diverse admixture of cells. Moreover, identification of the TCR VB of the malignant clone allows it to be further characterized with anti-Vb antibodies, and assessed after treatment as MRD. Disclosures Kirsch: Adaptive Biotechnologies: Employment, Equity Ownership. Williamson:Adaptive Biotechnologies: Employment, Equity Ownership. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties.

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