Differential use of FasL- and perforin-mediated cytolytic mechanisms by T-cell subsets involved in graft-versus-myeloid leukemia responses

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 1047-1055 ◽  
Author(s):  
Michael H. Hsieh ◽  
Robert Korngold
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Myeloid Leukemia ◽  
T Cell Subsets ◽  
Leukemia Cell Line ◽  
Deficient Mice

In graft-versus-leukemia (GVL) responses, the cellular subsets and effector mechanisms responsible for cytotoxicity against leukemic cells in vivo remain poorly characterized. A murine model of syngeneic GVL that features CD4+ and CD8+T-cell responses against the MMB3.19 myeloid leukemia cell line has been previously described. MMB3.19 expresses high levels of functional Fas and tumor necrosis factor (TNF) receptors that do not transduce proapoptotic signals. Through the use of perforin- and Fas ligand (FasL)-deficient mice, it was demonstrated that CD4+ T cells mediate anti-MMB3.19 effects in vivo primarily through the use of FasL and secondarily through perforin mechanisms. Conversely, CD8+ T cells induce GVL effects primarily through the use of perforin and minimally through FasL mechanisms. Although the in vivo observations of CD8+ T cells were reflective of their in vitro cytotoxic T lymphocyte (CTL) activity, for CD4+ T cells, in vitro responses were dominated by the perforin pathway. In addition, the diminished capacity of T cells from perforin- and FasL-deficient mice to lyse MMB3.19 target cells appeared directly related to their deficient cytotoxic functions rather than to defects in activation because these cells were fully capable of mounting proliferative responses to the tumor cells. These findings demonstrate that GVL responses of T-cell subsets can involve preferential use of different cytotoxic mechanisms. In particular, these findings identify a role for both FasL-employing CD4+CTLs and the more novel perforin-utilizing CD4+ T-cell subset in responses against a myeloid leukemia.

Differential use of FasL- and perforin-mediated cytolytic mechanisms by T-cell subsets involved in graft-versus-myeloid leukemia responses

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 1047-1055 ◽  
Author(s):  
Michael H. Hsieh ◽  
Robert Korngold
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Myeloid Leukemia ◽  
T Cell Subsets ◽  
Leukemia Cell Line ◽  
Deficient Mice

Abstract In graft-versus-leukemia (GVL) responses, the cellular subsets and effector mechanisms responsible for cytotoxicity against leukemic cells in vivo remain poorly characterized. A murine model of syngeneic GVL that features CD4+ and CD8+T-cell responses against the MMB3.19 myeloid leukemia cell line has been previously described. MMB3.19 expresses high levels of functional Fas and tumor necrosis factor (TNF) receptors that do not transduce proapoptotic signals. Through the use of perforin- and Fas ligand (FasL)-deficient mice, it was demonstrated that CD4+ T cells mediate anti-MMB3.19 effects in vivo primarily through the use of FasL and secondarily through perforin mechanisms. Conversely, CD8+ T cells induce GVL effects primarily through the use of perforin and minimally through FasL mechanisms. Although the in vivo observations of CD8+ T cells were reflective of their in vitro cytotoxic T lymphocyte (CTL) activity, for CD4+ T cells, in vitro responses were dominated by the perforin pathway. In addition, the diminished capacity of T cells from perforin- and FasL-deficient mice to lyse MMB3.19 target cells appeared directly related to their deficient cytotoxic functions rather than to defects in activation because these cells were fully capable of mounting proliferative responses to the tumor cells. These findings demonstrate that GVL responses of T-cell subsets can involve preferential use of different cytotoxic mechanisms. In particular, these findings identify a role for both FasL-employing CD4+CTLs and the more novel perforin-utilizing CD4+ T-cell subset in responses against a myeloid leukemia.

scholarly journals Selective anergy of V beta 8+,CD4+ T cells in Staphylococcus enterotoxin B-primed mice.

1990 ◽  
Vol 172 (4) ◽  
pp. 1065-1070 ◽  
Author(s):  
Y Kawabe ◽  
A Ochi
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
T Cell Anergy ◽  
Specific Cytotoxicity ◽  
Enterotoxin B ◽  
Staphylococcus Enterotoxin B

The cellular basis of the in vitro and in vivo T cell responses to Staphylococcus enterotoxin B (SEB) has been investigated. The proliferation and cytotoxicity of V beta 8.1,2+,CD4+ and CD8+ T cells were observed in in vitro response to SEB. In primary cytotoxicity assays, CD4+ T cells from control spleens were more active than their CD8+ counterparts, however, in cells derived from SEB-primed mice, CD8+ T cells were dominant in SEB-specific cytotoxicity. In vivo priming with SEB abrogated the response of V beta 8.1,2+,CD4+ T cells despite the fact that these cells exist in significant number. This SEB-specific anergy occurred only in V beta 8.1,2+,CD4+ T cells but not in CD8+ T cells. These findings indicate that the requirement for the induction of antigen-specific anergy is different between CD4+ and CD8+ T cells in post-thymic tolerance, and the existence of coanergic signals for the induction of T cell anergy is suggested.

scholarly journals T cell memory. Long-term persistence of virus-specific cytotoxic T cells.

1989 ◽  
Vol 169 (6) ◽  
pp. 1993-2005 ◽  
Author(s):  
B D Jamieson ◽  
R Ahmed
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Population ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Biological Properties ◽  
Antigenic Specificity ◽  
Ctl Response

This study documents that virus-specific CTL can persist indefinitely in vivo. This was accomplished by transferring Thy-1.1 T cells into Thy-1.2 recipient mice to specifically identify the donor T cell population and to characterize its antigenic specificity and function by using a virus-specific CTL assay. Thy-1.1+ T cells from mice previously immunized with lymphocytic choriomeningitis virus (LCMV) were transferred into Thy-1.2 mice persistently infected with LCMV. The transferred LCMV-specific CTL (Thy-1.1+ CD8+) eliminate virus from the chronically infected carriers and persist in the recipient mice in small numbers, comprising only a minor fraction of the total T cells. Upon re-exposure to virus, these long-lived "resting" CD8+ T cells proliferate in vivo to become the predominant cell population. These donor CD8+ T cells can be recovered up to a year post-transfer and still retain antigenic specificity and biological function. They kill LCMV infected H-2-matched cells in vitro and can eliminate virus upon transfer into a second infected host. In addition, these long-lived CD8+ T cells appear not to be dependent on help from CD4+ T cells, since depletion of CD4+ T cells has minimal or no effect on their biological properties (proliferation, CTL response, viral clearance). These donor CTL also exhibit an immunodominance over the host-derived LCMV-specific CTL response. When both host and donor T cells are present, the donor CTL response is dominant over the potential CTL response of the cured carrier host. Taken together, these results suggest that virus-specific CTL can persist for the life span of the host as memory cells.

scholarly journals P025 Identification and validation of predictors for tofacitinib through supervised and unbiased approaches in Inflammatory Bowel Disease

2021 ◽  
Vol 15 (Supplement_1) ◽  
pp. S141-S141
Author(s):  
B Liu ◽  
M Spalinger ◽  
L G Perez ◽  
A Machicote ◽  
N Gagliani ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Signaling Pathway ◽  
Cd4 T Cells ◽  
Cytokine Production ◽  
Stat Signaling ◽  
Deficient Mice

Abstract Background Inflammatory Bowel Disease (IBD) is characterized by an overwhelming gut inflammation, where CD4+ effector T cells are main mediators of the inflammatory response. Tofacitinib, a small molecular drug recently used in IBD patients, blocks the JAK/STAT signaling pathway necessary for CD4+ effector T-cell activation. However, clinical data show that a percentage of patients do not respond to the treatment. Our main goal is to identify biomarkers predicting the response of patients to tofacitinib. Methods Tofacitinib efficacy was studied in vivo in wild type (WT) and T-cell-specific PTPN2 deficient mice (CD4-Cre;Ptpn2 floxed) in which the JAK/STAT signaling pathway is over activated. WT and PTPN2 deficient mice were gavaged with tofacitinib (50mg/kg, twice daily) or vehicle. Acute DSS-colitis was induced. Colitis development was evaluated by weight loss, colonoscopy and histology. CD4+ T cells were isolated from the colon and analyzed by flow cytometry. To study the effect of tofacitinib on T-cell differentiation, we isolated naïve T cells from mouse spleen and polarized them in vitro to different T-cell subsets with or without tofacitinib. CD4+ T cells differentiation and cytokine production were analyzed by flow cytometry. To evaluate the influence of tofacitinib on human CD4+ T cells, human peripheral blood mononuclear cells (PBMCs) from healthy donors and IBD patients were stimulated in presence of tofacitinib, and analyzed by flow cytometry. Results While no protective effect was found after tofacitinib treatment in WT mice, PTPN2 deficient mice were protected from colitis based on less weight loss, lower endoscopic and histological scores. The expression of pro-inflammatory cytokines such as IL-17 and IFN-γ by colonic CD4+ T cells was also decreased by tofacitinib. Consistent with the in vivo observations, in vitro experiments revealed a strong impact of tofacitinib on CD4+ T-cells cytokine production. In PBMCs from IBD patients, IFN-γ and TNF-α expression was strongly impacted. In contrast, in healthy donors, IL-10 was the most impacted cytokine. Finally, tofacitinib decreased the in vitro differentiation of Th1, Th2, Th17, Th22, Treg and Tr1. Conclusion In the T-cell-specific PTPN2 deficient mice, tofacitinib exerts a protective effect after DSS-induced colitis. In line with the in vivo findings, in vitro experiments show that tofacitinib has a strong impact on pro-inflammatory cytokine production, especially in the IBD patients. Taken together, these data suggest that tofacitinib might be suitable primarily for IBD patients where the JAK/STAT signaling pathway is over activated.

Critical and Opposing Effects of T Cell-Derived TNFα and Interferon-γ on IL-17 Induction Assessed in Vitro and in Vivo Following Allogeneic Bone Marrow Transplantation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3482-3482
Author(s):  
Minghui Li ◽  
Kai Sun ◽  
Mark Hubbard ◽  
Doug Redelman ◽  
Angela Panoskaltsis-Mortari ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Th17 Cells ◽  
Allogeneic Bone Marrow Transplantation ◽  
Allogeneic Bone ◽  
Allogeneic Bmt

Abstract IL-17-producing CD4 T cells (Th17) are a recently identified T helper subset that plays a role in mediating host defense to extracellular bacteria infections and is involved in the pathogenesis of many autoimmune diseases. In vitro induction of IL-17 in murine CD4+ T cells has been shown to be dependent on the presence of the proinflammatory cytokines TGF-β and IL-6 whereas IFNγ can suppress the development of Th17 cells. In the current study, we examined the roles of TNFα and IFNγ on IL-17 production by purified T cells in vitro and in vivo after allogeneic bone marrow transplantation (BMT). We present findings that expression of TNFα by the T cell itself is necessary for optimal development of Th17 under in vitro polarizing conditions. A novel role for T cell-derived TNFα in Th17 induction was observed when in vitro polarization of Tnf−/−CD4+ T cells resulted in marked reductions in IL-17+CD4+ T cells compared to Tnf+/+CD4+ T cells. In marked contrast, T cell-derived IFNγ markedly inhibited Th17 development as more IL-17+CD4+ T cells were found in Ifnγ−/−CD4+ T cells than in Ifnγ+/+CD4+ T cells, and of particular interest was the dramatic increase in IL-17+CD8+ cells from Ifnγ−/− mice. To determine if T cell-derived TNFα or IFNγ can regulate Th17 development in vivo we examined the differentiation of alloreactive donor T cells following allogeneic BMT. We have found that donor-derived Th17 cells can be found in lymphoid tissues and GVHD-affected organs after allogeneic BMT. However, transfer of Tnf−/− CD4+ T cells after allogeneic BMT resulted in marked reductions in Th17 cells in the spleen (18×103 vs 7×103, P<0.05). In agreement with the in vitro data and in contrast to what was observed with transfer of Tnf−/− CD4+ T cells, transfer of donor Ifnγ−/− T cells resulted in marked increases in not only IL-17+CD4+ but also IL-17+CD8+ T cells infiltrating the liver (7×103 vs 14×103, P<0.05; 4×104 vs 12.5×104, P<0.05). These results suggest that the donor T cell-derived TNFα and IFNγ opposingly regulate IL-17 induction of both CD4+ and CD8+ T cells in vitro and after allogeneic BMT which correlates with GVHD pathology.

Artificial APC-Based Generation of IL-21 Secreting CD4+ T Cells That Can Provide Help to CD8+ T Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 466-466
Author(s):  
Makito Tanaka ◽  
Marcus Butler ◽  
Sascha Ansén ◽  
Osamu Imataki ◽  
Alla Berezovskaya ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Binding Assay ◽  
Cell Responses ◽  
Large Numbers ◽  
A Cell

Abstract Abstract 466 CD8+ T cells are thought to be major players in T cell immunity because of their potent direct effector function. However, many studies have demonstrated that CD4+ T cells also play a critical role by providing help which optimizes CD8+ T cell responses. In vivo experiments using murine models have suggested that common cytokine receptor γ-chain cytokines such as IL-2, IL-15 and IL-21 are mediators of this CD4+ T cell help. Previously, we generated K562-based artificial APC (aAPC) by transducing HLA-A2, CD80, and CD83. This aAPC can generate large numbers of antigen-specific CD8+ CTL with a central/effector memory phenotype and potent effector function. These CTL are surprisingly long-lived and can be maintained in vitro without any feeder cells or cloning. We are currently conducting a clinical trial where large numbers of anti-tumor CD8+ CTL generated ex vivo using this aAPC and IL-2/IL-15 are adoptively transferred to patients with advanced cancer. Early results have demonstrated that adoptively transferred anti-tumor CTL can expand and persist as memory T cells for longer than 6 months without lymphodepletion or cytokine administration. Furthermore, some patients have demonstrated objective clinical responses. These in vivo results suggest that K562-based aAPC might serve as a clinically important APC to generate large numbers of antigen-specific T cells for adoptive therapy. Based upon these observations, we have generated a K562-derived aAPC that can expand antigen-specific CD4+ T cells capable of providing help to CD8+ T cells. One challenge with the study of human HLA class II-restricted antigen-specific CD4+ T cells lies in the fact that there is no DR allele with a frequency greater than 25% in any race or ethnic extraction. To overcome this issue, we targeted HLA-DP0401 (DP4), which is positive in 64% of Caucasians and is the most frequent HLA allele in many other ethnic groups. aAPC was generated by sequentially transducing DPA1*0103, DPB1*0401, CD80 and CD83 to HLA class I-, class II-, CD54+, CD58+ K562. Using this aAPC and 57 overlapping peptides encompassing the full-length protein, we identified three DP4-restricted immunogenic epitopes derived from CMV pp65. One of the 3 epitopes, peptide #23 (aa 221-240) appeared to be an immunodominant epitope, since specific CD4+ T cells were expanded from all donors tested. A cell-based in vitro competitive binding assay confirmed that #23 binds DP4 molecules. #23-specific CD4+ T cells generated using aAPC and low dose IL-2/IL-15 were long-lived, up to 4 months in vitro without any feeder cells or cloning, and were able to recognize APC exogenously pulsed with pp65 protein. ELISPOT showed that #23-specific CD4+ T cells were able to secrete IL-2, IL-4, IFN-γbut not IL-10 in an antigen-specific manner. Interestingly, intracellular cytokine staining revealed that a fraction of IFN-γsecreting CD4+ T cells concurrently produced IL-4. Most importantly, using an aAPC expressing HLA-A2, DP4, CD80, and CD83, we were able to demonstrate that pp65-specific CD4+ T cells can provide help to pp65-specific CD8+ T cells in an antigen-specific way. Survivin is an attractive target antigen for tumor immunotherapy, since it is expressed by many tumor types and is indispensable for tumor growth. We have also successfully generated DP4-restricted Survivin-specific CD4+ T cells using this aAPC. Using a cell-based in vitro binding assay, 5 Survivin-derived peptides with high binding capacity to DP4 molecules were identified. Among these 5 peptides, peptide #90 (aa 90-104) bound DP4 most potently. aAPC pulsed with #90 was able to induce antigen-specific CD4+ T cell responses from cancer patients. These CD4+ T cells were also long-lived, up to 3 months in vitro and secreted IL-2, IL-4, and IFN-γbut not IL-10. Interestingly, IL-21 was also produced upon antigen-specific stimulation. It should be noted that our K562-based aAPC did not expand Foxp3+ regulatory T cells under the experimental conditions tested. Taken all together, we have established a K562-based aAPC to generate large numbers of HLA-DP4-restricted antigen-specific CD4+ T cells that possess longevity and functional competence. Based upon our previous success in clinical translation of K562-based aAPC for CD8+ T cells and the high prevalence of HLA-DP4, generating a clinical grade version of this aAPC for CD4+ T cells is of high priority. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Normal T-Cell Development and Immune Functions in TRIM-Deficient Mice

2006 ◽  
Vol 26 (9) ◽  
pp. 3639-3648 ◽  
Author(s):  
Uwe Kölsch ◽  
Börge Arndt ◽  
Dirk Reinhold ◽  
Jonathan A. Lindquist ◽  
Nicole Jüling ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
T Cell Development ◽  
Cell Development ◽  
T Cell Subsets ◽  
Immune Functions ◽  
Deficient Mice

ABSTRACT The transmembrane adaptor molecule TRIM is strongly expressed within thymus and in peripheral CD4+ T cells. Previous studies suggested that TRIM is an integral component of the T-cell receptor (TCR)/CD3 complex and might be involved in regulating TCR cycling. To elucidate the in vivo function of TRIM, we generated TRIM-deficient mice by homologous recombination. TRIM−/− mice develop normally and are healthy and fertile. However, the animals show a mild reduction in body weight that appears to be due to a decrease in the size and/or cellularity of many organs. The morphology and anatomy of nonlymphoid as well as primary and secondary lymphoid organs is normal. The frequency of thymocyte and peripheral T-cell subsets does not differ from control littermates. In addition, a detailed analysis of lymphocyte development revealed that TRIM is not required for either positive or negative selection. Although TRIM−/− CD4+ T cells showed an augmented phosphorylation of the serine/threonine kinase Akt, the in vitro characterization of peripheral T cells indicated that proliferation, survival, activation-induced cell death, migration, adhesion, TCR internalization and recycling, TCR-mediated calcium fluxes, tyrosine phosphorylation, and mitogen-activated protein family kinase activation are not affected in the absence of TRIM. Similarly, the in vivo immune response to T-dependent and T-independent antigens as well as the clinical course of experimental autoimmune encephalomyelitis, a complex Th1-mediated autoimmune model, is comparable to that of wild-type animals. Collectively, these results demonstrate that TRIM is dispensable for T-cell development and peripheral immune functions. The lack of an evident phenotype could indicate that TRIM shares redundant functions with other transmembrane adaptors involved in regulating the immune response.

scholarly journals CD25+FoxP3+Memory CD4 T Cells Are Frequent Targets of HIV InfectionIn Vivo

2016 ◽  
Vol 90 (20) ◽  
pp. 8954-8967 ◽  
Author(s):  
Mkunde Chachage ◽  
Georgios Pollakis ◽  
Edmund Osei Kuffour ◽  
Kerstin Haase ◽  
Asli Bauer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Hiv Infection ◽  
Cd4 T Cells ◽  
T Cell Subsets ◽  
Hiv Dna ◽  
Active Viral Replication ◽  
Memory Cd4 T Cells

ABSTRACTInterleukin 2 (IL-2) signaling through the IL-2 receptor alpha chain (CD25) facilitates HIV replicationin vitroand facilitates homeostatic proliferation of CD25+FoxP3+CD4+T cells. CD25+FoxP3+CD4+T cells may therefore constitute a suitable subset for HIV infection and plasma virion production. CD25+FoxP3+CD4+T cell frequencies, absolute numbers, and the expression of CCR5 and cell cycle marker Ki67 were studied in peripheral blood from HIV+and HIV−study volunteers. Different memory CD4+T cell subsets were then sorted for quantification of cell-associated HIV DNA and phylogenetic analyses of the highly variable EnvV1V3 region in comparison to plasma-derived virus sequences. In HIV+subjects, 51% (median) of CD25+FoxP3+CD4+T cells expressed the HIV coreceptor CCR5. Very high frequencies of Ki67+cells were detected in CD25+FoxP3+memory CD4+T cells (median, 27.6%) in comparison to CD25−FoxP3−memory CD4+T cells (median, 4.1%;P< 0.0001). HIV DNA content was 15-fold higher in CD25+FoxP3+memory CD4+T cells than in CD25−FoxP3−T cells (P= 0.003). EnvV1V3 sequences derived from CD25+FoxP3+memory CD4+T cells did not preferentially cluster with plasma-derived sequences. Quasi-identical cell-plasma sequence pairs were rare, and their proportion decreased with the estimated HIV infection duration. These data suggest that specific cellular characteristics of CD25+FoxP3+memory CD4+T cells might facilitate efficient HIV infectionin vivoand passage of HIV DNA to cell progeny in the absence of active viral replication. The contribution of this cell population to plasma virion production remains unclear.IMPORTANCEDespite recent advances in the understanding of AIDS virus pathogenesis, which cell subsets support HIV infection and replicationin vivois incompletely understood.In vitro, the IL-2 signaling pathway and IL-2-dependent cell cycle induction are essential for HIV infection of stimulated T cells. CD25+FoxP3+memory CD4 T cells, often referred to as regulatory CD4 T cells, depend on IL-2 signaling for homeostatic proliferationin vivo. Our results show that CD25+FoxP3+memory CD4+T cells often express the HIV coreceptor CCR5, are significantly more proliferative, and contain more HIV DNA than CD25−FoxP3−memory CD4 T cell subsets. The specific cellular characteristics of CD25+FoxP3+memory CD4+T cells probably facilitate efficient HIV infectionin vivoand passage of HIV DNA to cell progeny in the absence of active viral replication. However, the contribution of this cell subset to plasma viremia remains unclear.

Low Incidence of Post-Transplant EBV-Related Disease After Alemtuzumab-Mediated T Cell Depletion Is Explained by the Differential Susceptibility to Alemtuzumab of B Cells and Protective CD8 and CD4 T Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2330-2330
Author(s):  
Constantijn J.M. Halkes ◽  
Inge Jedema ◽  
Judith Olde Wolbers ◽  
Esther M van Egmond ◽  
Peter A. Von Dem Borne ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cell Depletion ◽  
T Cell Depletion

Abstract Abstract 2330 In vivo T cell depletion with anti-thymocyte globulin (ATG) or alemtuzumab (anti-CD52) before reduced intensity allogeneic stem cell transplantation (alloSCT) in combination with in vitro T cell depletion with alemtuzumab reduces the risk of GVHD. Detectable levels of circulating antibodies are present up to several months after the alloSCT, leading to a delayed immune reconstitution which is associated with an increased incidence of opportunistic infections and early relapses. Prior to 2007, combined in vitro (Alemtuzumab 20 mg added “to the bag”) and in vivo T cell depletion with horse-derived ATG (h-ATG) resulted in good engraftment without GVHD in the absence of GVHD prophylaxis after reduced intensity alloSCT using conditioning with fludarabine and busulphan. Due to the unavailability of h-ATG, rabbit-derived ATG (r-ATG) 10–14 mg/kg was introduced in the conditioning regimen in 2007. Strikingly, in this cohort of patients, early EBV reactivation and EBV-associated post-transplantation lymphoproliferative disease (PTLD) was observed in 10 out of 18 patients at a median time of 6 weeks after alloSCT (range 5 to 11 weeks) in the absence of GVHD or immunosuppressive treatment. Analysis of T and B cell recovery early after transplantation revealed preferential depletion of T cells as compared to B cells, thereby allowing unrestricted proliferation of EBV infected B cells. Due to this unacceptable high incidence of EBV-related complications, in the conditioning regimen r-ATG was replaced by low dose alemtuzumab (15 mg i.v. day -4 and -3) in 2008. In this cohort of 60 patients, only 2 patients experienced transient EBV reactivation during the first 3 months after alloSCT and one patient developed an EBV-associated lymphoma 4 weeks after alloSCT. To investigate the mechanisms underlying the low incidence of EBV reactivation using alemtuzumab for T cell depletion, we studied the in vivo and in vitro effects of alemtuzumab on different lymphocyte subsets. First, lineage-specific reconstitution was studied in 20 patients from the alemtuzumab cohort with known CD52 negative diseases (11 AML and 9 multiple myeloma) to exclude the confounding effect of antibody absorption by malignant cells. Whereas at 3 weeks after alloSCT detectable numbers of circulating NK cells and T cells were observed (medians 71 (range 6–378), and 12 (range 1–1164)E6/L, respectively), no circulating B cells could be detected (median 0, range 0–1 E6/L). At 6 weeks after alloSCT, NK and T cell numbers further increased (medians 212 (52-813), and 130 (range 25–1509)E6/L, respectively), whereas B cell numbers still remained low in the majority of patients (median 15, range 0–813E6/L). In all patients, T cells were detectable before the appearance of circulating B cells. Furthermore, the expression of CD52 and the sensitivity to alemtuzumab-mediated complement-dependent cell lysis (CDC) of B cells, T cells and NK cells was measured in vitro. The highest CD52 expression was observed on B cells (mean fluorescence intensity (MFI) 120), resulting in 95% lysis after incubation with 10ug/mL alemtuzumab and rabbit complement. NK cells showed a significantly lower CD52 expression (MFI 41), which was also reflected by a lower susceptibility to alemtuzumab-mediated CDC (62% lysis). Interestingly, differential expression of CD52 was observed on CD4 and CD8 T cells (MFI 120 and 101, respectively). Cytotoxicity analysis revealed relative protection of CD8 compared to CD4 T cells against alemtuzumab-mediated CDC, resulting in 52% and 90% lysis, respectively. Based on these results, we investigated in detail the presence and phenotype of the CD4 and CD8 subsets and EBV-specific CD8 T cells using tetramer staining at 6 weeks after alloSCT. In accordance with the in-vitro expression and susceptibility data, circulating CD52+ CD8 T cells including EBV-specific T cells were detectable. Interestingly, the majority of circulating CD4 T cells (64-93%, n=4) lacked CD52 expression, explaining their capacity to persist in the presence of alemtuzumab. We conclude that in vivo and in vitro T cell depletion with alemtuzumab is associated with a relatively low risk of EBV-associated PTLD because of efficient B cell depletion and persistent EBV immunity allowed by the relative insusceptibility for alemtuzumab of CD8 T cells and the development of CD52 negative escape variants of CD4 T cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Bone Marrow GZMK +IL7R + Progenitor-Exhausted CD8 + T Cells Correlate with Sustained Clinical Remission in Patients with Acute Myeloid Leukemia (AML) Undergoing Chemotherapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 521-521
Author(s):  
Francesco Mazziotta ◽  
Luca Biavati ◽  
Rupkatha Mukhopadhyay ◽  
Hanna A. Knaus ◽  
Ivan M. Borrello ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Myeloid Leukemia ◽  
Cd8 T Cell ◽  
T Cell Subsets ◽  
Spectral Flow

Abstract Introduction The role of T cells in chemotherapy response and maintenance of remission in acute myeloid leukemia (AML) patients is not fully understood. In solid tumors and chronic infections, exhaustion is a multistep process ranging from less differentiated progenitor exhausted (Tpex) to intermediate and terminally exhausted T cells (Beltra et al. 2020). High frequencies of Tpex correlate with response to immune-checkpoint blockade in solid tumors (Miller et al. 2019). In AML, where the backbone of treatment is chemotherapy, the role of dysfunctional T-cell subsets has yet to be elucidated. Methods Serial bone marrow (BM) samples from 16 AML patients (10 complete responders (Res) and 6 non-responders (NonRes)) at diagnosis and at response assessment after induction chemotherapy and 12 healthy donors (HD) were analyzed by flow cytometry using a 13-color panel. Moreover, we performed single-cell RNA sequencing (scRNAseq) (10X Genomics) on BM samples from 2 HD and 5 AML patients (3 Res, 2 NonRes) at baseline and after chemotherapy. Subsequently, we used a scRNAseq-guided 26-color spectral flow cytometry panel and explored T-cell phenotypes on BM of 22 AML patients (12 Res and 10 NonRes). Custom-made R scripts were employed for high-dimensional flow cytometry and scRNAseq analysis. Results Initial flow-cytometry analysis showed a significant increase in BM PD1 +CD28 + CD8 + T cell subset (p&lt;0.01) in Res vs NonRes at baseline and post-chemotherapy (data not shown). To further investigate these results, we performed 5' VDJ scRNAseq and used gene signatures mapped in two dimensions via UMAP to annotate the T-cell clusters as naive, Tpex, T effector CX3CR1 + (Teff CX3CR1pos), Terminally exhausted 1 (Term_exh1) and Terminally exhausted 2 (Term_exh2) (Fig 1A). Of note, the two most upregulated genes in Tpex were GZMK and IL-7R. We then performed differential abundance analysis to investigate differences in terms of clusters' frequencies across the three conditions (Res, NonRes, HD). At both timepoints Res had an increased frequency of Tpex and Teff CX3CR1pos compared to NonRes. Conversely, Term_exh2 cells were more abundant in NonRes (Fig. 1B). Next, we measured the magnitude of clonal expansion in antigen-experienced CD8 + T cells in Res and NonRes generating an overlay of the position of clonally expanded cells projected onto the UMAP. The most clonally expanded subsets were Tpex and Teff CX3CR1pos in Res (Fig. 1C) and Term_exh2 in NonRes (Fig. 1D) revealing a strong relationship between abundance and clonal expansion of the CD8 + T-cell subsets. Our scRNAseq results were then confirmed at the protein level with spectral flow-cytometry. The FlowSOM algorithm identified a CD8 + GZMK +CD127 + subset to be increased at baseline in Res vs NonRes (Fig. 1E). Remarkably, this cluster was also characterized by the expression of TIGIT, PD1 and TCF-1. These results were subsequently reproduced by manual gating of the GZMK +CD127 + subset which was significantly enriched (p&lt;0.01) in Res vs NonRes (Fig. 1F). Of note, patients with a higher-than-median frequency of GZMK +CD127 +CD8 + T cells experienced significantly (p&lt;0.02) prolonged overall survival after therapy (Fig. 1G). Conclusion Improving our understanding of the immune microenvironment in AML is critical for the rational integration of novel treatment strategies that seek to increase the response rate and/or maintain remission. We identified GZMK +IL7R + CD8 + cells as a distinct entity in the early differentiated CD8 + memory T cell pool that is clonally expanded and more abundant in Res compared to NonRes. This subset has a stem-like signature and may be associated with longer in vivo CD8 + T cell persistence and long-term AML control. An in-depth functional characterization with in vitro experiments and in vivo mouse models is currently ongoing. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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