Functional Signatures Revealed By Deep Phenotyping of CMV-Specific CD8+ T Cells Predict Risk of Early CMV Reactivation after Allogeneic Hematopoietic Cell Transplantation

Abstract CMV is the most clinically significant viral infection in HCT recipients. Control CMV reactivation after HCT is highly dependent on CMV-specific T cells. Despite dramatic technical advances, the clinical utility of functional assays of virus-specific T cells to predict CMV reactivation following HCT remains to be established. Using 13-color flow cytometry, we studied CD8+T cell responses to pp65 and IE-1 CMV peptide stimulation in cryopreserved PBMC from three clinically distinct subgroups (n=10 each) of HCT patients: 1) Elite Controllers (EC) : CMV seropositive (R+) recipients who never reactivated CMV based on weekly surveillance testing; 2) Spontaneous Controllers (SC): CMV R+ recipients who spontaneously resolved low-grade viremia (<200 IU/mL) without antiviral therapy; and 3) Non-controllers (NC): CMV R+ recipients who experienced high-grade CMV viremia (>1,000 IU/mL) requiring antiviral therapy. NC had lower numbers of CD8+ T cells that simultaneously produced 3-4 cytokines in response to CMV peptides compared to EC and SC (18, 26 and 34%, respectively) suggesting that progressive CMV viremia is associated with loss of CD8+ T cell polyfunctionality. Among 15 possible cytokine signatures, we identified two CMV-specific CD8+ T cell cytokine signatures, measured at day +30, that were strongly associated with the risk of CMV reactivation (Fig. 1): i) the non-protective signature (NPS) consisting of IL-2negIFNγposTNFαnegMIP-1βpos CD8+ T cells was positively associated with CMV reactivation (4.9% of CMV-specific CD8+ T cells vs. 19.4 P=0.002 for EC vs. SC/NC; 4.9 vs. 10.8 P=0.02 for EC vs. SC; 4.9 vs. 22.8 P=0.005 for EC vs. NC for pp65 stimulated cells; similar trends were observed in IE-1 stimulated cells); ii) the protective signature (PS) consisted of quadruple producers (IL-2posIFNγposTNFαposMIP-1βpos), and was significantly reduced among NC vs. SC following pp65 and IE-1 stimulation (0.05% of CMV-specific CD8+ T cells vs. 2.85 for pp65, P= 0.02; 0 vs. 1.25 for IE-1, P= 0.02); this association was also found in superantigen-stimulated cells. Production of IFNγ alone or in combination did not predict reactivation (P=0.49). Since NC trended toward more frequent recurrence of CMV viremia compared to SC (60 vs. 10%, respectively; P=0.06), we explored the association between PS and NPS and number of episodes of CMV reactivation (Fig. 1). We observed a significant stepwise increase in the levels of the NPS in pp65-stimulated cells in patients who experienced 0 vs. 1 and ≥2 episodes of CMV reactivation (4.9, 18.3 P=0.002, and 22.4 P=0.06). In addition, patients with ≥2 episodes of CMV had the lowest levels of the PS across groups (0 vs. 2.2% for ≥2 vs. 1 episode P=0.02). Similar trends were observed in IE-1 stimulated cells. Whereas T-cell depletion, aGVHD, lymphoid malignancy and CMV donor serostatus were not associated with risk of CMV in this small cohort, a NPS >10% was associated with increased risk of CMV reactivation (OR: 21, CI95 2-215; P=0.01) and need for treatment (OR: 14, CI95 1.5-137; P=0.02); and a PS >2% was associated with trend toward reduced risk of need for treatment (OR: 0.1, CI95 0.01-1.05; P=0.06). Multivariable modeling was not performed due to sample size. Time to event curves showed that high levels of NPS (>10%) predicted risk of CMV reactivation (log-rank P=0.0002). This remained true in analyses restricted to patients with CMV reactivation after day 30 (log-rank P=0.01). High levels of NPS or low levels of PS (<2%) predicted risk of need for treatment (log-rank P=0.003 and P=0.04, respectively). Combination of the PSlow/NPShigh had the highest predictive value for risk of need for treatment (log-rank P <0.0001; fig. 2). 18 graft products were available. As expected CMV-specific responses were not detected in grafts from CMV seronegative donors. Among the 9 CMV seropositive grafts, NPS expression was null across groups suggesting that the NPS is an immune phenotype that is absent in healthy donors; there was a stepwise decrease in the number of quadruple producer CD8+ T cells in CMV seropositive grafts for EC, SC and NC: 3.4 (n=3), 2 (n=4) and 0 (n=2), respectively, suggesting that donor PS might influence recipient reactivation. In conclusion, we have identified two novel CMV-specific CD8+ T cell cytokine signatures with robust predictive value for risk of CMV reactivation and need for treatment. These biomarkers might be useful in guiding clinical decision making in HCT recipients. Disclosures No relevant conflicts of interest to declare.

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Recovery of CMV-Specific T Cells Following Alternative Donor Allogeneic Transplant with Post-Transplant Cyclophosphamide

Blood ◽

10.1182/blood.v126.23.5462.5462 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5462-5462

Author(s):

Ayman Saad ◽

Samantha B Langford ◽

Shin Mineishi ◽

Lawrence S. Lamb

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cell Response ◽

Cd8 T Cell ◽

T Cell Response ◽

Cell Recovery ◽

Post Transplant ◽

Increased Risk ◽

Cmv Reactivation

Abstract Background: Post-transplant cyclophosphamide (PTCy) is increasingly used for GVHD prophylaxis after allogeneic hematopoietic stem cell transplantation (HCT) using alternative donors. However, immune reconstitution can be delayed posing an increased risk for CMV reactivation. We evaluated the outcomes of patients who received HCT-apheresis products comparing the impact of PTCy on lymphocyte recovery, CMV reactivation and CMV-specific CD8+ T cell recovery following haplo-identical (HAPLO), matched unrelated donor (MUD), and mismatched unrelated donor (mMUD) grafts vs. with conventional matched related donor (MRD) graft recipients. Methods: We examined 26 patients (median age, 49 years; range, 20-72 years) with advanced hematologic malignancies; n=5 (HAPLO); 6 (MRD); 15 (MUD). All patients received myeloablative conditioning regimens that was either busulfan- or total body irradiation (TBI)-based. PTCy (50 mg/kg/day) was administered on days +3 and +4 following HAPLO and on day +3 following MUD/mMUD transplant. Peripheral blood lymphocyte reconstitution and frequency of circulating CMV-directed CD8+ T cells was assessed (day ± 10 days) on post-transplant days +30, +60, and +90. Circulating anti-CMV T cell frequency was assessed using a phycoerythrin-tagged MHC dextramer against HLA-specific CMV pp65, IE-1, or pp50 peptides (Immudex; Copenhagen, DK) in combination with Tru-Count¨ tubes and fluorescent-labeled monoclonal antibodies against CD3, CD8, CD4, CD16/56, and CD19 (BD Biosciences; San Jose, CA). Anti-CMV CD8+ T cell immunity was defined as a CMV-dextramer (CMV/DEX) positive count of ≥7cells/ml. CMV reactivation was defined as a serologic titer of >500IU/mL. All patients with CMV reactivation received ganciclovir therapy until CMV titer became negative. Results: Day +30 total T cell recovery was significantly faster in MRD than CY-treated recipients (p=0.015) due principally to more robust CD8+ T cell recovery. CD4 T cell recovery remained below normal range in all groups through day +100. NK cells recovered to normal numbers at day +28 in all groups. Neither PTCy nor donor source significantly impacted the percentage of patients that recovered anti-CMV CD8+ T cells at each time interval (p = 0.8232). Excluding donors (D) and recipients (R) that were both negative, CMV/DEX+ T cells recovery was >7/mL in 4/5 MRD, 7/14 MUD, and 3/5 HAPLO by day +100. Among MRD recipients either D+ or R+ (n=5), 2 patients showed CMV reactivation within 40 days of transplant that was associated with <7 CMV/DEX+ T cells on day +30. Subsequent high (>90/mL) CMV/DEX T cell response in one patient shortened the duration of viremia to 10 days (vs. 16 days with poor responder) and 3 patients showed no CMV reactivation and a high CMV/DEX+ T cell response by day +60. For MUD CMV D+ and/or R+ recipients (n=14), 3 showed CMV reactivation within 50 days of transplant. All 3 patients had suboptimal CMV/DEX T cell response on day +30. Robust CMV/DEX+ T cell response on day +60 predicted shorter duration of viremia (20 days vs. average of 32 days). For HAPLO CMV D+ and/or R+ (n=5) recipients, 4 experienced CMV reactivation within 50 days of transplant. All patients had a <7 CMV/DEX+ T cells/mL +30. Robust CMV/DEX+ T cell response by day +60 was associated with shorter duration of viremia (range 7-21 days), while one patient with <7/mL CMV/DEX+ T cells had continued CMV viremia for 36 days. Conclusion: In this preliminary analysis, neither PTCy nor donor source significantly impacted the percentage of patients that recovered anti-CMV CD8+ T cells at each time interval. A weak CMV/DEX+ response (<7 cells/mL) on day +30 was consistent with increased risk of CMV reactivation (viremia) in all groups. A CMV/DEX+ T cell count ≥7 cells/mL was not immediately protective against CMV reactivation, but higher counts were associated with a shortened duration of viremia while on antiviral therapy. Conversely, subnormal counts were associated with a longer duration of viremia. This interim analysis suggests that CMV/DEX+ T cell enumeration is a useful biologic correlate for determining clinical response to antiviral therapy, and that donor-derived CMV specific T cell immunity is not further compromised with following PTCy in alternative donor HCT. Disclosures No relevant conflicts of interest to declare.

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Efficacy, safety, and immune activation with pegylated human IL-10 (AM0010) in combination with an anti-PD1 in advanced NSCLC.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.9091 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. 9091-9091

Author(s):

Deborah Jean Lee Wong ◽

Jeffrey Gary Schneider ◽

Raid Aljumaily ◽

Wolfgang Michael Korn ◽

Jeffrey R. Infante ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd8 T Cells ◽

Cell Activation ◽

Advanced Nsclc ◽

De Novo ◽

Cd8 T Cell ◽

Outcome Data ◽

Low Grade

9091 Background: Although IL-10 has anti-inflammatory properties, it stimulates cytotoxicity and proliferation of intratumoral antigen activated CD8+ T cell at higher concentrations. AM0010 is anticipated to activate antigen stimulated, intratumoral CD8 T cells while PD-1 inhibits them, providing the rationale for combining AM0010 and anti-PD-1 antibody. Methods: We treated a cohort of 34 NSCLC pts with AM0010 (10-20mg/kg QD, SC) and a PD-1 inhibitor [pembrolizumab (2mg/kg, q3wk IV; n=5) or nivolumab (3mg/kg, q2wk IV; n=29)]. Tumor responses were assessed by irRC every 8 weeks. Immune responses were measured by analysis of serum cytokines (Luminex), activation of blood derived T cells (FACS) and peripheral T cell clonality (TCR sequencing). Tumor PD-L1 expression was confirmed by IHC (22C3). Results: Pts had a median of 2 prior therapies. Median follow-up is 9.6 mo (range 0.5-77.3) in this fully enrolled cohort. AM0010 plus anti-PD-1 was well-tolerated. TrAEs were reversible and transient, with most being low grade, most commonly fatigue and pyrexia. G3/4 TrAEs were thrombocytopenia (7), anemia (6), fatigue (4), rash (3), pyrexia (2), hypertriglyceridemia (1) and pneumonitis (1). As of Jan. 31 2017, 22 pts had at least 1 tumor assessment. Partial responses (PRs) were observed in 8 pts (36.4%). 17 of these 22 pts had tissue for analysis of percent of tumor cells with PD-L1 expression (22C3): 58.8% had <1%, 17.7% had 1-49% and 23.5% had >50%. Best response data stratified for PD-L1 are shown in the table. Median PFS and OS for the entire cohort have not been reached. Updated outcome data that includes all enrolled pts will be available at the meeting. AM0010 plus anti-PD1 increased serum Th1 cytokines (IL-18, IFNγ), the number and proliferation of PD1+ Lag3+ activated CD8+ T cells and a de-novo oligoclonal expansion of T cell clones in the blood while decreasing TGFβ. Conclusions: AM0010 in combination with anti-PD1 is well-tolerated in advanced NSCLC pts. The efficacy and the observed CD8+ T cell activation is promising. Clinical trial information: NCT02009449. [Table: see text]

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Adoptive Transfer and Consequential Selective Reconstitution of Streptamer-Selected Cytomegalovirus-Specific CD8+ T Cells Leads to Enduring Virus Clearance in Patients after Allogeneic Stem Cell Transplantation

Blood ◽

10.1182/blood.v112.11.1181.1181 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1181-1181 ◽

Cited By ~ 3

Author(s):

Anita Schmitt ◽

Torsten Tonn ◽

Dirk Busch ◽

Götz Grigoleit ◽

Hermann Einsele ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Cd8 T Cells ◽

Adoptive Transfer ◽

Cd8 T Cell ◽

Adoptive T Cell Transfer ◽

T Cell Transfer ◽

Cmv Reactivation ◽

Cmv Antigenemia

Abstract Background: Cytomegalovirus (CMV) disease constitutes a serious complication after allogeneic peripheral blood stem cell transplantation (allo-PBSCT). For the clearance of CMV, CD8+ T cells are pivotal. Patients after allo-PBSCT with recurrent CMV reactivation usually lack such CMV specific T cells. Conventional antiviral therapy of CMV reactivation characteristically results in myelosuppression and further suppression of CMV specific T cells. Adoptive transfer of CMV specific T cells may help to overcome this problem. A novel technology designated “streptamers” allows the selection of CMVpp65 specific CD8+ T cell up to 98% purity without altering the functional properties of the selected T cells and without requiring cumbersome and time consuming T cell cultures. Materials and Methods: Here, the novel streptamer technology was used for adoptive transfer of CMV specific T cells into two acute leukemia patients with recurrent high CMV antigenemia after allo-PBSCT. Standard peripheral blood mononuclear cell apheresis was performed on the former stem cell donors of two patients with acute leukemia. Isolation of CMV specific donor lymphocytes was performed using a Good Manufacturing Product (GMP)-grade Streptamer selection kit on a CliniMacs™ device. Briefly, MHC-Streptamers (CMVpp65/HLA-B7 for patient 1; CMVpp65/HLA-A2 for patient 2) were labeled with beads overnight to obtain MHC-streptamer-bead complexes. Subsequently CMV specific T-lymphocytes were immunomagnetically labeled by incubating mononuclear cells with MHC-Streptamer-bead complexes. Cells were run on a CliniMacs™ device. The positive fraction was then incubated with biotin to detach the steptamers from the T cells. Results: A single specific donor lymphocyte infusion (sDLI) of 0.4 or 2.2 ×105 CMVpp65 specific T cells per kg body weight was performed in an AML or ALL patient respectively, after allogeneic PBSCT developing a CMVpp65 antigenemia with a maximum of 959 or 716 CMVpp65 positive/500,000 cells and treatment with foscarnet, ganciclovir and valganciclovir. After sDLI, the CMV antigenemia was cleared and remained persistently controlled even after discontinuation of valganciclovir therapy in both patients. No acute or chronic toxic side effect, particularly no aggravation of graft-versus-host disease (GvHD) was observed. A strong and sustained increase of the absolute count of CMV-specific CD8+ T cells in concordance with the increase of CD3+CD8+ T cells up to 440/μl was detected. CMV-specific CD8+ T cells showed no significant expression of CCR7, CD62L or CD107, but stained increasingly positive for CD45RA, indicating a preferential effector T cell phenotype. Results from stimulation experiments of CD3+ T cells with HLA-B7 versus HLA-A2 restricted CMVpp65 derived peptides demonstrate late reconstitution of HLA-A2-restricted CMV-specific T cells, whereas the adoptively transferred HLA-B7-restricted CMV-specific T-cell response augmented very early und was maintained over time. The chimerism analysis of the in vivo expanded CMV-specific CD8+ T cells demonstrated a 100% donor chimerism. T cell receptor excision circle (sjTRECs) analysis revealed a frequency of sjTRECs two logs lower than expected, indicating peripheral expansion rather than thymic proliferation of CMV specific CD8+ T cells. cDNA generated from FACS-purified donor-derived CMV B7 pp65-specific CD8+ T cells was probed with the indicated 5′ Vß14-specific and 3′ CDR3-specific primers for the presence of clonotypic T cells. The respective CDR3 region sequence was identical for both donor T cells and CMVpp65 specific T cells in the patients at different time points after the adoptive T cell transfer, thus clearly indicating that the expanded CMV specific T cell were of clonogenic donor origin. Conclusion: Streptamer technology offers the advantage of selecting CMV specific CD8+ T cells at GMP level for adoptive T cell transfer. Two CMVpp65 specific T cell transfers resulted in a marked increase of CMV-specific CD8+ T cells and induced long-lasting CD8+ T cell responses, which allowed the patients to discontinue toxic antiviral drug therapy without further high level reactivation of CMV.

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Impact of Donor Serostatus on CMV Reactivation and Reconstitution of Multi-Function CMV-Specific T Cells in CMV-Positive Transplant Recipients

Blood ◽

10.1182/blood.v112.11.4339.4339 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4339-4339

Author(s):

Wendi Zhou ◽

Jeff Longmate ◽

Simon F Lacey ◽

Joycelynne Palmer ◽

Ghislaine Gallez-Hawkins ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Cd8 T Cells ◽

Cd8 T Cell ◽

Donor Group ◽

Functional Profile ◽

Tnf Α ◽

Ifn Γ ◽

Cmv Reactivation

Abstract CMV reactivation remains a significant cause of morbidity and mortality due to the extended period of immunodeficiency after allogeneic hematopoietic stem cell transplantation (HCT), despite great strides in management of the infection in the past two decades. Reconstitution of cytomegalovirus (CMV)-specific CD8+ T cells is essential to control of CMV infection in CMV-seropositive recipients (R+) after HCT. The CMV serologic status of the recipient before HCT has a strong influence on outcome. Key questions addressed in this study are the impact of donor CMV serostatus on the reconstitution of effective CMV immunity or risk of CMV reactivation and GCV usage in CMV R+ recipients. Betts and colleagues have reported that HIV-specific CD8+ T-cells which simultaneously degranulated and produced IFN-γ, TNF-α, MIP-1β and IL2 were associated with lower viral load and HIV long term non-progressor status. These findings in the context of HIV infection motivated us to investigate whether levels of multi-functional CMV-specific CD8+ T-cells in HCT recipients correlated with the CMV serostatus of the donor and the differentiation state of transferred CMV-specific memory T-cells. We hypothesize that a mature CD8+ T-cell functional profile leads to a lower incidence of CMV viremia in R+ recipients of T-cell replete HCT with a D+ donor. A total of 183 R+ HCT recipients were enrolled. CMV reactivation was defined as any positive CMV blood culture or plasma PCR obtained during a monitoring period that began at d40 post-HCT and continued thereafter twice weekly until d100. After d100, CMV in plasma was monitored in patients at high risk because of GVHD or immunosuppressive medication. Peripheral blood mononuclear cells from R+ HCT recipients were collected at intervals between d90 and d360 post-HCT. A subset (n=123) of the subjects limited only by sample availability were evaluated for CMV-specific CD8+ T-cells producing IFN-g (IFN-γ-CD8+). We used a well-described pp65 peptide library as a stimulatory antigen to evaluate the ex vivo functional profile of pp65-specific CD8+ T-cells from R+ recipients either receiving CMV seropositive (D+/R+) or seronegative (D−/R+) T-cell replete donor grafts. D+ status was associated with higher IFN-γ-CD8+ during the sampling time-course by fitting linear generalized estimating equation models on a logarithmic scale (p=0.0004). A significant interaction was found between prior CMV reactivation and donor serostatus on IFN-γ-CD8+ levels (p=0.0001). Comparing the subset of IFN-γ-CD8+ measurements prior to reactivation, the D− donor group produced lower levels of IFN-γ-CD8+ than the D+ donor group (p=0.0002), although both donor groups have similar levels of IFN-γ-CD8+ post-reactivation. Similar results were obtained when adjusting for the 9 pre-transplant covariates, and none were associated with IFN-γ-CD8+ levels, except donor serostatus and its interaction with CMV reactivation. Six-color flow cytometry was used to assess the functional profile of CMV-specific CD8+ T-cells in 62 of 183 HCT recipients prospectively followed for CMV reactivation. R+ recipients receiving grafts from D− donors (D−/R+) reconstituted fewer multi-functional CD8+ T-cells expressing IFN-γ, TNF-α, MIP-1β and CD107 compared to D+/R+ recipients. Unlike mono-functional CD8+ T-cells secreting IFN-g, which were abundantly generated during CMV reactivation in D−/R+, the relative lack of multi-functional CD8+ T-cells persisted until at least one year post-HCT. In addition, D−/R+ recipients had more CMV reactivation than D+/R+ recipients. D+/R+ transplants have elevated levels of multi-functional CD8+ T cell levels and lower hazard for CMV reactivation. Virologic and immunologic outcomes were robust to adjustment for pre-transplant factors affecting HCT, including donor type, stem cell source, recipient age, and preparative regimen. Statistical modeling to account for pre- and post-transplant factors including GVHD and its treatment by steroids had minimal effect on the contribution of serostatus to the risk of CMV reactivation and differences in multi-functional CD8+ T cell levels.

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Successful treatment of posttransplantation lymphoproliferative disorder (PTLD) following renal allografting is associated with sustained CD8+ T-cell restoration

Blood ◽

10.1182/blood-2002-01-0210 ◽

2002 ◽

Vol 100 (7) ◽

pp. 2341-2348 ◽

Cited By ~ 39

Author(s):

Pierluigi Porcu ◽

Charles F. Eisenbeis ◽

Ronald P. Pelletier ◽

Elizabeth A. Davies ◽

Robert A. Baiocchi ◽

...

Keyword(s):

T Cells ◽

Renal Transplantation ◽

T Cell ◽

B Cell ◽

Antiviral Therapy ◽

Cd8 T Cells ◽

Lymphoproliferative Disorder ◽

Cd8 T Cell ◽

Related Factors ◽

Posttransplantation Lymphoproliferative Disorder

Posttransplantation lymphoproliferative disorder (PTLD) is a life-threatening Epstein-Barr virus (EBV)–associated B-cell malignancy occurring in 1% to 2% of renal transplantation patients. Host- and PTLD-related factors determining the likelihood of tumor response following reduction of immune suppression (IS) and antiviral therapy remain largely unknown. Standard therapy for PTLD is not well established. Eleven consecutive renal transplantation patients who developed EBV-positive PTLD 8 to 94 months after allografting were uniformly treated with acyclovir and IS reduction. All PTLDs were EBV-positive diffuse large B-cell lymphomas. Ten patients (91%) obtained a durable complete response (CR), and 9 (82%) have remained in continuous CR with a median follow-up of 29 months. Five patients (45%) lost their allograft. Of these, 4 patients had PTLD affecting the transplanted kidney. Peripheral blood CD8+ T cells increased significantly (P = .0078) from baseline in 8 responders available for analysis. One of 2 patients whose absolute CD8+ T-cell count subsequently dropped to baseline after IS reduction relapsed. The expanded CD8+ T cells from 2 responders specifically recognized an immunodominant peptide from the EBV lytic gene BZLF-1. Another lytic EBV gene, thymidine kinase, was expressed in all 8 PTLDs tested. IS reduction and antiviral therapy for PTLD after renal transplantation is a highly successful therapeutic combination, but the risk of graft rejection is significant, particularly in patients with PTLD involving the renal allograft. A sustained expansion of CD8+ T cells and a cellular immune response to EBV lytic antigens may be important for PTLD clearance in renal transplantation patients.

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Quantitative Assessment of T Cell Repertoire Recovery after Double Unit Cord Blood Transplantation Demonstrates Early T Cell Diversity and Correlation Between CMV Reactivation and CD8 Clonal Domimance

Blood ◽

10.1182/blood.v124.21.1158.1158 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1158-1158

Author(s):

Miguel Perales ◽

Ying Taur ◽

Ingrid Leiner ◽

Marissa N Lubin ◽

Boze Susac ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cord Blood ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Cord Blood Transplantation ◽

Cd8 T Cell ◽

Cell Repertoire ◽

Tcr Diversity ◽

Cmv Reactivation

Abstract Introduction: Double-unit cord blood transplantation (DCBT) is a viable therapy for adults with high-risk hematologic malignancies who lack an adult donor. However, lack of transfer of memory T cells in the graft is associated with an increased risk of viral infections. To study immune reconstitution, we recently described a novel method that combines 5' rapid amplification of complementary DNA ends (RACE) PCR and deep sequencing to quantify T cell receptor (TCR) diversity after allogeneic hematopoietic stem cell transplant (van Heijst, Nat Med 2013). In that study, we showed that recipients of DCBT recover TCR diversity comparable to healthy donors by 12 months. We now report results of a prospective analysis of CD4+ and CD8+ T cell repertoire recovery in DCBT recipients and correlation with clinical outcomes. Methods: We prospectively collected samples from 33 DCBT recipients. The median age was 45 years (range 26-71), 18 (55%) were CMV seropositive, and the majority (n = 17, 52%) had non-European ancestry. Diagnoses included 20 (61%) acute leukemias and 13 (39%) lymphomas. Conditioning was myeloablative (n=1, 3%), reduced intensity (n=28, 85%), or non-myeloablative (n=4, 12%), and all patients received GVHD prophylaxis with cyclosporine-A and mycophenolate mofetil and no ATG. Patients received double-unit CB grafts (4-6/6 HLA-A,-B antigen, -DRB1 allele donor-recipient matched); this was supplemented with haploidentical CD34-selected PBSC in 18 patients. The 66 units had a median donor-recipient HLA-allele match of 6/8 (range 3-8). Infused total nucleated cell doses were 2.3 (1.7-3.3) and 1.9 (1.3-2.5) for the larger and smaller units, respectively. Samples were collected from the DCB grafts, recipient day+21 bone marrow, and peripheral blood at days +30, 60, 90, 120, 180 and 365 post-transplant. TCR-β sequences from each sample were amplified and sequenced using the Illumina/MiSeq sequencing platform after isolation of CD4+ and CD8+ T cells. TCR abundances were assessed at the level of clonotype and TCR diversity was calculated using inverse Simpson index. Results: Of the 33 patients, long-term samples were obtained in 25 patients, short-term samples (≤ day 100) in 6 patients who died early after DCBT, and no samples other than the graft for 2 patients. The remainder of the results focuses on 25 patients with complete samples. As previously shown, there is a 1-2-log increased diversity in CD4+ vs. CD8+ T cells (Figure). Furthermore, median CD4+ steady-state diversity is achieved early by day 60. In contrast, there is a higher rate of clonal dominance in CD8+ compared to CD4+ T cells (24/25 vs. 11/25, p=0.0001 by Fisher test). Several patterns of clonal dominance were observed, including two main patterns in CD8+ T cells. In 7/24 patients, clonal dominance is established by day 60 and persists throughout, whereas in 12/24 patients, clonal dominance fluctuates throughout follow-up. In CD4 +T cells, where less dominance is observed, a similar distribution is seen, though prolonged clonal dominance is rare. Interestingly, some of the dominant clones can be detected in the graft and are present in the day 21 marrow sample. Persistent clonal dominance in CD8+ T cells was seen 6/9 patients with CMV reactivation, whereas ongoing fluctuation was seen in 9/12 patients without CMV reactivation. In 2 patients with fluctuating clones who reactivated CMV, 1 had low level and the other a late viremia. In contrast, no link to a specific pattern was observed in patients with HHV6 viremia or acute GVHD. Finally, when we assessed similarity in clonal distribution between time points, there was more similarity in CD8+ than CD4+ T cells. Conclusions: This novel deep TCR repertoire sequencing provides a quantitative picture of T cell recovery after DCBT and supports the following: 1) separate analysis of CD4+ and CD8+ T cell populations is critical given different patterns of recovery in T cell subsets; 2) there is significant turnover in CD4+ clones but with overall limited dominance, whereas there is less turnover in CD8+ clones; 3) although the grafts contain predominantly naïve T cells, the clonal evolution of CD8+ T cells strongly suggests generation of virus-specific T cells that control viral infection; and 4) CMV appears to be an important driver of CD8+ T cell clonal expansion after CBT. Ongoing analyses are correlating immune recovery with cord blood unit dominance as well as the biology of GVHD and relapse. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Imprint Of Early CMV Reactivation On The Reconstituting T-Lymphocyte Compartment One and Two Year After Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood.v122.21.3295.3295 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3295-3295

Author(s):

Gertjan Lugthart ◽

Monique M. van Ostaijen-Ten Dam ◽

Cornelia M. Jol-van der Zijde ◽

Tessa C. van Holten ◽

Michel G.D. Kester ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd8 T Cell ◽

Cell Compartment ◽

Hematopoietic Stem ◽

Cell Numbers ◽

One Year ◽

The Impact ◽

Cmv Reactivation

Abstract Background Cytomegalovirus (CMV) reactivation frequently occurs during early immune reconstitution after hematopoietic stem cell transplantation (HSCT). Although CMV is associated with accelerated immune ageing in healthy individuals, the impact of early CMV reactivation on T-cell immunity long term post HSCT is unknown. In this study, we report the impact of early CMV reactivations on the reconstitution and composition of the T-lymphocyte compartment one to two year after HSCT in a large cohort of pediatric HSCT recipients. Methods We analyzed the lymphocyte compartment one and two year after transplantation in 131 consecutive (2002 - 2011) pediatric HSCT recipients that were eligible for follow up one year post HSCT. Viral infections were routinely monitored by weekly serum viral DNA PCR in the first 100 days. Peripheral blood mononuclear cells were routinely analyzed by multicolor flow cytometry. Six patients with early CMV reactivation and an HLA type for which CMV-tetramers were available were analyzed for the presence and phenotype of CMV-specific CD8+ T-lymphocytes. Results One year post HSCT, patients with early CMV reactivation (n = 46, PCR ≥ 1x ≥ 200 copies / mL) had significantly higher lymphocyte counts compared to patients without CMV reactivation (n = 85, PCR always < 200 copies / mL). This could be attributed to a significant increase of CD3+ T-lymphocytes while NK- and B-cell numbers did not differ. Within the T-cell compartment, a three-fold expansion of CD8+ T-cells (median 1323 vs. 424 cells / μL, p < 0.0001, Mann-Whitney) and an increase in gamma-delta T-cells (median 104 vs. 47 cells / μL, p = 0.0005) was observed, while absolute numbers of CD4+ T-cells did not differ between the groups. This effect was not observed in relation to Epstein-Barr virus (EBV) or Adenovirus (HAdV) reactivation. In multivariate analysis, CD8+ T-cell numbers one year post HSCT were highly influenced by CMV reactivation (p < 0.0001, linear regression) but not affected by pre-transplant CMV serostatus of donor (p = 0.22) or recipient (p = 0.14). In a representative subcohort (n = 53, 2008 - 2010), we more closely analyzed the differentiation stages of T-cells based on expression of CD45RA and CCR7. In both the CD4+ and CD8+T-cell subset, the proportion of Effector Memory (EM) and EMRA T-cells was enlarged in patients with (n = 18) compared to patients without (n = 35) early CMV reactivation. In the CD8+ T-cell compartment, this was caused by a major expansion of CD8+ EM and EMRA T-cells (median 485 vs. 141, p < 0.0001 and 509 vs. 114 cells / μL, p < 0.0001, Figure A), while the Naive (median 105 vs. 169 cells / μL, p = 0.17) and Central Memory (CM) compartment did not differ. In the CD4+ compartment, a non-significant increase of EM and EMRA cells was accompanied by a non-significant decrease of N and CM cells. CMV-specific CD8+ T-cells (median 4.1, range 0.3 - 25.6% of CD8+ T-cells) were detected within the EM and EMRA compartment. Two year after HSCT, data were available for 76 patients. Both in patients with (lymphocyte subsets: n = 34 / T-cell differentiation: n = 12) and without (n = 42 / n = 19) early CMV reactivation, a further reconstitution of CD4+ and CD8+ T-cells was observed, reflected by an expansion of Naive and CM cells (Figure B). However, the total number of CD8+ T-cells decreased in patients with early CMV reactivation, caused by a contraction of late differentiated CD8+ EM and EMRA cells (median 825 to 618, p = 0.0068 and 555 to 378 cells / μL, p = 0.0342, Wilcoxon). Conclusion Early CMV reactivation leaves a virus-specific and dynamic imprint on the reconstituting immune system 1 and 2 year after HSCT. The marked expansion of CD8 + EM and EMRA T-cells was not seen in patients with early EBV or HAdV reactivation and did not compromise the reconstitution of the Naive and CM compartment available to respond to neo- and recall antigens. Pediatric HSCT recipients differed from solid organ transplantation recipients in which CMV has been correlated to an accelerated and ongoing accumulation of late differentiated T-cells. The dynamic contraction of the CD8+ late differentiated memory T-cell compartment in the second year after HSCT implies that an ongoing process of immune-regulation and further reconstitution is modeling the cellular immune system after discontinuation of immunosuppressive medication. Disclosures: No relevant conflicts of interest to declare.

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Pathogenic CD8 T cells defined by longitudinal liver sampling in chronic hepatitis B patients starting antiviral therapy

10.1101/2021.12.16.21267870 ◽

2021 ◽

Author(s):

Shirin Nkongolo ◽

Deeqa Mahamed ◽

Adrian Kuipery ◽

Juan D. Sanchez Vasquez ◽

Samuel C. Kim ◽

...

Keyword(s):

T Cells ◽

Chronic Hepatitis ◽

T Cell ◽

Hepatitis B ◽

Antiviral Therapy ◽

Chronic Hepatitis B ◽

Liver Damage ◽

Cd8 T Cells ◽

Human Liver ◽

Cd8 T Cell

Accumulation of activated immune cells results in non-specific hepatocyte killing in chronic hepatitis B (CHB), leading to fibrosis and cirrhosis. We enrolled 15 CHB patients with active liver damage to receive antiviral therapy, and performed longitudinal liver sampling using fine-needle aspiration to investigate mechanisms of CHB pathogenesis in the human liver. Single-cell sequencing of total liver cells revealed a distinct liver-resident, polyclonal CD8 T cell population that was enriched at baseline and displayed a highly activated immune signature during liver damage. Cytokine combinations, identified by in silico prediction of ligand-receptor interaction, induced the activated phenotype in healthy liver CD8 T cells, resulting in non-specific Fas ligand-mediated killing of target cells. These results define a CD8 T cell population in the human liver that can drive pathogenesis, and a key pathway involved in their function in CHB patients.

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Profound T Cell Depletion Does Not Eradicate CMV Specific CD8 T Cells Responsible for Protective Immunity to CMV after T Cell Depleted Allogeneic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v108.11.2920.2920 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2920-2920

Author(s):

Pim L.J. van der Heiden ◽

Jayant S. Kalpoe ◽

Wilmy M.E. Haarman ◽

Sonja M. Opdam ◽

Roelof Willemze ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Protective Immunity ◽

Cd8 T Cell ◽

T Cell Response ◽

Cell Depletion ◽

Cmv Infection ◽

T Cell Depletion ◽

Cmv Reactivation

Abstract CMV infection is inversely correlated with T cell immunity and frequently observed after T cell depleted allogeneic stem cell transplantation (TCD alloSCT). In case of profound T cell depletion of the graft, it may be anticipated that donor immunity is not of influence on immunity to CMV after TCD alloSCT, in contrast to conventional alloSCT. To address this hypothesis, we prospectively analyzed CMV load and T cell reconstitution in 20 consecutive CMV+ patients transplanted with a CMV+ or CMV− donor. The grafts were T cell depleted using Campath 20 mg “in the bag” and infused without further manipulation. No post transplantation GVHD prophylaxis had to be given, illustrating the intensity of T cell depletion. CMV DNA load was weekly monitored. As expected, by day 120 no difference in incidence of CMV reactivation was present between CMV+ patients with CMV+ or CMV− donors (8/9 vs 11/11). However, the area under the curve (AUC) representing total CMV DNA load was significantly higher in patients with a CMV− donor (AUC log 6.4 vs 5.5, p=0.03). 2 patients with a CMV− donor developed fatal CMV pneumonitis, whereas no CMV disease was present in patients with a CMV+ donor. To analyze whether this difference in the course of CMV infection was due to a difference in CMV specific T cell reconstitution, we characterized T cells populations after transplantation. At day 90, total T cell numbers in CMV+ patients were significantly increased in patients with a CMV+ donor compared to patients with a CMV− donor (1,2 vs 0.5*10E9/L, p=0.025). This difference was caused by an increase in CD8 T cells (1.0 vs 0.3*10E9/L, p=0.009) but not CD4 T cells (0.2 vs 0.2*10E9/L). To investigate whether this difference was due to expansion of CMV specific T cells, we analyzed 16 patients for whom HLA restricted CMV specific tetramers were available. All patients with a CMV+ donor developed a substantial tetramer positive (tet+) CD8 T cell response (>5*10E6/L) compared to only 33% of patients with a CMV− donor (7/7 vs 3/9, p=0.006). 13% (range 2 – 26%) of the CD8 T cells were tet+ in patients with a CMV+ donor at the peak of the response. This percentage does not include CD8 T cell responses directed against CMV epitopes with other HLA restriction. When extrapolated, this suggests that the increase of CD8 population was largely due to CMV specific T cells. In patients with a CMV− donor the CMV specific CD8 response could have developed from naive donor cells or from residual patient memory cells. We determined the origin of tet+ T cells in 3 patients with a CMV− donor. In all patients, the tet+ cells were of patient origin, indicating absence of a de novo primary donor response. In summary, although the incidence of CMV reactivation was identical in TCD alloSCT with a CMV+ or a CMV− donor, CMV load was lower in patients with a CMV+ donor. In CMV+ patients with a CMV+ donor a CMV specific CD8 response was always present, in contrast to most CMV+ patients with a CMV− donor. Furthermore, when tet+ cells developed early after SCT in CMV+ patients with a CMV− donor, they were of patient origin. We conclude that no primary CMV specific CD8 T cell response of donor origin develops shortly after TCD alloSCT, and that despite profound T cell depletion CMV specific memory CD8 T cells from donor or patient survive the conditioning regimen and provide protective immunity to CMV.

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Longitudinal Changes in Cd4+, Cd8+ T Cell Phenotype and Activation Marker Expression Following Antiretroviral Therapy Initiation among Patients with Cryptococcal Meningitis

Journal of Fungi ◽

10.3390/jof5030063 ◽

2019 ◽

Vol 5 (3) ◽

pp. 63

Author(s):

Alice Bayiyana ◽

Samuel Okurut ◽

Rose Nabatanzi ◽

Godfrey Zziwa ◽

David R. Boulware ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Antiretroviral Therapy ◽

Cell Surface ◽

Cd8 T Cells ◽

Cryptococcal Meningitis ◽

Cd8 T Cell ◽

Cd4 T Cell ◽

Effector Memory ◽

Memory Subset

Despite improvement in the prognosis of HIV/AIDS (human immunodeficiency virus/acquired immune deficiency syndrome) patients on antiretroviral therapy (ART), cryptococcal meningitis (CM) still causes 10–15% mortality among HIV-infected patients. The immunological impact of ART on the CD4+ and CD8+ T cell repertoire during cryptococcal co-infection is unclear. We determined longitudinal phenotypic changes in T cell subsets among patients with CM after they initiated ART. We hypothesized that ART alters the clonotypic phenotype and structural composition of CD4+ and CD8+ T cells during CM co-infection. For this substudy, peripheral blood mononuclear cells (PBMC) were isolated at four time points from CM patients following ART initiation during the parent study (ClinicalTrials.gov number, NCT01075152). Phenotypic characterization of CD4+ and CD8+ T cells was done using T cell surface marker monoclonal antibodies by flow cytometry. There was variation in the expression of immunophenotypic markers defining central memory (CD27+CD45R0+), effector memory (CD45R0+CD27–), immune activation (CD38+ and Human Leucocyte Antigen DR (HLA-DR+), and exhaustion (Programmed cell death protein one (PD-1) in the CD4+ T cell subset. In comparison to the CD4+ T cell population, the CD8+ central memory subset declined gradually with minimal increase in the effector memory subset. Both CD4+ and CD8+ T cell immune exhaustion and activation markers remained elevated over 12 weeks. The relative surge and decline in the expression of T cell surface markers outlines a variation in the differentiation of CD4+ T cells during ART treatment during CM co-infection.

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