Familial Haploidentical (FHI) Allogeneic Stem Cell Transplantation (AlloSCT) Utilizing CD34 Enrichment and PB MNC Addback in Children and Adolescents with High Risk Sickle Cell Disease (SCD): Rapid Engraftment, Immune Cell Reconstitution, and Sustained Donor Chimerism (IND 14359)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1245-1245 ◽  
Author(s):  
Janet Ayello ◽  
Yaya Chu ◽  
Carolyn A. Keever-Taylor ◽  
Julie-An Talano ◽  
Rona Weinberg ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
High Risk ◽  
Whole Blood ◽  
Graft Failure ◽  
Immune Reconstitution ◽  
Immune Cell ◽  
Nk Cell ◽  
Granzyme B ◽  
Donor Chimerism

Abstract Background: SCD is characterized by chronic vaso-occlusive crises and multiorgan failure resulting in poor quality of life and early mortality (Bhatia/Cairo et al, BMT 2014). There is presently no curative therapy for patients with high risk SCD other than HLA-identical sibling AlloSCT. (Freed/Cairo et al BMT 2012). However, less than 15% of eligible SCD patients have an unaffected MSD with a 10-15% increase of graft failure and TRM (Talano/Cairo et al, EJH, 2015). Similarly, most patients lack a matched related donor and UCB is an inferior source in SCD recipients (Radhakrishman/Cairo et al, BBMT 2013). Haploidentical familial donors with SCD trait offers an opportunity for a new donor source for children with high risk SCD. To overcome HLA barriers, Geyer/Cairo et al (BJH, 2012) demonstrated that T cell depletion using CD34 enriched HPC products with PB MNC addback transplanted in pediatric recipients utilizing MUD was associated with sustained engraftment, low risk of aGVHD but limited by delayed immune reconstitution. Efforts to use FHI donors and T replete AlloSCT in patients with SCD were associated with high rates of graft failure (Bolanes-Meade J et al Blood 2012; Ruggieri et al BBMT 2011). We previously reported FHI CD34 enriched/PB MNC addback AlloSCT is feasible and well tolerated in patients with high risk SCD (Abikoff/Cairo, ASBMT 2015). Objective: To characterize immunological reconstitution following FHI AlloSCT with CD34 enriched grafts with PB MNC addback in children and adolescents with high risk SCD. Methods: 15 patients were evaluatedpretransplant at D+30, 60, 100 and 180 following FHI AlloSCT. GCSF mobilized HPC were collected by apheresis (Spectra OPTIA, Terumo BCT) and products underwent CD34 enrichment using the CliniMACS cell separation system (materials generously supplied by Miltenyi Biotec, Cambridge , MA) with a PB MNC addback dose of 2x10*5 CD3/kg. Immune cell and subset reconstitution was assessed by flow cytometry. NK function was determined by cytotoxic activity against K562 tumor targets at 10:1 E:T ratio by europium release assay and intracellular LAMP-1 (CD107a) and granzyme B expression by flow cytometry. Whole blood, T cell and RBC chimerism (CD71) determined by flow cytometry and by STR. Results: Patients achieved neutrophil and platelet engraftment in a median time of 10 and 16 days, respectively. By D+30, median whole blood donor chimerism was ≥93% and ≥95% at most recent followup (D+30-730). Median donor chimerism in the erythroid lineage was 95% by D+60, with 7 of 13 patients ≥99% at D+30. This was maintained at most recent followup (D+30-730). Median T cell chimerism was 90% (D+60-550) and median NK cell chimerism was 90% by D+30 and maintained at ≥95% through D+730. NK (CD3-/56+) and NKT (CD3+/56+) cell reconstitution following FHI AlloSCT was rapid and peaked at D+30 (35.5±8.6%, 271x10*3/ul; 14.2±4%, 179x10*3/ul, respectively). Moreover, there was robust NK cell receptor expression reconstitution with high levels of activating receptors, NKp46, NKG2D and KIR2DS and inhibitory receptors NKG2A, CD94 and KIR2DL2/3 at D+30 [Fig 1]. NK cytotoxicity against K562 at E;T 10:1 peaked at D+30 (26±3%) and D+180 (28±3%) compared to pretransplant (16±2%, p<0.01). NK activation marker, CD107a, peaked at D+30 (37±9%) and D+180 (41±6%) and there was robust granzyme B degranulation at D+30. CD3+, CD4+, CD8+ and CD19+ immune reconstitution occurred between D+180 and D+270. One year absolute (mean±SEM) cells/ul of CD3+, CD4+, CD8+, CD19+ and CD56+ was 795±168, 408±102, 375±90, 815±352 and 204±37, respectively. [Fig 2] Conclusion: Immune reconstitution and donor chimerism was relatively rapid after FHI AlloSCT with CD34 enriched grafts with PB MNC addback in high risk SCD patients. The donor MNC addback after CD34 selection may in part contribute to rapid engraftment and immune reconstitution along with sustained donor chimerism. This research was supported by FDA grant 5R01FD004090. Disclosures Cairo: Celgene: Research Funding.

scholarly journals CD45RA-Depleted Haploidentical Transplantation Combined with NK Cell Addback Results in Promising Long-Term Outcomes in Pediatric Patients with High-Risk Hematologic Malignancies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 172-172
Author(s):  
Swati Naik ◽  
Aimee C Talleur ◽  
Ying Li ◽  
Renee M. Madden ◽  
Ewelina Mamcarz ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
High Risk ◽  
Cumulative Incidence ◽  
Graft Failure ◽  
Immune Reconstitution ◽  
Nk Cell ◽  
Entire Cohort ◽  
Active Disease

Abstract Novel graft engineering strategies that allow selective T-cell depletion have made haploidentical donor (haplo) hematopoietic cell transplantation (HCT) feasible for patients lacking a matched donor. We hypothesized that selective CD45RA+ cell depletion can limit graft-versus-host disease (GVHD) through removal of naive T cells but allow for rapid immune reconstitution through adoptive transfer of memory T cells, minimizing risk of graft failure, infections and relapse. We performed a prospective clinical trial utilizing CD45RA+ depleted haplo HCT followed by donor NK cell addback to maximize the graft-versus-leukemia effect in children with high-risk hematological malignancies (NCT01807611), and present here an interim analysis. Between 2013 to 2019, 72 patients (42 males, 30 females) were enrolled. The median age was 8.1 years (range 0.6-20.8). Twenty-nine patients had acute lymphoblastic leukemia (ALL), 39 had myelodysplastic syndrome/acute myeloid leukemia (MDS/AML), 3 had biphenotypic leukemia and 1 had Non-Hodgkin's Lymphoma. At time of HCT, 25 patients were in CR1, 24 in CR2, 4 in CR3/&gt; and 19 had active disease. Of the 53 patients in CR, 22 had evidence of minimal residual/detectable disease (MRD) at HCT. Donors used were mothers (n=30), fathers (n=35), or sibling/other (n=6) with majority (n=62) being KIR-ligand mismatched to the recipient. All patients received serotherapy-free, reduced intensity conditioning with fludarabine, melphalan, cyclophosphamide and Total Lymphoid Irradiation. GCSF mobilized peripheral blood grafts were infused on i) Day 0 CD34 + selected graft (median CD34+: 9.85 x10 6cells/kg, range 1.96-44.64) and ii) Day +1 CD45 RA+ depleted graft (median: CD34+: 5.82 x10 6cells/kg, range 0.58-39.43); providing a large number of CD45RO+ T-cells (median CD3+ : 60.1 x10 6cells/kg, range 16.08-528.43) after depletion of CD45RA+ cells (CD3+CD45RA+ median 0, range 0-0.2 x10 6cells/kg). NK cells (median: 11.7 x10 6cells/kg; range: 1.65-99.2), isolated from a non-mobilized apheresis by CD3 depletion/CD56 selection, were infused on day +6. GVHD prophylaxis included MMF (n=61) and/or Sirolimus (n= 8) until day +42 post-HCT. The median time for neutrophil and platelet engraftment was 11 (range 9-13) and 17 days (range 10-100) respectively. One patient had primary graft failure and was successfully re-transplanted. Immune reconstitution was rapid and robust and recapitulated CD45RA depleted graft content with mean CD3, CD4 and CD8 T-cell count at day +30 of 1080, 640 and 230 cells/ul respectively. TCR Vbeta spectratyping revealed a broad repertoire by day +100 prior to the emergence of naïve T cells. The incidence of Cytomegalovirus and Adenovirus viremia was 38% (28/72) and 13% (10/72) respectively. The cumulative incidence of aGVHD was 36.1% (25.1-47.2%), aGVHD grade III-IV was 29.2% (19.1-39.9%) and chronic GVHD was 20.8% (CI: 12.3-30.9%). With a median follow-up of 3.1 years (range 0.04-8.0 years) the 3-year leukemia free survival (LFS) was 88% (CI: 76.1-100%) for patients in CR1, 70.8% (CI: 54.8-91.6%) for patients in CR2, 50% (CI: 18.8-100%) for patients in CR3/&gt; and 21.1% (CI: 8.81-50.3) for patients with active disease (p = &lt;0.0001). Amongst patients in CR, those with no detectible disease had 3-year LFS of 84.4% (CI: 72.7-97.9%) as compared to 68.2% (CI: 51.3-90.7%) for patients with detectible MRD at time of HCT (p=0.087). Three-year LFS was comparable for ALL and AML at 62.1% (CI 46.7-82.5%) and 58.5% (CI 44.8-76.4%) respectively (p=0.95). For the entire cohort, the 3-year OS and LFS was 68.9% and 62.2% (CI 58.9-80.6% and 51.9-74.6%), respectively. Despite the inclusion of patients with active disease and detectible MRD the cumulative incidence of relapse for the entire cohort was 26.5% (CI:16.8-37.1%), comparable to other studies with patients transplanted in MRD-negative CR. The cumulative incidence of non-relapse mortality was 11.5%, (CI, 5.3-20.4%), with 5/10 deaths occurring in the 19 patients with active disease. In conclusion, selective CD45RA+ depletion allowed for adoptive transfer of abundant memory T cells that along with NK cell addback was associated with rapid, functional immune reconstitution, resulting in low rates of graft failure, viral reactivation and relapse. Despite a higher incidence of aGVHD compared to other T-cell depleted grafts, our approach translated into promising long-term outcomes in this high-risk patient population. Disclosures Sharma: Vindico Medical Education: Honoraria; CRISPR Therapeutics: Other, Research Funding; Vertex Pharmaceuticals/CRISPR Therapeutics: Other: Salary support paid to institution; Novartis: Other: Salary support paid to institution; Spotlight Therapeutics: Consultancy; Medexus Inc: Consultancy. Gottschalk: Novartis: Consultancy; Catamaran Bio: Consultancy; Other: Other: patents and patent applications in the field of cancer cell and gene therapy ; Immatics: Membership on an entity's Board of Directors or advisory committees; Tidal: Consultancy; Tessa Therapeutics: Consultancy. Triplett: Miltenyi: Other: Travel, meeting registration.

scholarly journals Sustained Donor Chimerism and Rapid Immune Cell Reconstitution Following Familial Haploidentical (FHI) CD34 Enriched Stem Cell Transplantation with Pbmnc Addback in Patients with High Risk Sickle Cell Disease (SCD) (IND 14359)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1990-1990
Author(s):  
Yaya Chu ◽  
Julie-An Talano ◽  
Lee Ann Baxter-Lowe ◽  
Carolyn A. Keever-Taylor ◽  
Erin Morris ◽  
...  
Keyword(s):  
High Risk ◽  
Immune Cell ◽  
Nk Cell ◽  
Cell Depletion ◽  
High Risk Patients ◽  
Donor Chimerism ◽  
Cd34 Cells ◽  
Risk Patients ◽  
Grade Ii ◽  
Nk Cytotoxicity

Background: Allogeneic stem cell transplantation (AlloSCT) from an HLA-matched sibling donor is the only known curative therapy in patients with high-risk SCD (Talano/Cairo, EJH, 2015). Unfortunately only about 15% of high risk patients with SCD have an HLA-matched unaffected sibling donor. T cell depletion has been employed to reduce AGVHD e.g., CD3/CD19 cell depletion (Barfiled RC, et al, Cytotherapy, 2004), αβ T-cell/CD19 cell depletion (Locatelli F, et al, Blood, 2017), CD34+ positive selection (Aversa F, et al, NEJM, 1998). MUD transplantation in high-risk SCD recipients has shown unexpectedly high rates of CGVHD (Shenoy et al, Blood, 2016). We reported a very low incidence of acute and chronic GVHD in pediatric recipients receiving CD34 enriched HPC products with PB MNC addback with 2 x 105 CD3/kg from MUD donors (Geyer/Cairo et al, BJH, 2012). Furthermore, rapid NK cell reconstitution after AlloSCT is associated with a significant improvement in 1yr OS (Pical-Izard, BBMT, 2015; Dunbar et al, Hematologica, 2008). Recently, we reported promising results for high-risk SCD patients at 1 year follow-up after FHI CD34 enriched/PBMNC with addback AlloSCT with the probability of 1-year overall survival (OS) n=17; 88.2% (CI95: 60.6-96.9) (Talano/Cairo, ASH, 2017), expanding the donor pool and hopefully improving outcomes for high-risk patients with SCD. Objective: To investigate donor chimerism, immune cell reconstitution and NK cell function in high-risk patients with SCD following AlloSCT using FHI CD34 enrichment/PBMNC (2 x 105 CD3/kg) addback. Methods: Twenty-one eligible SCD patients (2-<21 yrs) were enrolled. Nineteen patients received hydroxyurea, azathioprine, fludarabine, busulfan, thiotepa, cyclophosphamide, R-ATG, and TLI followed by FHI AlloSCT to date (Talano/Cairo, ASH, 2017). CD34 cells were enriched using the CliniMACS® system, kindly provided by Miltenyi Biotec, with a target dose of 10 x 106 CD34+ cells/kg with a PBMNC addback dose of 2x10*5 CD3/kg in the final product. Whole blood and RBC chimerism (estimated using CD71 to isolate an eythroid lineage-enriched fraction) were determined by STR. Immune cell and subset reconstitution was assessed by flow cytometry as previously described (Geyer/Cairo et al. BJH, 2012). NK function was determined by cytotoxic activity against K562 tumor targets at 10:1 E:T ratio by europium release assay and intracellular LAMP-1 (CD107a) and granzyme B expression by flow cytometry as previously described (Chu/Cairo et al, Can Imm Res, 2015). Results: There was 100% engraftment of neutrophils and platelets. The median day post-HISCT to neutrophil and platelet engraftment was +9 and +19, respectively. Whole blood donor chimerism (mean±SEM) at 1-year, 2-year, and 3-year post-HISCT was 97±1%, 97±1%, 97±1%, respectively (Fig.1). Donor chimerism for CD71+ RBCs (mean±SEM) at 1-year, 2-year, 3-year post-HISCT was 97±2%, 98±1%, 98±1%, respectively (Fig.1). Immune reconstitution of CD3, CD4, CD8, and CD19 was evaluated. The time to recovery of minimally normal levels post-HISCT of CD3 (800 cells/ul), CD4 (400 cells/ul), CD8 (200 cells/ul), and CD19 (200 cells/ul), was approximately 365, 365, 270, and 60 days post-HISCT (Fig.2), respectively. Probability of Grade II-IV AGVHD, CGVHD and 1 year EFS/OS was 6.2%, 6.7% and 90%, respectively. NK reconstitution was rapid and peaked at d+30 (36±9%, 2710cells/ml). NK cytotoxicity against K562 at a E:T=10:1 peaked at d+30 (26±3%) and d+180 (28±3%) vs at pre-t (16±2%) (p<0.01) (Fig. 3A). Consistent with increased NK cytotoxicity, CD56dimCD3- subset was increased at d+30 vs pre- HISCT (p<0.05). The NK activation marker, CD107a peaked at d+30 (38±9%) and d+180 (41±6%) (Fig.3B). More over, reconstituted NK cells expressed higher level of activating receptors NKp46 (24±9%), NKG2D (32±9%) and KIR2DS (8±3%) and inhibitory receptors NKG2A (33±9%), CD94 (28±9%) and KIR2DL2/3 (11±2%) at d+30 compared to other time points. Conclusion: Despite a 5 log depletion of T cells, the PBMNC addback (fixed at 2 x 105 CD3/kg) facilitated rapid donor chimerism and immune reconstitution with a low probability of Grade II-IV AGVHD. The rapid NK reconstitution may have in part contributed to the excellent 1yr OS in the FHI study. (Supported by FDA R01FD004090 (MSC)). Disclosures Cairo: Jazz Pharmaceuticals: Other: Advisory Board, Research Funding, Speakers Bureau; Osuka: Research Funding; Miltenyi: Other: MTA.

scholarly journals Mapping and Characterization of HCMV-Specific Unconventional HLA-E-Restricted CD8 T Cell Populations and Associated NK and T Cell Responses Using HLA/Peptide Tetramers and Spectral Flow Cytometry

2021 ◽  
Vol 23 (1) ◽  
pp. 263
Author(s):  
Amélie Rousselière ◽  
Laurence Delbos ◽  
Céline Bressollette ◽  
Maïlys Berthaume ◽  
Béatrice Charreau
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Immune Responses ◽  
Nk Cells ◽  
Hcmv Infection ◽  
Immune Cell ◽  
Nk Cell ◽  
Spectral Flow ◽  
Cell Responses

HCMV drives complex and multiple cellular immune responses, which causes a persistent immune imprint in hosts. This study aimed to achieve both a quantitative determination of the frequency for various anti-HCMV immune cell subsets, including CD8 T, γδT, NK cells, and a qualitative analysis of their phenotype. To map the various anti-HCMV cellular responses, we used a combination of three HLApeptide tetramer complexes (HLA-EVMAPRTLIL, HLA-EVMAPRSLLL, and HLA-A2NLVPMVATV) and antibodies for 18 surface markers (CD3, CD4, CD8, CD16, CD19, CD45RA, CD56, CD57, CD158, NKG2A, NKG2C, CCR7, TCRγδ, TCRγδ2, CX3CR1, KLRG1, 2B4, and PD-1) in a 20-color spectral flow cytometry analysis. This immunostaining protocol was applied to PBMCs isolated from HCMV- and HCMV+ individuals. Our workflow allows the efficient determination of events featuring HCMV infection such as CD4/CD8 ratio, CD8 inflation and differentiation, HCMV peptide-specific HLA-EUL40 and HLA-A2pp65CD8 T cells, and expansion of γδT and NK subsets including δ2-γT and memory-like NKG2C+CD57+ NK cells. Each subset can be further characterized by the expression of 2B4, PD-1, KLRG1, CD45RA, CCR7, CD158, and NKG2A to achieve a fine-tuned mapping of HCMV immune responses. This assay should be useful for the analysis and monitoring of T-and NK cell responses to HCMV infection or vaccines.

Adoptive Immunotherapy with Tregs Prevents GvHD and Favours Immune Reconstitution After HLA Haploidentical Transplants for Hematological Malignancies.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4-4 ◽  
Author(s):  
Mauro Di Ianni ◽  
Franca Falzetti ◽  
Alessandra Carotti ◽  
Adelmo Terenzi ◽  
Elisabetta Bonifacio ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
High Risk ◽  
Immune Reconstitution ◽  
Nk Cell ◽  
Immune Recovery ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Cd34 Cells

Abstract Abstract 4 Transplantation of large numbers of highly purified CD34+ cells from haploidentical relatives is a viable strategy for the cure of acute leukaemia at high risk of relapse (Aversa et al., NEJM 1998; JCO 2005). As extensive T cell depletion is required to prevent GvHD, the very narrow T cell repertoire in the inoculum delays recovery of immune response against pathogens, leading to a high incidence of infection-related deaths. Thus the key challenge is to improve immune recovery by administering allogeneic donor T cells without causing GvHD. Preclinical studies demonstrated that freshly isolated or ex vivo expanded T regulatory cells (Tregs) could be used to control GvHD following bone marrow transplantation. The present phase I/II clinical trial evaluated the impact of early infusion of freshly isolated donor CD4/CD25+ Tregs, followed by an inoculum consisting of donor mature T cells (Tcons) and positively immunoselected CD34+ cells, on GvHD prevention and immunological reconstitution. Twenty-two patients (10 male; 12 female; median age 40.5, range, 21 to 60) with AML (n=17; 8 in CR1 at high risk, 7 in ≥CR2 and 2 in relapse), ALL (n=4; 3 in CR1; 1 in relapse) and 1 with high grade NHL in relapse were enrolled from September 2008 onwards. The conditioning regimen consisted of 8Gy single fraction TBI, thiotepa (4 mg/kg×2), fludarabine (40mg/m2×4), and cyclophosphamide (35 mg/kg×2). All patients received CD4/CD25+ GMP immunoselected Tregs (CliniMACS, Miltenyi Biotec) (21/22 2×106/kg bw; 1/22 1×106/kg bw). Three days later they received positively immunoselected CD34+ cells (median 8.2, range 5.0-19.1) together with Tcons (16/22 1×106/kg bw; 4/22 0.5 ×106/kg bw; 2/22 did not receive Tcons). Immunoselected CD4/CD25+ Tregs (purity 91.5±4.5) consisted of CD25high 25.6%±11.2; CD25int 57.4%±5.9; CD25low 8.5%±6; FoxP3 64%±1; CD127 14.9%±13.7 (mean±SD). As suggested by in vitro immunosuppressive assays and by immunophenotypic analysis, the contaminating cells in the Treg fraction were 50% of the CD25int and 100% of the CD25low, so that the infused Tregs:Tcons ratio was established at 1:1.5. No post-transplant prophylaxis against GvHD was used. 20/22 patients engrafted. Neutrophils reached 1×109/L at a median of 15 days (range, 11 to 39 days). Platelets reached 25×109/L and 50×109/L at median of 13 and 15 days, respectively (range, 11 to 48 days, and 13 to 60 days). All engrafted patients showed persistent full donor-type chimerism in peripheral blood and bone marrow. Strikingly, no GvHD was observed in 17/20 valuable patients, 2/20 developed grade I cutaneous self-limited untreated GvHD and 1/20 developed grade III GvHD. This patient had received the fewest Tregs. Six patients died (1 bacterial sepsis, 2 VOD, 1 fungal pneumonia, 1 CNS aspergillosis and 1 GvHD/systemic toxoplasmosis). In contrast with our previous experience, the speed of immune recovery was enhanced. The CD4 and the CD8 counts reached, respectively, 50/μL medianly on days 34 (range, 19 to 63 days) and 24 (range, 15 to 87); 100/μL medianly on days 47 (range, 28 to 100 days) and 34 (range, 19 to 95); 200/μL on days 70 (range, 41 to 146 days) and 61 (range, 21 to 95). We also observed a rapid development of a wide T-cell repertoire and detection of high frequencies of specific CD4+ and CD8+ for opportunistic pathogens such as Aspergillus, Candida, CMV, ADV, HSV, VZV, Toxoplasma. In KIR ligand-mismatched transplants, speed of NK cell reconstitution/maturation and size of donor vs recipient alloreactive NK cell repertoires were preserved (Ruggeri et al., Science 2002). In conclusion, in the setting of haploidentical transplantation infusion of freshly purified Tregs makes administration of high dose of T cells feasible for the first time. This strategy provides a long-term protection from GvHD and robust immune reconstitution. Treg-based cellular therapy may represent an innovative strategy to improve the outcome of haploidentical transplants. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Immune Monitoring of CD34+ Placental Cell Derived Natural Killer Cell Therapy (PNK-007) in Phase I Study of Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4457-4457
Author(s):  
William Van Der Touw ◽  
Lin Kang ◽  
Joseph Dennis Tario ◽  
Bhavani Stout ◽  
Vanessa Voskinarian-Berse ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Cell Therapy ◽  
Phase I ◽  
Nk Cells ◽  
Immune Reconstitution ◽  
Nk Cell ◽  
Killer Cell ◽  
Cell Counts ◽  
White Blood Cell Counts

Background: Natural Killer (NK) cells are innate immune lymphocytes with cytotoxic function and are critical for immune surveillance. Unlike T cells which rely on antigen-specific responses, NK cells recognize transformed cells through a variety of NK cell-specific receptor-ligand interactions. Clinical studies have highlighted the therapeutic potential of NK cell-based therapies and Celularity has developed a GMP procedure for generating PNK-007: an off-the-shelf and allogeneic NK cell product culture-expanded and differentiated from human placental CD34+ stem cells. PNK-007 was evaluated as a single-dose allogeneic cell therapy in a phase I dose-escalation study for multiple myeloma (MM) patients receiving autologous stem cell transplant (ASCT) (NCT02955550). 12 of 15 patients received subcutaneous IL-2 every 48 hours for 10 days following PNK-007 cell infusion while 3 patients in a separate cohort did not receive IL-2. Here, we report translational studies evaluating post-treatment immune reconstitution, minimal residual disease (MRD) detection at 10-5 threshold, and serum profiling of cytokines and chemokines. Methods: Patient bone marrow aspirate was collected for EuroFlow MRD assessment and immune phenotyping by flow cytometry at baseline, 90-100 days, 6 months and 1 year post-ASCT. Peripheral blood was collected at baseline, then weekly for six weeks following PNK-007 infusion, at 6 months and at 1 year post-ASCT. PNK-007 persistence, leukocyte populations, and HLA antibody panels were determined by flow cytometry. Serum was analyzed by Luminex for panel cytokines, chemokines and soluble cytokine receptors. All patients and PNK-007 drug product were typed for HLA and killer-cell immunoglobulin-like (KIR) genotype. Results: PNK-007 infusion did not interfere with immune reconstitution kinetics post-ASCT. Cohorts receiving PNK infusion 14 days post-ASCT had already recovered white blood cell counts to normal levels. One cohort receiving 3x107 PNK cells/kg 7 days post-ASCT showed an immune deficient environment (0.05x103 ± 0.004x103 leukocytes/ml, n=3), but recovered their white blood cell counts by day 21 (6.8x103 ± 1.8x103 leukocytes/ml, n=3). Using a validated Euro-flow MRD assay, 4/15 patients were MRD(-) at pre-ASCT baseline, and by day 90, 10/15 pts were MRD(-). PNK-007 persistence was not detected in patients by flow cytometry with the earliest timepoint tested being 7 days post-infusion. Panel HLA serum antibodies were not detected at any timepoint indicating the absence of alloantibodies. Withholding IL-2 administration in one cohort allowed us to evaluate its potential effects on NK cell recovery and immune reconstitution. We found that subcutaneous IL-2 q.o.d. for 10 days following PNK-007 infusion did not affect recovery kinetics and concentration of endogenous NK cells. However, these patients showed a 5-10 fold increase in serum soluble IL-2RA from baseline and elevated regulatory T cell (Treg) count in the blood versus baseline (167 ± 107 Treg/mL vs. 61 ± 33 Treg/mL, p=0.0075). Patients not receiving IL-2 saw no change in serum soluble IL-2RA or blood Treg level from baseline. CD4 and CD8 T cells from all patients retained their ability to become activated in response to ex vivo stimulation and favored release of IL-2 and IFNg. T cell effector function was maintained in all patients post-ASCT but was lost in a subset of patients at 1 year. Serum analysis showed low levels of free IL-15 at the time of PNK dosing despite the transient lymphodepleted state associated with ASCT. Cytokines associated with myeloid inflammation and T cell immunity in the month post dosing were within normal homeostatic level. Serum TGFβ was significantly lower at the day of PNK infusion compared to normal homeostatic levels. Conclusion: Translational data from a Phase I study of PNK-007 in post-ASCT MM established that dosing up to 3x107 cells/kg at 14 or 7 days post-transplant did not impair engraftment or immune reconstitution. EuroFlow MRD assessment of the bone marrow showed conversion of 6/11 patients from MRD(+) to MRD(-) by day 90 post transplant. The administration of IL-2 in this clinical study did not appear to benefit NK cell reconstitution but instead favored soluble IL2RA antagonism and increased systemic levels of Treg. These results demonstrate the feasibility of allogeneic NK cell therapy in the MM + ASCT setting and will help inform the design of further clinical studies. Disclosures Van Der Touw: Celularity, Inc.: Employment, Patents & Royalties. Kang:Celularity, Inc.: Employment, Patents & Royalties. Stout:Celularity, Inc.: Employment. Voskinarian-Berse:Celularity, Inc.: Employment. Rousseva:Celularity, Inc.: Employment. Hariri:Celularity Inc: Employment. Zhang:Celularity Inc: Employment.

scholarly journals Dynamical Systems Modeling of Early-Term Immune Reconstitution with Different Anti-Thymocyte Globulin (ATG) Administration Schedules in Allogeneic Stem Cell Transplantation.

2021 ◽  
Author(s):  
Viktoriya Zelikson ◽  
Amir Ahmed Toor ◽  
Gary Simmons ◽  
Natasha Raman ◽  
Elizabeth Krieger ◽  
...  
Keyword(s):  
T Cell ◽  
Clinical Outcomes ◽  
Cell Transplantation ◽  
Immune Reconstitution ◽  
Cellular Therapy ◽  
Immune Cell ◽  
Chronic Gvhd ◽  
T Cell Response ◽  
Cell Recovery ◽  
Early Term

Alloreactivity forms the basis of allogeneic hematopoietic cell transplantation (HCT), with donor derived T cell response to recipient antigens mediating clinical responses either in part or entirely. These encompass the different manifestations of graft vs. host disease (GVHD), infection risk as well as disease response. Whilst the latter is contingent upon disease biology and thus may be less predictable, the former two are more likely to be directly proportional to the magnitude of donor derived T cell recovery. Herein we explore the quantitative aspects of immune cell recovery following allogeneic HCT and clinical outcomes in two cohorts of HLA matched allograft recipients who received rabbit anti-thymocyte globulin (ATG) on different schedules (days -9 to -7 vs. -3 to -1). Monocyte as well as donor derived T cell (ddCD3) recovery was superior in those given ATG early in their course (days -9/-7). This difference was related to a more rapid rate of ddCD3 recovery, largely driven by CD3+/8+ cells in the first month following transplantation. Early monocyte recovery was associated with later T cell recovery, improved survival, and less chronic GVHD. In contrast rapid and early ddCD3 expansion out of proportion to monocyte recovery was associated with a high likelihood of acute GVHD and poor survival. This analytic methodology demonstrates that modeling 'early-term immune reconstitution' following HCT yields insights that may be useful in management of post-transplant immunosuppression and adaptive cellular therapy to optimize clinical outcomes.

scholarly journals Single-Cell RNA Sequencing Reveals the Expansion of Cytotoxic CD4+ T Lymphocytes and a Landscape of Immune Cells in Primary Sjögren’s Syndrome

2021 ◽  
Vol 11 ◽  
Author(s):  
Xiaoping Hong ◽  
Shuhui Meng ◽  
Donge Tang ◽  
Tingting Wang ◽  
Liping Ding ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Immune Cell ◽  
Susceptibility Genes ◽  
Primary Sjögren’S Syndrome ◽  
Healthy Controls ◽  
Immune Cell Subsets ◽  
Single Cell Rna Sequencing

ObjectivePrimary Sjögren’s syndrome (pSS) is a systemic autoimmune disease, and its pathogenetic mechanism is far from being understood. In this study, we aimed to explore the cellular and molecular mechanisms that lead to pathogenesis of this disease.MethodsWe applied single-cell RNA sequencing (scRNA-seq) to 57,288 peripheral blood mononuclear cells (PBMCs) from five patients with pSS and five healthy controls. The immune cell subsets and susceptibility genes involved in the pathogenesis of pSS were analyzed. Flow cytometry was preformed to verify the result of scRNA-seq.ResultsWe identified two subpopulations significantly expand in pSS patients. The one highly expressing cytotoxicity genes is named as CD4+ CTLs cytotoxic T lymphocyte, and another highly expressing T cell receptor (TCR) variable gene is named as CD4+ TRAV13-2+ T cell. Flow cytometry results showed the percentages of CD4+ CTLs, which were profiled with CD4+ and GZMB+ staining; the total T cells of 10 patients with pSS were significantly higher than those of 10 healthy controls (P= 0.008). The expression level of IL-1β in macrophages, TCL1A in B cells, as well as interferon (IFN) response genes in most cell subsets was upregulated in the patients with pSS. Susceptibility genes including HLA-DRB5, CTLA4, and AQP3 were highly expressed in patients with pSS.ConclusionsOur data revealed disease-specific immune cell subsets and provided some potential new targets of pSS. Specific expansion of CD4+ CTLs may be involved in the pathogenesis of pSS, which might give valuable insights for therapeutic interventions of pSS.

scholarly journals Early Immune Reconstitution after Haplo-Identical Hematopoietic Stem Cell Transplantation (HCT) with Post-Transplant Cyclophosphamide (PTCY) Does Not Improve Overall Survival Compared to Matched Unrelated Donor HCT Recipients Receiving Thymoglobulin Prophylaxis (ATG)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5676-5676
Author(s):  
Yasser Khaled ◽  
Joshua Boss ◽  
Poojitha Valasareddy ◽  
Arnel Pallera ◽  
Robert Johnson ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Overall Survival ◽  
T Cell ◽  
Graft Failure ◽  
Immune Reconstitution ◽  
T Cell Proliferation ◽  
First Year ◽  
Post Transplant ◽  
Allogeneic Hct ◽  
Primary Graft Failure

Recent retrospective studies demonstrated similar overall survival (OS) and relapse rate after allogeneic HCT using matched unrelated or haplo-identical donors. However, differences in graft versus host disease (GVHD) prevention protocols using ATG or PTCY may have influenced the results. In addition, there is little knowledge about immune reconstitution after PTCY compared to ATG. We examined the outcomes of 73 consecutive patients who received allogeneic HCT from 5/2015 to 4/2019 (39 Haplo, 34 MUD). Patient's Characteristics shown in table-1. The two groups matched except for donor age, CD34 dose infused and race. Conditioning regimens shown in table-1. MUD recipients received GVHD prophylaxis with Tacrolimus/ Mycophenolate (Tacro/MMF) in addition to ATG (24 Patients) or PTCY (10 Patients) while Haploidentical patient received Tacro/MMF with PTCY. A panel of immune reconstitution markers collected at day 100 post- transplant for CD3, CD4, CD8, Activated T cell ( HLA- DR3+ CD3+)and NK cells ( CD56+) was obtained for 29 MUD and 28 Haploidentical recipients. We observed pronounced proliferation and recovery in all T cell subsets in Haploidentical patients compared to MUD patients at day 100 as shown in Fig-1. This robust T cell recovery in Haploidentical transplant patients with PTCY was statistically significant for CD3, CD4 and CD8. When Immune reconstitution for Haploidentical patients compared to MUD patients who received PTCY, it maintained its robust effect on T cell proliferation (Fig-2) although it did not reach statistical significance. The overall survival at one-year with median duration of follow up of 22.6 months was 61.5% and 82.3% for Haploidentical and MUD recipients respectively; P=0.14. There were 15 deaths during the first year in the Haploidentical patients (3 = relapse, 5 = severe cytokine release syndrome (CRS), 1=Veno-occlusive disease, 3= infection, 2=GVHD and 1 = primary graft failure). In contrast there were only six deaths in MUD patients (2= relapse, 3= GVHD and 1= infection). There was no deaths in MUD PTCY patients in the first year. There was no primary graft failure in either arm, however secondary graft failure occurred in 2 Haploidentical and 1 MUD patients. Median time to engraftment was 18 days for Haploidentical (range, 12-57) and 11.6 days for MUD (range, 10-18). Acute GVHD grade 2-4 developed in 35% in MUD and 23% in Haploidentical patients. Conclusions: We found robust early immune recovery after Haploidentical HCT compared to MUD HCT. The degree of HLA mismatch with Haploidentical HCT and antigen presentation may have contributed to pronounced T cell proliferation as the same effects was not observed in MUD HCT with PTCY. Despite the early recovery of T cells after Haploidentical HCT the overall survival did not exceed the overall survival with MUD HCT. Severe CRS contributed to the increased mortality seen in Haploidentical HCT patients. Further strategies are needed to decrease treatment related mortality with Haploidentical HCT. Disclosures No relevant conflicts of interest to declare.

scholarly journals P073 Compartmentalised human gut immunity: studies of innate and adaptive lymphocyte distribution in the epithelium, lamina propria and GALT of healthy gut mucosa by flow cytometry

2020 ◽  
Vol 14 (Supplement_1) ◽  
pp. S172-S172
Author(s):  
A Carrasco Garcia ◽  
A Rao ◽  
E Kokkinou ◽  
S Haapaniemi ◽  
U Lindforss ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Lamina Propria ◽  
Immune Cell ◽  
Cd8 T Cell ◽  
Peyer's Patches ◽  
Peyer’S Patches ◽  
Human Gut ◽  
Cell Ratio ◽  
Health And Disease

Abstract Background The human gut mucosal immune system is compartmentalised in distinct and specialised immune niches. The epithelium and the lamina propria have been proposed as effector sites, while gut-associated lymphoid tissues (GALTs) constitute inductive immune niches. The major mucosal GALTs are the Peyer’s patches in the ileum and the colonic isolated lymphoid follicles (ILFs), scattered in the submucosa of the colon. The majority of studies of human gut immune function in health and disease have analysed unfractionated mucosal tissue samples. Hence, in contrast to mice, little is known about compartmentalised immune cell specialisation in the human gut. The aim of this study was to use novel dissection methods to analyse separate human gut immune niches. Methods Macroscopically healthy margins from colorectal cancer colectomies were obtained at a minimum distance of 10 cm from the tumour border. After faeces, mucus, fat and muscle removal, Peyer’s patches were identified and dissected using a stereomicroscope (based on Keita et al., Lab Invest, 2006). Colonic mucosa and submucosa (containing ILFs) fractions were mechanically separated by forceps (based on the method developed by Fenton et al., Immunity, under revision). Isolation of epithelial and lamina propria fractions from the mucosal compartment was performed by calcium chelation (DTT and EDTA) and enzymatic digestion (Collagenase II and DNAse), respectively. Cell suspensions from each fraction were analysed by flow cytometry (BD LSR-Fortessa and BD FACSymphony). Results As expected, mucosal GALTs were characterised by an enrichment of germinal centre B cells (CD19+CD20+CD38+), lymphoid tissue-like innate lymphoid cells (Lin−CD127+CD117+Nrp1+) and a higher CD4+/CD8+ T-cell ratio vs. mucosa, whereas the mucosal fraction was enriched for plasma cells (CD19+CD20−CD38high) and distinguished by a decreased CD4+/CD8+ T-cell ratio as compared with the GALT in both ileum and colon. CD19+/CD3+ ratios were only higher in Peyer’s patches but not in colonic submucosa enriched with ILFs, possibly due to the smaller size of the B-cell follicles in the latter. The intraepithelial compartment lacked B cells and contained more γδ-T cells as compared with the GALT and lamina propria. Conclusion We have used novel dissection methods in human intestinal tissues that reveal a compartmentalised immune cell specialisation that is in line with what has previously been described in mice. The method will allow for future deeper analysis of the human gut immune niches in health and disease, such as in inflammatory bowel disease.

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