RESEARCH OF RESTORATION PROCESSES AFTER INDUCTION OF LYMPHOPENIA BY 
CYCLOPHOSPHANE IN AN EXPERIMENTAL MODEL ON MOUSE

Objective: to evaluate the dynamics of T-cell recovery after exposure to cyclophosphamide (CP). CP was injected at a dose of 125 mg/kg, followed by killing of mice on days 0, 10, 20, 30, 60 and determination of the subpopulation composition by flow cytometry. It was shown that thymocytes are more vulnerable to the action of the CP than spleen lymphocytes. Cell restoration in the thymus begins earlier with delayed recovery of spleen lymphocytes. Due to CP, there is an increase in the conversion of the naive T-cells into central memory T-cells with a relative accumulation of the latter.

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Circulating Memory T Cells Isolated from Hodgkin Lymphoma Patients Display Evidence of Exhaustion and Chronic Activation

Blood ◽

10.1182/blood.v124.21.4400.4400 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4400-4400

Author(s):

Catherine S. Diefenbach ◽

Bruce G. Raphael ◽

Kenneth B. Hymes ◽

Tibor Moskovits ◽

David Kaminetzky ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Memory T Cells ◽

Central Memory ◽

Effector Memory ◽

T Cell Exhaustion ◽

Differentiation Markers ◽

Cell Markers ◽

Cell Exhaustion ◽

Central Memory T Cells

Abstract Background: In Hodgkin lymphoma (HL) the malignant Hodgkin Reed-Sternberg (HRS) cells comprise only a small fraction of the total cellular tumor population. These HRS cells orchestrate an inflammatory microenvironment of reactive cells that propagate a permissive milieu for HL growth, contributing to an ineffective local anti-tumor immune response. The peritumoral CD4 and CD8 T cells in HL patients show high expression of the receptor programmed death-1 (PD-1), involved in the functional impairment and “exhaustion” of T cells. Growing data suggests that this HL-mediated immune suppression may have effects that extend beyond the tumor microenvironment. High systemic levels of inflammatory cytokines and chemokines in HL patients has been reported. We characterized the systemic immune profile of HL patients with both newly diagnosed (ND) and relapsed (R) disease. Methods: Informed consent for correlative blood testing was obtained from patients with ND (n=8) or R (n=5) HL treated at the NYU Perlmutter Cancer Center or NY Presbyterian/Weil Cornell since January of 2013. Blood samples were drawn pre-treatment, and at sequential timepoints during and after therapy. Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll separation method and cells were frozen for subsequent analysis. The frozen PBMC were then stained with fluorescent-conjugated antibodies against T cell surface molecules in 10-color FACS analysis. The analyses were performed after gating live cells for CD4, CD8 and memory and effector T cell markers. Patient samples were compared to normal controls matched for age and sex (n=18). Results: The median HL patient age was 32 (22-72), and 8 subjects were male. All ND HL patients were treated with ABVD (range 4-6 cycles) +/- consolidative radiation; R patients had median of 3 prior therapies. One patient out of 5 had prior autologous stem cell transplant (SCT), and 1 had prior allogeneic SCT, but was not on immunosuppression. Eight patients (6ND, 2R) responded to therapy (8 CR); 5 patients (1ND, 4R) progressed on therapy or had stable disease. HL patients displayed a high frequency of the exhaustion marker PD-1 on CD4 central memory T cells (CD4+CD45RO+CD27+) compared to normal matched controls (NC): mean 41, standard error (SE) 4.8 for HL patients vs. mean 22.2, SE 1.3 for NC (p = 0.0002) (Figure 1A). PD-1 expression was similarly elevated on CD8 central memory T cells (CD8+CD45RO+CD27+) of HL patients: mean 55, SE 3.3 vs. NC: mean 40, SE 3.3 (p = 0.003) (Figure 1B). HL patients also displayed an increased frequency of PD-1 expression on CD27 negative CD4 effector T cells: mean 43, SE 4, vs. NC: mean 28.5, SE 2.4 (p = 0.003) (Figure 2). In 4 of the HL patients who responded to therapy, PD-1 expression on central memory CD4+ cells declined after therapy: mean 30.1 vs. mean increase of +2.67 in 3 patients who progressed on therapy (p< 0.009). A higher number of subjects in prospective analysis is underway, to confirm whether a response to therapy may be correlated with a reversal of the suppressed phenotype of T cells in these patients. Conclusion: HL patients have evidence of chronic activation/exhaustion in their central memory and effector T cells, suggesting that ineffective immune clearance of the HRS cells may be a systemic rather than local phenomenon. In patients with progressive disease for whom this phenotype persists it is worthy of investigation whether this immune dysfunction is a cause or consequence of resistance to therapy. This may be rationale for immune targeted therapy in patients with relapsed or resistant disease. Figure 1. Evidence for increased levels of T cell exhaustion in central memory T cells of HL patients. PBMC were stained with specific fluorescent conjugated antibodies against T cell markers (CD3, CD4, CD8) together with differentiation markers (CD45RO, CD27) and PD1 and analyzed using FACS (LSR-II). The proportion of PD1+ T cells were determined in: A) CD4+CD45RO+CD27+ and B) CD8+CD45RO+CD27+ T cells. Figure 1. Evidence for increased levels of T cell exhaustion in central memory T cells of HL patients. PBMC were stained with specific fluorescent conjugated antibodies against T cell markers (CD3, CD4, CD8) together with differentiation markers (CD45RO, CD27) and PD1 and analyzed using FACS (LSR-II). The proportion of PD1+ T cells were determined in: A) CD4+CD45RO+CD27+ and B) CD8+CD45RO+CD27+ T cells. Figure 2. Evidence for increased levels of T cell exhaustion in effector memory CD 4+ T cells of HL patients. PBMC were stained with specific fluorescent conjugated antibodies against T cell markers (CD3, CD4) together with differentiation markers (CD45RO, CD27) and PD1 and analyzed using FACS (LSR-II). The proportion of PD1+ T cells was determined in CD4+RO+CD27- T cells Figure 2. Evidence for increased levels of T cell exhaustion in effector memory CD 4+ T cells of HL patients. PBMC were stained with specific fluorescent conjugated antibodies against T cell markers (CD3, CD4) together with differentiation markers (CD45RO, CD27) and PD1 and analyzed using FACS (LSR-II). The proportion of PD1+ T cells was determined in CD4+RO+CD27- T cells Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

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Oligoclonal Expansion and T-Cells Subpopulations in Patients with Aplastic Anemia at Different Stages of the Disease

Blood ◽

10.1182/blood-2018-99-119414 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3864-3864

Author(s):

Anastasia V. Abramova ◽

Elena A. Mikhaylova ◽

Zalina T. Fidarova ◽

Yuliya O. Davydova ◽

Nikolay M. Kapranov ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Aplastic Anemia ◽

Memory T Cells ◽

Clonal Expansion ◽

Th Cells ◽

Healthy Donors ◽

Oligoclonal Expansion

Abstract Background. The main mechanism of the bone marrow (BM) failure in idiopathic aplastic anemia (AA) has an immunomediated character. Researching the T-cell clone's effect in the AA pathogenesis is very relevant at the present time. Oligoclonal expansion of T cells is frequent in AA and the identification of immunodominant T-cell clones can correlate with the disease activity and may possibly serve as response predictor to immunosuppressive therapy (IST). The aim. To identify T-cells subpopulations, expression of PD-1 and PD-L1 on T-cells and TCR-Vβ repertoires by flow cytometry in different groups of AA patients. Methods. Thirty AA patients (pts) with median age of 30.5 (19-71), m/f ratio 1:1,3 were divided in 3 groups: pts with newly diagnosed (ND) AA (n=13), pts with overall response to IST (OR) (n=10), non-response pts (NR) for 2 and more lines of IST (n=7). Flow cytometry was performed with BD FACS Canto II. We used commercial kit (IOTest® Beta Mark TCR Vb Repertoire) for evaluation of TCR-Vβ repertoire in the bone marrow (BM) of these patients. We performed analysis of BM samples from healthy donors as a control group (n=8). Due to low amount of donor samples the maximal value each of the 24 subclones (for CD4+ (T-helpers - Th) and CD8+ cells (T-cytotoxic cells - TCL)) was accepted as threshold. We concluded the presence of clonal expansion if TCR subclone exceeded this threshold. We identified different T-cell subpopulations in all 3 groups of AA and healthy donors by flow cytometry: double positive T-cells (CD3+CD4+CD8+), double negative T-cells (CD3+CD4- CD8-), Th (CD3+CD4+), TCL (CD3+CD8+), NK-T-cells (CD3+CD56+) out of CD3+ cells. Among Th and TCL cells was determined naive T-cells (CD28+CD95-), effector T-cells (CD28-CD95+), memory T-cells (CD28+CD95+), regulatory T-cells (CD4+CD127-CD25high) and subpopulations Th and TCL co-expressed PD-1 and PD-L1. Multiple comparisons were assessed by ANOVA or Kruskal Wallis test by GraphPad Prism software. Results. In our study all 30 AA patients had an immunodominant TCR-Vβ clones among Th and/or TCL cells. We identified the most common clonotypes in comparison with healthy donors - Vβ1, Vβ2, Vβ3 among the Th cells and Vβ3, Vβ9, Vβ13.1 among the TCL cells. In ND group Vβ1 was highly expanded in 5 (38.5%), Vβ3 - in 7 (53.8%) pts among Th, and Vβ3 - in 3 (23.1%) and Vβ9 - in 4 (30.8%) out of 13 pts among TCL. In OR group Vβ2 expansion was in 4 (40%) and Vβ3 - in 5 (50%) pts among Th; Vβ3 in 6 (60%) and Vβ9 in 6 (60%) out of 10 pts among TCL. In NR group the most frequent was Vβ13.1 clone in TCL - in 3 (42.9%) out of 7 pts. In NR group in overall clonal expansion was less frequent than in ND and OR groups. We also analyzed the previously mentioned subpopulations of T-cells in patients with AA in three groups (ND, OR, NR) compared to healthy donors (table 1). We obtained significant differences in the count of naive Th and TCL cells, memory T-cells in all three groups of AA patients compared to donors: proportion of naive Th and TCL cells was significantly higher and proportion of memory Th cells was lower in the donor group than in AA pts. The percent of TCL effectors was higher in ND AA pts compare to donors. We also found that cell count of activated Th (CD4+CD25+) was higher in the group of refractory pts. In OR pts proportion of PD-1-positive Th was higher than in donors. In NR pts Th and TCL co-expressed with PD-L1 were lower compare to donors (table 1). Conclusions. In our study we found immunodominant clonotypes in different AA pts and depletion of the pool of naive T cells. Dynamic observation of changes in the most common clonotypes in AA pts during treatment will provide suitable therapy tactics (allogenic bone marrow transplantation or IST). Disclosures No relevant conflicts of interest to declare.

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Mobilization of CD8+ Central Memory T-Cells with Enhanced Reconstitution Potential in Mice By a Combination of G-CSF and GMI-1271-Mediated E-Selectin Blockade

Blood ◽

10.1182/blood.v126.23.512.512 ◽

2015 ◽

Vol 126 (23) ◽

pp. 512-512 ◽

Cited By ~ 1

Author(s):

Ingrid G Winkler ◽

Valerie Barbier ◽

Kristen J Radford ◽

Julie M Davies ◽

Jean-Pierre Levesque ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Research Funding ◽

Memory T Cells ◽

Central Memory ◽

Donor Blood ◽

Central Memory T Cells

Abstract T-cells are critical mediators of immune defense against pathogens and cancer. Adoptive T cell immunotherapy and T-cell engineering have promising clinical applications but T cell survival and exhaustion are current limitations. Central memory cells (TCM CD62L+ CCR7+) and their precursors, stem central memory T-cells (TSCM) possess the stem-like properties needed to reconstitute and prolong an effective immune response long-term. These cells have been shown to significantly improve therapeutic efficacy of adoptive T-cell therapy. The challenge remains to harvest good quality TCM-cells for these immunotherapy approaches. The bone marrow (BM) is the major reservoir of CD8+ TCM and their precursors. We have previously shown that E-selectin is expressed in the BM vasculature and drives activation and differentiation of hematopoietic stem cells during G-CSF induced mobilization to the blood. We find therapeutic blockade of E-selectin promotes HSC self-renewal and reconstitution in vivo. We now examine the impact of E-selectin blockade on CD8+ T cell mobilization from the bone marrow to the blood and hypothesize that E-selectin blockade may also dampen the activation/differentiation of this subset. First we administered a standard G-CSF regime (filgastim 250ug/kg/day for 3 days) to mice and then dosed some cohorts with GMI-1271 (40mg/kg BID) from 12 to 72 hours within this 3 day period. Administration of G-CSF alone results in a near complete disappearance of bone marrow resident CD8+ TCM cells, and their apparent migration (increase in numbers) to the blood, while CD8+ subsets in the lymph nodes and spleen were barely affected by G-CSF. Furthermore among T-cell subsets, CD8+ but not CD4+ TCM were specifically mobilized into the blood when GMI-1271 was co-administered for the last 12 to 24 hours of G-CSF. These findings are consistent with reports demonstrating the bone marrow to be a major reservoir for CD8+ but not CD4+ central memory T-cells. Administration of GMI-1271 caused a marked enhancement in mobilization into the blood of CD8+ TCM/SCM (CD62Lhi, CCR7+) cells over treatment with G-CSF alone (p<0.05). To determine the functional consequences of this skewed mobilization following GMI-1271 co-administration, 25 uL of mobilized blood was transplanted into irradiated congenic B6.SJL recipients together with 2x105 congenic BM cells to analyze long-term donor T-cell engraftment in the recipient mice. We found G-CSF mobilized donor blood did not contribute CD8+ TCM cells that can persist post-transplant (<0.5% at 20 weeks post-transplant). In contrast when donor mice were mobilized with G-CSF together with E-selectin blockade (GMI-1271), we found elevated levels of donor blood derived CD8+ T-cells demonstrating robust long-term CD8+ T-cell persistence / regeneration (5.3 ±3.2% of total recipient T-cells, p=0.04). This dramatic boost in donor CD8+ T-cell reconstitution in mobilized blood following GMI-1271 co-administration is likely to be due to the long-term persistence and in vivo amplification of CD8+ TCM cells from donor mobilized blood. Similar in vivo enhancing effects of GMI-1271 were also observed with other mobilizing agents such as combined CXCR4 and VLA-4 blockade and GM-CSF resulting in a significant 4.9-fold boost in donor CD8+ reconstitution with GMI-1271. Importantly, only 12 hours of E-selectin blockade was sufficient to achieve this boost in CD8+ TCM numbers in the blood following G-CSF. In a previous report we have shown that therapeutic blockade of E-selectin promotes HSC self-renewal in vivo. Thus, it is possible that E-selectin blockade boosts mobilization of CD8+ TCM/SCM with stem-like properties into the blood by loosening factors retaining CD8+ TCM/SCM in the bone marrow and/or blocking the E-selectin-mediated activation and differentiation of this T-cell subset. In summary, our studies identify E-selectin blockade as a novel target to improve harvesting of CD8+ TCM/SCM cells with stem-like properties. Blockade of this target with GMI-1271 significantly improves the in vivo reconstitution potential and regenerative properties of CD8+ T-cells from donor blood allowing a valuable source of desired T-cells for use in adoptive immunotherapy and T-cell engineering. Disclosures Winkler: GlycoMimetics Inc: Research Funding. Barbier:GlycoMimetics Inc: Research Funding. Davies:GlycoMimetics Inc: Research Funding. Smith:GlycoMimetics, Inc.: Employment. Fogler:GlycoMimetics, Inc.: Employment. Magnani:GlycoMimetics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Targeted in-vitro-stimulation reveals highly proliferative multi-virus-specific human central memory T cells as candidates for prophylactic T cell therapy

PLoS ONE ◽

10.1371/journal.pone.0223258 ◽

2019 ◽

Vol 14 (9) ◽

pp. e0223258 ◽

Cited By ~ 2

Author(s):

Benjamin Faist ◽

Fabian Schlott ◽

Christian Stemberger ◽

Kevin M. Dennehy ◽

Angela Krackhardt ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

Memory T Cells ◽

Central Memory ◽

T Cell Therapy ◽

Central Memory T Cells

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Fludarabine Treatment Is Associated with Depletion of Host CD4+CD25high, FOXP3+, CTLA-4+ Cells and Increased Incidences of Full Donor Chimerism and GVHD in Non-Myeloablative Haploidentical Hematopoietic Cell Transplant Recipients.

Blood ◽

10.1182/blood.v106.11.2898.2898 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2898-2898

Author(s):

Juanita M. Shaffer ◽

Jean Villard ◽

Terry Means ◽

David Dombkowski ◽

Bimalangshu Dey ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Hematopoietic Cell ◽

Cell Depletion ◽

Cell Recovery ◽

Myeloablative Conditioning ◽

T Cell Depletion ◽

Donor Chimerism

Abstract Purpose: To evaluate T cell recovery and donor chimerism following haploidentical hematopoietic cell transplantation (HCT) with a non-myeloablative conditioning approach that includes T cell depletion of host and donor and delayed DLI. Methods: Eighteen patients, 3 cohorts of 4 patients each and 1 cohort of 6 evaluable patients/10 transplanted, with chemorefractory hematologic malignancies, received related HLA 1–3 of 6, A, B, or DR antigen mismatched donor HCT after non-myeloablative conditioning with Medi-507 (anti-CD2 humanized mAb; Biotransplant, Inc.), cyclophosphamide, thymic irradiation and peritransplant cyclosporine. The patients in Protocols A received a MEDI-507 test dose of 0.1 mg/kg on Day -2 followed by 0.6 mg/kg on Days −1, 0 and +1 and transplantation of unmanipulated bone marrow. In Protocol B, the timing and dose of Medi-507 was modified. The patients in Protocol C and D received the latter Medi-507 protocol, but were given Isolex ®-selected CD34+ cells from G-CSF mobilized PBSC. Protocol D differs from Protocol C with the addition of fludarabine to more reliably achieve sustained chimerism. Donor leukocyte infusions were administered in an effort to convert mixed to full donor chimerism and to achieve a graft-versus-tumor effect. Chimerism was measured by peripheral blood microsatellite markers or by flow cytometry using HLA-specific mAbs. T cell recovery and phenotype were followed by flow cytometry. Because a high percentage of CD4 T cells post- transplant were CD25high, we performed quantitative RTPCR for Foxp3 and CTLA-4 on sorted PBMC populations. Results: T cell depletion early post-HCT was detected in all patients. There was a marked difference in the percentage of graft acceptance/loss, GVHD prevalence, and T cell phenotype related to each protocol modification. The majority (>90%) of CD4 T cells appearing in the first 100 days post-SCT were CD45RO+/CD45RA- “memory” cells and CD8 T cells were CD45RO+CD45RA-/CD62L-. In addition, a remarkably high percentage (19.5–75%, mean 38.1%) of CD4 T cells expressed high levels of CD25 in recipients of Protocols A, B, and C early post-HCT. CD25 expression decreased as T cell counts increased. By quantitative RTPCR, we found that sorted CD25highCD4 T cells expressed Foxp3 and CTLA-4, consistent with a regulatory phenotype. The addition of fludarabine in Protocol D resulted in an overall reduction in the percentage of peripheral CD4CD25high T cells compared to Protocol C at 4 weeks post-HCT (C 23.43% +/−4.7% versus D 2.1% +/− 0.9%, p<0.00005). The addition of fludarabine improved sustained engraftment from 2/4 in Protocol C to 5/6 in Protocol D. In Protocol C, T cell chimerism conversion occurred following DLI in 2/4 patients with no or grade II skin-limited GVHD. In Protocol D, full or near full donor chimerism was achieved in 5/6 patients, 3 spontaneously and 2 following DLI, and grade I–IV GVHD developed in 5/6 patients. Conclusions: These data suggest that the addition of fludarabine may increase the incidence of sustained and full donor chimerism. Fludarabine efficiently depleted host CD4+CD25high Foxp3+ cells, which may have a regulatory role, preventing spontaneous chimerism conversion and associated GVHD.

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Activation of T and NK Cells Following Lenalidomide Therapy in Patients with Follicular Lymphoma.

Blood ◽

10.1182/blood.v120.21.2766.2766 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2766-2766

Author(s):

Seema Rawal ◽

Nathan Fowler ◽

Min Zhang ◽

Zhiqiang Wang ◽

Tariq Muzzafar ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Nk Cells ◽

Research Funding ◽

Memory T Cells ◽

T Cell Subsets ◽

Central Memory T Cells

Abstract Abstract 2766 Background: Lenalidomide plus rituximab therapy is a highly effective and well-tolerated therapy in patients (pts) with follicular lymphoma (FL). In a Phase II trial, this combination induced a complete remission rate of 87% in pts with advanced stage untreated FL (Fowler et al, Ann Oncol, 2011; 22; suppl 4:137). A randomized Phase III trial was recently initiated to compare this combination with current standard of care therapies in pts with FL. Although lenalidomide is known to be an immunomodulatory drug with effects on a variety of immune cells in vitro, its effects have not been well studied in vivo in humans. Understanding the in vivo effects of lenalidomide could lead to novel combination strategies to enhance the efficacy and improve clinical outcome in FL and other malignancies. Methods: Pts received lenalidomide 20 mg/day on days 1–21 of each 28-day cycle and rituximab was given at 375 mg/m2on day 1 of each cycle. Peripheral blood mononuclear cells (PBMC) were phenotyped by multiparametric flow cytometry at baseline, on cycle 2 day 15 (C2D15), and at the end of cycle 6. In addition, peripheral blood (PB) samples were collected in PAXgene Blood RNA tubes at baseline and on C2D15 for whole genome gene expression profiling (GEP). Results: Immunophenotyping of baseline and end of cycle 6 PBMC (n=17) showed that the percentages and absolute numbers of CD3+, CD4+, CD8+, TCRgd, and Foxp3+ regulatory T cells; and NK, NKT, and myeloid dendritic cells were not significantly different between the two time points. However, a significant increase in CD4+CD45RO+ (p<0.01) and CD8+CD45RO+ (p=0.04) memory T cells was observed post-therapy. Further characterization of CD4+ T cells showed a significant increase in central memory T cells (p<0.001) and a decrease in naïve (p<0.01) and terminally differentiated (p<0.01) T cells, but no change in effector memory T cells. The increase in CD8+ central memory T cells was marginally significant (p=0.06). Plasmacytoid dendritic cells (PDC) were also significantly increased (p=0.02). In contrast, no such changes in T cell subsets or PDC were observed in FL pts (n=9) treated with 6 cycles of R-CHOP chemotherapy that received equal number of rituximab doses and analyzed at similar time points (baseline and end of cycle 6). To understand lenalidomide-induced changes on a molecular level, we compared GEP data at C2D15 vs. baseline for 7 pairs of PB samples. The paired significance analysis of microarrays method, based on Student's t test, identified 1,748 differentially expressed genes (DEG; 713 up, 1035 down), without a fold-change threshold, in C2D15 samples vs. baseline. Results were influenced by rituximab-induced depletion of B cells in C2D15 samples, but there were many changes that suggested altered PBMC physiology. Noteworthy up-regulated genes (>1.5 fold) included genes associated with T and NK cell activation including BATF, CCR2, CD1B, CD2, CD160, CTLA4, CXCR3, ICOS, and LAG3; and CD163 and CD209, phagocytic receptors expressed on monocytes/macrophages. Down-regulated genes (>1.5 fold) included CXCR5, which mediates B cell migration into follicles; and IL1B and TNFSF13B (BAFF), which are produced by activated macrophages and induce B cell proliferation. Gene set enrichment analysis of all GEP results, and Ingenuity Pathway Analysis of DEGs, indicated up regulation of multiple pathways and processes including ribosomal and mitochondrial components involved in translation and oxidative phosphorylation, CTLA4 signaling in cytotoxic T cells, and differentiation and signaling by ICOS and CD28 in T helper cells. We confirmed up regulation of CTLA4, ICOS, and LAG3 at the protein level in C2D15 PBMC by flow cytometry. Furthermore, treatment of PBMC derived from untreated FL pts with lenalidomide in vitro resulted in up regulation of these molecules in T and/or NK cells consistent with our in vivo results. Conclusions: In FL pts, lenalidomide induced multiple changes in the immune system including increases in PDC and memory T cell subsets, activation of T and NK cells, and down-regulation of certain genes mediating B cell migration and proliferation. These results provide insights into the mechanism of action of lenalidomide and suggest that it can be combined with other immunostimulatory agents such as therapeutic vaccines, adoptive T cell therapy strategies, and immune checkpoint inhibitors to further enhance its efficacy in FL and other malignancies. Disclosures: Fowler: Celgene: Research Funding. Heise:Celgene Corporation: Employment, Equity Ownership. Lacerte:Celgene: Honoraria. Samaniego:Celgene: Research Funding. Neelapu:Celgene Corporation: Research Funding.

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Enhanced Antitumor Efficacy of Adoptively Transferred CD19-Redirected CMV Specific Central Memory T Cells by CMV Vaccine.

Blood ◽

10.1182/blood.v120.21.3014.3014 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3014-3014

Author(s):

Xiuli Wang ◽

Winnie Wong ◽

Wen-Chung Chang ◽

Don Diamond ◽

Michael C. Jensen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Antitumor Activity ◽

Adoptive Transfer ◽

Viral Antigen ◽

Memory T Cells ◽

Central Memory T Cells ◽

Tumor Bearing ◽

Nsg Mice

Abstract Abstract 3014 Development of T cell products that have engineered specificity for CD19 has broad application to adoptive transfer therapy for B-lineage lymphoma and leukemia. Clinical studies have demonstrated the safety and feasibility of cloned and bulk T cell transfer as a therapy for patients. But potency of this strategy has proven challenging, primarily due to issues relating to a lack of persistence of the adoptively transferred cells in patients. In contrast, the adoptive transfer of viral specific T cells has shown efficient efficacy for preventing progressive viral infections and exhibited long term persistence in patients, in part due to the viral specific T cells received optimal co-stimulation after engagement of their native receptors. Conceptually, engineering CMV specific T cells with CD19CAR to provide them with a second specificity for a tumor antigen may enable the transferred T cells (bi-specific T cells) to persist or numerically expand in vivo by stimulation of the endogenous TCR by virus antigen. Moreover, bi-specific T cell can be used in treatment for B cell malignancies in allo-settings without causing GVHD due to the pre-defined non-alloreactive TCR specificity. In this study, we explored the use of CMVxCD19CAR bi-specific T cells in CD19+tumor bearing NSG mice and evaluated their antitumor activity in response to CMVpp65 antigen stimulation as a consequence of CAR transduced T cell expansion. CMV specific T cells derived from central memory T cells were selectively expanded by 2 rounds of stimulation with cGMP grade pp65 protein followed a rapid expansion containing OKT3 and feeder cells. The established CMV specific Tcm, in which majority of them are CMVpp65 tetramer positive, were then transduced with cGMP grade SIN lentivirus expressing CD19R:CD28:z/EGFRt. After stimulation with CD19 positive LCL, 40% of the resultant cells co-express pp65 tetramer and CAR as detected by EGFRt/Erbitux analysis. Functionally, the bi-specific T cells exhibit specific cytolytic activity and secret IFNg, IL2 and TNFα upon engagement with pp65 or CD19 antigen, indicating that the effector function of the bi-specific T cells can be induced through endogenous TCR or the introduced CAR. To evaluate the in vivo viral antigen driven anti-tumor efficacy of the adoptively transferred bi-specific T cells, CD19+LCL expressing GFPffluc were inoculated (i.v) into huIL-15 reconstituted NSG mice. Once the tumor engraftment was confirmed by in vivo imaging, bi-specific T cells were adoptively transferred (i.v) into the tumor bearing mice. Anti- tumor activity was observed 14 days post T cell infusion. As expected, this effect is transient and tumor re-progression occurred. In order to deliver CMV antigen for vaccine, we generated T-APC by loading CMVpp65 peptide into autologous T cells and injected the CMV T-APCs (I.v) into the bi-specific T cell treated mice, Influenza specific MP1 peptide pulsed autologous T cells were used as control T-APCs. CMV T-APC induced a second wave of antitumor activity 2 weeks post vaccine and mice survived for more than 2 months post adoptive transfer of T cells, while tumor grew vigorously when MP1-T-APCs were given as stimulators. The findings demonstrated that CD19CAR modified CMV specific T cells are capable of responding to viral antigen reactivation through their endogenous TCR, which could be used to magnify the antitumor activity of CAR transduced T cells in vivo. Disclosures: No relevant conflicts of interest to declare.

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Chemokine Receptor Expression Identifies Pre–T Helper (Th)1, Pre–Th2, and Nonpolarized Cells among Human CD4+ Central Memory T Cells

Journal of Experimental Medicine ◽

10.1084/jem.20040774 ◽

2004 ◽

Vol 200 (6) ◽

pp. 725-735 ◽

Cited By ~ 205

Author(s):

Laura Rivino ◽

Mara Messi ◽

David Jarrossay ◽

Antonio Lanzavecchia ◽

Federica Sallusto ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Memory T Cells ◽

Cell Receptor ◽

Central Memory ◽

Th2 Cells ◽

T Helper ◽

Central Memory T Cells ◽

Th 1

We previously reported that central–memory T cells (TCM cells), which express lymph node homing receptors CCR7 and CD62L, are largely devoid of effector functions but acquire characteristics of effector–memory T cells (TEM cells) (i.e., CCR7− T helper [Th]1 or Th2 cells) after stimulation with T cell receptor agonists or homeostatic cytokines. Here we show that three chemokine receptors identify functional subsets within the human CD4+ TCM cell pool. TCM cells expressing CXCR3 secreted low amounts of interferon γ, whereas CCR4+ TCM cells produced some interleukin (IL)-4, but not IL-5. In response to IL-7 and IL-15, CXCR3+ TCM and CCR4+ TCM cells invariably generated fully differentiated CCR7− Th1 and Th2 cells, respectively, suggesting that they represent pre-Th1 and pre-Th2 cells. Conversely, CXCR5+ TCM cells lacking CXCR3 and CCR4 remained nonpolarized and retained CCR7 and CD62L expression upon cytokine-driven expansion. Unlike naive cells, all memory subsets had a low T cell receptor rearrangement excision circle content, spontaneously incorporated bromodeoxyuridine ex vivo, and contained cells specific for tetanus toxoid. Conversely, recall responses to cytomegalovirus and vaccinia virus were largely restricted to CXCR3+ TCM and TEM cells. We conclude that antigen-specific memory T cells are distributed between TEM cells and different subsets of TCM cells. Our results also explain how the quality of primary T cell responses could be maintained by TCM cells in the absence of antigen.

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Low-dose controlled release of mTOR inhibitors maintains T cell plasticity and promotes central memory T cells

Journal of Controlled Release ◽

10.1016/j.jconrel.2017.02.034 ◽

2017 ◽

Vol 263 ◽

pp. 151-161 ◽

Cited By ~ 11

Author(s):

Joshua M. Gammon ◽

Emily A. Gosselin ◽

Lisa H. Tostanoski ◽

Yu-Chieh Chiu ◽

Xiangbin Zeng ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Controlled Release ◽

Low Dose ◽

Memory T Cells ◽

Mtor Inhibitors ◽

Central Memory ◽

Cell Plasticity ◽

Central Memory T Cells

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Results from a First-in-Human Study with Bnz-1: A Novel Peptide Inhibitor of IL-2, IL-9 and IL-15 for the Treatment of T-Cell Malignancies That Safely and Selectively Decreases Regulatory T-Cells, Natural Killer Cells, and CD8+ Central Memory T-Cells

Blood ◽

10.1182/blood.v130.suppl_1.695.695 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 695-695

Author(s):

Paul Frohna ◽

Yutaka Tagaya ◽

Anoshie Ratnayake ◽

Nick Doerr ◽

Asjad Basheer ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Nk Cells ◽

Memory T Cells ◽

Human Study ◽

Central Memory ◽

Post Dose ◽

Central Memory T Cells ◽

T Cell Malignancies

Abstract Background: The γc-family of cytokines (IL-2, IL-4, IL-7, IL-9, IL-15, IL-21) control major immune responses and lymphocyte development. However, aberrant γc cytokine activity contributes to or pathologically drives human diseases including T-cell malignancies, graft-versus-host disease, and numerous autoimmune diseases (e.g., alopecia areata), where IL-2, IL-9, and IL-15 have been specifically implicated. Importantly, these multi-cytokine diseases are not effectively and safely treated by the currently available anti-cytokine approaches. Methods: BNZ-1 is the first of a novel class of rationally-designed, multi-cytokine inhibitors that selectively inhibits IL-2 and IL-15, and to a lesser degree, IL-9 signaling through the γc receptor, without affecting IL-4, IL-7, or IL-21. Preclinical proof-of-concept has been demonstrated in several animal models of T-cell malignancies (Nata et al., 2015 J Biol Chem), including Large Granular Lymphocyte Leukemia (LGL) and Adult T-cell Leukemia. In this open-label, single-dose, dose-escalation, first-in-human study conducted at a single center in the United States (NCT03046459), 18 healthy adults (n=3/cohort) received a single intravenous dose of 0.2, 0.4, 0.8, 1.6, 3.2 or 6.4 mg/kg infused over ≤5 minutes on Day 1 and were followed for safety and sample collection for 30 days. Results: All subjects completed the study. BNZ-1 was considered well-tolerated with a good safety profile with no serious or severe adverse events (AEs), no dose-limiting toxicities, and no clinically-significant changes on clinical labs (serum biochemistry, hematology, liver enzymes), vital signs or electrocardiograms. Headache was the only AE reported in ≥1 subject (n=3). BNZ-1 exposure was generally dose proportional with an elimination half-life of ~5 days across the range of doses tested, which is consistent with other PEGylated peptides and supports weekly or every other week dosing. The pharmacodynamic activity of BNZ-1 was characterized by flow cytometry of PBMCs obtained on Days 4, 15 and 31 and used to calculate the maximum change from baseline (Emax) for regulatory T-cells (Tregs; IL-2 effect), NK cells (IL-15 effect) and CD8+ central memory T-cells (Tcm; IL-2 & IL-15 effect). For Tregs, a ~50-60% decrease was observed on Day 4 after doses of 0.4 to 1.6mg/kg, while doses of 3.2 and 6.4mg/kg produced an 80-93% decrease from baseline that peaked on Day 15. NK cells were decreased from baseline on Day 4 by 20%, 40% and 60% at 0.2, 0.4 and 0.8mg/kg, respectively, and plateaued at 70 to 80% decrease at doses ≥1.6 mg/kg. Tcm were decreased at Day 4 for the three highest dose cohorts that continued to decline to Day 15 when all doses, except 0.2 mg/kg, showed a mean decrease ranging from 10 to 81% that generally trended with dose. Tregs, NK cells, and Tcm returned to/toward baseline by 30 days post dose. Post-dose counts of CD4+ and CD8+ T-cells, B-cells, and monocytes were unchanged at all time points studied. Conclusions: These preliminary clinical data suggest that BNZ-1 is a highly-active, selective immunomodulator that safely decreases Tregs, NK cells and Tcm, while leaving the major leukocyte populations unaffected. These data support the planned Phase 1/2 dose-ranging study in LGL and Cutaneous T-cell Lymphoma. Disclosures Frohna: Bioniz Therapeutics: Employment. Tagaya: Bioniz Therapeutics: Equity Ownership. Ratnayake: Bioniz Therapeutics: Consultancy. Doerr: Bioniz Therapeutics: Employment. Basheer: Bioniz Therapeutics: Employment. Al-Mawsawi: Bioniz Therapeutics: Employment. Kim: Bioniz Therapeutics: Employment. Azimi: Bioniz Therapeutics: Employment.

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