Clinical Trial Evaluating DC/AML Fusion Cell Vaccination Alone and in Conjunction with PD-1 Blockade in AML Patients Who Achieve a Chemotherapy-Induced Remission

Abstract Abstract 948 Patients with acute myeloid leukemia (AML) achieve remission following chemotherapy; however, curative outcomes remain elusive due to relapse with chemotherapy-resistant disease. Allogeneic transplantation remains a potentially curative therapy for AML patients, but is associated with significant morbidity and mortality due to the lack of specificity of the alloreactive response. A promising area of investigation is the development of cancer vaccines that educate host immunity to more selectively target leukemia cells, including the stem cell compartment. Our group has developed a cancer vaccine model in which dendritic cells (DCs) are fused to autologous tumor cells, resulting in the presentation of multiple tumor antigens with the capacity to elicit a broad anti-tumor response. A fundamental challenge to developing a more effective tumor vaccine is overcoming the immunosuppressive milieu by which tumor cells evade host immunity. Key elements contributing to tumor-mediated immune suppression are the increased presence of regulatory T cells in patients with malignancy, and upregulation of the PD-1/PDL1 pathway. Tumor expression of PD-L1 promotes T cell tolerance by binding PD-1 on activated T cells and suppressing their capacity to secrete stimulatory cytokines. In addition, the PD-1/PDL-1 pathway has been shown to inhibit T cell-mediated lysis of tumor cells, potentially preventing a clinically meaningful immunologic response to vaccination. We are conducting a clinical trial in which AML patients who are in a first or second complete remission following chemotherapy receive three monthly doses of DC/AML fusion cells alone (Cohort 1) or in conjunction with anti-PD1 antibody, CT-011 (cohort 2). To date, 16 patients (9 males, 7 females; mean age 55 years) have been enrolled to the first cohort. All patients underwent successful tumor collection from either a bone marrow aspirate (N=12), collection of 20 cc of peripheral blood (N=3), or leukapheresis product (N=1) at the time of presentation with newly diagnosed AML (N=15) or first relapsed AML (N=1). The mean yield was 1.45×108 cells, and the mean viability was 90%. Tumor cells were subjected to immunohistochemical analysis to identify antigens unique to the leukemia fusion partner. Those patients achieving complete remission following 1–2 cycles of induction chemotherapy underwent leukapheresis for dendritic cell generation. Adherent peripheral blood mononuclear cells were isolated, cultured in the presence of GM-CSF and IL-4 for 5–7 days, and then exposed to TNFα for 48–72 hours to generate mature DCs. Mean viability of the DC preparation was 92%. DCs strongly expressed the co-stimulatory molecule CD86 (mean 75% expression). One patient died during remission induction chemotherapy and 3 patients were removed from study after induction chemotherapy to undergo allogeneic transplantation. Vaccine was successfully generated in 9 patients at a dose of 5×106 fusions cells, mean fusion efficiency of 30%, and viability of 87%. As a measure of their activity as antigen presenting cells, the capacity of the fusion cell preparation to stimulate allogeneic T cell proliferation ex vivo was quantified. In contrast to the leukemia preparation (mean stimulation index (SI) 3.7), the DC and fusion cell preparation were potent stimulators (mean SI 20.8 and 13.1, respectively). Vaccination with the DC/leukemia fusion vaccine was initiated within 12 weeks from count recovery following their final cycle of chemotherapy. 4 patients have completed vaccinations and are 2, 4, 5 and 6 months following the final vaccine. One patient was taken off study for disease progression one week after receiving his first vaccine. 4 patients experienced grade 1 vaccine site reactions. Biopsy of a vaccine site reaction demonstrated a dense T cell infiltrate. Additional vaccine related adverse events have included grade 1 ankle pain and edema. The remaining patients are undergoing chemotherapy, and when complete, will initiate vaccination. Peripheral blood samples are being collected prior to each vaccination and at 1, 3, and 6 months following completion of vaccination. Immune response targeting leukemia cells, leukemic stem cells, and leukemia associated antigens will be assessed. Levels of circulating regulatory T cells and T cell expression of PD1 will be measured. Time to disease progression will also be determined. Disclosures: Avigan: Curetech: Research Funding.

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Clinical Trial Evaluating DC/AML Fusion Cell Vaccination In AML Patients

Blood ◽

10.1182/blood.v122.21.3928.3928 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3928-3928 ◽

Cited By ~ 6

Author(s):

Jacalyn Rosenblatt ◽

Richard M. Stone ◽

Lynne Uhl ◽

Donna S. Neuberg ◽

Baldev Vasir ◽

...

Keyword(s):

Clinical Trial ◽

Bone Marrow ◽

T Cells ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Ex Vivo ◽

Fold Increase ◽

Vaccine Site ◽

Fusion Cell ◽

The Mean

Abstract We have developed a promising leukemia vaccine in which patient derived AML cells are fused with autologous dendritic cells (DCs), presenting a broad array of antigens. We are conducting a clinical trial in which AML patients who are not candidates for allogeneic transplantation undergo vaccination with DC/AML fusion cells following chemotherapy induced remission. Twenty-six patients (14 males, 12 females) underwent collection of AML cells at disease presentation for vaccine generation and immune monitoring studies. Median age of the patients is 66 years. Tumor was collected from either a bone marrow aspirate (N=16), 20 cc of peripheral blood (N=7), or leukapheresis product (N=3) at the time of presentation with newly diagnosed AML (N=25) or first relapsed AML (N=1). The mean yield of AML cells was 109 x 106 cells with a mean viability of 91%. Eligible patients achieving CR following chemotherapy (N=16) underwent leukapheresis for DC generation and vaccine preparation. Adherent peripheral blood mononuclear cells were isolated, cultured in the presence of GM-CSF and IL-4 for 5-7 days, and exposed to TNFα for 48-72 hours to generate mature DCs. The mean yield of DCs was 177 x106 cells with a mean viability of 89%. Fusion cells were generated by co-culture of DCs with AML cells in the presence of 50% polyethylene glycol and identified as cells co-expressing antigens that were unique to the DC and tumor population. Mean fusion efficiency and viability was 38% and 85%, respectively. As a measure of their activity as antigen presenting cells, the capacity of fusion cells to stimulate allogeneic T cell proliferation ex vivo was quantified. In contrast to the leukemia preparation (mean stimulation index (SI) 3.81), the DC and fusion cell preparation were potent stimulators (mean SI 19.61 and 13.48, respectively). Vaccination with DC/leukemia fusion cells was initiated within 12 weeks from count recovery following the final cycle of chemotherapy. 13 patients received at least two monthly vaccinations at a dose of 5x106 fusion cells. 8 patients had intermediate risk cytogenetics, 3 patients had good risk cytogenetics, and 2 patients had a complex karyotype. Vaccination was well tolerated, and importantly, was not associated with clinically significant auto-immunity. Possibly related adverse events were transient and of grade 1-2 intensity, including vaccine site reactions, pruritis, arthalgias, myalgias, eosinophilia, leukopenia, thrombocytopenia. Biopsy of vaccine site reactions demonstrated a dense infiltrate of CD4 and CD8 T cells consistent with recruitment of reactive T cell populations to the vaccine bed. To date, 9 patients remain in remission (69%), with a mean follow up of 23 months. Peripheral blood samples were collected prior to each vaccination and at 1, 3, and 6 months following completion of vaccination. Vaccination resulted in the potent induction of leukemia specific immunity as measured by an increase in CD8 T cells expressing IFNγ in response to ex vivo exposure to autologous leukemia cell lysates (mean fold increase 8, n=6). Bone marrow derived T cells were isolated prior to and following vaccination in patients who are HLA2.1+. Vaccination resulted in the expansion of bone marrow infiltrating T cells recognizing MUC1 (9 fold increase), WT1 (5 fold increase), PRAME (12 fold increase) tumor antigens by tetramer analysis (n=2). In conclusion, DC/AML fusion cell vaccination results in the potent expansion of leukemia reactive T cells and durable remissions following chemotherapy. Enrollment to a second cohort is being initiated, in which patients with be treated with DC/AML fusion cell vaccination in conjunction with PD1 blockade. Disclosures: No relevant conflicts of interest to declare.

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TIGIT Induces (CD3+) T Cell Dysfunction by Inhibiting Glucose Metabolism and its Reversal by TIGIT and/or PD-1 Blockade in Colorectal Cancer

10.21203/rs.3.rs-641407/v1 ◽

2021 ◽

Author(s):

qi shao ◽

Lei Wang ◽

maoling yuan ◽

Xiaohong Jin ◽

changping wu

Keyword(s):

Colorectal Cancer ◽

T Cells ◽

T Cell ◽

Glucose Metabolism ◽

Tumor Growth ◽

Tumor Cells ◽

Peripheral Blood ◽

Cancer Tissue ◽

Cell Dysfunction ◽

T Cell Dysfunction

Abstract Background: T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is an immunosuppressive receptor expressed on the surface of immune cells, suppressing immune responses by activating the intracellular negative regulatory signals. TIGIT plays an important role in the pathogenesis of various tumors, but its immune escape in colorectal cancer remains unclear.Methods: In this study, TIGIT expression in the peripheral blood and tissue microarrays was detected flow cytometry and immunofluorescence and its relationship with prognosis was evaluated. The proliferation and cytokines of TIGIT+ T cells were measured. Glucose metabolism and key enzymes were detected by qPCR or western blot. After establishing the co-cultured system and xenotransplant models, TIGIT antibody alone or combined with PD-1 antibody was blocked to observe the tumor growth.Results: We found that the proportion of CD3+TIGIT+ T cells was increased in peripheral blood and cancer tissue in colorectal cancer patients when compared with the healthy donors. These cells exhibited functional defects, low proliferative activity, impaired cytokine production and reduced glucose metabolism. A strong association was also observed between the elevated TIGIT expression and poor prognosis. In the in vitro co-culture assays of T cells and tumor cells, the suppressed glucose metabolic activity of T cells was reversed by TIGIT blockade. In addition, this blockade induced the apoptosis and reduced G2/M transit in tumor cells. The antitumor efficacy of TIGIT Ab therapy was further demonstrated in a human colorectal xenograft mice model while co-blockers of TIGIT and PD-1 exhibited synergistic suppressing effects on tumor growth.Conclusions: It is suggest that while TIGIT induces CD3+ T cell dysfunction in colorectal cancer, co-targeting TIGIT and PD-1 can lead to an effective antitumor response and may serve as a novel therapeutic strategy for colorectal patients.

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T-cell-subset characterization of human T-CLL

Blood ◽

10.1182/blood.v53.6.1066.1066 ◽

1979 ◽

Vol 53 (6) ◽

pp. 1066-1075 ◽

Cited By ~ 61

Author(s):

EL Reinherz ◽

LM Nadler ◽

DS Rosenthal ◽

WC Moloney ◽

SF Schlossman

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Peripheral Blood ◽

Lymphoblastic Leukemia ◽

Cell Subset ◽

T Cell Subsets ◽

Terminal Deoxynucleotidyl Transferase ◽

Human T Cell ◽

Malignant T Cells

Abstract Circulating peripheral blood tumor cells in four cases of chronic lymphoproliferative disease were immunologically characterized. By the use of T-cell-specific heteroantisera and indirect immunofluorescence, all were shown to involve proliferation of malignant T cells. Three cases demonstrated morphologic and clinical features consistent with chronic lymphocytic leukemia (CLL), and one case presented as a lymphosarcoma cell leukemia. Antisera specific for normal human T-cell subsets defined the malignant T cells in each case as arising from the TH2--subset. This subset normally constitutes approximately 80% of human peripheral blood T cells. Terminal deoxynucleotidyl transferase (TdT) was not detected in any of the T-cell CLL cases, thus supporting the notion that T-cell CLL represents a malignancy of a mature phenotype. The one patient with lymphosarcoma whose tumor cells were TdT-positive subsequently developed T-cell acute lymphoblastic leukemia (ALL). Moreover, la-like antigen (p23,30) was detected on two of these tumor cell populations. In addition, it was shown that not all tumor cells were E-rosette-positive, since only cells from 3 of 4 patients were capable of forming spontaneous rosettes. These findings demonstrate that heteroantisera can provide an additional important tool for dissecting the heterogeneity of T-cell leukemias and for relating them to more differentiated normal T cells.

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DAP10 Costimulates Chimeric Receptor-Mediated T Cell Responses to Tumor Antigen.

Blood ◽

10.1182/blood.v106.11.3052.3052 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3052-3052

Author(s):

Bianca Altvater ◽

Sibylle Pscherer ◽

Heribert Juergens ◽

Claudia Rossig

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

T Cell Activation ◽

Adoptive Immunotherapy ◽

Peripheral Blood ◽

Cell Activation ◽

Human Peripheral Blood ◽

Cytokine Secretion ◽

Immunotherapy Of Cancer

Abstract Chimeric receptors (chRecs) combining extracellular recognition domains with the T cell receptor ζ an redirect the cellular immune response of primary T-cells to tumor cells. T cell activation by chRec induces efficient cytokine release and cytotoxicity, however, it fails to mediate proliferative responses, limiting the usefulness of chRec-gene-modified T cells for adoptive immunotherapy of cancer. Inclusion of a CD28 costimulatory signaling component in the chRec endodomain enhances antigen-specific proliferation. Whereas the signal mediated by ligation of CD28 is of crucial importance for the activation of resting CD4+ T cells, further molecules with costimulatory functions have contributory roles. NKG2D is a stimulatory receptor that was first identified in NK cells, but is also expressed in cytotoxic T cells and positively modulates CD8+ T cell immune responses. We hypothesized that inclusion of the NKG2D-associated signaling domain DAP10 would enhance the capacity of chRecs to induce tumor-specific activation and proliferation of in vitro expanded effector T cells. Based on a GD2-specific scFv, we generated chRecs containing either the DAP10 signaling chain alone (14.G2a-DAP10) or combined with TCRζ 14.G2a-DAP10ζ), and expressed them in nonspecifically activated human peripheral blood T cells of three individual donors by retroviral gene transfer. As controls, T cells were transduced with 14.G2a-ζ and -CD28ζ chRec. High chRec surface expression was obtained with all four constructs (55±11%, ζ; 85±3, CD28ζ; 68±5%, DAP10; 78±1%; DAP10ζ). Immunophenotypes were dominated by a CD3+CD8+ population in all cell cultures. Whereas DAP10 alone failed to mediate specific tumor cell lysis, 51Cr release assays revealed efficient and comparable lysis of GD2+ tumor targets by T cells transduced with all ζ-containing constructs, with 49±8% (ζ), 52±7% (CD28ζ), and 52±18% (DAP10ζ) cytolysis at an effector-to-target ratio of 40:1. Intracellular cytokine secretion by chRec+ T cells was induced in response to tumor targets by 14.G2a-ζ (up to 37% IFN-γ secreting cells), CD28ζ, and DAPζ (both up to 22%), but not by DAP10 alone (0,2%). Weekly stimulation with tumor cells for 6 weeks induced only limited expansion of T cells transduced with 14.G2a-ζ (7–45fold) or with 14.G2a-DAP10 (14–26-fold). Adding CD28 or DAP10 domains significantly enhanced expansion by a comparable degree (270–483-fold and 126–436-fold, respectively). Thus, while neither CD28 nor DAP10 enhances antigen-specific cytokine secretion and cytolysis, DAP10 signaling can completely replace CD28 signaling in costimulating antigen-specific proliferation of peripheral blood T cells. DAP10-containing chRec may be a powerful new tool for adoptive immunotherapy of cancer.

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T-cell-subset characterization of human T-CLL

Blood ◽

10.1182/blood.v53.6.1066.bloodjournal5361066 ◽

1979 ◽

Vol 53 (6) ◽

pp. 1066-1075

Author(s):

EL Reinherz ◽

LM Nadler ◽

DS Rosenthal ◽

WC Moloney ◽

SF Schlossman

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Peripheral Blood ◽

Lymphoblastic Leukemia ◽

Cell Subset ◽

T Cell Subsets ◽

Terminal Deoxynucleotidyl Transferase ◽

Human T Cell ◽

Malignant T Cells

Circulating peripheral blood tumor cells in four cases of chronic lymphoproliferative disease were immunologically characterized. By the use of T-cell-specific heteroantisera and indirect immunofluorescence, all were shown to involve proliferation of malignant T cells. Three cases demonstrated morphologic and clinical features consistent with chronic lymphocytic leukemia (CLL), and one case presented as a lymphosarcoma cell leukemia. Antisera specific for normal human T-cell subsets defined the malignant T cells in each case as arising from the TH2--subset. This subset normally constitutes approximately 80% of human peripheral blood T cells. Terminal deoxynucleotidyl transferase (TdT) was not detected in any of the T-cell CLL cases, thus supporting the notion that T-cell CLL represents a malignancy of a mature phenotype. The one patient with lymphosarcoma whose tumor cells were TdT-positive subsequently developed T-cell acute lymphoblastic leukemia (ALL). Moreover, la-like antigen (p23,30) was detected on two of these tumor cell populations. In addition, it was shown that not all tumor cells were E-rosette-positive, since only cells from 3 of 4 patients were capable of forming spontaneous rosettes. These findings demonstrate that heteroantisera can provide an additional important tool for dissecting the heterogeneity of T-cell leukemias and for relating them to more differentiated normal T cells.

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Tumor-derived activated cells: preliminary laboratory and clinical results.

Clinical Chemistry ◽

10.1093/clinchem/35.8.1576 ◽

1989 ◽

Vol 35 (8) ◽

pp. 1576-1580 ◽

Cited By ~ 2

Author(s):

R K Oldham ◽

J R Maleckar ◽

C S Friddell ◽

W M Lewko ◽

W H West ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Peripheral Blood ◽

Tumor Antigens ◽

Interleukin 2 ◽

Limiting Factors ◽

Limiting Factor ◽

Clinical Effects ◽

Lymphokine Activated Killer Cells

Abstract It is well known that T lymphocytes can mediate significant anti-tumor responses. A limiting factor has always been the ability to expand T cells, whether from the peripheral blood, spleen, or tumor. The recent availability of recombinant interleukin-2 (r-IL2) has demonstrated the feasibility of expanding T cells and the clinical efficacy of these cells as anti-tumor effectors in murine models. Concomitantly, researchers discovered that lymphokine-activated killer cells--peripheral blood cells functionally distinct from T cells--could be cultured, expanded, and re-infused in patients, with significant clinical effects. For many years, the infiltrating lymphocytes have been recognized in tumor biopsies and known to be cytolytically active. Major limiting factors were the ability to culture large numbers of these infiltrating cells and the limited understanding of the tumor antigens involved for T-cell stimulation. Restimulation by antigen (tumor cells) appears to provide the ongoing antigen stimulation needed to maintain selective killing of tumor cells. By defining various factors in the medium that support and enhance T-cell growth and activation, the components are becoming available to develop a broad attack on advanced cancer by using this laboratory-based technology of stimulation and expansion of tumor-derived activated cells.

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Leukemia Derived Dendritic Cells (LDCs) Are Functionally Deficient and Inferior to DC/Leukemia Fusion Cells as a Tumor Vaccine for AML.

Blood ◽

10.1182/blood.v106.11.2788.2788 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2788-2788

Author(s):

Jacalyn Rosenblatt ◽

Richard Stone ◽

Corrine Lenahan ◽

Zekwui Wu ◽

Baldev Vasir ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Interferon Gamma ◽

Tumor Immunity ◽

Peripheral Blood ◽

Tumor Antigens ◽

Tumor Vaccine ◽

Costimulatory Molecules ◽

Leukemia Cells

Abstract We have previously demonstrated that dendritic cells (DCs) fused with malignant cells stimulate anti-tumor immunity by presenting a braod array of tumor antigens in the context of DC mediated costimulation. DCs differentiated from leukemia cells (LDCs) are also being explored as cancer vaccines in which leukemia associated antigens are presented. We examined the phenotypic and functional characteristics of DC/Leukemia fusions and LDCs to assess their potential as tumor vaccines. Leukemia blasts were isolated from peripheral blood of patients with AML. CD34 selection was performed on a subset of samples by magnetic bead separation. LDCs were generated by culturing blasts in the presence of GM-CSF, IL-4 and TNFα for 7 days. Alternatively, leukemia cells were fused with DC by coculture in the presence of polyethylene glycol. Differentiation of leukemic blasts into LDCs resulted in increased expression of HLA-DR and CD 11c. Unlike normal peripheral blood mononuclear cells, differentiation of leukemic blasts resulted in only modest expression of the costimulatory molecules, CD80 and CD86 (mean expression 12% and 30%) and no increase in expression of the maturation marker, CD83 (mean expression 4%). In addition, expression of the leukemia associated antigen c-kit (CD117) was lower on LDCs than on blasts (mean expression 34% on blasts, 15% on LDCs). To assess the capacity of the primitive leukemia clonal population to differentiate into DCs, CD34+ cells were isolated from the blast population and assessed after cytokine differentiation. Cytokine differentiation did not result in upregulation of CD80, CD83, or CD86 expression in the CD34+ population (mean expression 5%, 2%, 17%). In contrast, differentiation of the CD34- population resulted in moderate expression of CD80, CD83 and CD86 (mean expression 15%, 14%, 48%). In contrast to LDCs which do not strongly express co-stimulatory molecules and lose expression of leukemia associated antigens, fusion cells expressed both DC and tumor associated antigens (mean fusion efficiency 27%). The functional characteristics of DC derived from leukemic blasts were examined. Allogeneic T cell proliferation in response to stimulation by LDCs was not significantly higher than after stimulation with undifferentiated blasts (ratio 10:1, mean SI 17% with LDCs vs 9% with undifferentiated blasts, p=0.19). Neither stimulation with blasts nor with LDCs induced T cell production of interferon gamma. In contrast, interferon gamma production by T cells in response to stimulation with fusion cells was higher than after stimulation with undifferentiated blasts. In summary, LDCs do not demonstrate normal upregulation of costimulatory molecules, and lose expression of tumor antigens. In contrast, DC/leukemia fusions coexpress tumor and DC associated markers. While LDCs stimulate interferon gamma production by T cells poorly, fusion cells more potently stimulate interferon gamma production by allogeneic T cells than do undifferentiated blasts. This suggests that LDC may be ineffective as a tumor vaccine in AML, and that fusion cells may be superior to LDC in generating effective anti-tumor immune responses. Strategies to enhance the ability of both LDC and of fusion cells to stimulate anti-tumor immunity are being explored.

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Combining Early Heat Shock Protein Vaccination with Directed IL-2 Leads to Effective Anti-Tumor Immunity in Autologous Hematopoietic Cell Transplantation Recipients

Blood ◽

10.1182/blood.v118.21.998.998 ◽

2011 ◽

Vol 118 (21) ◽

pp. 998-998

Author(s):

Robert G. Newman ◽

Eckhard R. Podack ◽

Robert B. Levy

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Cd8 T Cells ◽

Hematopoietic Cell Transplantation ◽

Cd8 T Cell ◽

Tumor Bearing ◽

Donor T Cells ◽

Vaccine Site ◽

Autologous Hematopoietic Cell Transplantation

Abstract Abstract 998 Tumor relapse is still the major cause of morbidity and mortality in patients with hematologic cancers that undergo aggressive chemo-radiotherapy followed by autologous hematopoietic cell transplantation (auto-HCT). Hence, there is a critical need for new anti-tumor therapies. Heat shock protein (HSP) based vaccines elicit innate and adaptive immune responses in murine studies and have shown promise in clinical trials. The pre-clinical studies here investigated the efficacy of vaccination with tumor cells secreting the HSP fusion gp96-Ig together with directed IL-2 in tumor bearing auto-HCT recipients. To mimic clinical T cell replete auto-HCT, transplanted donor T cells were obtained from congenic tumor bearing mice (C57BL/6 CD45.2+ CD90.1+) that had been previously inoculated intraperitoneally (ip) with 4×106 OVA expressing lymphoma cells (E.G7). Some of these donor mice received 0.5×106 CD8 T cells specific for OVA257–264 (OT-I) to allow for tumor antigen specific T cell monitoring. Three weeks later, T cells were harvested from these animals bearing progressively growing tumor for use in T cell replete auto-HCT. Recipient mice (C57BL/6 CD45.2+ CD90.2+) received 9.5 Gy TBI with subsequent infusion of 5×106 congenic T cell depleted bone marrow cells (C57BL/6 CD45.1+ CD90.2+) supplemented with 2×106 enriched T cells from the tumor bearing donors. The following day, recipients were inoculated ip with 1×105 viable E.G7 lymphoma cells. Based on our prior findings, a multiple vaccination protocol was employed utilizing 1×107 irradiated E.G7 cells transfected to secrete the HSP fusion gp96-Ig (E.G7-gp96-Ig). Some recipients were administered IL-2 via specific antibody-cytokine complexes comprised of IL-2 and αIL-2 mAb clone S4B6 (IL-2/αIL-2CD122). This specific IL-2 complex has been shown to interact with cells expressing the β chain (CD122) of the IL-2 receptor, such as memory CD8 T cells and NK cells, but not with cells expressing the α chain (CD25). Compared to recipients of T cell replete auto-HCT vaccinated with parental E.G7 tumor cells who exhibited virtually no increase in antigen-specific CD8 T cells, marked expansion was detected in the blood after 2 vaccinations with E.G7-gp96-Ig, i.e. within 1 week of auto-HCT. This response reached a plateau after 3 vaccinations, and persisted throughout the 5 vaccine protocol. To quantitate this vaccine induced CD8 T cell expansion, analysis of the vaccine site, splenic and lymph node compartments was performed following 3 vaccinations, i.e. 2 weeks post-HCT. In contrast to the modest 25× increase observed after vaccination with parental E.G7 cells, a 175× expansion was detected following E.G7-gp96-Ig vaccination (6.8×106 vs. 3.8×104 input). Moreover, 75% of these gp96-Ig expanded CD8 T cells at the vaccine site were bifunctional, expressing IFN-γ and TNF-α following antigen specific stimulation ex vivo. Strikingly, combined treatment with vaccine cells secreting gp96-Ig together with IL-2/αIL-2CD122 complex resulted in a 1000× enhancement of antigen specific CD8 T cell numbers in all compartments analyzed. Tumor bearing auto-HCT recipients exhibited a median survival time (MST) of 1 month if not vaccinated or if vaccinated with parental E.G7 cells (Figure). However, vaccination with E.G7-gp96-Ig extended the MST by more than 2 weeks and ∼20% of recipients survived long term (>100 days). This effect was dependent on T cells since gp96-Ig vaccination alone without donor T cells resulted in no MST extension. Combination therapy with tumor cells secreting gp96-Ig and IL-2/αIL-2CD122 complex markedly elevated total CD8 T cells as well as NK cells at the vaccine site and in secondary lymphoid tissues, two populations that have been shown to facilitate HSP based vaccines. Notably, this strategy resulted in a MST >100 days with ∼60% of mice surviving indefinitely. We propose that 3 components are required together with auto-HCT to avoid relapse related mortality: (1) transplanted autologous T cells, (2) a pan-antigen vaccination approach that induces potent antigen presentation and activation of multiple antigen specific T cells, i.e. tumor cells secreting gp96-Ig, and (3) an adjuvant that potentiates this vaccine induced response, i.e. IL-2 delivered in the form of an antibody-cytokine complex. In total, this combinatorial protocol represents a promising regimen that could be translated into the clinic for patients with hematologic cancers. Disclosures: Podack: Heat Biologics, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

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Blocking of EphA2 on Endometrial Tumor Cells Reduces Susceptibility to Vδ1 Gamma-Delta T-Cell-Mediated Killing

Frontiers in Immunology ◽

10.3389/fimmu.2021.752646 ◽

2021 ◽

Vol 12 ◽

Author(s):

Robert Hudecek ◽

Barbora Kohlova ◽

Ingrid Siskova ◽

Martin Piskacek ◽

Andrea Knight

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Cell Lines ◽

Peripheral Blood ◽

Peritoneal Fluid ◽

Γδ T Cells ◽

Gamma Delta ◽

Healthy Donors ◽

Endometrial Tumor

BackgroundEndometriosis is a common gynecological disease characterized by the presence of endometrial tissue outside the uterus causing chronic inflammation, severe pain, and infertility. However, the innate immunity of gamma-delta (γδ) T lymphocytes in endometriosis has not been characterized. Women with endometriosis present numerous endocrine and immune dysfunctions and elevated risk for endometrial, ovarian, and breast cancers. The tyrosine kinase EphA2 is often overexpressed in cancer including endometrial carcinoma.MethodsWe analyzed Vδ1 and Vδ2 γδ T cells in peripheral blood and paired peritoneal fluid samples in endometriosis patients (n = 19) and compared the counts with that of age- and sex-matched healthy donors (n = 33) using flow cytometry. Vδ1 and Vδ2 T cells isolated from healthy donors were used against KLE, RL-95, and Ishikawa endometrial tumor cells in 4 h flow cytometric cytotoxicity assays. The EphA2 blocking studies were performed using antibody, small-molecule inhibitor ALW-II-41-27, and the CRISPR/Cas9.ResultsWe determined Vδ1 T cells substantially reduced in patients’ peripheral blood (p < 0.01) and peritoneal fluid (p < 0.001). No differences were found for circulating Vδ2 T cells compared with peritoneal fluid samples. We observed inherent cytotoxic reactivity of Vδ1 and Vδ2 γδ T lymphocytes against endometrial tumor cells. Importantly, we found reduced specific lysis of EphA2-positive cell lines KLE and RL-95 by Vδ1 T cells in the EphA2 antibody blocking studies and by the EphA2 inhibitor. Furthermore, Vδ1 T-cell-mediated killing was significantly decreased in RL-95 cell EPHA2 knockout. Finally, potent cytolytic activity exerted by Vδ1 T cells was significantly reduced in EPHA2 knockouts in renal A-498 and colon HT-29 carcinoma cell lines.ConclusionsWe determined variable levels of Vδ1 and Vδ2 γδ T cells in endometriosis patients. We observed inherent cytotoxic reactivity of γδ T-cell subsets against endometrial cell lines. Specifically, we found that blocking of EphA2 expression resulted in significant inhibition of endometrial tumor killing mediated by Vδ1 γδ T cells. These results suggest that EphA2 is involved in tumor cell lysis and contributes to susceptibility to Vδ1 γδ T cells cytotoxic reactivity.

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Intensive Immunosuppression Plus T-Cell Depleted Autologous Peripheral Blood Stem Cell Transplantation for Systemic Sclerosis.

Blood ◽

10.1182/blood.v104.11.5205.5205 ◽

2004 ◽

Vol 104 (11) ◽

pp. 5205-5205

Author(s):

Diane BuchBarker ◽

Albert D. Donnenberg ◽

Thomas A. Medsger ◽

Michael P. Carroll ◽

Deborah L. Griffin ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

Systemic Sclerosis ◽

T Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Peripheral Blood ◽

Peripheral Blood Stem Cell ◽

The Mean ◽

Blood Stem Cell Transplantation

Abstract Autologous peripheral blood stem cell transplantation (PBSCT) may be therapeutic in patients (pts) with severe autoimmune disorders. A pre-PBSCT regimen that optimizes immunosuppression without excessive myelotoxicity would potentially have favorable safety and efficacy profiles in these pts. To test this hypothesis we are conducting a phase I trial of cyclophosphamide (CY) dose escalation (initial CY dose 200 mg/m2/day x 5, days −7 to −3) plus fludarabine (25 mg/m2/day x 5, days −7 to −3) and rabbit anti-thymocyte globulin (ATG; 2.5 mg/kg/day x 3, days −5 to −3) followed by T-cell depleted (TCD) autologous PBSCT in adults with systemic sclerosis (SSc). To date, 5 pts (2 male, 3 female; median age, 44 yr; range, 39–59 yr) have undergone TCD PBSCT. Each pt had satisfactory PBSC collection with one 4-hr large-volume leukapheresis (LVL) after mobilization with CY (2.0 g/m2) plus G-CSF. Mean yields of CD34+ and CD3+ cells by LVL were 18.8 (range, 10.5–33.0) x 106/kg and 1.23 (range, 0.37–2.45) x 108/kg, respectively. To deplete T cells in the PBSC product we used a combination of CD34+ selection (Isolex 300i v 2.5; Baxter Oncology) and ex vivo incubation with anti-CD3 antibody (OKT3; Ortho Biotech), which resulted in a mean 5.2-log reduction of T cells (range, 4.7–6.3 log). The final PBSC products contained a mean of 9.94 (range, 4.8–13.0) x 106 CD34+ cells/kg and 1.34 (range, 0.06–4.54) x 103 CD3+ cells/kg. The daily dose of CY was 200 mg/m2 in the first 3 pts and 400 mg/m2 in the next 2 pts. Using rare-event flow cytometry, we found that the mean half-life (t 1/2) of CD3+ cells in these pts was biphasic; t 1/2 was 0.8 days from day −7 to −5 and 0.2 days from −5 to −3 days, correlating with ATG administration. In contrast, the mean t1/2 of CD3+ cells was 2.5 days in pts with hematologic malignancies receiving a myeloablative preparative regimen of CY, busulfan and etoposide. After PBSCT, the median nadir of WBC, neutrophil and platelet levels were 1.2 (range, 0.4–2.0), 0.9 (range, 0.1–1.7) and 135 (range, 114–185) x 109/L, respectively. One pt at the second CY dose level developed pericardial effusion and tamponade at day +5 and required surgical intervention. No pts developed opportunistic infections. All pts are alive at a median of 6.5+ months (range, 4.9+–15.5+ months) after PBSCT. Compared with pre-PBSCT levels, Rodnan total skin score (TSS) decreased by 10, 11 and 15 points, respectively, in 3 pts and increased by 5 and 8 points in 2 pts. One pt with worsening TSS at 12 months after PBSCT developed SSc renal crisis and requires hemodialysis. Of 3 pts with improved TSS, 1 has worsening polymyositis and 1 has recurrence of palpable tendon friction rubs. Even at the lowest CY dose, this immunosuppressive regimen provides significantly greater and more rapid T-cell kill than a conventional myeloablative regimen. The efficacy of this regimen and TCD-PBSCT in SSc is encouraging but requires longer followup and experience with a larger number of pts.

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