Mean Specific Lysis Research Articles

SAT297 Major Immunophenotypic Abnormalities In Patients With Primary Adrenal Insufficiency Of Different Etiology

Abstract Disclosure: H.F. Nowotny: None. T. Marchant Seiter: None. J. Ju: None. A. Gottschlich: None. H. Schneider: None. S. Zopp: None. F. Vogel: None. L. Tschaidse: None. M.K. Auer: None. C. Lottspeich: None. S. Kobold: None. S. Rothenfusser: None. F. Beuschlein: None. M. Reincke: None. L. Braun: None. N. Reisch: None. Background: Increased risk of infection, adrenal crises and a higher mortality rate have been reported for patients with primary adrenal insufficiency (PAI). Such dismal outcomes have been inferred to immune cell dysregulation as a consequence of unphysiological cortisol replacement. As the immune landscape of patients with different types of PAI has not been systematically explored, we set out to immunophenotype PAI patients with different causes of glucocorticoid deficiency. Methods: The present cross-sectional single center study includes 28 patients with congenital adrenal hyperplasia (CAH), 27 patients after bilateral adrenalectomy due to Cushing’s syndrome (BADx), 21 patients with Addison’s disease (AD) and 52 healthy controls. All patients with PAI were on a stable glucocorticoid replacement regimen using a median dose of 25 mg hydrocortisone (IQR 5 mg for BADx and AD and IQR 10 mg for CAH) per day. Heparinized blood samples were processed to isolate peripheral blood mononuclear cells. All samples were processed for analysis of immune cell subsets using multicolor flow cytometry after four-hour stimulation with phorbol myristate acetate (PMA)/Ionomycin, as well as analysis of natural killer (NK-) cell cytotoxicity and clock gene expression. Results: Analysis of immune cell profiles revealed reduced percentages of IFNγ-secreting T helper (Th1) in AD (p = 0.0024) and cytotoxic (Tc1) cells in CAH (p = 0.0055) and AD patients (p = 0.0075) compared to controls. Moreover, we observed a downregulation of the percentage of IL-4+ Th2 cells in AD patients (p = 0.0157) and Tc2 cells in AD (p = 0.0154) and CAH patients (p = 0.0012) compared to controls. Th17 and Tc17 cells were also downregulated in patients with AD compared to the other patient groups and controls (p < 0.0001). NK-cell cytotoxicity (NKCC) using bioluminescence measurements was reduced in all subsets of PAI patients compared to controls with the lowest change in patients with CAH (mean specific lysis 57.5% in controls, 21.7% in CAH, -0.5% in AD and -28.7% in BADx). The percentage of activated NK-cells was upregulated in BADx (p = 0.0008) and AD patients (p = 0.0348) compared to controls, also expressed as an increase in CD107a expression as a marker of NK-cell degranulation (BADx p < 0.0001; AD p = 0.0002). While the percentage of NK-cell activating receptor expressing NK-cells did not differ from controls, expression percentage of NK-cell inhibiting receptor CD94 was upregulated in both BADx and AD patients (p < 0.0001). Conclusion: In patients with three different etiologies of PAI, distinct differences in T- and NK-cell-phenotypes became apparent despite the use of same GC preparation and dose. Our results highlight so far unsuspected differences in immune cell composition and NK-cell function in PAI patients of different causes and suggest disease-specific alterations that might necessitate disease-specific treatment. Presentation: Saturday, June 17, 2023

Targeting GD2 after allogeneic SCT: effector cell composition defines the optimal use of ch14.18 and the bispecific antibody construct NG-CU (GD2-CD3).

We investigated whether T cell-recruiting bispecific anti-CD3/GD2 antibody NG-CU might be an alternative to therapeutic anti-GD2 monoclonal antibody (mAb) ch14.18, mediating complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) through natural killer (NK) cells for immunotherapy in high-risk/relapsed neuroblastoma after autologous/allogeneic stem cell transplantation (auto/alloSCT). Different antibody concentrations and effector-to-target ratios (E:T) were evaluated using xCELLigence RTCA system, peripheral blood mononuclear cells (PBMCs) (healthy donors and patients after alloSCT), and neuroblastoma cell lines (LS/LAN-1). Mean specific lysis of LS cells utilizing PBMCs from healthy donors and ch14.18 (1µg/ml) was 40/66/75% after 12/24/48 h compared to 66/93/100% in the presence of NG-CU (100 ng/ml). NG-CU showed enhanced cytotoxicity compared to ch14.18, even at lower concentrations and E:T ratios, and completely eradicated LS cells after 72 h. To decipher the influence of effector cell subsets on lysis, different ratios of T and NK cells were tested. At a ratio of 1:1, ch14.18 was more effective than NG-CU. Using patient PBMCs taken at different time points posttransplant, significant lysis with both constructs was detectable depending on percentages and total numbers of T and NK cells; in the early posttransplant phase, NK cells were predominant and ch14.18 was superior, whereas later on, T cells represented the majority of immune cells and NG-CU was more effective. Our study highlights the importance of analyzing effector cell subsets in patients before initiating antibody-based therapy. Consequently, we propose an adjusted administration of both antibody constructs, considering the state of posttransplant immune recovery, to optimize anti-tumor activity.

T-cell exhaustion induced by continuous bispecific molecule exposure is ameliorated by treatment-free intervals

AbstractT-cell–recruiting bispecific molecule therapy has yielded promising results in patients with hematologic malignancies; however, resistance and subsequent relapse remains a major challenge. T-cell exhaustion induced by persistent antigen stimulation or tonic receptor signaling has been reported to compromise outcomes of T-cell–based immunotherapies. The impact of continuous exposure to bispecifics on T-cell function, however, remains poorly understood. In relapsed/refractory B-cell precursor acute lymphoblastic leukemia patients, 28-day continuous infusion with the CD19xCD3 bispecific molecule blinatumomab led to declining T-cell function. In an in vitro model system, mimicking 28-day continuous infusion with the half-life–extended CD19xCD3 bispecific AMG 562, we identified hallmark features of exhaustion arising over time. Continuous AMG 562 exposure induced progressive loss of T-cell function (day 7 vs day 28 mean specific lysis: 88.4% vs 8.6%; n = 6; P = .0003). Treatment-free intervals (TFIs), achieved by AMG 562 withdrawal, were identified as a powerful strategy for counteracting exhaustion. TFIs induced strong functional reinvigoration of T cells (continuous vs TFI-specific lysis on day 14: 34.9% vs 93.4%; n = 6; P < .0001) and transcriptional reprogramming. Furthermore, use of a TFI led to improved T-cell expansion and tumor control in vivo. Our data demonstrate the relevance of T-cell exhaustion in bispecific antibody therapy and highlight that T cells can be functionally and transcriptionally rejuvenated with TFIs. In view of the growing number of bispecific molecules being evaluated in clinical trials, our findings emphasize the need to consider and evaluate TFIs in application schedules to improve clinical outcomes.

Augmenting Efficacy of T-Cell Bispecific Antibodies in AML through a Tumor Stroma-Targeted 4-1BB Agonist

Bispecific antibodies represent a promising treatment option for acute myeloid leukemia (AML). We have recently described a novel T-cell bispecific antibody (TCB) targeting the intracellular tumor antigen Wilms tumor 1 (WT1) in the context of HLA-A*02 (Augsberger et al. Blood 2021). Based on these findings a multicenter first-in-human clinical trial was initiated in relapse/refractory AML (NCT04580121). Possible immune escape mechanisms against T-cell based immunotherapy are provided by the tumor microenvironment (TME) of the bone marrow by co-inhibition of T cells or stromal cells shielding leukemic cells from immune effector cells. To overcome the immunosuppressive effect of the TME and to enhance T-cell responses, we evaluated the combination of the WT1-TCB with an antibody fusion protein that targets a stromal antigen (Fibroblast-activation protein; FAP) and provides a positive costimulatory signal (4-1BBL) to T cells. FAP is upregulated on cancer-associated fibroblasts after remodulation of the bone marrow niche by leukemic cells, and the FAP specificity of the molecule therefore provides T-cell co-stimulation tightly restricted to the tumor niche.Efficacy of the combination (WT1-TCB + FAP-4-1BBL antibody fusion protein) was evaluated in co-culture assays over 4 days with primary HLA-A*02 + AML cells, healthy donor (HD) T cells and three NIH-3T3 fibroblast cell lines. NIH-3T3 cell lines were genetically modified to express low and high levels of FAP, respectively. Wild-type NIH-3T3 cells were included as control. Additionally, a control (Ctrl)-TCB and a Ctrl-4-1BBL antibody fusion protein recognizing a non-tumor target derived from the human germline repertoire were included. Enhancement of T-cell mediated cytotoxicity by the FAP-4-1BBL antibody fusion protein was evaluated by (1) specific lysis of primary AML cells, (2) upregulation of the T-cell activation markers CD25 and 4-1BB, (3) T-cell expansion calculated as fold change compared to day 0, and (4) Granzyme B-expression which was evaluated by intracellular staining.After 4 days of co-culture, with an E:T ratio of 1:2, we observed a mean specific lysis of 55.1±8.2% (±SEM; n=4) of primary AML cells mediated by HD T cells and WT1-TCB. Notably, this was reduced to 19.4±5.9% (±SEM; n=4) in the presence of NIH-3T3 cells. However, AML cell lysis was restored by the addition of the FAP-4-1BBL antibody fusion protein in the presence of high FAP expressing NIH-3T3 cells (mean specific lysis: 62.8±7.3%; ±SEM; n=4). Concomitantly, the FAP-4-1BBL antibody fusion protein led to increased expression of the activation molecules CD25 (MFI ratio: 22.1±5.3 vs. 10.4±1.3; ±SEM; n=4) and 4-1BB (MFI ratio: 10.4±6.0 vs. 2.1±0.3; ±SEM; n=4) on CD3 + T cells. Furthermore, lysis was accompanied by increased frequencies of granzyme B expressing T cells (45.0±2.5% vs. 16.1±5.3%; n=3). Importantly, the FAP-4-1BBL antibody fusion protein led to improved T-cell proliferation, especially of CD8 + T cells (fold change on day 4 vs day 0: 5.7±2.2 vs. 1.0±0.3; ±SEM; n=4). Overall similar observations were made in the presence of low FAP expressing NIH-3T3 cells.Taken together, we have established an in vitro model system mimicking the immunoprotective bone marrow TME using NIH-3T3 cells resulting in impaired AML cell lysis. Providing additional T-cell co-stimulation by a tumor-stroma targeted 4-1BB agonist, however, restored WT1-TCB-mediated cytotoxicity of primary AML cells in the presence of FAP expressing cell lines. Importantly, the combination overcame the immunosuppressive effect of the NIH-3T3 cells on T cells as further demonstrated by improved T-cell activation and expansion. The tumor-stroma targeted 4-1BB agonist therefore represents a promising combinatorial approach to enhance T-cell activity at the local tumor site and warrants further investigations in an in vivo model system. DisclosuresPulko: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Claus: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Buecklein: Pfizer: Consultancy, Honoraria, Speakers Bureau; Kite/Gilead: Consultancy, Honoraria, Other: Congress and travel support, Research Funding; Novartis: Consultancy, Other: congress and travel support, Research Funding, Speakers Bureau; Miltenyi: Research Funding; BMS/Celgene: Consultancy, Research Funding; Amgen: Consultancy, Honoraria. Umana: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Klein: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Subklewe: Novartis: Consultancy, Research Funding, Speakers Bureau; Klinikum der Universität München: Current Employment; Roche: Research Funding; Seattle Genetics: Consultancy, Research Funding; Pfizer: Consultancy, Speakers Bureau; Janssen: Consultancy; Takeda: Speakers Bureau; MorphoSys: Research Funding; Miltenyi: Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau.

Targeting Intracellular WT1 in AML Utilizing a T Cell Bispecific Antibody Construct: Augmenting Efficacy through Combination with Lenalidomide

Antibody-based immunotherapy represents a promising strategy to target chemo-resistant leukemic cells. However, current antibody-based approaches are restricted to cell lineage surface antigens. Targeting intracellular antigens enables to enlarge the number of suitable tumor-associated target antigens with a more restricted expression profile. In this study we evaluated a 2+1 T Cell Bispecific (TCB) antibody for immunotherapy of acute myeloid leukemia (AML). The T cell receptor (TCR)-like TCB targets the intracellular tumor antigen Wilms tumor 1 (WT1) by bivalent recognition of the peptide RMFPNAPYL in the context of human leukocyte antigen allele A*02 (HLA-A2). Complementary binding to CD3ε recruits T cells irrespective of their TCR-specificity. We further analyzed enhancement of TCB-mediated T cell cytotoxicity through combination with the immune-modulatory drug lenalidomide. WT1 expression levels in cancer cell lines and primary AML patient samples at different time points during course of the disease were determined by quantitative real-time PCR, western blot and immunohistochemical staining. WT1-TCB-mediated cytotoxicity was analyzed by co-cultivation of WT1-expressing HLA-A2+ cancer cell lines with T cells from healthy donors. Specific lysis was assessed by flow cytometry. TCR downstream signaling was measured by co-cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1-TCB-mediated cytotoxicity against primary AML cells and combination with 10 μM lenalidomide was evaluated in our pre-established feeder layer-based ex vivo long-term culture system. For in vivo testing, NSG mice (NOD.Cg-Prkdcscid-Il2rgtm1Wjl/SzJ) were humanized with human HLA-A2+ CD34+ cord blood cells. After successful engraftment and development of human T cells, WT1-expressing HLA-A2+ SKM-1 tumor cells were subcutaneously inoculated followed by weekly administration of the WT1-TCB. In accordance with previous reports, we observed WT1 expression in 79% (n=38) of cancer cell lines and in 92% (n=65) of AML patient samples at the time of initial diagnosis. Moreover, WT1 expression levels correlated with the percentage of AML blasts: no significant WT1 expression was observed at time of CR (n=26), whereas WT1 was expressed again at time of relapse (n=21). WT1-TCBs elicited antibody-mediated T cell cytotoxicity against peptide-pulsed T2 cells and AML cell lines in a WT1 and HLA-restricted manner. Equally, TCR downstream signaling was observed in a WT1-restrictive manner by co-cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1-TCBs further mediated specific lysis of primary AML cells upon addition of allogenic T cells from healthy donors (mean specific lysis: 67±6% after 13-14 days; ±SEM; n=18). Correspondingly, up-regulation of T cell activation and surrogate exhaustion markers was observed (MFI fold change CD69: 9.3±1.5, PD-1: 5.1±0.7, TIM-3: 4.7±0.6; ±SEM; n=22). WT1-TCBs also mediated killing of primary AML cells in an autologous setting (mean specific lysis: 38±13% after 13-14 days; ±SEM; n=5). In comparison with WT1RMF-specific T cells, only bivalent binding by WT1-TCB induced efficient lysis of primary AML cells. Interestingly, combination of WT1-TCB with lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean specific lysis on day 3-4: 32±10% vs 59±9%; p=0.0017; ±SEM; n=13). This was accompanied by an increased secretion of the proinflammatory cytokines IL-2, IFN-γ and TNF-α and promoted the differentiation of naïve T cells towards a memory phenotype characterized by a downregulation of CD45RA. Furthermore, WT1-TCB-treated humanized mice bearing SKM-1 tumors showed a dose dependent and significant reduction in tumor growth resulting in tumor control. TCR-like TCBs targeting intracellular tumor antigens are a promising tool for cancer immunotherapy. Notably, the 2+1 TCB molecular format for bivalent binding facilitates potent in vitro, ex vivo and in vivo killing of AML cell lines and primary AML samples which present low numbers of the RMF peptide-MHC complex on the cell surface validating WT1-TCB as a promising therapeutic agent for the treatment of AML. Our results further indicate that the combinatorial approach with lenalidomide leads to increased TCB-mediated T cell cytotoxicity. Disclosures Klein: Roche: Employment, Equity Ownership, Patents &amp; Royalties. Xu:Roche: Employment, Equity Ownership, Patents &amp; Royalties. Heitmüller:Roche: Employment. Hanisch:Roche: Employment, Equity Ownership, Patents &amp; Royalties. Sam:Roche: Employment, Equity Ownership, Patents &amp; Royalties. Pulko:Roche: Employment, Equity Ownership, Patents &amp; Royalties. Schönle:Roche: Employment, Equity Ownership, Patents &amp; Royalties. Challier:Roche: Employment, Equity Ownership, Patents &amp; Royalties. Carpy:Roche: Employment, Equity Ownership, Patents &amp; Royalties. Lichtenegger:Roche: Employment. Umana:Roche: Employment, Equity Ownership, Patents &amp; Royalties. Subklewe:Roche: Consultancy, Research Funding; Miltenyi: Research Funding; Oxford Biotherapeutics: Research Funding; Morphosys: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; AMGEN: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Janssen: Consultancy.

PF207 TARGETING WILMS TUMOR 1 WITH A T CELL BISPECIFIC ANTIBODY (WT1‐TCB): EX VIVO AND IN VIVO POTENCY BY BIVALENT RECOGNITION OF PEPTIDE‐MHC COMPLEXES FROM AN INTRACELLULAR TUMOR ANTIGEN

Background:Antibody‐based immunotherapy represents a promising strategy to target chemo‐resistant leukemic cells. However, classical antibody‐based approaches are restricted to cell surface antigens, even though targeting of intracellular antigens would allow further access to a large number of tumor‐specific target structures.Aims:In this study we evaluated a 2+1 T Cell Bispecific (TCB) antibody based on T cell receptor (TCR)‐like antibodies for immunotherapy of acute myeloid leukemia (AML). The TCB targets the intracellular tumor antigen Wilms Tumor 1 (WT1) by bivalent recognition of the peptide RMFPNAPYL in the context of human leukocyte antigen allele A∗02:01 (HLA‐A2). Complementary binding to CD3ε recruits T cells irrespective of their TCR‐specificity.Methods:WT1 expression levels in cancer cell lines and primary AML patient samples at different time points during course of the disease were determined by quantitative real‐time PCR, western blot and immunohistochemical staining. WT1‐TCB‐mediated cytotoxicity was analyzed by co‐cultivation of WT1‐expressing HLA‐A2+ cancer cell lines with T cells from healthy donors. Specific lysis was assessed by flow cytometry. TCR downstream signaling was measured by co‐cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1‐TCB‐mediated cytotoxicity against primary AML cells was evaluated in our pre‐established feeder layer‐based ex vivo long‐term culture system. For in vivo testing, NSG mice (NOD.Cg‐Prkdcscid‐Il2rgtm1Wjl/SzJ) were humanized with human HLA‐A2+ CD34+ cord blood cells. After successful engraftment and development of human T cells, WT1‐expressing HLA‐A2+ SKM‐1 tumor cells were subcutaneously inoculated followed by weekly administration of the WT1‐TCB.Results:In accordance with previous reports, we observed WT1 expression in 79% (n = 38) of cancer cell lines and in 92% (n = 65) of AML patient samples at the time of initial diagnosis. Moreover, WT1 expression levels correlated with the percentage of AML blasts: no significant WT1 expression was observed at time of CR (n = 26), whereas WT1 was expressed again at time of relapse (n = 21). WT1‐TCBs elicited antibody‐mediated T cell cytotoxicity against peptide‐pulsed T2 cells and AML cell lines in a WT1 and HLA‐restricted manner. Equally, TCR downstream signaling was observed in a WT1‐restrictive manner by co‐cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1‐TCBs further mediated specific lysis of primary AML cells upon addition of allogenic T cells from healthy donors (mean specific lysis: 67 ± 6% after 13–14 days; ± SEM; n = 18). Correspondingly, up‐regulation of T cell activation and surrogate exhaustion markers was observed (MFI fold change CD69: 9.3 ± 1.5, PD‐1: 5.1 ± 0.7, TIM‐3: 4.7 ± 0.6; ± SEM; n = 22). WT1‐TCBs also mediated killing of primary AML cells in an autologous setting (mean specific lysis: 38 ± 13% after 13–14 days; ± SEM; n = 5). In comparison with WT1RMF‐specific T cells, only bivalent binding by WT1‐TCB induced efficient lysis of primary AML cells. Furthermore, WT1‐TCB‐treated humanized mice bearing SKM‐1 tumors showed a dose dependent and significant reduction in tumor growth resulting in tumor control.Summary/Conclusion:TCR‐like TCBs targeting of intracellular tumor antigens are a promising tool for cancer immunotherapy. Notably, the 2+1 TCB molecular format for bivalent binding facilitates potent in vitro, ex vivo and in vivo killing of AML cell lines and primary AML samples which present low numbers of the RMF peptide‐MHC complex on the cell surface validating WT1‐TCB as a promising therapeutic agent for the treatment of AML.

Use of a Fc-Optimized CD19 Antibody for Treatment of MRD in Pediatric Patients with B-Lineage Acute Lymphoblastic Leukemia

AbstractAbstract 581Primary refractory or relapsed B-lineage acute lymphoblastic leukemia can be successfully treated by allogeneic stem cell transplantation (SCT). However, relapse remains still a major risk and will be significantly influenced by residual disease prior to or after SCT. In addition to standard chemotherapy, immunotherapeutic approaches are assumed to be able to reduce or clear persistent minimal disease. Here we investigated an Fc-optimized chimerized CD19 antibody on a compassionate use basis in pediatric patients at very high risk prior to and after allogeneic, HLA matched (n=2) or mismatched (n=9) first or subsequent SCT. Through its improved capability to recruit FcγRIIIA bearing effector cells, this mAb mediates enhanced ADCC by NK cells but no complement lysis. 11 patients with B-lineage ALL (CR2 with MRD>10−4, n=3; ≥CR3, n=4; active disease, n=4; 8/11 had previous SCTs) received a mean number of 10 (range 1–30) infusions with an anti-CD19 mAb (4G7SDIE) over 6 hours weekly or every second week posttransplant. In 7 patients, additional infusions were given prior to transplant. Dosages ranged from 5 to 50 mg/m2. The infusions were well tolerated without any severe side effects; only fever or headache was observed in a few patients (n=4). Minimal residual disease (MRD) or overt relapse (>50% blasts) was detectable in bone marrow aspirates prior to therapeutic mAb administration in 5/11 and 2/11 patients, respectively. Prophylactic CD19mAb infusions were given posttransplant without measurable MRD levels in 4/11 patients and in another 2 patients (who did not respond to pretransplant CD19mAb because of overt relapse but reached MRD negativity after SCT) due to an extremely high relapse risk. In 4 out of 5 patients with detectable MRD, leukemic load was reduced for at least 1 log or eradicated by the mAb. 2 out of those patients relapsed, 2 other patients are in molecular remission since 270 and 350 days. Both patients with overt relapse did not respond. 2/6 patients with prophylactic mAb infusions relapsed after 121 and 186 days, 4 other patients remain in molecular remission since 52–180 days. Taken together, a response could be documented in 4/7 patients; a total of 6/11 patients are in molecular remission (median follow up 157 days). Concomitant in vitro investigations showed that NK cells of the patients exerted insufficient lysis of primary B-lineage ALL blasts (mean specific lysis: 4,00%). However, lysis could be significantly increased by adding autologous patient serum taken after antibody treatment (mean specific lysis: 20,33 %, p = 0.0001, student’s t-test) or by adding 1 μg/ml 4G7SDIE (mean specific lysis: 34.84 %, p < 0.0001). Activity of patient NK cells was similar to NK cells of healthy donors. ADCC relevant serum titers of biologically active CD19mAb were detectable up to 7 days after antibody infusion. In conclusion, promising antileukemic effects have been observed in vitro and in vivo in this pilot study and a further clinical trial has to address the question whether this approach will be able to reduce relapse rates. Disclosures:No relevant conflicts of interest to declare.

Infusion of CD19-Directed and Multivirus Specific Cytotoxic T Lymphocytes After Allogeneic Hematopoietic Stem Cell Transplantation for B Cell Malignancies,

Abstract 4083Allogeneic hematopoietic stem cell transplant (HSCT) may increase long term disease free survival in patients with high risk B-cell malignancies. The delayed immune reconstitution associated with the procedure is, however, associated with significant morbidity and mortality from infections with cytomegalovirus (CMV), Epstein Barr virus (EBV), and adenoviruses (AdV). Disease relapse is another common cause of treatment failure. We reasoned that a single T cell platform mediating both antiviral and antileukemic activity could benefit these patients. We prepared cytotoxic T lymphocytes with specificities through their native receptors that were directed toward EBV, CMV and adenovirus; we then engineered these cells to express a chimeric antigen receptor (CAR) directed against CD19 (an antigen commonly present on B cell malignancies) and containing the co-stimulatory endodomain derived from CD28. We used donor derived antigen presenting cells (monocytes for the initial stimulation, EBV- lymphoblastoid cell lines, EBV-LCLs, for subsequent stimulations) that expressed adenovirus antigens and transgenic CMVpp65 following transduction with the adenoviral vector Ad5f35CMVpp65. After the third stimulation, multivirus-specific T cells were transduced with a retroviral vector encoding the CAR-CD19.28zeta transgene. Ten CTL cultures have been generated for clinical use, and contain a median of 71% (range 3–98%) CD8+ and 23% (range 0.1–92%) CD4+ cells, with a median of 14% (range 1–83%) CD45RA-/CD62L+ T cells. Transduction efficiency ranged from 20–55% (median 41%). These CTLs showed specific activity against B-cell leukemia/lymphoma targets in 51Cr release and/or IFNγ Elispot assays (mean specific lysis 48% at E:T ratio of 20:1, range 4–87%) and against CMV, EBV, and adenovirus peptide expressing target cells (mean specific lysis 28% at E:T ratio of 20:1, range 4–100%) but not recipient PHA blasts (<10%). Three patients (23/F, 58/M, and 56/M) with relapsed Ph+ALL (n=1) or B-CLL (n=2) have been treated with CAR-CD19.28z-CTLs. Patients received between 1.5 to 3 × 107CTLs/m2 without infusion related toxicity. In patient 1, who has the longest follow up, a three-fold increase in CMV–specific T cells was seen by 4 weeks post CTL infusion as detected by IFNγ Elispot assay. Responses to CMV peptides increased from 225 IFNγ-secreting cells (SFC/2.5 × 105 PBMC) pre-T cells to 782 SFC/2.5×105PBMC 6 weeks post CTLs and responses to CD19+ targets increased from 3 SFC/2.5×105 PBMC 1 week post T cells to 35 SFC/2.5×105 PBMC 6 weeks post CTLs. Detection of Transgenic T cells: CAR-CD19-transduced T cells have been detected by quantitative real-time PCR amplification in the peripheral blood of Patients 1, 2, and 3 up to 4, 1, and 0 weeks post infusion, respectively. Although already absent from peripheral blood, the transgenic cells were found at sites of disease. In Patient 1, T cells were detected in the bone marrow (44.8 copies/1000 ng of DNA) 9 weeks post T cell transfer. Patient 2 developed fever, diarrhea and hypotension 4 weeks post T cell therapy. Findings were consistent with ileitis at a known previous site of disease. Biopsy of the gut showed an abnormal absence of normal and malignant B cells, but significant levels of CAR-CD19.28z T cells by quantitative PCR (55.44 copies/1000 ng of DNA). Anti-Viral Activity of Transgenic Cells: No patient developed viral infection post CTL. Anti-Tumor Activity of Transgenic Cells: Patient 1 with Ph+ ALL had 4% blasts detectable in the peripheral blood at the time of the first infusion of CTLs which cleared within two weeks. She received a second infusion two months later. She became bcr abl negative but subsequently relapsed and died of progressive disease seven months post CTL. Patient 2 with CLL had resolution of lymphadenopathy within two weeks but following the disappearance of CTLs from peripheral blood showed evidence of disease progression two months post CTL. Patient 3 is too early in the course of therapy to evaluate for disease response. In summary, our initial findings show that immunotherapy with CTL targeting both CD19 through a chimeric receptor and multiple viral antigens through their native receptor can be well tolerated after allogeneic stem cell transplantation and may have both anti-viral and anti-tumor activity. Disclosures:No relevant conflicts of interest to declare.

Genetic Modification of Multi Leukemia Antigen-Specific Cytotoxic T Lymphocytes (CTL) to Enhance In Vivo Safety and Persistency

Abstract 644Adoptive transfer of ex-vivo expanded leukemia-specific T cells has been clinically evaluated for its ability to produce anti tumor effects without causing graft-versus-host disease (GVHD). Previous protocols, however, allowed the expansion only of HLA class I-restricted CD8 T cells, which were short-lived and permitted tumor immune escape since they recognized only a single epitope/antigen target.To minimize tumor immune escape and extend this therapeutic alternative to more patients irrespective of HLA type we have developed a novel strategy to produce single cultures of in vitro generated T cell lines containing tumor-cytotoxic T cells from both CD4+ and CD8+ populations with specificity for a multiplicity of epitopes on several tumor associated antigens (TAAs) frequently expressed by myeloid leukemias.Tumor-directed T cells were activated in vitro using dendritic cells (DCs) loaded with peptide libraries (pepmixes) spanning Proteinase 3 (Pr3), PRAME, and WT1 and expanded in the presence of a cocktail of Th1-polarizing, pro-survival and pro-proliferative cytokines (IL7, 12, 15 and 27). This approach consistently generated TAA-specific cytotoxic T lymphocytes (CTLs) with simultaneous reactivity against PRAME (mean 651+/−69 spot forming units (SFU)/1×106 CTLs), WT1 (mean 156+/−20 SFU/1×106 CTLs) and PR3 (mean 52.3+/−8.8 SFU/1×106 CTLs) in IFNg ELIspot assays (n=10). These lines were polyclonal, comprising antigen-specific CD4+ (mean 32.5+/−2.3%) and CD8+ (mean 38.3+/−4%) T cells. Functionally, these CTLs killed autologous PHA blasts pulsed with PRAME (mean specific lysis 67+/−5% SEM), WT-1 (mean 54+/− 5%) and PR3 pepmixes (mean 16+/−6%, n=6) (E:T of 40:1). In addition, TAA CTLs killed the partially (1-3 alleles) HLA class I or HLA class II matched whole antigen-expressing AML cell line THP1 in coculture assays (n=5).The in vivo anti-tumor activity of multiTAA-CTLs was tested using a SCID mouse tumor model. After subcutaneous engraftment of FFLuc transduced THP-1 cells, mice were treated with either 1×107 multiTAA-CTLs or control EBV-CTLs generated from the same partially HLA-matched donor (sharing HLA-A2, DRB1-01,DQB1-05) plus 1000U of IL2 (5 times per week). Tumor signals as measured by bioluminescence using an in vivo imaging system rapidly increased in untreated and EBV-CTL treated groups, whereas mice treated with TAA specific CTLs were able to control tumor growth (p<0.05) and significantly improve overall survival (log rank p=0.008).Next, we transduced multiTAA-CTL with a retroviral vector (SFG-hIL-7R/iCasp9) encoding IL7Ralpha and the inducible suicide gene iCaspase9. This genetic modification improved the in vivo persistence, anti-tumor potency of multiTAA-CTL by restoring their response to homeostatic IL7. Furthermore we improve safety with the potential to control unwanted proliferation of the transgenic cells and to treat potentially occurring autoimmune disease caused by targeting self antigens through the administration of a small molecule chemical inducer of dimerization (CID) (AP20187) which activates the suicide gene.Transduction of multiTAA-CTLs did not adversely affect the antigen specificity of the product as evaluated by IFNg ELIspot (528.5 SFU/1×106 CTL transduced vs. 573.9 SFU/1×106 CTL non transduced, PRAME, 86.7 vs.46.5 SFU/1×106 CTL, Pr3 and 129.2 vs 130.2 SFU/1×106 CTL and WT1 transduced vs. non transduced); and cytolytic activity against peptide-pulsed PHA blasts as well as whole antigen presenting tumor cell lines was maintained. In the presence of IL-7, transgenic multiTAA-CTLs expanded significantly (transduced cells increased from 55%+/1% to 89%+/−1%). Such expansion was not observed when these cells were cultured in the presence of IL-15 (decrease 55%+/1% to 34.6%+/−3.5%) (n=8). Proliferation assays confirmed that only IL7/casp9 engineered multiTAA-CTLs were able to proliferate in the presence of IL-7 (84638 CPM vs. 14923 CPM transduced vs non-transduced). Upon activation of iCaspase9 by of CID (AP20187), the proliferation of multiTAA-CTLs was halted and a decrease of transgenic cells from 89% to 3% with was detected within 24 hrs (n=7).Hence CTLs can be prepared that simultaneously target multiple TAA antigens expressed by myeloid malignancies, reducing the risk of tumor immune escape. In addition these tumor CTLs can be genetically modified to safely improve their in vivo survival, and persistence. Disclosures:No relevant conflicts of interest to declare.

Assessment of physiologic natural killer cell cytotoxicity in vitro

Here, we describe an improved (51)chromium release assay (CRA) to compare donor natural killer (NK) cell activity. To validate the assay, we analyzed sample preparation, incubation, and cryopreservation of NK cells. The effector-to-target ratio was corrected for the percentage of NK cells. A logarithmic curve was fitted to the data of the CRA for calculation of the maximum activity. The specific lysis was standardized to a reference sample and normalized to the mean specific lysis of the reference. We found that a longer time span involved with both the addition and the removal of DMSO increased the recovery of NK cell activity. Freezing and thawing reduced the cytotoxicity of NK cells but sustained the relative differences that were seen between freshly prepared NK cells. In contrast, medium incubation of thawed cells markedly increased the cytotoxic potential but also deranged these relative differences. Those were widely equalized when cells were stimulated with IL-2. In conclusion, we established a standardized assay with cryopreserved peripheral blood mononuclear cells as an appropriate tool for investigation of individual physiologic NK cell activity. This assay may help to predict donor NK cell activity in vivo, to reconcile conflicting data about NK cells obtained in transplantation studies.

Ex Vivo IL-2 Expansion of CB-NK Cells Promotes Synergistic LFA-1 and CD2 Engagement at the NK Cell Lytic Immune Synapse; Implications for Adoptive CB-NK Cell Therapy in Acute Myeloid Leukemia.

Abstract 3029Poster Board II-1005Donor peripheral blood (PB) natural killer (NK) cell have shown clinical promise in cancer immunotherapy. Tightly regulated receptor signaling between NK cells and susceptible tumor cells is essential for NK cell-mediated cytotoxicity. Umbilical cord blood (CB) represents an important alternative source of NK cells for adoptive immune therapy. We first demonstrated that cord blood (CB) derived NK cells have poor cytolytic activity and deficiency in the formation of the F-actin immunological synapse with HLA class I deficient target K562 cells and primary AML blasts compared to PB-NK cells. In this study, we explored the cellular mechanism of these dysfunctions. We hypothesized that adhesion and signaling molecules may be defective in unmanipulated CB NK cells. Activating receptor Both CD2 and the integrin lymphocyte function-associated antigen (LFA-1) play important roles in both T lymphocyte and NK cell immune synapse formation and their trafficking to the immune synapse regulates both T and NK cell function. We now show that unmanipulated CB NK cells exhibit reduced LFA-1 mediated adhesion to mobilized ICAM-1 compared to IL-2 expanded CB NK cells (CB NK 29.7+/- 3.2 %, vs expanded CB NK 78.5+/- 6.1%, n=6). Moreover, unmanipulated CB-NK cells demonstrated reduced surface expression of CD2, and high affintyLFA-1 detected by the specific antibody (MHM24). There was decreased recruitment of CD2 and LFA-1 to the NK cell immune synapse site as quantified by confocal microscope analysis (RRI CD2 CB NK 2.02 vs PB NK 4.98, n=3). Furthermore, defective LFA-1 trafficking lead to a decrease in downstream cytotoxic granules that traffic to the immunological synapse as demonstrated by decreased perforin trafficking to the CB-NK synapse site (> 60% reduction).We next wanted to confirm that CD2 or LFA-1 play a role in restoring the immune synapseformation for IL-2 expanded CB NK cells. We incubated expanded CB NK cells with blocking antibodies specific for LFA-1 or CD2 prior to conjugation to the K562 target cells. After CD2 or LFA-1 blocking there was decreased synapse formation, with a resultant decrease in cytotoxic function. When monoclonal antibodies against both CD2 and LFA-1 were used there was significant blockade of the formation of the immune synapse, and a marked reduction of CB NK cell cytolytic activity (Mean specific lysis of K562 targets at E:T ratio 20:1 was 81% IgG control vs 22% with anti-CD2; and 29% with anti-LFA-1, n=6, P<0.001). This data shows that CD2 and LFA-1 are defective in unmanipulated CB NK cells resulting in impaired immune synapse formation. In contrast, ex vivo IL-2 expansion of CB-NK cells enhanced lytic synapse formation with the synergistic repair of CD2 and LFA-1 localization and activity. We believe our results provide important mechanistic insights for the potential use of IL-2 expanded CB-derived NK cells for adoptive immune therapy in leukemia. DisclosuresNo relevant conflicts of interest to declare.

Intrahepatic Murine CD8 T-Cell Activation Associates With a Distinct Phenotype Leading to Bim-Dependent Death

Chronic infections by hepatotropic viruses such as hepatitis B and C are generally associated with an impaired CD8 T-cell immune response that is unable to clear the virus. The liver is increasingly recognized as an alternative site in which primary activation of CD8 T cells takes place, a property that might explain its role in inducing tolerance. However, the molecular mechanism by which intrahepatically activated T cells become tolerant is unknown. Here, we investigated the phenotype and fate of naïve CD8 T cells activated by hepatocytes in vivo. Transgenic mouse models in which the antigen is expressed in lymph nodes and/or in the liver were adoptively transferred with naïve CD8 T cells specific for the hepatic antigen. Liver-activated CD8 T cells displayed poor effector functions and a unique CD25(low) CD54(low) phenotype. This phenotype was associated with increased expression of the proapoptotic protein Bim and caspase-3, demonstrating that these cells are programmed to die following intrahepatic activation. Importantly, we show that T cells deficient for Bim survived following intrahepatic activation. This study identifies Bim for the first time as a critical initiator of T-cell death in the liver. Thus, strategies inhibiting the up-regulation of this molecule could potentially be used to rescue CD8 T cells, clear the virus, and reverse the outcome of viral chronic infections affecting the liver.

Mature T Cells in the Graft Improve NK Cells Reconstitution after Haploidentical Hematopoietic Stem Cell Transplantation.

We previously demonstrated that natural killer (NK) cells generated after haploidentical stem-cell transplantation (SCT) are blocked at an immature state characterized by phenotypic features and impaired functioning, a blockage that may affect transplantation outcome (Nguyen et al. Blood 2005). Hypothesizing that the absence of mature donor T cells in the graft may affect NK cell differentiation and function, we examined NK cells from 21 patients who received haploidentical SCT from relatives for advanced malignant hematopoietic disease and underwent either partial (pTCD) (CD3+ in the graft >1x105/Kg, mean: 6.9x105/Kg; n=11) or extensive (e-TCD) (CD3+ in the graft<1x105/Kg; mean: 0.35x105/Kg; n=10) T cell depletion and compared them with NK cells from their healthy donors. As previously described, compared with donor cells, recipient NK cells expressed lower levels of inhibitory KIR (in particular KIR2DL1 and KIR2DL2) and higher levels of CD94/NKG2A receptors after transplant (mean expression of CD94/NKG2A on recipient NK cell at 3 months post-transplant: 93.4%±7.2% versus 49.6%±10.9% on donor NK cells, p<0.0001), but these levels did not differ significantly between the pTCD and eTCD groups. However, the frequency of the immunoregulatory CD3−CD56bright NK subset was sharply lower in the pTCD than eTCD groups after transplantation (25.0%±9.6% versus 53.3%±18.0 at 3 months; p<0.001). The level of NKp30 receptors on NK cells was also higher after pTCD than eTCD transplantation (70.3%±7.1% versus 58.0%±6.5%, p=0.013) and that of pTCD patients resembled the donor NK repertoire. NK cytotoxicity against primary haplomismatched AML blasts was significantly more pronounced after pTCD than eTCD transplants (29.0%±8.9% specific lysis versus 6.7%±4.1% at a ratio Effector/Target (E/T):20/1, p=0.002), although still lower than in donor NK cells (mean specific lysis of donor NK cells from both groups against AML blasts: 43.5%±13% at a ratio E/T: 20/1). This more mature phenotypic and functional profile of NK cells after pTCD transplant was clinically associated with a lower rate of relapse and superior survival (1/11 relapse, 3/11 patients alive in complete remission at 11, 10 and 3 years) than in eTCD group (8/10 relapse; no patient alive at 1 year). These results support a model in which mature donor T cells in the graft may play a key role, in vivo, in NK cell differentiation by improving NK cell maturation and cytolytic function against leukemic blasts. They point to the dilemma of haploidentical hematopoietic SCT in leukemic patients: on the one hand, extensive T-cell depletion is associated with a risk of fatal leukemia relapse due to the loss of the GvL effect T cells, which can not be replaced by immature NK cells; on the other hand, partial T-cell depletion might increase the risk of GvHD but also improves the GvL effect mediated by NK cells. New treatments infusing mature haploidentical NK cells in leukemic patients should be used to test the efficiency of NK alloreactivity.

Human complement activation via the alternative pathway on porcine endothelium initiated by IgA antibodies.

The role of the classical pathway (CP) and the alternative pathway (AP) of complement activation in hyperacute xenograft rejection remains a matter of considerable debate. In addition, it is unknown whether IgG and IgA antibodies activate complement, although these antibodies have been found in hyperacutely rejected xenografts. This study was initiated to assess a possible role of the AP of complement activation in a pig-to-human transplantation model using fresh human sera and isolated antibodies with cultured porcine endothelial cells (PEC) as targets. IgM, IgG, monomeric IgA, and dimeric IgA (dIgA) antibodies with reactivity toward PEC as determined by ELISA were isolated from pooled normal human sera. Serum from patients with agammaglobulinemia was used as a source of human complement. C3 and C4 deposition on nonfixed PEC during CP (1% serum) or AP activation (10% serum with MgEGTA) was analyzed using an ELISA. Complement-mediated PEC lysis was tested in a 51Cr release assay. Using normal human sera as the source of antibodies and complement, C3 and C4 deposition was already found after 10 min of incubation in the CP, whereas an increasing amount of C3 was found in the AP. During AP activation, no C4 deposition was observed, indicating that CP activation did not contribute to the observed AP-mediated C3 deposition. Moreover, dIgA antibodies caused deposition of C3 and not C4. Purified IgM and dIgA antibodies (1 mg/ml) in the presence of 10% agammaglobulinemic serum showed a mean specific PEC lysis of 31% and 28%, respectively. Agammaglobulinemic serum alone or with IgG or monomeric IgA antibodies had no detectable lytic activity. In conclusion, dIgA antibodies might play an additional role in pig-to-human xenograft rejection by activating human complement via the AP.

Peripheral blood γ‐δ T cells lyse fresh human brain—Derived oligodendrocytes

T cells are postulated to contribute to the injury of the oligodendrocyte-myelin complex underlying the demyelinating disease multiple sclerosis (MS). The apparent lack of class I or II major histocompatibility complex (MHC) expression in situ on human oligodendrocytes and the consistent failure to identify a universal myelin antigen in MS suggest that the immune damage might be mediated by effector T cells that are capable of reacting in an antigen-nonspecific and possibly MHC-unrestricted manner, such as T cells expressing the gamma-delta T-cell receptor. Since gamma-delta T cells are reported to be present in MS plaques and an increased number are found in the cerebrospinal fluid of patients with MS, we directly examined whether gamma-delta T cells are capable of inducing injury to human oligodendrocytes. We found, using a 6-hour 51Cr release assay, that oligodendrocytes cultured from surgically resected human brain specimens were effectively lysed in a dose-dependent manner by human gamma-delta T cells (28 +/- 5% mean specific lysis, n = 6, at an effector-target ratio of 20:1). Although heat shock protein HSP72, a putative gamma-delta T-cell recognition molecule, could be induced in vitro in our oligodendrocytes, an antibody to HSP72 did not inhibit gamma-delta T cell-mediated lysis of oligodendrocytes. These results suggest that gamma-delta T cells gaining entry into the central nervous system may be deleterious to oligodendrocytes and thus may contribute to the pathogenesis of MS.

Assessment of human natural killer and lymphokine-activated killer cell cytotoxicity against Toxoplasma gondii trophozoites and brain cysts.

Because previous work has suggested that NK cells may be important in host resistance against the intracellular parasite Toxoplasma gondii we examined whether human NK cells and lymphokine-activated killer (LAK) cells have activity against trophozoites and cysts of this organism in vitro. A method to radiolabel Toxoplasma trophozoites with 51Cr was developed and direct cytotoxic activity was determined by using modifications of the standard 51Cr release assay. Viability of 51Cr-labeled trophozoites assessed by both methylene blue staining and trypan blue exclusion was greater than 90%. Significantly more 51Cr was released by anti-Toxoplasma antibody and C than by antibody in the absence of C. Incubation of trophozoites with freshly isolated human NK cells or NK cells activated with either rIL-2 or rIFN-alpha did not result in significant release of 51Cr (specific lysis was 0 to 2.3%). In contrast, the average specific lysis of radiolabeled trophozoites by LAK cells was significant (specific lysis was 7.8% +/- 1.1, p less than 0.01). In a series of separate experiments, preincubation of radiolabeled trophozoites with heat-inactivated normal or Toxoplasma antibody-positive human serum increased the cytotoxicity of LAK cells from a mean specific lysis of 15% +/- 4.5 to 39% +/- 8.5, respectively (p less than 0.05), as assessed by 51Cr release. Because previous work has shown that radioisotope release from parasites may be nonspecific, separate experiments were performed to determine the cytotoxicity of LAK cells against antibody-coated trophozoites by using ethidium bromide-acridine orange staining to assess effector cell damage. LAK cells had a mean specific lysis of 51% against antibody-coated trophozoites by ethidium bromide-acridine orange staining. Preincubation with heat-inactivated Toxoplasma-antibody positive human serum did not increase activity of rIL-2-activated NK cells against 51CR-labeled trophozoites. Neither human NK cells (freshly isolated or activated by rIL-2 or rIFN-alpha) nor LAK cells were cytotoxic for purified preparations of cysts of Toxoplasma isolated from the brains of chronically infected mice.

Cellular and antibody mediated cytotoxicity in autoimmune thyroid disease.

Antibody-dependent cell-mediated cytotoxicity (ADCC) and natural killer (NK) cell-mediated cytotoxicity was measured in patients with Hashimoto's thyroiditis (HT) and Graves' disease (GD) using a cytotoxicity assay against thyroid target cells. In the ADCC assay, mean +/- SD specific lysis produced by sera from patients with HT was 21.7 +/- 10% compared t 6.2 +/- 3.9% from normal subjects. In the NK assay, cytotoxicity was significantly increased using lymphocytes from HT patients as effector cells. At effector: target (E:T) cell ratios of 50:1 and 25:1, mean specific lysis +/- SD was 18.3 +/- 14.3% and 14 +/- 11.6%, respectively, compared to 3.7 +/- 2.1 and 3.1 +/- 2.1, respectively, for normals. In Graves' disease, 9 of 19 patients had elevated cytotoxicity, whereas no significant changes of ADCC could be found either, as determined in thyrotoxic patients, after 6 months and at the end of a one-year antithyroid drug treatment. Eight of 19 patients showed normal cytotoxicity (mean % specific lysis 2.5 +/- 3.1% compared to 2 +/- 2.9% in normal controls) and low titres of microsomal antibodies (Mab), 3 patients had significantly increased cytotoxicity (mean specific lysis 27.6 +/- 10%) in the presence of high titres of Mab, whereas 8 patients evidenced high values for cytotoxicity (mean specific lysis 24.5 +/- 14.1%) but low titres of Mab. NK cell activity, determined in euthyroid Graves' disease patients either under antithyroid drug therapy or in remission, was not significantly different than that of normal subjects at all E:T cell ratios.(ABSTRACT TRUNCATED AT 250 WORDS)

CELLULAR INTERACTIONS IN THE LYSIS OF VZV INFECTED FIBROBLASTS

Human blood mononuclear cells (MNC) lysed varicella zoster virus (VZV) infected targets in an 18 hour 51Cr release assay at effector:target (E:T) ratios of 50:1 to 10:1. Mean specific lysis at 50:1 E:T was 45% when effectors were autologous with targets; 35% when 2 or 3 HLA A or B antigens were shared and 18% when no HLA antiens were shared between E and T. Addition of antibody to HLA inhibited lysis by 65%. Inhibition of lysis following panning to deplete subsets selectively was: 52% for OKT 8; 44% for OKT 4; 30% for HNK 1 and OKM 1. Inhibition by OKT 4 was entirely, and by OKT 8 partly, reversed by addition of a PHA induced T cell supernatant (SUP). Density gradient seperated NK cells lysed targets in the presence of SUP. In combined depletion experiments the inhibition of lysis by anti-HLA summated with inhibition by HNK 1. The inhibition studies suggest the existence of at least two pathways for VZV-fibroblast lysis: one HLA restricted and presumably mediated by cytotoxic T lymphocytes and another dependent on lymphokine and mediated by NK cells. Studies in patients receiving leukemia remission maintenance treatment indicate that both pathways may be severely inhibited by chemotherapy.

Absence of allogeneic restriction in human T-cell-mediated cytotoxicity to Epstein-Barr virus-infected target cells. Demonstration of an HLA-linked control at the effector level.

Peripheral T lymphocytes from patients with infectious mononucleosis (IM) are sensitized in vivo against the Epstein-Barr virus (EBV). The expression of HLA-A, B, or C molecules at the target cell surface is necessary for the cytotoxic reaction because (a) EBV-positive Daudi cells lacking HLA-A, B, and C determinants are resistant to anti-EBV T-cell lysis, (b) cytolysis of EBV-positive target cells can be consistently inhibited by anti-HLA-A, B, and C and anti-beta 2 microglobulin antibodies. However, no evidence for allogeneic restriction in this system was apparent as (a) cytotoxic T lymphocytes (CTL) from one given individual could exert a cytotoxicity of a similar magnitude on different EBV-positive target cells, regardless of the number of HLA-A or B specificities shared by the effectors and targets; (b) CTL from IM patients were able to kill target cells without any HLA-A or B antigen in common; and (c) T5-1 variants lacking one or two HLA antigens at the A, B, or D locus are killed to the same extent as the parental cells. 7 of the 9 IM patients with detectable circulating anti-EBV CTL carried the HLA-A1 antigen, whereas none of the 16 IM patients lacking detectable peripheral CTL were HLA-A1 positive (mean specific lysis of T5-1 target cells by T cells from HLA-A1 positive patients: 29.3 vs. 0.6% in HLA-A1-negative patients) (P less than 10(-9)). These data suggest an HLA-A1-linked gene control of the magnitude of the anti-EBV CTL response. Thus, the HLA region appears to act at two different level sin the T-cell-mediated lysis of EBV-infected cells by controlling first, the development of anti-EBV and second, the expression of HLA-A, B, and C molecules involved as recognition structures at the target cell surface.