Normal Donor Peripheral Blood Mononuclear Cells Research Articles

AML Immunotherapy Using a Novel Tcrm-Based Bispecific Antibody That Targets a Leader Sequence Peptide Derived from Cathepsin G

Introduction: Myeloid azurophil granules provide a rich source of intracellular leukemia antigens. Cathepsin G (CG) is a serine protease that has higher expression in AML blasts in comparison to normal myeloid progenitors. Based on the unique biology of HLA-A*0201 (HLA-A2), in which presentation of leader sequence (LS)-derived peptides is favored, we focused on the LS-CG-derived peptide CG1 (FLLPTGAEA). LS peptides are naturally processed and loaded on HLA-A2, providing an abundant source of surface peptide/HLA (pHLA) targets. We eluted CG1 from the surface of primary HLA-A2 + AML blasts and AML cell lines, and demonstrated CG1-targeting immunity in leukemia patients following allogeneic stem cell transplant, hence highlighting the potential for CG1 to be a promising immunotherapeutic target in AML. Numerous platforms have been developed to target cell surface pHLA complexes. T cell receptor (TCR) mimic (m) antibodies are immunotherapeutic antibodies that target pHLA, which are the natural ligands for the TCR. TCRm antibodies have been a breakthrough in immunotherapy, as they: (1) target intracellular antigens expressed on HLA; (2) are engineered to have high affinity for HLA; (3) can elicit their cytotoxic effect independent of the TCR; and (4) can be produced in large quantities providing an off-the shelf-product for patient use. Here we report on the engineering, preclinical efficacy, and safety evaluation of a novel CG1-targeting, T cell-engager, bispecific antibody (CG1xCD3e), which incorporates a novel TCRm construct that recognizes cell surface CG1/HLA-A2 complexes. Methods: To generate the CG1/HLA-A2-binding monoclonal antibodies, H2L2 human transgenic mice were immunized with CG1/HLA-A2 monomers subcutaneously. The resulting paired heavy and light chains were cloned from the antigen-sorted memory B cells into the single pcDNA3.1(+) vector with both human (h) immunoglobulin (Ig)G1 and hKappa for subsequent ExpiCHO high-throughput transient expression. Antibody binding was assessed by Bio-Layer Interferometry (BLI) and T2 cellular binding assays. Xencor anti-CD3e scFv and Knobs-in-holes technology was applied for the bispecific antibody engineering for xMab format. In vitro activity of CG1xCD3e and T cell cytokine secretion was confirmed using flow cytometry-based T cell-dependent cellular cytotoxicity (TDCC). The specificity of CG1xCD3e for leukemia was assessed using colony forming unit assays. In vivo efficacy was evaluated in NSG mice engrafted with AML cell lines and treated with CG1xCD3e with normal donor peripheral blood mononuclear cells (ND-PBMC). Results: Using BLI, binding avidity of the CG1xCD3e to CD3-Fc, KD= 7.52 x 10-10 M, and affinity to CG1/HLA-A2, KD= 1.28 x 10-10 M were calculated. Using flow cytometry, we confirmed high affinity binding of CG1xCD3e to CG1/HLA on the CG1- expressing EM2 HLA-A2 + cell line, and also demonstrated binding of CG1xCD3e to CD3 using jurkat T cell line. After co-culturing GFP/Luciferase + HLA-A2 + AML (U937-A2 and ML-2) or CML (EM2) cell lines with ND-PBMC and different concentrations of CG1xCD3e bispecific antibody for 24 or 48 hours, flow cytometry analysis demonstrated highly specific CG1/HLA-A2 dependent killing of AML cells by T cells cultured with CG1xCD3e bispecific antibody, in comparison with control bispecific antibody with a disabled CG1 binding arm. This correlated with both tumor- and bispecific antibody- dependent T cell activation and cytokine secretion. To study the in vivo anti-leukemia activity of CG1xCD3e, we engrafted NSG mice with the HLA-A2 + ML-2 and U937-A2 AML cell lines, and treated mice after tumor engraftment with ND-PBMC (1 x 107 cells) and weekly intraperitoneal injections of CG1xCD3e bispecific antibody (0.01 mg/Kg, 0.05 mg/Kg, and 0.1 mg/Kg) or PBS. Bioluminescence imaging demonstrated that the mice treated with ND-PBMC + CG1xCD3e had significantly lower levels of leukemia burden compared to the mice treated with ND-PBMC alone (Figure 1). Lastly CFU assays using HLA-A2 + normal donor marrow in a semi-solid matrix of methylcellulose in the presence of T cells and either CG1xCD3e bispecific antibody or isotype antibody, confirmed specificity of CG1xCD3e bispecific antibody for leukemia. Conclusion: Our study provides strong pre-clinical evidence supporting the targeting of LS- derived peptides, specifically CG1, in the setting of AML using a novel TCRm-based bispecific antibody.

Increased immune infiltration and chemokine receptor expression in head and neck epithelial tumors after neoadjuvant immunotherapy with the IRX-2 regimen

ABSTRACTIRX-2 is an injectable cancer immunotherapy composed of cytokines purified from stimulated normal-donor peripheral blood mononuclear cells. In a phase 2a trial (n = 27), neoadjuvant IRX-2 significantly increased lymphocyte infiltration (LI) into resected head and neck tumors and was associated with changes in fibrosis and necrosis. Event-free survival was 65% at 2 years, and overall survival 65% at 5 years. Overall survival was longer for patients with LI greater versus lower than the median. This substudy of the mechanisms responsible for the increase in LI with neoadjuvant IRX-2 employed multiplex immunohistochemistry (IHC) and transcriptome analysis to interrogate matched pre- and post-treatment tumor specimens from 7 available phase 2a trial patients. Multiplex IHC showed substantial increases in CD68-expressing cells (5 patients), T-cell density (4 patients), and PDL1 mean fluorescent intensity (4 patients). Consistent with IRX-2 activation of multiple immune cells, transcriptome analysis showed mean increases in expression of genes associated with NK cells, B cells, CD4+ T cells, CD8+ T cells, and dendritic cells, but not of genes associated with neutrophils. There were increases in mean expression of genes for most immune subsets, most markedly (2- to 3-fold) for B cells and dendritic cells. Mean increases in gene expression for chemokines suggest that tumor LI may be driven in part by IRX-2-induced production of chemo-attractants. Upregulation of checkpoint genes including PDL1 and CTLA4 along with increased T-cell infiltration suggests a functional antitumor immune response such that the efficacy of IRX-2 may be enhanced by combination with immune checkpoint inhibitors.

Abstract 1252: Random unrelated donor peripheral blood mononuclear cells (PBMC) stimulate cytolytic host T cell activity against leukemia.

Abstract Introduction: In the treatment of acute leukemia, much of the therapeutic effect of allogeneic stem cell transplantation is centered on engraftment of donor cells into the host, with a resultant graft versus leukemia effect. Work previously done by our group and others has shown the infusion of haploidentical stem cells in patients with acute leukemia results in a cytokine storm and exhibits an anti-tumor effect in the absence of engraftment. Whether the underlying clinical response was due to this cytokine storm or secondary to host or donor effector cell interactions was not determined. We present in vitro data collected from leukemia patients in which patient CD3+ cells are stimulated with randomly selected unrelated normal donor peripheral blood mononuclear cells (PBMC). The stimulated patient CD3+ cells are then collected and tested for their ability to lyse leukemia cells from the patient. Methods: New leukemia patients were identified through standard pathologic diagnostic criteria. Patient samples were collected and separated into CD3+ and CD3- fractions. Patient CD3+ cells were then placed in a mixed lymphocyte culture and stimulated with mitomycin C treated unrelated normal donor PBMC. Cell proliferation was measured on Day 5. On Day 7, the stimulated patient CD3+ cells were collected and then cultured with Chromium 51 labeled CD3- cells, the bulk of which were leukemic blast cells. Cytolytic activity was measured through Cr51 release assays. Cell lysis was recorded as lytic units/million cells (LU/106), which is inversely related to the effector to target ratio at which 30% cell lysis occurs. Patient demographic data including sex and age in addition to leukemia type, cytogenetics, and molecular markers were recorded. Results: 10 total patients were enrolled in the study; 8 AML, 1 CML, and 1 CMML. 9 patients underwent ex vivo CD3+ stimulation with 2 separate donor PBMC samples while 1 sample underwent only 1 donor stimulation for 19 total assays. The average white blood cell count was 57,640 cells/uL (range 7,100-336,500 cells/ul) with 4-80% peripheral blasts. 7 out of 19 (37%) assays showed leukemia cell cytolytic activity greater than 1 lytic unit. All 7 successful assays were derived from patients with AML (Table 1). Conclusions: Patient CD3+ cells stimulated with random unrelated donor PBMC are able to generate a cytolytic anti-leukemia response in a donor dependant fashion. This cytolytic activity appears to be unrelated to host lymphocyte proliferation upon donor antigenic stimulation. Interestingly, of the seven assays with cytolytic activity greater than one lytic unit, four were performed in the two patients with known FLT3 ITD positive leukemia, which has a notoriously poor prognosis. Further studies to elucidate a mechanism of action for cellular therapy are warranted. Citation Format: John L. Reagan, Loren D. Fast, Martha Nevola, Peter J. Quesenberry. Random unrelated donor peripheral blood mononuclear cells (PBMC) stimulate cytolytic host T cell activity against leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1252. doi:10.1158/1538-7445.AM2013-1252

OMIP‐006: Phenotypic subset analysis of human T regulatory cells via polychromatic flow cytometry

This panel was optimized for the enumeration and phenotypic characterization of T regulatory cells (Tregs) within the CD4⁺ T-cell pool using human peripheral blood mononuclear cells (PBMC) using intranuclear and intracellular staining methods. The panel was optimized for HIV⁺ clinical trial specimens through the use of HIV-infected and normal donor PBMC. Because the panel is to be used in the context of testing cryopreserved PBMC obtained from multiple sites participating in clinical trials, it was essential to develop an assay that performed well using cryopreserved PBMC. Other tissue types have not been tested.

Enhancing Natural Killer (NK) Cell Mediated Killing of Non-Hodgkin's Lymphoma.

Abstract 2706Poster Board II-682Follicular lymphoma is incurable with the current chemo- or chemoimmunotherapy with median survival of 8–12 years. Relapse free survival after each subsequent therapy steadily decreases, resulting in an expected median survival of 4.5 years following initial relapse. Hence new treatment strategies are needed. Natural killer (NK) cells are important effector cells in mediating the anti-lymphoma effect of rituximab. Indeed, antibody-dependent cell-mediated cytotoxicity (ADCC) is a major mechanisms of action of rituximab with NK cells being important effector cells. However, in addition to ADCC, NK cells also exert natural cytotoxicity against tumor cells, which is modulated by a balance of inhibitory and activating signals through NK cell receptors. NK cell function is inhibited when their inhibitory killer immunoglobulin-like receptors (KIR) are ligated by their cognate MHC class I antigens on tumor targets. The novel agent IPH2101 (1-7F9) is a fully human monoclonal antibody directed against KIR2DL receptor that blocks the interaction of KIR with its HLA-C ligands breaking NK cell tolerance to autologous tumor cells. We investigated whether the combination of the IPH2101and Rituximab will augment the NK cell mediated cytotoxicity against CD20+ lymphoma targets as compared to rituximab alone.Raji cells are human CD20+ Burkitt lymphoma cell line cells that expresses HLA-A*03,- (ligand to inhibitory KIR3DL2); -B*71[Bw6] (no inhibitory KIR-Ligand) and -Cw*03,w*04 (group 1 and 2 of HLA-C ligands to inhibitory KIR2DL2/3 and KIR2DL1), and were chosen for study because they have HLA-C antigens that ligate the inhibitory KIR2DL2/3 and KIR2DLI receptors, making them a good target to test our hypothesis of inhibiting inhibitory KIR. NK cells were isolated from normal donor PBMC (peripheral blood mononuclear cells) with the Miltenyi NK isolation Kit. Using LDH release based cytotoxicity assay, we show (Figure 1) that the treatment of target Raji cells with Rituximab significantly enhanced natural cytotoxicity of the purified NK cells against Raji cells. IPH2101alone treatment of NK cells also significantly enhanced the cytotoxicity of Raji cells, however, the combination of IPH2101treated NK cells against Rituximab treated Raji cells significantly enhanced cytotoxicity beyond that observed with each agent alone. Effector: Target (E:T) ratios of 14:1 or less, from more than 5 random donors showed similar results indicating a synergistic, or at least and additive effect ( representative experiment shown Figure 1) . In these experiments purified NK cells were treated with 30ug/ml of IPH2101for 30 min and Raji targets were treated with 0.1-30ug/ml of Rituximab for 30 min. NK cells in the presence or absence of IPH2101were co-cultured with Raji cells in the presence or absence of Rituximab for 4 hour in a 96 well plate. NK cytotoxicity was assessed with an LDH release based assay. Our results suggest that there is a positive cooperation between natural cytotoxicity mediated through KIR-MHC blockade and that mediated by ADCC. Indeed, wee have shown that the blockade of KIR-MHC class I interaction by anti-KIR blocking antibody (IPH2101) augments the cytotoxicity of freshly isolated normal donor NK cells against CD20+ lymphoma cell lines as compared to rituximab alone, providing a rationale for the clinical investigation of the combination of IPH2101 (1-7F9) and rituximab in non-Hodgkin's lymphoma [Display omitted] Disclosures:Squiban:Innate pharma: Employment.

Characteristics of PBMC obtained from leukapheresis products and tumor biopsies of patients with non-small cell lung cancer

The current study characterized peripheral blood mononuclear cells (PBMC) obtained from leukapheresis products of patients with non-small cell lung cancer (NSCLC) for cytokine release, the ability to incorporate tritiated thymidine following stimulation using PHA as well as the levels of both CD4 and CD8 regulatory T cells (Tregs) as defined by FoxP3 expression. Results were compared to normal donor PBMC obtained from buffy coat products. Heterogeneous levels of Th1 (gamma interferon and IL-2), Th2 (IL-10 and IL-13), pro-inflammatory (TNF-alpha and IL-6) and the hematopoietic inducing cytokine GMCSF were detected from both populations of PBMC as measured using ELISA. Overall, we observed that combined levels of Th1 and Th2 cytokines were higher in lung cancer patients compared to that seen in normal donor PBMC. The increased cytokine production was coupled with an observed decrease in the ability of lung cancer patient PBMC to incorporate tritiated thymidine. Furthermore, cytokine containing supernatants obtained from patients inhibited the incorporation of tritiated thymidine from PBMC obtained from normal donors. Thus, the combined cytokines which included high levels of IL-10, appeared to exhibit suppressive functional activity. While not statistically significant, the overall trend toward a Th2 cytokine environment was supported by an increased level of Tregs observed in the leukapheresis products of lung cancer patients. These levels were variable and were accompanied by higher than normal levels of CD8+ cells co-expressing FoxP3. Finally, tumor biopsies were examined from lung cancer patients along with autologous normal adjacent tissue (NAT). In these studies, both gamma interferon and IL-10 were detected. The levels of IL-10 in the LPS stimulated cultures were statistically greater from the cancer biopsies compared to the NAT. The current study confirms many earlier results in a comprehensive manner and extends the analysis to leukapheresis products. An environment is described in cancer patients which is characterized by increased cytokine production and decreased proliferative potential likely under the influence of a significant population of regulatory T cells (Tregs). Taken together, these results are discussed as they relate to the potential implications in lung cancer patients immune response to their disease.

Severely burned patient peripheral linage– CD34+ cells are inhibitory on the antimicrobial peptide production by human epidermal keratinocytes (133.10)

Abstract The importance of epidermal keratinocyte-produced antimicrobial peptides in the control of skin infections has been described. However, these peptides are not produced sufficiently in tissues surrounding the burn area. In this study, linage- CD34+ cells that infiltrated into burn area tissues were shown to be inhibitory on the antimicrobial peptide production by human epidermal keratinocytes (NHEK). Peripheral blood mononuclear cells (PBMC) taken from 5 severely burned patients (3rd degree, more than 40% total body surface area burn) enrolled in these experiments. NHEK (1 x 105 cells/ml, lower chamber) were cultured with 1 x 105 cells/ml of healthy donor PBMC or patient PBMC in a dual-chamber transwell. Forty-eight hrs after cultivation, cells in the lower chamber were assayed for human β-defensin-1 (HBD-1) and cathelicidin (CAP-18) mRNAs by RT-PCR. Without any stimulation, mRNAs for HBD-1 and CAP-18 were consistently expressed by NHEK. mRNA expression for HBD-1 and CAP-18 by NHEK was not influenced when peptide producer cells were transwell-cultured with normal donor PBMC. However, the expression of HBD-1 and CAP-18 mRNAs by NHEK was greatly suppressed when they were transwell-cultured with patient PBMC. Suppressor cells in patient PBMC for the peptide production by NHEK were determined to be linage- CD34+ cells. Linage- CD34+ cells isolated from healthy donor PBMC did not have any suppressor cell activities. These results indicate that linage- CD34+ cells isolated from severely burned patient PBMC are suppressor cells on the antimicrobial peptide production by epidermal keratinocytes. This study was supported by SHC NA #8610.

PD-1 Expression Is Markedly Upregulated on Intratumoral CD4+ and CD8+ T Cells in Follicular Lymphoma and Is Associated with T-Cell Exhaustion.

Follicular lymphoma is one of the most immune-responsive of all human malignancies. However, immunoregulatory mechanisms in the tumor microenvironment may impair the efficacy of immunotherapies such as vaccines and adoptive T-cell therapy. The inhibitory receptor programmed death 1 (PD1), a negative regulator of activated T cells was recently shown to be upregulated on the surface of HIV-specific CD4+ and CD8+ T cells in humans and was associated with impaired T-cell function. Blockade of the immunoregulatory PD-1/PD-ligand 1 (PD-L1) pathway with antibodies against the PD-L1 augmented the function of HIV-specific CD4+ and CD8+ T cells (Day CL et al, Nature, 2006). To investigate the role of PD-1 in lymphoma, we examined PD-1 expression on peripheral blood mononuclear cells (PBMC) and intratumoral T cells in patients with follicular lymphoma prior to therapy. We observed that PD-1 expression is significantly upregulated on peripheral blood and intratumoral CD4+ and CD8+ T cells in patients with follicular lymphoma as compared with normal donor PBMC. Furthermore, PD-1 expression was significantly higher on intratumoral (mean 61%, range 34% to 86%) compared with peripheral blood CD4+ T cells (mean 25%, range 9 to 40%). Likewise, PD-1 expression was significantly higher on intratumoral (mean 44%, range 31% to 69%) compared with peripheral blood CD8+ T cells (mean 16%, range 9 to 31%). PD-1 expression on CD4+ and CD8+ T cells was associated with impaired type 1 cytokine production (IL-2, TNFa, and IFNg) and blockade of the PD-1/PD-ligand pathway with antibodies against PD-1 significantly enhanced T-cell function. These data indicate that the immunoregulatory PD-1/PD-ligand pathway is operative in patients with follicular lymphoma. Moreover, this pathway of reversible T-cell impairment provides a potential target for enhancing the function of exhausted T-cells in follicular lymphoma in combination with other immunomodulatory strategies such as vaccines and adoptive T-cell therapy.

Lenalidomide and Bortezomib Inhibit Osteoclast Differentiation and Activation in Multiple Myeloma: Clinical Implications.

Osteolytic bone disease in Multiple Myeloma (MM) is caused by enhanced osteoclast (OCL) activation and inhibition of osteoblast function. The proteasome inhibitor bortezomib (PS341, Velcade) has potent anti-myeloma activity with impressive clinical responses. A recent study indicated that bortezomib has inhibitory effects on OCL (ASH 2005, Abstract #2488). Lenalidomide (CC-5013, Revlimid) is an immunomodulatory derivative of thalidomide that has shown promising anti-MM effects in patients with relapsed or refractory MM (Richardson et. al, Blood Jul 06). Significantly, a phase I clinical trial showed that lenalidomide and bortezomib could achieve responses in the majority of patients with MM, refractory to either agent alone (ASH 2005, Abstract #365). However, the effect of lenalidomide on human OCL lineage is unknown. Here we investigated the effect of lenalidomide and bortezomib on human OCL. Peripheral blood mononuclear cells (PBMC) from MM patients (n=11) and healthy donors (n=5) were stimulated with receptor activator of NFk-B ligand (RANKL) (50ng/ml) and M-CSF (25ng/ml) for two weeks to induce OCL formation, in the presence or absence of lenalidomide or bortezomib. OCL were identified by flow cytometric analysis using anti-aVb3 integrin. Lenalidomide and bortezomib inhibited OCL differentiation in a dose-dependent manner (n=13, median control: 70.9% at 0 μM; 63% at lenalidomide 2μM and 45% at 10μM; 35% at bortezomib 2nM and 11% at 5nM). TRAP staining (tartrate-resistant acid phosphatase) was performed to identify OCL and confirm OCL activity. Lenalidomide, as well as bortezomib inhibited OCL in a dose-dependent manner, as evidenced by a marked decrease in TRAP+ cells. To assess bone resorption activity, OCL were cultured with dentine discs, in the presence or absence of lenalidomide and bortezomib, followed by light microscopic analysis and additional measurement of soluble collagen I fragments from the supernatant. Both lenalidomide and bortezomib inhibited bone resorption in a dose-dependent manner. We next asked whether mature OCL were affected. OCL were induced by cytokine stimulation for 3 weeks and treated for 72h, followed by flow cytometry. Neither lenalidomide nor bortezomib altered total number of aVb3 integrin-expressing mature OCL (n=6). In addition, OCL culture supernatants were collected, and two major MM growth and survival factors produced by OCL, B-cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL), were measured by specific ELISA. Both lenalidomide and bortezomib strongly inhibited secretion of BAFF and APRIL. Finally, we determined whether lenalidomide and bortezomib inhibited expression of transcriptional factors important for OCL differentiation and survival. Cell lysates of CD14-expressing monocytic cells from normal donor PBMCs were subject to immunoblotting. Importantly, lenalidomide inhibited OCL differentiation by downregulation of PU.1 expression. These results therefore indicate, that lenalidomide and bortezomib inhibit OCL differentiation, thereby directly preventing the development of new osteolytic lesions. Moreover, BAFF and APRIL secretion by OCL is downregulated, thereby inhibiting MM cell survival in the bone marrow microenvironment.

Evidence for the cofactor role of human T-cell lymphotropic virus type 1 in mycosis fungoides and Sézary syndrome.

The aetiology of mycosis fungoides (MF) and Sézary syndrome (SS) is unknown. A pathogenic role for the human T-cell lymphotropic virus type 1 (HTLV-1) has been suggested but remains controversial. We used an animal model to test the possibility that peripheral blood mononuclear cells (PBMC) obtained from MF patients harbour the HTLV-1 virus which may be infective. The polymerase chain reaction (PCR) was used to detect HTLV-1 proviral DNA sequences in PBMC of 27 MF patients and one SS patient of non-Iranian origin. Positive results were found in six of the patients. Twelve of the 28 patients tested by Western blot showed HTLV-1 antibodies. Twenty-eight immunosuppressed inbred Fisher F344 rats were inoculated intravenously with cultures of PBMC obtained from the 28 patients. Eight of these 28 rats showed antibodies to HTLV-1 while the proviral genome was demonstrated in the blood of only two of the rats. PBMC from two MF patients, in spite of showing negative results for the proviral genome by PCR, still induced HTLV-1 antibody formation in the F344 rat model. None of 10 control rats inoculated with normal donor PBMC showed antibodies to HTLV-1, nor the proviral genome. The present study suggests that HTLV-1 plays a cofactor role in MF/SS patients.

In vitro apoptosis and expression of apoptosis-related molecules in lymphocytes from patients with systemic lupus erythematosus and other autoimmune diseases.

To analyze factors related to apoptosis in systemic lupus erythematosus (SLE) peripheral blood mononuclear cells (PBMC) and to compare the findings in SLE PBMC with those in normal donor PBMC or PBMC from patients with other autoimmune diseases. PBMC from normal healthy donors or patients with SLE, mixed connective tissue disease (MCTD), rheumatoid arthritis (RA), or various vasculitides were isolated. The percentage of apoptosis after activation through different signaling pathways was quantified using propidium iodide staining. Protein expression of Fas/APO-1 or bcl-2, and messenger RNA (mRNA) expression of bcl-2, bcl-xL, bax, bak, Fas/APO-1, Fas ligand (Fas-L), c-myc, mad, or max were determined. We confirmed previous findings of increased numbers of apoptotic cells in SLE PBMC compared with normal donor cells after in vitro incubation. After activation of PBMC with CD28 monoclonal antibody plus phorbol myristate acetate (CD28 MAb/ PMA), staphylococcal enterotoxin B (SEB), or phytohemagglutinin (PHA), the percentage of apoptotic cells was unchanged (SEB) or diminished (CD28 MAb/PMA, PHA) in SLE cells, and the difference between normal donor and SLE cells was less pronounced. On the mRNA level, expression of apoptosis-related gene products did not differ between SLE cells and normal donor cells. Expression of Fas/APO-1 protein was increased in freshly isolated SLE T lymphocytes compared with normal donor T lymphocytes, whereas bcl-2 protein was up-regulated after a 3-day culture period. Cellular activation further increased bcl-2 protein levels, eliminating differences between normal donors and SLE patients. In RA cells, the percentage of apoptosis was similar to that in normal donor PBMC, whereas results using cells from patients with other autoimmune diseases (MCTD, Wegener's granulomatosis, Takayasu arteritis, polyarteritis nodosa) were comparable with those found using SLE PBMC. Addition of growth factors such as interleukin-2 (IL-2), IL-4, or IL-15 to culture medium decreased the percentage of in vitro apoptosis in both normal donor and SLE cells. Based on these data, we conclude that accelerated in vitro apoptosis and increased Fas/ APO-1 and bcl-2 protein expression in SLE are nonspecific for the disease, and might be explained at least in part by the increased in vivo activation levels of PBMC from patients with SLE, MCTD, or autoimmune vasculitides combined with in vitro incubation under "noninflammatory" conditions and growth factor withdrawal.

HIV-1 sensitivity to zidovudine: A consensus culture technique validated by genotypic analysis of the reverse transcriptase

In order to select and standardize a reliable assay for the analysis of sensitivity of HIV isolates to AZT, we have compared two culture methods. The first assay (Cell-Associated Isolate Sensitivity Assay: CAISA) quantified AZT-resistant HIV isolates by end-point dilution cultures of peripheral blood mononuclear cells (PBMCs) in the presence of various concentrations of AZT. In the second assay (Cell-Free Isolate Sensitivity Assay: CFISA), following a conventional isolation of HIV, dilutions of infected cell-free supernatants were cultivated with fresh normal donor PBMCs in the presence of increasing concentrations of AZT. Samples from 64 untreated and AZT-treated patients were studied by CAISA (41), CFISA (43) or both assays (20). The CFISA, which allows the determination of titration parameters with respect to various kinetics patterns of viral replication was selected, and some of the CFISA phenotypically characterized isolates were further studied by nucleotide sequence analysis of the reverse transcriptase gene. CFISA showed that isolates from untreated patients were susceptible to AZT while the frequency of resistance increased with the duration of therapy. Genotypic analysis of CFISA-resistant isolates exhibited mutations at crucial positions, particularly at residue 215. We consider CFISA as a consensus culture technique for longitudinal studies of isolates from patients receiving AZT or other analogs of nucleosides.

Similar replication capacities of primary human immunodeficiency virus type 1 isolates derived from a wide range of clinical sources

Numerous studies have suggested that there are significant differences in replication capacities and cytopathicities among human immunodeficiency virus type 1 (HIV-1) isolates and that these differences correlate with the clinical status and geographical origin of infected individuals. However, it has been difficult to assess whether reported distinctions could be attributed to the methods used or whether they imply a true disparity between viral isolates. We thus attempted to characterize the replication properties of HIV-1 isolates directly recovered from infected patients (primary isolates) by using a standardized infection assay. Viruses were isolated from patients' peripheral blood mononuclear cells (PBMC) by a single coculture with normal donor PBMC stimulated with phytohemagglutinin. Replication curves and cytopathic effect of a standard inoculum (1 ng of p24) of 66 primary HIV-1 isolates were similar regardless of clinical stage of the patient (asymptomatic, AIDS-related complex, or AIDS) and evolutive feature (rate of progression to AIDS). There was no difference between viruses derived from patients sensitive to zidovudine and those derived from patients resistant to zidovudine. Moreover, no difference was found among viral isolates of different geographical origins (Central Africa, Zaire, Brazil, or France). Similarly, the replication patterns and cytopathicities of isolates from bronchoalveolar lymphocytes did not differ from those of isolates derived from PBMC. In contrast, the same amount of viral inoculum of five laboratory HIV-1 strains (HIV-1, EL1, SF, MN, and RF) produced different replication curves and were much less cytopathic. In contrast to laboratory viral strains, it appears that the primary HIV-1 isolates tested, whatever their clinical status and source, exhibited similar replication capacities and cytopathicities in allogeneic donor PBMC.

High Titers of Cytopathic Virus in Plasma of Patients with Symptomatic Primary HIV-1 Infection

Primary infection with the human immunodeficiency virus (HIV-1) frequently causes an acute, self-limited viral syndrome. To examine the relations among viral replication, the immune response of the host, and clinical illness during this initial phase of infection, we undertook a quantitative, molecular, and biologic analysis of infectious HIV-1 in the blood and plasma of three patients with symptomatic primary infection and of a sexual partner of one of them. During an eight-week period of primary infection, HIV-1 was cultured frequently in dilutions of plasma and peripheral-blood mononuclear cells (PBMC), and levels of HIV-1 antigen and antibody were determined sequentially by enzyme-linked immunosorbent assay and immunoblotting. Replication-competent HIV-1 proviruses were cloned and characterized biologically. Six to 15 days after the onset of symptoms, high titers of infectious HIV-1 (from 10 to 10(3) tissue-culture-infective doses per milliliter of plasma) and viral p24 antigen were detected in the plasma of all three patients. These titers fell precipitously by day 27, and the decline coincided with an increase in the levels of antiviral antibodies and the resolution of symptoms. Sequential isolates of virus from plasma and PBMC obtained throughout the period of primary infection, as well as virus derived from two molecular proviral clones, were highly cytopathic for normal-donor PBMC and immortalized T cells, despite the marked reduction in the titers of virus in plasma. Primary, symptomatic HIV-1 infection is associated with high titers of cytopathic, replication-competent viral strains, and during such infection potential infectivity is enhanced. Effective control of HIV-1 replication during primary infection implies the activation of clinically important mechanisms of immune defense that merit further examination in relation to the development of antiviral therapy and vaccines.

Natural killer cell-mediated inhibition of bone marrow colony formation (CFU-GM) in refractory anaemia (preleukaemia): evidence for patient-specific cell populations.

The role of peripheral blood mononuclear cells (PB-MNC) on the growth of bone marrow (BM) CFU-GM was investigated in refractory anaemia (RA) patients. Whereas normal donor PB-MNC were found to inhibit autologous day 7 CFU-GM, PB-MNC from RA patients exhibited little modulatory effect on autologous or allogeneic day 7 CFU-GM. In contrast, patient PB-MNC inhibited autologous CFU-GM at day 10 at a time where no significant inhibition was seen in the PB-MNC/RA CFU-GM combination. The identity of the inhibitory cells was investigated using anti-T8+ and anti-N901+ subsets purified by immune-rosette depletion with a panel of monoclonal antibodies. The activity of these subsets was tested on immature myeloid cells enriched for MY7+ cells, and it was found that cells highly enriched for NK cells were responsible for the inhibition. Further support for NK cells as the inhibitory cells was obtained in experiments where a positive correlation between the level of PB NK cytotoxicity against K562 cells and the degree of CFU-GM inhibition was demonstrated. Thus, these data suggest the presence of a specialized subset of NK cells with a capacity to inhibit autologous CFU-GM. Since RA is a potentially premalignant disease, in which a significant number of cases transform into AML, these findings also suggest a physiological role for NK cells in suppression of newly arisen clonogenic cells at least in early stages of the disease.