Preclinical targeting of aggressive T-cell malignancies using anti-CD5 chimeric antigen receptor.

The outlook for T-cell malignancies remain poor due to the lack of effective therapeutic options. Chimeric antigen receptor (CAR) immunotherapy has recently shown promise in clinical trials for B-cell malignancies, however, designing CARs for T-cell based disease remain a challenge due to the shared surface antigen pool between normal and malignant T-cells. Normal T-cells express CD5 but NK (natural killer) cells do not, positioning NK cells as attractive cytotoxicity cells for CD5CAR design. Additionally, CD5 is highly expressed in T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphomas (PTCLs). Here, we report a robust anti-CD5 CAR (CD5CAR) transduced into a human NK cell line NK-92 that can undergo stable expansion ex vivo. We found that CD5CAR NK-92 cells possessed consistent, specific, and potent anti-tumor activity against a variety of T-cell leukemia and lymphoma cell lines as well as primary tumor cells. Furthermore, we were able to demonstrate significant inhibition and control of disease progression in xenograft mouse models of T-ALL. The data suggest that CAR redirected targeting for T-cell malignancies using NK cells may be a viable method for new and complementary therapeutic approaches that could improve the current outcome for patients.

Preclinical targeting of human T-cell malignancies using CD4-specific chimeric antigen receptor (CAR)-engineered T cells.

Peripheral T-cell lymphomas (PTCLs) are aggressive lymphomas with no effective upfront standard treatment and ineffective options in relapsed disease, resulting in poorer clinical outcomes as compared with B-cell lymphomas. The adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) is a promising new approach for treatment of hematological malignancies. However, preclinical reports of targeting T-cell lymphoma with CARs are almost non-existent. Here we have designed a CAR, CD4CAR, which redirects the antigen specificity of CD8+ cytotoxic T cells to CD4-expressing cells. CD4CAR T cells derived from human peripheral blood mononuclear cells and cord blood effectively redirected T-cell specificity against CD4+ cells in vitro. CD4CAR T cells efficiently eliminated a CD4+ leukemic cell line and primary CD4+ PTCL patient samples in co-culture assays. Notably, CD4CAR T cells maintained a central memory stem cell-like phenotype (CD8+CD45RO+CD62L+) under standard culture conditions. Furthermore, in aggressive orthotropic T-cell lymphoma models, CD4CAR T cells efficiently suppressed the growth of lymphoma cells while also significantly prolonging mouse survival. Combined, these studies demonstrate that CD4CAR-expressing CD8+ T cells are efficacious in ablating malignant CD4+ populations, with potential use as a bridge to transplant or stand-alone therapy for the treatment of PTCLs.

Progesterone promotes the acrosome reaction in capacitated human spermatozoa as judged by flow cytometry and CD46 staining.

The acrosome reaction is a necessary prerequisite for spermatozoa to acquire fertilizing ability. Several different moieties appear to promote the acrosome reaction through different pathways, including solubilized zona pellucidae, recombinant zona protein ZP3, follicular fluid, calcium ionophores, and mannosylated bovine serum albumin (BSA). Although many investigators have presented evidence that progesterone also promotes the acrosome reaction through the mediation of a non-genomic cell membrane receptor, this concept has been challenged. Other workers have suggested that progesterone does not promote an acrosome reaction in human spermatozoa, as judged by the detection of CD46, a complement regulatory protein present on the inner acrosome membrane, through flow cytometric analysis of large numbers of spermatozoa. Prior investigations were criticized by the limited numbers of spermatozoa enumerated visually, the use of non-specific staining techniques, and the failure to eliminate dead spermatozoa during the scoring of the acrosome reaction. We have repeated these experiments, using both a supravital dye to eliminate dead spermatozoa from flow cytometric analysis, and anti-CD46 monoclonal antibody to score acrosome-reacted spermatozoa. Care was taken to validate the adequacy of capacitation conditions, which were proven by the ability of spermatozoa to acrosome react in response to mannosylated BSA and to penetrate zona-free hamster eggs. Confocal microscopy was used to confirm that CD46 immunostaining was limited to the acrosomal region of the spermatozoon head. Our results indicate that progesterone does promote an acrosome reaction within capacitated spermatozoa.

Measurements of the linear energy transfer spectra on the Mir orbital station and comparison with radiation transport models.

A tissue equivalent proportional counter designed to measure the linear energy transfer spectra (LET) in the range 0.2-1250 keV/micrometer was flown in the Kvant module on the Mir orbital station during September 1994. The spacecraft was in a 51.65 degrees inclination, elliptical (390 x 402 km) orbit. This is nearly the lower limit of its flight altitude. The total absorbed dose rate measured was 411.3 +/- 4.41 microGy/day with an average quality factor of 2.44. The galactic cosmic radiation (GCR) dose rate was 133.6 microGy/day with a quality factor of 3.35. The trapped radiation belt dose rate was 277.7 microGy/day with an average quality factor of 1.94. The peak rate through the South Atlantic Anomaly was approximately 12 microGy/min and nearly constant from one pass to another. A detailed comparison of the measured LET spectra has been made with radiation transport models. The GCR results are in good agreement with model calculations; however, this is not the case for radiation belt particles and again points to the need for improving the AP8 omni-directional trapped proton models.

In-flight radiation measurements on STS-60.

A joint investigation between the United States and Russia to study the radiation environment inside the Space Shuttle flight STS-60 was carried out as part of the Shuttle-Mir Science Program (Phase 1). This is the first direct comparison of a number of different dosimetric measurement techniques between the two countries. STS-60 was launched on 3 February 1994 in a nearly circular 57 degrees x 353 km orbit with five U.S. astronauts and one Russian cosmonaut for 8.3 days. A variety of instruments provided crew radiation exposure, absorbed doses at fixed locations, neutron fluence and dose equivalent, linear energy transfer (LET) spectra of trapped and galactic cosmic radiation, and energy spectra and angular distribution of trapped protons. In general, there is good agreement between the U.S. and Russian measurements. The AP8 Min trapped proton model predicts an average of 1.8 times the measured absorbed dose. The average quality factor determined from measured lineal energy, y, spectra using a tissue equivalent proportional counter (TEPC), is in good agreement with that derived from the high temperature peak in the 6LiF thermoluminescent detectors (TLDs). The radiation exposure in the mid-deck locker from neutrons below 1 MeV was 2.53 +/- 1.33 microSv/day. The absorbed dose rates measured using a tissue equivalent proportional counter, were 171.1 +/- 0.4 and 127.4 +/- 0.4 microGy/day for trapped particles and galactic cosmic rays, respectively. The combined dose rate of 298.5 +/- 0.82 microGy/day is about a factor of 1.4 higher than that measured using TLDs. The westward longitude drift of the South Atlantic Anomaly (SAA) is estimated to be 0.22 +/- 0.02 degrees/y. We evaluated the effects of spacecraft attitudes on TEPC dose rates due to the highly anisotropic low-earth orbit proton environment. Changes in spacecraft attitude resulted in dose-rate variations by factors of up to 2 at the location of the TEPC.

Results of time-resolved radiation exposure measurements made during U.S. Shuttle missions with a tissue equivalent proportional counter.

Time-resolved radiation exposure measurements inside the crew compartment have been made during recent Shuttle missions with the USAF Radiation Monitoring Equipment-III (RME-III), a portable four-channel tissue equivalent proportional counter. Results from the first six missions are presented and discussed. The missions had orbital inclinations ranging from 28.5 degrees to 57 degrees, and altitudes from 200-600 km. Dose equivalent rates ranged from 40-5300 micro Sv/dy. The RME-III measurements are in good agreement with other dosimetry measurements made aboard the vehicle. Measurements indicate that medium- and high-LET particles contribute less than 2% of the particle fluence for all missions, but up to 50% of the dose equivalent, depending on the spacecraft's altitude and orbital inclination. Iso-dose rate contours have been developed from measurements made during the STS-28 mission. The drift rate of the South Atlantic Anomaly (SAA) is estimated to be 0.49 degrees W/yr and 0.12 degrees N/yr. The calculated trapped proton and Galactic Cosmic Radiation (GCR) dose for the STS-28 mission were significantly lower than the measured values.

Cell-mediated immunity in allopurinol-induced hypersensitivity.

Allopurinol may induce severe hypersensitivity characterized by hepatitis, interstitial nephritis, and skin rash. The mechanisms for this hypersensitivity syndrome are incompletely elucidated. Immunologic studies were performed on tissue and peripheral blood lymphocytes from a patient with allopurinol hypersensitivity. Immunohistochemistry was performed on sections of the liver biopsy utilizing monoclonal antibodies for T and B lymphocytes. Peripheral blood lymphocyte immunophenotyping by flow cytometry and peripheral blood lymphocyte stimulation studies with either allopurinol or oxypurinol measured as tritiated thymidine uptake were performed in the hypersensitive patient and compared to a group of six patients treated with allopurinol without hypersensitivity and eight normal control patients. Additional single- and dual-color immunophenotyping by flow cytometry of oxypurinol-stimulated lymphocytes was performed in the hypersensitive patient and compared to normal controls. The liver biopsy demonstrated predominantly a T lymphocyte infiltrate. The number of peripheral blood lymphocytes expressing activation antigens was significantly greater in the hypersensitive patient compared to that of both control groups. Lymphocytes from the hypersensitive patient were moderately stimulated by allopurinol and markedly stimulated by oxypurinol compared to both control groups. Oxypurinol-stimulated lymphocytes from the hypersensitive patient demonstrated enhanced expression of activation antigens compared to unstimulated lymphocytes from this patient and normal controls. These studies suggest that cell-mediated immunity directed toward allopurinol and more importantly to its oxypurinol metabolite is involved in the pathogenesis of allopurinol-induced hypersensitivity.

Radiation dosimetry measurements during U.S. Space Shuttle missions with the RME-III.

Time-resolved radiation dosimetry measurements inside the crew compartment have been made during recent Shuttle missions with the U.S. Air Force Radiation Monitoring Equipment-III (RME-III), a portable battery-powered four-channel tissue equivalent proportional counter. Results from the first six missions are presented and discussed. Half of the missions had orbital inclinations of 28.5 degrees with the remainder at inclinations of 57 degrees or greater; altitudes ranged from 300 to 600 km. The determined dose equivalent rates ranged from 70 to 5300 microSv/day. The RME-III measurements are in good agreement with other dosimetry measurements made aboard the vehicles. Measurements indicate that medium- and high-LET particles contribute less than 2% of the particle fluence for all missions, but up to 50% of the dose equivalent, depending on the spacecraft's altitude and orbital inclination. Isocontours of fluence, dose and dose equivalent rate have been developed from measurements made during the STS-28 mission. The drift rate of the South Atlantic Anomaly is estimated to be 0.49 degrees W/yr and 0.12 degrees N/yr. The calculated trapped proton and GCR dose for the STS-28 mission was significantly lower than the measured values.

Analysis of radiation risk from alpha particle component of solar particle events.

The solar particle events (SPE) will contain a primary alpha particle component, representing a possible increase in the potential risk to astronauts during an SPE over the often studied proton component. We discuss the physical interactions of alpha particles important in describing the transport of these particles through spacecraft and body shielding. Models of light ion reactions are presented and their effects on energy and linear energy transfer (LET) spectra in shielding discussed. We present predictions of particle spectra, dose, and dose equivalent in organs of interest for SPE spectra typical of those occurring in recent solar cycles. The large events of solar cycle 19 are found to have substantial increase in biological risk from alpha particles, including a large increase in secondary neutron production from alpha particle breakup.

An acute form of T gamma lymphoproliferative disease presenting with massive splenomegaly--importance of immunophenotyping for diagnosis.

We describe a case of T gamma lymphoproliferative disease (T gamma LPD) which presented in an uncustomary acute onset in an adult with massive splenomegaly. Morphologically the cells represented monocytic leukemia. Karyotyping and equivocol special stain results suggested hairy cell leukemia. Gene rearrangement indicated a T lymphocytic malignancy. Immunocytochemistry stains were not definitive. Immunophenotyping by flow cytometry defined the cells as consistent with T gamma LPD (CD45+, CD56+, CD2+, CD3+, CD11b+, and CD38+; some cells CD8+; and CD57-). Although the cells did not have spontaneous activity, which is often the situation for most cases of T gamma LPD, the cells could be partially induced with exogenous interleukin 2 to exhibit in vitro cytotoxicity against a natural killer lymphocyte-susceptible target cell line (K562) but not a lymphocyte-activated killer target cell line (HEPG2). This report hopefully continues to increase the awareness of T gamma LPD as well as demonstrates an unusual acute form which could have been misdiagnosed unless a multidisciplinary approach, especially including flow cytometric immunophenotyping, was used to evaluate the patient.

Nonprotein antigens of Borrelia burgdorferi.

Preparative thin-layer chromatograms of chloroform-methanol extracts of Borrelia burgdorferi (B31) sonicates showed four fractions (Rf values of 0.84, 0.81, 0.66 and 0.61) that stained with iodine vapors, orcinol, or phospray, suggesting the presence of lipid-, carbohydrate-, and phosphorus-containing compounds. Sera from patients with Lyme disease showed IgM or IgG antibody reactivity to hydrophobic fractions, designated F1 and F2, in both early and late stages of the disease. Lack of constitutive amino acids in these fractions was shown by protein, amino acid, and peptide detection analyses. Sera from patients with syphilis, systemic lupus erythematosus, and antiphospholipid syndrome reacted to one or both of the fractions. Adsorption of sera from Lyme disease patients with intact B. burgdorferi resulted in significantly different pre- and postadsorption patterns of reactivity by whole cell ELISA, whereas adsorption with F1 and F2 resulted in similar pre- and postadsorption patterns. These fractions may not be present in aqueous whole cell or whole cell lysate ELISA antigens or in immunoblots.

In vitro enhancement of natural cytotoxicity by atrial natriuretic peptide fragment 4-28.

The presence of atrial natriuretic peptide (ANP) binding sites in the thymic cortex, medulla, and splenic white pulp suggests that this peptide may have immunoregulatory activity. We examined the effect of ANP on human natural killer (NK) cell activity. ANP significantly augmented NK cell cytotoxicity after twenty-four hours of incubation but had no effect on NK activity after short-term incubations of one hour. In addition, atrial natriuretic peptide did not effect the expression of natural killer or T cell surface markers. This study demonstrates that atrial natriuretic fragment 4-28 enhances natural killer cell activity.

Natural killer lymphocyte blast crisis of chronic myelogenous leukemia.

We describe for the first time a case report documenting a chronic myelogenous leukemia (CML) patient who developed a blast crisis of natural killer (NK) lymphocytes. Many of the blasts exhibited large granular lymphocytic (LGL) morphology. Single parameter immunophenotyping results determined that the granulated as well as the agranulated blast cells were NK lymphocytes (CD45, NKH1, CD2, LEU 17, and CD16 positive; CD3, CD8, and LEU 7 negative). Dual parameter flow cytometric testing also determined that some of the blasts expressed the CD11b and CD11c markers as reported for some types of NK lymphocytes. Approximately 10% of the cells were in the S phase of the cell cycle as determined by a modified Vindelov DNA content analysis test and may theoretically reflect some of those cells expressing CD11b and CD11c. The cells did not express in vitro NK lymphocyte functional activity against a K562 target and therefore similar to other reported cases of presumably immature NK lymphocytic leukemias. The NK lymphocyte blast crisis was successfully treated with vincristine and prednisone. The patient's disease eventually relapsed and transformed to a progenitor stem cell before she died (CD45, 13, CD38, and CD34 positive). The flow cytometric immunophenotyping results contributed significantly as an important adjunct in determining the appropriate diagnosis, helping to select the type of therapy, and monitoring the patient with this unusual type of blast crisis.

Inverted T helper/T suppressor lymphocyte ratio is not a reliable indicator of coexistent HIV infection in the presence of carcinoma: report of a patient with ovarian carcinoma and inverted TH/TS ratio.

Patients with non-HIV (Human Immunodeficiency Virus) related cancers may also have HIV infection. Inverted peripheral blood lymphocyte T helper/T suppressor ratios with selective loss of T helper cells may be used as a clinical screening test for HIV infection in these patients since they may be seronegative for retrovirus infection early in the course of infection. We describe a case in which carcinoma alone appeared to induce systemic changes that resembled coexistent HIV infection. Many of these abnormalities, including inverted TH/TS ratio with selective loss of T helper cells, improved in the immediate postoperative period, indicating that HIV infection was not present. We conclude then, that diagnosis of HIV infection should not be made without more definitive evidence of its presence than an inverted TH/TS ratio in a patient with carcinoma.

Seronegative Lyme disease. Dissociation of specific T- and B-lymphocyte responses to Borrelia burgdorferi.

The diagnosis of Lyme disease often depends on the measurement of serum antibodies to Borrelia burgdorferi, the spirochete that causes this disorder. Although prompt treatment with antibiotics may abrogate the antibody response to the infection, symptoms persist in some patients. We studied 17 patients who had presented with acute Lyme disease and received prompt treatment with oral antibiotics, but in whom chronic Lyme disease subsequently developed. Although these patients had clinically active disease, none had diagnostic levels of antibodies to B. burgdorferi on either a standard enzyme-linked immunosorbent assay or immunofluorescence assay. On Western blot analysis, the level of immunoglobulin reactivity against B. burgdorferi in serum from these patients was no greater than that in serum from normal controls. The patients had a vigorous T-cell proliferative response to whole B. burgdorferi, with a mean ( +/- SEM) stimulation index of 17.8 +/- 3.3, similar to that (15.8 +/- 3.2) in 18 patients with chronic Lyme disease who had detectable antibodies. The T-cell response of both groups was greater than that of a control group of healthy subjects (3.1 +/- 0.5; P less than 0.001). We conclude that the presence of chronic Lyme disease cannot be excluded by the absence of antibodies against B. burgdorferi and that a specific T-cell blastogenic response to B. burgdorferi is evidence of infection in seronegative patients with clinical indications of chronic Lyme disease.

Leukemia derived from intermediately differentiated lymphocytic lymphoma.

The availability of monoclonal antibodies has facilitated the immunophenotypic characterization of malignant lymphocytes from patients with lymphoma and leukemia. The chronic lymphocytic leukemias are diseases of both clinical and morphological diversity and the application of monoclonal antibodies can prove helpful in their classification. Enzyme cytochemistry, surface markers, mouse rosetting, and electron microscopy were used to determine the phenotype of cells from an atypical case of B-CLL. The use of monoclonals Leu-1, CALLA and BA-2 on bone marrow and peripheral blood provided the opportunity to diagnose this patient's disease as intermediately differentiated lymphoma. Leu-1 was found to be a useful alternative to mouse rosetting, a technique not easily performed in a routine setting. Ultrastructural studies helped to prove the prolymphocytic component of this patient's disease. It was concluded that phenotypic characterization of lymphoid cells using monoclonal antibodies directed against membrane antigens facilitated the assessment of this patient's disease.

A murine IgM monoclonal antibody binds an antigenic determinant in outer surface protein A, an immunodominant basic protein of the Lyme disease spirochete.

A hybridoma cell line formed by the fusion of the P3x63-Ag8.653 myeloma cell line with splenocytes from BALB/c mice immunized with Borrelia burgdorferi produced an IgM monoclonal antibody (mAb-11G1) with kappa-light chains which detected an antigenic determinant in a major spirochetal protein of m.w. approximately 31,000, also known as outer surface protein A (OSP-A). Apparent saturation was reached in approximately 35 min with 34 ng of mAb-11G1 binding to 5 X 10(7) spirochetes giving an estimated 4.8 X 10(2) IgM molecules per spirochete and thus a minimum of 480 binding sites per organism. Enzymatic digestion studies suggest that the antigenic determinant to mAb-11G1 is contained within the peptide chain of OSP-A as binding could be eliminated by treatment of the spirochetes with proteinase K, Pronase and pepsin (100 to 200 micrograms/ml of enzyme) but not by trypsin or bromelain treatment. Periodate oxidation as well as mixed and endoglycosidase treatment of the spirochetes did not alter the binding of mAb-11G1. Two-dimensional gel electrophoresis of whole spirochetal cell lysates disclosed that OSP-A is a heterogeneously charged basic protein with an apparent isoelectric point range from 8.5 to 9.0. Amino acid analysis of OSP-A showed a 10% lysine component which could provide the basic nature to the protein. OSP-A with the intact antigenic determinant for mAb-11G1 can be found in the urine of hamsters experimentally infected with B. burgdorferi.

An IgE response to spirochete antigen in patients with Lyme disease.

Most but not all Lyme disease patients produce specific IgE antibodies to Borrelia burgdorferi. Development of IgE antibodies paralleled that of other immunologic classes and appeared to be directed against a polypeptide with a molecular weight of 41,000. Total serum IgE levels in Lyme disease patients were usually within the normal range in all stages of the disease. However, highly elevated total serum IgE in certain patients were not correlated to any particular disease stage nor to specific antibody titers. Spirochetes and spirochetal sonicates in high concentration induced release of histamine from basophils derived from both patients and controls. At lower antigen concentrations, histamine release could be induced only from basophils derived from patients. Synovial fluids from patients with Lyme arthritis contained IgE but only negligible amounts of histamine.

Selective inhibition of natural killer activity by the monoclonal antibodies OKT10 and VEP10 at the single cell level.

The monoclonal antibodies, VEP10 and OKT10, which have been shown to recognize determinants on human natural killer (NK) cells, inhibit large granular lymphocyte (LGL) NK activity against K562, MOLT4, and CEM tumor target cells in the single cell conjugate agarose assay. Inhibition of NK activity by monoclonal antibodies was expressed independently of effector-target cell binding, as inhibitory activity could be demonstrated when the monoclonal antibodies VEP10 and OKT10 were added to preformed conjugates or to the LGLs and targets prior to the binding event. In addition, this inhibition was exerted on the effector cell and not the target cell since VEP10 and OKT10 did not react with determinants on K562 target cells. Furthermore, the 4F2 monoclonal antibody, which reacted with determinants on the LGL and all of the targets used, effected no inhibition of NK activity. Inhibition of killing by OKT10 and VEP10 was specific to endogenous NK activity since the same antibodies did not inhibit antibody-dependent cellular cytotoxicity (ADCC), mixed lymphocyte-generated NK, or cytotoxic T lymphocyte (CTL) activities.

Potentiation of the cytolytic activity of peripheral blood monocytes by lymphokines and interferon.

Human peripheral blood monocytes obtained by EDTA-reversible adherence to plastic surfaces precoated with autologous serum can rapidly lyse a variety of tumor cells. That the effector cells in this system are indeed monocytes has been demonstrated (1). Using a short-term (3 to 4 hr) 51Cr-release assay and the single cell conjugate cytotoxic assay, we studied the effects of lymphokine-rich supernatants containing gamma-interferon and partially purified fibroblast interferon on the monocyte cytolytic activity. Overnight incubation of the monocytes in fetal bovine serum-containing medium resulted in a relatively small decrease in cytotoxic activity compared to the one obtained with monocytes incubated in autologous serum. The addition of lymphokines or interferon under both incubation conditions resulted in augmented activity as measured in the 51Cr-release assay. However, the proportions of binding and cytotoxic monocytes, determined in the single cell conjugate assay, did not increase. These results suggest that augmented activity is not due to recruitment of inactive cells. Kinetics studies of tumor cell lysis indicate the increase in killing efficiency is probably due to both an increase in the rate of killing and in the recycling ability of the cytotoxic cells. Using the conjugate/agarose technique, we also demonstrated that excess tumor cells could impair the lytic machinery of freshly isolated monocytes, whereas monocytes treated with lymphokines or interferon partially lost their sensitivity to this inhibitory effect. The ability of tumor cells to impair the lytic machinery of monocytes could be one of the mechanisms by which tumors escape immunosurveillance.