From www.bloodjournal.org by guest on December 29, 2014. For personal use only. Comparative Density Peripheral Blood of the T Cells By A 65.000 dalton strated to nant be T cells, the but surface tory. not (CLI). cells and demon- density nonsecre- to that on cases. normal as well the CLI immunoglobulins flow a usually bearing with of light ent on all normal circulating T lymphocytes, as well greater than 95% of thymocytes. By IF microscopy finding dual size analysis, between (вЂ�ells and (вЂ�dl Heparinized with donors. The RPMI-l640 cell cells.вЂ™ thymus Blood. Vol. from in this fetal the samples (a kind obtained abortion. 10% have presence calf gift 57. No. 4 (April), were serum characteristics a fetus thymus CEM,67 study. of human Child lines, thymus of 14-16 tissue 1981 was wk from 20 isotype (RPC-5. was used adult MoIt-4,68 IIPB- at 37#{176}Cin (FCS) without antibiotics. of original malignant the UCSD, gestation obtained j.g/ml). used in this La Jolla, following during with of cell (8402) to implications of differentiation (a kind gift La Jolla, of Dr. Calif.). IgG used protein of the same Md.) 20 zg/mI). a the (concen- Kensington, Fluores- (FL-RAMIG, as IgG28 Miles secondary Labo- antibody for staining. Preparation Fresh heparinized (Roger bated 20 for centrifuged venous Bellon at 400 g for at Technicon Biological humid atmosphere. minced into the incubated layer over and was collected with 10% fetal N.Y.) was calf serum Veterans FCS, Administration San 5% CO2 finely mononuclear cells Medical of Hematology/Oncology, of (вЂ�alifornia, in (l0% (Grand in 10% viable and overnight at 37#{176}C and placed and 5 ml centri- penicillin-streptomycin tissue Service, layered N.J.) by incubation l% Reagent. for 30 mm at 37#{176}C shed suspension. Division then was then Island. Thymus Research the Department Diego, School of of Medicine, (вЂ�alif Supported in part by the Veterans NOI-(вЂ™B-84250-31. (ontract (;rant and Grand cell collected. Piscataway, lymphocyte were incu- was N.Y.) mixture Inc.. supplemented University La Jolla, Tarrytown. 3 ml of and layer Separator proteins a single and leukocyte and at 400 g. The Co.. with France) ( Lymphocyte This 1-glutamine, Island mixed resuspended, cells. media was Neuilly, 7 mm, Corp.. Cytophilic ml) solution (Pharmacia for 40 mm (вЂ�ancer filing phagocytic Ficoll-I1)paque (10 37#{176}C.The Instruments 1% blood Laboratories, mm 3 ml of an iron Administration. and American National Cancer Society IM-207. Submitted Address Calif.) 92093. (i myeloma (concentration was the to determine [.aboratories, anti-mouse Ind.) to used on the cell surface mouse control rabbit belonging was dalton) Bionetics immunofluorescent Medicine, saline antibody purified as a negative Elkhart. study. corrective patient Foundation, T cells.вЂќ (65.000 A Plasmagel with mouse human [.itton cein-conjugated FCS). 10 7 healthy propagated were of Dr. S. Sarkar. obtained other staining The lymphocyte on a 9-yr-old antigen tration RPMI-1640 a relative T lymphofrom staining Research and for ofT65 From were and the performed specific washed. METHODS CLL and The Clinic a monoclonal fuged hematopoietic cell human thymocytes samples T-cell used with lines o Two Fetal was blood diagnosed leukemic and 8402,69i0 surgery Medicine, clinically to surface cells (Molt-4). regard were low T cells. heterogeneous staining with R. Fox, Scripps of Lines four cardiac (вЂ�enter, peripheral patients ALL,6 AND CLI with discussed. to remove detected normal For purposes of comparison, of T-cell lineage as well as analyzed. MATERIALS The also and the intensity Thymocytes two to normal bright. findings (вЂ�elI as it flow cytometry. we CLL resembling from fluorescence T cells. populations. low-density indirect analyses of peripheral blood lymphofrom patients with CLL were performed to those of normal donors. Utilizing parameter difference cytes. lines were with median circulating similar varied ratories. the density of the T65 surface antigen CLL cells than on normal peripheral In this study, we have confirmed and this a lower three of T65 TIOl. . Histogram cytes (PBL) and compared of subclass, (MoAb) termed TIOl In contrast, T65 was not found on normal B cells, B cell lines, or secretory CLL associated with a circulating M-protein, but was pres- extended Cells Antisera to either K or A type.вЂ™ immunofluorescence (IF) and cytofluorographic analysis4 to differ from normal B lymphocytes by the presence of significantly lower densities of slg. Recently, another antigen, T65, was demonstrated on nonsecretory CLL cells with the use of a previously described monoclonal antibody appeared that was lower on blood T cells. are Normal Leukemia normal density lines on more is a monothe composed these lines had of uniform. peripheral T65 I. Royston and that higher antigen as cell cells (slg), in each case restricted cells were shown by T65 and density lymphocytic the L. Collins, homogeneous on and of Antigen Lymphocytic than malig- detected with lymphocytic leukemia of B lymphocytes surface chain CLL patients thymocytes. all been chronic surface human In HRONIC proliferation clonal from compared lineage. C has M. and of immunofluorescence relative was T cells T-ceII B cells, cells B. Wormsley, of normal T-CeII Chronic previously surface normal By means the blood antigen the and S. immunoglobulin-positive cytometry. CLI specific on of leukemic surface leukemia on T-cell present Human September reprint V-Il 2, 1980; requests lE, 1981 by Grune U(вЂ™SD & Stratton. accepted November to Ivor Royston. School of 18, 1980. M.D.. Medicine, Department La Jolla. of Calif. Inc. ()006--4971/8I/5704--0005$0I.00/0 657 From www.bloodjournal.org by guest on December 29, 2014. For personal use only. WORMSLEY, 658 COLLINS, AND ROYSTON 500 NORMAL 250 I 0: LU 500 -J -J LU B : CLL ____ THYMUS (-) I 250 I 0 0 510 510 0 FLUORESCENCE INTENSITY Fig. 1 . Fluorescence profiles (control versus positive) of peripheral (B). and normal thymocytes (C) stained with TiOl . The arrows designate blood lymphocytes from a normal the median intensity fluorescence donor (A). values. a patient with 400 >- (I) z uJ I- 300 2nd Peak w L) w L) C,) w 0: 200 0 :D -J 1i z C 100 Is, Peak uJ 0 LU Q->-x> ck:( ED a L) tf :i: L) c: L) J Lu NORMDAL Fig. 2. Summary of median :i: :t > -, -вЂ� fluorescence values THYMUS for normal вЂ�aвЂ™ OD LiJ Li CLL intensity - J I- PBL. CLI cells. thymocytes. CELL and cell 0 () LINES lines. CLI From www.bloodjournal.org by guest on December 29, 2014. For personal use only. T65 ANTIGEN ON T CELLS separated by including cell cases Ficoll-Hypaque lines, viability Imni washed were aliquots each resuspended of TIOl FCS and and resuspended secondary sample (positive) inactivated and through and FCS Analysis and was used signal for as analyzed. sis, with were fluorescence determined. the the the percent positive (y-axis). dual axis each cells stained nm) of scatter cells were and analyscatter were on centrally presented (x-axis) as versus representing sample was antibody was TIOl cells with stained with broadly revealed with median blood TiOl are tion of TIOl shows a more cence intensity TIOI shown fluorescence T cells (Fig. distribution. normal PBL cells profiles in Fig. In this study, was 60%-77%. вЂ� consisted fluorescence in patients uniform intensity was of heterogeneous stained point to the Normal peripheral a broad heterogeneous range ofTIOl In contrast, the with nonsecretory show IC). The cells in distribu- two first peaks peak CLL of was was fluoresof low, (median channel 84), and the other higher intensity (median chan- nd 332) staining. Greater were positive for TIOI. median is shown the arrows than 95% of the thymocytes fluorescence intensity of each case in Fig. 2. All CLL cases had a lower value than did normal PBL, while the values for lines varied widely. The median value for the intense thymocyte subpopulation was comparable that for CLL, subpopulation whereas the was comparable more intense to the normal of light (vertical) are presented scatter sedimentation at unit had PBL thymocyte T cells does versus fluoin Fig. 3. not directly gravity.вЂ™3 The cytogram low fluorescence has light In all cases a greater degree and tended to of light be more for thymocytes intensity peak of two distinct size populations. The intensity peak consisted of a single higher popu- case ( Fig. 4 A and B), considered to be of non-T, non-B origin (TIOl slg revealed no TIOl cells above background control. Analysis of the second case , (Fig. 4 C and lating M-protein (4.8%) ofTIOl D), ), which was associated with a circuof lgMK, revealed a small population PBL with a fluorescence distribution for normal T cells. DISCUSSION A 65,000 cells thymocytes lation of cells midway between the other two in relative size. We also examined two cases of CLL shown to be TIOl (Fig. 4). Analysis of the PBL from the first TI 01. cells in patients with CLL (Fig. IB) homogeneous and decreased fluoresthan do normal PBL. The range of 70%-90%. Thymocytes cence intensity (Fig. The studied I (the channels). lA) show of TIOl the RESULTS immunolluorescence degree distributed. that the expected channel positive with positive of (488 located channels reactive percent 50H parameter were The of passed in 2 ml 20,000 profiles 510 of to each as staining. as generation case the the CLL cells than normal then source Although has The of the coincide with the cell size, a positive correlation been found between relative size measured by and description laser signals over of cells percent peak. tested, scatter FCS-AZ. were day horizontal intensity percent first scatter heat- Cytotluorograf by using fluorescence channel in the ml added A detailed each scatter intensity by subtracting from In on fall a 30-mm resuspended Ortho as well size. PBL in each The determined an obtained fluorescence cells 50 il microliters was finally Mass.). intensity relative of cells median control cell Normal Fifty 1:30, Two I 1640-10T. A 5 W argon In addition, of and activation Cytograms histograms number using of for convenience. the out published.вЂ™2 After on the same rsis fluorescence axis. with The azide with over FCS-AZ. Anal a measure the vertical in all sodium stained (control). diluted Wcstwood, has been and washed analyzed carried 0.02% layered as above were Instrunients. the system and x 10 cells/mI. were I640-10. washed Cells was RPC-5 at 0#{176} for 30 mm. Cviofluorographic (Ortho removed FL-RAMIG incubated + of 5.0 were samples in 25 l of 1вЂ™7. formalin. FCS centrifuged. antibody, sample PBL. Seventy-five preparations. Cytograms displaying scatter rescence intensity (horizontal) in l640-l0 0#{176}C,the at cell to staining. 95.. to a concentration of each incubation prior Staining FCS-AZ) 25-il 659 All 3 times than unofluorescent Cells CLL CELLS centrifugation. were was greater ( 1640-l0T AND cell less to in been dalton T-cell-specific shown by Royston et al. antigen termed T65 to be present on slg cells of patients with nonsecretory (absence lating M-protein) CLL. We have compared fluorographic analysis using a monoclonal of circuby cytoantibody, termed TIOI, the relative density of T65 on the surface of normal peripheral blood T lymphocytes and cells from patients with CLL. For further comparison, four cell lines of T-cell lineage and normal human thymocytes were examined. The CLL cells, in all cases studied, had lower surface density of T65 and were of a more homogeneous nature than normal circulating T lymphocytes. The cell lines varied from high-density heterogeneous staining of 8402 to the low-density uniform staining of Molt-4. InvestigatorsвЂ™4вЂ™5 have previously suggested that CLL cells represent immature cells arrested early in B-cell differentiation, manifested in part by the faint immunofluorescent compared to normal nonsecretory CLL staining of slg on the B cells. The presence cells, also in decreased provides further evidence and may indicate arrest lymphocyte diflerentiation. The absence ofTiOl from other B-cell CLL cells of T65 on density, in support of this hypothesis at an even earlier stage reactivity proliferative with diseasesвЂќ abnormal of cells is consistent From www.bloodjournal.org by guest on December 29, 2014. For personal use only. WORMSLEY, 660 I9G2A C0NTF0L COLLINS, AND ROYSTON 1101 Normal PBL CLL 0 C-) Cl) LU N-i Cl) -J -J LU Child Thymus (-) FLUORESCENCE with the hypothesis that these other cies probably represent proliferations tiated B cells.15вЂ™6 Based al.,5 postulate that any further B-cell with a serum gen. (Fig. Consistent 4 C and INTENSITY B-cell malignanof more differen- on this reasoning, CLL that shows differentiation, M-protein, will such as not carry with this hypothesis D), who has вЂњCLLвЂќ Royston evidence cells. (Leu et of an association the T65 antiwas patient associated LK with serum monoclonal 1gM (WaldenstromвЂ™s macroglobulinemia). His leukemic cells stained brightly for 1gM (not shown) and had less than 5% cells reacting with TIOl, cence which profile. Other monoclonal resembled investigators anti-human normal have T cells Fig. 3. Dual parameter analysis of narrow forward angle scatter (y-axis) versus fluorescence intensity bc-axis) of normal PBL. CLI cells, and thymocytes stained with lgG2 (control) and TiOl (positive). in their confirmed the T-cell antibodies fluores- binding of to CLL 95% with Wang et al.вЂ™7 have described a p69,71 I detected by immunofluorescence, ), of normal human sheep erothrocytes on antigen 80%- T cells, which form rosettes (E), and on slg CLL cells from I I of 14 patients. Boumsell et al.вЂ™5 have described a monoclonal antibody, termed A50, which reacts by complement-mediated cytotoxicity with 70%-90% slg CLL ship Leu of E PBL and with greater cells from 17 of 39 patients. of monoclonal 1 await further Recently, Reinherz thymic differentiation escent T-cell staining subset antibodies study. TlOl, than 70% of The relationA50, and anti- et al.вЂ™9 have postulated an intrascheme based on immunofluor- with a series of monoclonal antibodies. Their studies anti-human suggest that From www.bloodjournal.org by guest on December 29, 2014. For personal use only. T65 ANTIGEN ON T CELLS AND 661 CLL CELLS IQG2A T 101 CONTROL Null cell CLL b. gammopathy 0. 1gm Fig. 4. Dual parameter analysis of narrow forward angle scatter (y-axis) versus fluorescence intensity (x-axis) of cells from patients with null cell CLI and monoclonal gammopathy stained with lgG2 (control) and TiOl (positive). d. C. three major compartments of thymic differentiation exist in humans. By virtue ofthe fact that TlOl reacts with greater than 95% of thymocytes, we feel that T65 is present on regardless based all thymus of the stage on our cells in varying of differentiation. own hypothesis fluorescence analysis with anti-Thy-l.2. The majority of mouse thymocytes are small (90%) and large (5%) cells with high and intermediate levels of Thy-l.2, respectively. The third population of medium sized cells (5%) has low Thy- I .2 surface density. Weissman amounts Furthermore, that cells with less surface T65 are arrested at an earlier stage of differentiation, Molt-4 may be considered to be less differentiated than the other T-cell lines examined (Fig. 2). This notion is consistent with the findings of Reinherz et al.вЂ™9 that Molt-4 expresses tiation antigens than CEM. Our studies showed three cytes, the majority uniform, low-density medium sized cells staining, subgroups strated fewer size being small T65 and with higher, resembling of mouse by Fathman monoclonal stage II differen- populations of thymo- and large cells with a third population of more heterogeneous circulating T cells. Similar thymocytes have been demonet al.вЂ™3 using light scatter and et al.2#{176} have cytes high surface and Thy- further characterized the mouse thymo- shown that the small and large cells with I .2 are derived from the cortex and have low H-2, while the medium sized cells are medullary thymic lymphocytes and have the same phenotype (low Thy-l.2, high H-2) as peripheral T cells. We propose that the weaker staining populations in the human thymus may also represent cortical thymocytes and are in early stages more mature thymocytes, cytes, make up now underway frozen sections studies. the of differentiation, perhaps medullary brighter population. while the thymoStudies are to both localize of thymus and these subpopulations on sort them for functional marker lymphoproliferative REFERENCES I . 2. tors. 3. AC. Aisenberg neoplastic Pernis Ann Bloch lymphocytes. B, Forni NY Acad PreudвЂ™homme KJ: N EngI L, Amante Sci 190:420, JL. 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For personal use only. 1981 57: 657-662 Comparative density of the human T-cell antigen T65 on normal peripheral blood T cells and chronic lymphocytic leukemia cells SB Wormsley, ML Collins and I Royston Updated information and services can be found at: http://www.bloodjournal.org/content/57/4/657.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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