Isolation and electrophoretic characteristics of 3 lymphocyte populations of normal human blood]

Cell electrophoresis allows separation of normal human blood lymphocytes into two main groups which are a function of their relative rates of migration, with regard to the reference speed (1 mum.sec.-1V-1.cm): the lymphocytes which have a greater mobility than this value seem to be T-lymphocytes (80,1 per cent for 42 healthy adults); on the contrary, B-lymphocytes have an inferior mobility (19,9 per cent). Two known methods are used for the selection of the lymphoid populations: spontaneous rosetting with sheep's red blood cells, which are characteristic of T lymphocytes, and adherence to nylon wool columns, which is dominant in the case of B-lymphocytes. This method confirms the fact that T-lymphocytes have a rapid migration and B-lymphocytes a slow migration. We have isolated a third population, having neither the T markers, nor the B markers. It has a very homogeneous migration, centered on the two classes 1,05 and 1,10 mum.sec.-1.V.-1.cm.     

Electrophoretic mobility of human lymphocytes purified by nylon columns and spontaneous rosettes]

Cell electrophoresis enables the separation of the lymphocytes in normal human blood into two principal groups, as a function of their migration speed in relation to 1 mum.sec -1..v-1.cm. In 42 healthy adults, 19,9 % of the lymphocytes have a slower migration, and 80,1 % a faster migration than the reference speed. Two known methods are used for the selection of the lymphocytic populations : spontaneous rosetting with sheep red blood cells, a property of the T lymphocytes, and the adherence to nylon wool columns, which is dominant in the case of B lymphocytes. The cells which do not form spontaneous rosettes, and the cells adhering to nylon wool columns have above all a slow migration. On the contrary, cells which do not adhere to nylon columns have a fast migration. These arguments are in favour of the T nature of the rapid migrating lymphocytes, and of the B nature of the slow migrating lymphocytes.     

人和猴T淋巴细胞表面TRBC受体及其配体不同于E2分子和CD2的配体

CD2 (E receptor, LFA-3 receptor) and E2 molecules (Bernard, 1988) on human T lymphocytes, CD58 (LFA-3, lymphocyte function associated antigen 3) on human erythrocytes and S14,S42,S110-220 molecules (Bernard, 1987) of sheep erythrocytes are involved in rosette formation of human T lymphocytes with human or sheep erythrocytes. Rosette formation of human and macaque pan-T lymphocytes with tree shrew (Tupaia belangeri) red blood cells (TRBC) (TRBC rosette) has shown different physicochemical properties from that of rosette formation with sheep red blood cells (E rosette) (Ben, 1985). CD2, CD3/TCR complex, CD5, CD6, and CD7 are not involved in TRBC rosette formation (Zheng, 1990). In order to know whether E2, LFA-3,S14,S42 and S110-220 molecules are involved in TRBC rosette formation or human and macaque T lymphocytes, rosette inhibition and antigenic modulation or co-modulation were performed with relevant monoclonal antibodies (McAbs), and hemolytic assay and slide agglutination were also conducted. TRBC rosette formation of human and rhesus monkey PBL was not blocked by E2 McAb (inhibition rate 2.8% and 2.1%, respectively). In contrast, human E rosette formation was obviously blocked at inhibition rate of 49.8% and macaque E rosette formation was slightly inhibited (13.3%). The modulation or co-modulation of E2 molecule with E2 McAb did not affect human TRBC rosette formation. Similar results were shown in rosette formation inhibition of Jurkat cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histidine-rich glycoprotein blocks T cell rosette formation and modulates both T cell activation and immunoregulation

Histidine-rich glycoprotein (HRGP) is a plasma and platelet protein with undefined function in vivo. It has been reported to inhibit rosette formation between murine T cells and erythrocytes. We have shown that HRGP binds specifically to human T lymphocytes but not sheep erythrocytes and have demonstrated a 56-kDa HRGP-binding protein on the T cell surface which is distinct from the CD2 sheep erythrocyte receptor. We have now investigated whether HRGP can inhibit human T cell-sheep erythrocyte rosette formation and whether HRGP can modulate T cell activation. HRGP at physiologic concentrations specifically inhibited rosette formation between human T lymphocytes and sheep erythrocytes. HRGP suppressed proliferation of antigen receptor (CD3)-triggered T cells induced by interleukin 2; this suppression was specifically reversed by prior incubation of HRGP with affinity-purified anti-HRGP IgG. Addition of HRGP 12-24 h after CD3 triggering no longer suppressed T cell proliferation, suggesting HRGP suppressed T cell division by interfering with one or more early events in the process of T cell activation. Human serum (containing 100-150 micrograms/ml HRGP) was also capable of suppressing T cell proliferation; serum which had been immunodepleted of HRGP no longer inhibited T cell proliferation. Furthermore, HRGP inhibited interleukin 2 receptor expression on activated T cells, causing decreased T cell interferon-gamma release and altered T cell-dependent inhibition of erythropoiesis. HRGP is thus capable of modulating T cell activation and T cell immunoregulation; HRGP may function as a natural suppressive regulator of human T lymphocyte activation.     

Human autologous rosette-forming cells. III. Binding of erythrocytes from different species to the T-cell receptors for autologous red blood cells

Spontaneous rosette formation in humans is restricted to a subpopulation of the circulating T cells. We have previously shown that the interaction between lymphocytes and autologous red blood cells (auto-RBC) is not mediated by a self-recognition mechanism, since allogeneic (allo-) RBC bind to T cells through the same receptors. In this work, we have extended these observations to thymocytes. Using a mixed-rosette assay in which one type of erythrocyte was identified by FITC labeling, we have shown that almost all the thymocytes which attached auto-RBC could also fix allo-RBC. However, as for the peripheral blood lymphocytes (PBL), binding of human RBC to thymocytes occurred with varying affinities according to the erythrocyte's origin. In order to further study the specificity of the erythrocyte to lymphocyte binding in rosette formation, PBL were mixed with auto-RBC and erythrocytes of xenogeneic (xeno-) origin. Although very disparate incidences of rosettes were found according to the species from which the RBC were derived, most of the autorosetting lymphocytes also had receptors for xeno-RBC. In addition, preincubation of PBL with monoclonal antibody OKT11A (directed against the sheep RBC receptors on T cells) completely abrogated rosette formation with all the erythrocytes tested (human auto- and allo-, sheep, pig, and rabbit) except mouse RBC. Taken together these data strongly suggest that human auto- or allo-, as well as sheep or some other xeno-RBC, bind to T lymphocytes by a single receptor and that the combining sites are expressed with different densities or varying affinities depending upon the RBC's origin. Therefore, spontaneous autorosettes may represent T lymphocytes having high-affinity receptors for sheep RBC.     

Identification of human peripheral T cell antigens.

A new type of differentiation antigens on human T cells was demonstrated by using a heterologous anti-human T cell serum (ATS). This type of antigen, referred to as human peripheral T cell antigen (HPTA), was found on peripheral T cells and medullary thymocytes, but not on cortical thymocytes and B cells. The percentage of ATS-reactive lymphocytes in human peripheral lymphoid organs was correlated with that of cells rosetting with sheep erythrocytes, but contrasted with the number of B cells defined by the presence of a complement (C) receptor or by rabbit anti-human B cell serum (ABS). ATS also reacted with T cells purified by nylon fiber column filtration but ABS did not. Chronic lymphocytic leukemia cells rosetted with either sheep erythrocytes or erythrocyte-antibody-complement complexes were lysed by ATS and ABS, respectively. Mitogenic responses of blood lymphocytes to phytohemagglutinin (PHA) and concanavalin-A (Con A) were abrogated by treating them with ATS and C, whereas ABS suppressed only their response to Con A. Although numerous thymus cells rosetted with SRBC, only 14% were reactive with ATS. Quantitative absorption studies demonstrated that HPTA content of the thymus cells was much lower than that of lymph node cells. Anatomical localization of ATS-reactive lymphocytes in human lymphoid organs studied by immunofluorescence indicated that they were present in the thymus-dependent paracortical areas of lymph node and in the medullary region of thymus. ABS, on the other hand, did not stain thymocytes but reacted selectively with the cells located in the lymphoid follicles of lymph node. These data, together with that from cell suspension studies, confirmed that HPTA were shared between medullary thymocytes and peripheral T cells.     

A human lymphoid cell line with receptors for both sheep red blood cells and complement

The human lymphoid cell line MOLT 4, from a patient with acute lymphocytic leukemia, was initially considered to be derived from T lymphocytes, on the basis of rosette formation with sheep erythrocytes (E). This cell line has now also been found to form rosettes with sheep erythrocytes sensitized with rabbit antibody and mouse complement (EAC). Evidence is presented that the formation of both E and EAC rosettes is due to two separate receptors on the MOLT cells: (a) EAC rosettes were formed more rapidly and were more stable than E rosettes; (b) preincubation of MOLT with an EAC membrane preparation inhibited resetting with EAC and not with E; (c) MOLT formed rosettes with EAC prepared from trypsinized E, but did not bind to trypsin-treated E alone. The implications of this finding, in regard to the derivation of this cell line, are discussed.     

Detection of human B cells and chronic myelocytic leukemic cells by rosette formation with sheep erythrocytes and fresh human sera.

A new method of detecting the C3b receptor is reported. A particular merit of this method is that anti-RBC rabbit antiserum is not required. Rosettes were formed with human B lymphocytes, B lymphoblasts and granulocytes, using sheep erythrocytes (SRBC) sensitized with fresh human serum (FHS). T lymphocytes and T lymphoblasts did not form rosettes. The percentage of cells forming rosettes with this method approximated the percentage of rosettes formed with EACm. However, FHS coated SRBC did not react with most cells of B cell type chronic lymphocytic leukemia (CLL), whereas EACm rosette formations showed a definite reaction. On the other hand, 34--58% of cells of chronic myelocytic leukemia (CML) bound with the indicator red cells. SRBC sensitized with fresh rabbit or guinea pig serum formed rosettes with PBL, tonsil cells, B lymphoblasts and granulocytes. Complement and IgM antibody were required for this reaction, as in EAC rosette formation.     

Serologic cross-reactivity of T11 target structure and lymphocyte function-associated antigen 3. Evidence for structural homology of the sheep and human ligands of CD2

T11 target structure (T11TS) and lymphocyte function-associated antigen (LFA) 3 are the cell-surface glycoproteins on sheep and human erythrocytes (E) binding to cluster differentiation 2 (the E-receptor) on T cells in E rosette formation. Here we show that this functional cross-reactivity is most likely due to a structural homology of these molecules. A rabbit antiserum to sheep T11TS is shown to cross-react with LFA-3 in several independent assays: (a) rabbit anti-T11TS antiserum blocks the formation of E rosettes by human T cells with both autologous and xenogeneic (sheep) E by binding to the respective E; (b) the antiserum blocks the binding of anti-LFA-3 monoclonal antibody to human E; and (c) it reacts with purified LFA-3 in Western blotting. Together, these findings demonstrate that T11TS on sheep E and LFA-3 on human E are serologically related, providing further support for the notion that T11TS and LFA-3 are the sheep and human forms of the same cell interaction molecule.     

Human T and B lymphocyte rosette tests: Effect of enzymatic modification of sheep erythrocytes (E) and the specificity of neuraminidase-treated E

Sheep erythrocytes (E) were treated with papain or neuraminidase to evaluate what effect these enzymes would have on the E rosette test for human T lymphocytes. Few or no rosettes were detected with sheep erythrocytes treated with papain (E_P). Sensitization of E_P with rabbit antibody and mouse complement resulted in a rosette indicator (E_PAC) which could be used to detect peripheral blood complement receptor lymphocytes (CRL) and thymocyte CRL without having to perform the rosette assay at 37 °C. Neuraminidase treatment of E (E_N) enabled the detection of approximately 20% more rosette-forming cells (RFC) in human peripheral blood lymphocyte (PBL) suspensions compared to untreated E. Rosette studies on a patient with severe combined immunodeficiency disease and on human lymphoblastoid cell lines showed that the additional rosettes detected with E_N were T lymphocytes.     

Regulation of self-recognition by T-cell hybrids

Somatic cell hybrids were prepared between BW 5147, an AKR T lymphoma, and purified T cells from three sources: spleen cells exposed to sheep red blood cells, lymph node cells from mice sensitized to ovalbumin, and spleen cells of mice injected with azobenzenearsonate-IgG. Hybrid lines expressed constitutive markers of both parents which include H-2 antigens and the isoenzymes glucose phosphate isomerase and isocitrate dehydrogenase. Furthermore, they expressed both parental alleles of Thy 1, a differentiation antigen. Many of the hybrid lines formed rosettes with mouse erythrocytes. T-cell hybrids did not bind human or chicken red blood cells, though they did rosette with sheep erythrocytes to the same extent as with mouse red cells. We interpret the latter reaction as due to recognition of shared antigens by the murine T cells. This form of self-recognition is influenced by culture conditions and is expressed optimally by cells in late logarithmic phase of growth.     

The active E rosette test: correlation with delayed cutaneous hypersensitivity.

An active subpopulation of peripheral blood T lymphocytes, characterized by rapid (5-min) rosette formation with sheep erythrocytes (A-RFC), was measured in normal individuals after they were skin tested with microbial antigens. A significant rise in A-RFC occurred in all individuals who developed positive delayed cutaneous hypersensitivity (DCH) reactions, whereas skin test nonresponders showed no significant rise in A-RFC. No similar consistent changes occurred in populations of total T cells, characterized by longer (60-min) rosette formation with sheep erythrocytes, or in B cells, measured by immunofluorescence of surface immunoglobulin. The A-RFC response paralleled the DCH response in timing, but not in intensity. These results provide in vivo evidence for a biologically distinct T cell subpopulation, and focus attention on the A-RFC as immunologically active cells.     

Trypanosoma cruzi: Effect on B-cell-responsive and -responding clones

During the course of Trypanosoma cruzi infection in C57BL/6 mice, which are relatively resistant to the parasite, the hosts developed antibody activity against previously unencountered antigens. The anti-sheep erythrocyte and antitrinitrophenyl antibody levels increased rapidly from Day 7 of infection, reached a peak by the 21st day, and were maintained at this level through 120 days postinfection in these mice. In contrast, highly susceptible C3H(He) mice did not have demonstrable antibody responses to SRBC or TNP during the 24-day infection period. Autoantibody activity against the selfantigens presented on isologous erythrocytes or thymocytes, however, were reduced in infected C57BL/6 mice. No significant reduction in autoreactivity to the self-antigens on erythrocytes or thymocytes was observed in C3H(He) mice infected with T. cruzi although a trend of reduced autoresponsiveness toward erythrocytes appeared to be developing by the time of death. C57BL/6 mice immunized with sheep erythrocytes as neonates and infected with T. cruzi as adults, or adult mice primed with low doses of sheep erythrocytes prior to infection, had elevated antibody responses to sheep erythrocytes unless the mice were immunized with sheep erythrocytes during the course of infection, in which case suppression of the response against sheep erythrocytes resulted. The nonspecific synthesis of immunoglobulins in infected C57BL/6 mice was, in part, a result of the lymphocyteactivating properties of T. cruzi-associated antigens. The T. cruzi-associated antigens induced proliferative and differentiative responses in spleen cells in vitro. It is proposed that the T. cruzi-associated antigens differentially affect lymphocytes capable of responding to antigen and those lymphocytes previously stimulated by antigen.     

Migration inhibitory factor (MIF) and proliferative responses by human lymphocyte subpopulations separated by sheep erythrocyte rosette formation.

Human peripheral blood lymphocytes were separated by a combination of rosette formation with sheep erythrocytes and differential density centrifugation into subpopulations of rosette positive (T-enriched) cells and rosette negative (T depleted) cells. These were then tested in vitro for the production of macrophage migration inhibitory factor (MIF) and for incorporation of ³H-thymidine in response to specific antigens. Both T enriched and T depleted cell populations produced MIF but only T enriched cells exhibited significant antigen-induced ³H-thymidine incorporation. These findings using a T cell surface marker as the basis for cell separation, a technique which should not alter the B cell surface, confirm an earlier report in which human cells were separated on the basis of surface immunoglobulin, a B cell marker.     

Lymphocyte populations of Callithrix jacchus marmosets.

A lymphocyte population of common marmosets (Callithrix jacchus) was identified by rosette formation with African green monkey erythrocytes; the rosette-forming cells appeared to be T lymphocytes, as approximately 62% of circulating lymphocytes and 85% of thymus cells formed rosettes with African green monkey erythrocytes. In addition, common marmoset lymphoid cells carrying T-lymphotropic Herpesvirus saimiri or Herpesvirus ateles formed rosettes with African green monkey erythrocytes and treatment of common marmoset circulating lymphocytes with an anti-T cell serum and complement (C′) eliminated rosette-forming cells. Common marmoset T lymphocytes apparently carry a surface receptor for African green monkey erythrocytes, but unlike humans and other closely related nonhuman primates, T lymphocytes of common marmosets fail to form rosettes with sheep erythrocytes.     

New electrophoretic information on the surface composition of the rat erythrocyte

Microelectrophoretic determinations of erythrocyte surface charge at low pH conducted in different laboratories have given conflicting results with regard to the existence of a positive mobility branch in the mobility-pH curve. The electrokinetic properties of glutaraldehyde-fixed rat erythrocytes below their isoelectric point (pl ≈ 2) have, therefore, been studied by means of an independent method—stable-flow free boundary (STAFLO) electrophoresis. All experiments were conducted in NaClHCl solutions adjusted to pH 1.2 and ionic strength 0.145. Using transverse scattered light to view the migration pattern of the single-cell sample band, the electrophoretic mobilities for single cells were found to be in the range +0.96 to +1.01 μm/sec/V/cm. This was in good agreement with the value +0.84 ± 0.21 (SD) μm/sec/ V/cm measured by conventional microelectrophoresis using a laterally oriented rectangular chamber. The existence of a large positive surface charge was confirmed by two additional STAFLO optical methods. From the measured STAFLO migration pattern, cellular sedimentation velocities were also calculated and found to be in the range 1.05–1.12μm/sec.     

Numbers of rosette formine cells in human peripheral blood.

Thymus-derived (T-cell) and “bursal” derived (B-cell) lymphocytes in human peripheral blood were quantitated by assaying percentages of cells forming erythrocyte rosettes. T-cell rosettes were formed with neuraminidase treated sheep erythrocytes. B-cell rosettes were formed with complement coated sheep erythrocytes. Large differences in the percentages of T-rosette forming cells were noted depending on the method used to assay these cells. When rosette forming cells (RFC) and non-RFC were counted concurrently the percentage of T-cell rosettes were 53–75% whereas methods involving the separate counting of RFC and total cells gave T-cell RFC percentages of 23–40%. These differences were due to the “co-rosetting” of non-RFC into the T-cell rosette clusters. This occurred because of the gentleness required to resuspend the fragile T-cell rosettes. “Co-rosetting” was demonstrated by forming stable complement receptor rosettes with complement-coated human erythrocytes and resuspending them either gently or vigorously. Significantly higher percentages of rosettes were noted with gentle cell suspension than with vigorous resuspension. The percentages of rosette forming T-cells in human peripheral blood are therefore lower than previously estimated.     

Human Antigen-Presenting Cells: Characterization of the Cells in the T-Lymphocyte Proliferative Response

Human antigen-presenting cells (APC) which present the antigen to T lymphocytes resulting in a T-lymphocyte proliferative response were found among peripheral mononuclear cells (MNC), by employing purified protein derivative (PPD) as soluble antigen. To assess the adherence capacity of human antigen-presenting cells, MNC were separated by plastic Petri dishes or nylon wool columns. Plastic nonadherent cells were almost equivalent to unseparated cells in antigen-presenting ability. Plastic adherent cells, however, showed better antigen-presenting ability than unseparated cells. On the other hand, cells passed over nylon wool columns showed essentially no ability to present PPD to T lymphocytes. Removal of phagocytic cells by carbonyl iron resulted in about 50–70% reduction in antigen-presenting ability. Carrageenan, which is known to be toxic to macrophages, had no effect on APC. By using both rabbit anti-human Ia-like antiserum and alloantiserum specific for HLA-DR phenotype and complement, it was shown that APC possessed Ia-like antigens, whereas they did not bear surface immunoglobulins. These results indicate that the human APC is probably a cell in the monocyte-macrophage lineage. Allogeneic MNC were used as APC in order to determine whether any genetic restriction exists between MNC as APC and responding T lymphocytes. Optimal stimulation was shown to require identity of mixed leukocyte reaction (MLR)-activating determinants between APC and T lymphocytes. It is, however, obscure whether an HLA-D region restriction exists in these combinations because PPD-pulsed allogeneic MNC lost their ability to elicit even MLR. It is possible that this failure to elicit MLR was caused by T lymphocytes among the MNC used as APC.     

Rosetting of human T lymphocytes with sheep and human erythrocytes. Comparison of human and sheep ligand binding using purified E receptor

Previous studies have shown that the purified T lymphocyte glycoprotein, cluster differentiation 2 (CD2) (also known as T11, lymphocyte function-associated antigen (LFA)-2, and the erythrocyte (E) rosette receptor) interacts with the LFA-3 molecule on human E. We have examined the interaction of the purified CD2 molecule with the T11 target structure (T11TS) molecule on sheep E, and compared the two interactions. Purified, 125I-labeled CD2 bound to sheep E and the binding was inhibited by anti-T11TS monoclonal antibody (mAb). Reciprocally, the binding of T11TS mAb to sheep E was inhibited by pretreatment of sheep E with purified CD2. High concentrations of purified CD2 aggregated sheep E, possibly by inserting into the membrane, and the aggregation was inhibited by T11TS mAb. The affinity and number of binding sites for purified CD2 on sheep and human E was found to be similar, with Ka of 9 X 10(7)/M and 6 X 10(7)/M and 9800 and 8300 CD2 binding sites/E, respectively. Thus, the human T lymphocyte CD2 molecule is a receptor that cross-reacts between LFA-3 on human E and T11TS on sheep E, suggesting that LFA-3 and T11TS are functionally homologous ligands. As measured by saturation mAb binding, there are 8100 and 3900 ligand molecules/sheep and human E, respectively. Human and sheep E have surface areas of 145 and 54 micron 2, respectively. The 3.2- to 5.6-fold higher ligand density on sheep E appears to account for the ability of sheep but not human E to rosette with certain types of human T lymphocytes.     

Effects of anti-beta-2 microglobulin antibodies on human lymphocytes.

Anti-β2 microglobulin antisera prepared in rabbits immunized with β2m purified from the urine of a single patient were cytotoxic for human T and B lymphocytes of all donors tested; lymphocytotoxicity could be fully inhibited by all human sera tested, not by serum from other animal species. Anti-β2 microglobulin antibodies and their F(ab′)2 fragments had little effect on E and EAC rosette formation, suggesting that β2m is not closely associated with receptors for sheep erythrocytes on T lymphocytes or receptors for C3 on B cells. Anti-β2m IgG and F(ab′)2 fragments inhibited EA rosette formation though the latter did not impair lysis of antibody-coated xenogeneic erythrocytes by lymphocytes bearing receptors for the Fc portion of IgG. Some of the antisera had a mild mitogenic effect, all of them inhibited mitogen and antigen-induced lymphocyte proliferation at high concentrations whereas they potentiated these responses at low concentrations. In mixed lymphocyte cultures pretreatment of responding cells markedly depressed the response whereas coating of stimulating cells with β2m antibodies had little or no effect.