Pilar Garcia-Peñarrubia

A study of CD33 (SIGLEC-3) antigen expression and function on activated human T and NK cells: two isoforms of CD33 are generated by alternative splicing

The expression of CD33, a restricted leukocyte antigen considered specific for myeloid lineage, has been studied extensively on lymphoid cells. We demonstrated that wide subsets of mitogen‐ or alloantigen‐activated human T and natural killer (NK) cells express CD33 at protein and nucleic acid levels. CD33+ and CD33– T and NK cell populations showed identical surface expression of activation markers such as CD25, CD28, CD38, CD45RO, or CD95. Myeloid and lymphoid CD33 cDNA were identical. However, lymphoid CD33 protein had lower molecular weight, suggesting cell type‐specific, post‐translational modifications. Additionally, reverse transcriptase‐polymerase chain reaction and Northern blot analysis showed an unknown CD33 isoform (CD33m) expressed on all CD33+ cell lines or T cell clones tested. CD33m was identical to CD33 (CD33M) in the signal peptide, the immunoglobulin (Ig) domain C2, the transmembrane, and the cytoplasmic regions but lacked the extracellular ligand‐binding variable Ig‐like domain encoded by the second exon. CD33m mRNA was mostly detected on NKL and myeloid cell lines but poorly expressed on B cell lines and T lymphocytes. The CD33m extracellular portion was successfully expressed as a soluble fusion protein on transfected human cells, suggesting a functional role on cell membranes. Cross‐linking of CD33 diminished the cytotoxic activity of NKL cells against K562 and P815 target cells, working as an inhibitory receptor on NK cells. These data demonstrate that CD33 expression is not restricted to the myeloid lineage and could exist as two different splicing variants, which could play an important role in the regulation of human lymphoid and myeloid cells.

Epitope mapping, expression and post-translational modifications of two isoforms of CD33 (CD33M and CD33m) on lymphoid and myeloid human cells

We have tested the usefulness of several commercial anti-CD33 monoclonal antibodies (mAb) to determine the expression and localization of the two CD33 isoforms on several hematopoietic cell lines. The expression of the isoform CD33m, a CD33 transmembrane splice variant lacking the ligand-binding V immunoglobulin (Ig)-like domain, was detected by RT-polymerase chain reaction, western blot, confocal microscopy and flow cytometry on the membrane of several human cell types. CD33m was only detected by the anti-CD33 mAb HIM3-4 on the cell surface, whereas WM53, P67.6, 4D3, HIM3-4, WM54, D3HL60.251 or MY9 detected the CD33M isoform, indicating that HIM3-4 is the only mAb recognizing CD33 C(2) Ig domain. Accordingly, HIM3-4 binding to CD33 did not interfere with the binding of other antibodies against the CD33 V-domain. P67.6 mAb interfered with recognition by the rest of antibodies specific for the V domain. HIM3-4 staining could be increased after the sialidase treatment of all CD33(+) cells. However, this increase was stronger in activated T cells, suggesting a CD33 masking state in this cell population. Confocal microscopy analysis of CD33m HEK 293T-transfected cells revealed that this protein is expressed on the cell membrane and also detected in the Golgi compartment. CD33 is constitutively located outside the lipid raft domains, whereas cross-linked CD33 is highly recruited to this signaling platform. The unique ability of HIM3-4 mAb to detect the masking state of CD33 on different cell lineages makes it a good tool to improve the knowledge of the biological role of this sialic acid-binding Ig-like lectin.

Effect of conjugate size on the kinetics of cell-mediated cytotoxicity at the population level

We developed a model for the kinetics of target cell lysis by cytotoxic T-lymphocytes which accounts for most facts observed at the population level. In contrast to previous models, the following facts: conjugate frequency of cytotoxic T-lymphocytes bound to target cell, dependence of this frequency on the lymphocyte-to-target ratio (R), variation of R with time as target cells are destroyed, and population distributions of the different types of conjugates formed between lymphocytes and target cells, which are involved in the kinetics of these kinds of effector-target systems have been contemplated in the model. The relationship with effector-kinetic analogy models for the lytic process has been discussed. Predictions of the model have been explored and compared with experimental observations about target cell lysis reported in the literature.

Inflammatory status in human hepatic cirrhosis

This review focuses on new findings about the inflammatory status involved in the development of human liver cirrhosis induced by the two main causes, hepatitis C virus (HCV) infection and chronic alcohol abuse, avoiding results obtained from animal models. When liver is faced to a persistent and/or intense local damage the maintained inflammatory response gives rise to a progressive replacement of normal hepatic tissue by non-functional fibrotic scar. The imbalance between tissue regeneration and fibrosis will determine the outcome toward health recovery or hepatic cirrhosis. In all cases progression toward liver cirrhosis is caused by a dysregulation of mechanisms that govern the balance between activation/homeostasis of the immune system. Detecting differences between the inflammatory status in HCV-induced vs alcohol-induced cirrhosis could be useful to identify specific targets for preventive and therapeutic intervention in each case. Thus, although survival of patients with alcoholic cirrhosis seems to be similar to that of patients with HCV-related cirrhosis (HCV-C), there are important differences in the altered cellular and molecular mechanisms implicated in the progression toward human liver cirrhosis. The predominant features of HCV-C are more related with those that allow viral evasion of the immune defenses, especially although not exclusively, inhibition of interferons secretion, natural killer cells activation and T cell-mediated cytotoxicity. On the contrary, the inflammatory status of alcohol-induced cirrhosis is determined by the combined effect of direct hepatotoxicity of ethanol metabolites and increases of the intestinal permeability, allowing bacteria and bacterial products translocation, into the portal circulation, mesenteric lymph nodes and peritoneal cavity. This phenomenon generates a stronger pro-inflammatory response compared with HCV-related cirrhosis. Hence, therapeutic intervention in HCV-related cirrhosis must be mainly focused to counteract HCV-immune system evasion, while in the case of alcohol-induced cirrhosis it must try to break the inflammatory loop established at the gut-mesenteric lymph nodes-peritoneal-systemic axis.

Effector-target interactions: saturability, affinity and binding isotherms a study of such interactions in the human NK cell-K562 tumour cell system

Effector-target interactions at the cell-to-cell level have been studied. This has revealed that saturability, i.e., the existence of a finite number of specific receptor sites, applies to both the effector and target cell populations and plays a key role in the formation of conjugates. As a result, two maximum conjugate frequencies, alpha max and beta max, are recognised for the effector and target cell populations, respectively. The dissociation constant of the conjugates formed, KD, characterizes effector-target affinity. This constant, together with the maximum conjugate frequencies, are the three parameters which make it possible to describe the binding process quantitatively. The existence of binding isotherms for effector-target interactions has been demonstrated. These isotherms contain all the relevant information necessary to interpret quantitatively the formation of conjugates. Quantitative procedures to determine the three binding parameters are described together with the modifications necessary to use Scatchard plots in the analysis of conjugate frequencies in these kinds of cell-to-cell interactions. A quantitative study of these interactions in the NK-K562 tumour cell system has been performed. For this purpose, nine different cell source donors were used to test the model proposed. Relationships with related phenomena--CMC and the adhesion process--are also discussed.

The maximum conjugate frequency (αmax) characterizes killer cell populations

Abstract A quantitative procedure to characterize NK cell populations based on the dependence of the frequency of conjugation (α) on the effector-to-target ratio ( R ) is shown. To this end, a detailed study of the influence exerted by: (a) the value of R ; (b) the number of effector and target cells ( N , T ); and (c) the source (donor) and enrichment of the effector cell population on the frequency of conjugation between NK effector and K562 target cells has been performed. This has demonstrated that for a given value of R large differences in the values of α can be obtained for different donors and/or N values. Hence, the usual practice of reporting the frequency of conjugation at a given value of R cannot be used as a valid criterion for comparison, and this could explain the differences in the α values reported in the literature for the same effector-target system. Moreover, the frequency of conjugation depends on the enrichment of the effector cell populations, although it has been shown that in all cases a plot of 1/α vs. R for N = constant is always linear with intercept 1/ α max . α max represents the maximum frequency of conjugation for an effector-target system and remains constant for all values of R and N , and is also independent on the donor of the cell source. These characteristics make that the values of α max can be used as an easy criterion to determine with accuracy conjugate frequencies in an effector-target system, and could also be applied to characterize the activation or inhibition of effector cell populations by monoclonal antibodies or other agents. This criterion was applied to characterize the enrichment of NK cell populations and so, a value of α max = 58 ± 3% has been obtained when highly purified (⩾ 99%) NK effector cells obtained by panning with the monoclonal antibodies Leu-2, Leu-3 and Leu-4 are used. However, the corresponding value for MDC (14% NK cells) was lowered to 26 ± 1%.

Determination of parameters that characterize effector-target conjugation of human NK and LAK cells by flow cytometry.

Effector-target conjugation between different cell populations of human NK cells and K562 tumor cells has been studied from binding isotherms obtained from data of effector (alpha) and target (beta) conjugate frequencies measured by flow cytometry analysis at different effector-to-target ratios. Non-linear and linear regression methods were applied to these isotherms to calculate the binding parameters that characterize the process of conjugation, namely, the maximum effector and target conjugate frequencies, the dissociation constant of the conjugates formed, the binding units and the area under the binding isotherms. The results obtained show that: (1) flow cytometry analysis of effector-target conjugation is faster, unbiased and more suitable than microscopic counting of conjugates, thereby permitting the analysis of larger number of conjugates in shorter times, (2) the binding parameters derived from conjugate frequencies obtained by flow cytometry analysis differ from those obtained by microscopy, (3) the discrepancies between the two methods are due to the presence of several cells engaged in multicellular conjugates that are detected as single particles by flow cytometry and (4) the analysis of population distributions of the conjugates formed at different values of the effector-to-target ratio permit the above discrepancies to be corrected.

Model for population distributions of lymphocyte-target cell conjugates

A quantitative model for the population distributions of the different types of conjugates formed between cytotoxic T lymphocytes and target cells has been developed. The comparison of the theoretical predictions with data of the literature reveals that the transit populations among the different types of conjugates depends on the lymphocyte-to-target ratio, R, and two constants, k and k1. These constants (where k greater than k1) govern, respectively, the transit populations among conjugates of the type LTi (LTn----LTn-1----...LT), and among LjT conjugates (LT----L2T----...----LmT). We have found that high ratios are necessary to obtain conjugates where multiple T lymphocytes are bound to one target cell, and that under these conditions the predominant conjugate, LjT, varies according to j = 1 + k1R. Conversely, for low values of R the predominant population is of the type LTi, where i also shows a linear dependence on R. Our model explains also why the conjugate LT is normally the predominant population under the experimental conditions reported in the literature. A discussion of the influence exerted by the population distributions of lymphocyte-target cell conjugates on the kinetic of the lytic process for these kinds of effector-target systems has also been made.

Study of the physical meaning of the binding parameters involved in effector–target conjugation using monoclonal antibodies against adhesion molecules and cholera toxin

In earlier work, we established a mathematical model to characterize the binding properties of cytotoxic cells to target cells. These properties can be described by the values of the maximum effector and target conjugate frequencies, alpha(max) and beta(max), respectively, and the dissociation constant of the conjugates formed, K(D) (Garcia-Peñarrubia, P., Cabrera, L., Alvarez, R., and Galvez, J., J. Immunol. Methods 155 (1992) 133). Here, we address the problem of exploring the physical meaning of these parameters and their relationships with cytotoxicity. With this purpose, conjugation between a human leukemic NK cell line (NKL) and K562 tumor cells has been studied from binding isotherms obtained from data of effector (alpha) and target (beta) conjugate frequencies measured by flow cytometry analysis at different effector-to-target ratios (R). The results have been compared to those obtained after target cells treatment with monoclonal antibodies recognizing adhesion molecules ICAM-1 (CD54) and LFA-3 (CD58) (which are able to block some of the receptors implicated in conjugation), as well as with cholera toxin (CTX) that can modify the state of affinity of some adhesion molecules such as LFA-1 (CD11a/CD18). The results show that: (1) blocking adhesion receptors CD54 and CD58 on the surface of target cells leads to a significant decrease of alpha(max) and beta(max), indicating that these parameters are related to the density of expression of receptors implicated in effector-target adhesion; (2) treatment of effector cells with CTX induced an increase of K(D), demonstrating that this parameter is associated with the effector-target affinity of the system; and (3) parallel experiments of conjugation and cytotoxicity showed that effector-target affinity and saturability influence the cytotoxic activity of the effector population.

Binding units (BU) and the area under binding isotherms (AUI). New indices of effector-target conjugation.

New methods for simplified quantitation of effector-target conjugation have been developed. The binding unit (BU) is defined as the number of target cells required to bind a specified percentage of effector cells. The number of binding units is determined from binding isotherms in which effector conjugate frequencies are measured by holding constant the number of effector cells and by varying the number of target cells. Alternately, a binding unit can be defined as the number of effector cells required to bind a specified percentage of target cells. In this case, BU is computed from binding isotherms in which target conjugate frequencies are measured at different values of effector cells by holding constant the number of target cells. Also, the area under the curve (AUI) of these isotherms is another index that can be used as an overall measure of the binding capacity in an effector-target system. The experimental values of BU and AUI determined from effector and target isotherms agree well with theoretical predictions based on our previously developed binding model (J. Immunol. Methods (1992) 155, 133-147). The relationship between BU and AUI, and procedures to determine these parameters are shown. The value of these indices to express effector-target conjugation quantitatively has been confirmed by determining the values of BU and AUI for the NK-K562 effector-target system.