Resultados Generales que aplican a todos los Procesos de la Gestión Financiera

As discussed above, on activation of resting T cells a vast number of molecules are up regulated on the T cells which consequently allows a distinction to be made between naive and effector/memory cells.

1.24a The use of CD45 splice variants as markers of memory

The most commonly used distinctions of naive and memory T cells is based on the differential expression of the leukocyte common antigen CD45 expressed on all haemopoietic cells. Several lines of evidence suggested its importance in T cell activation. Thus crosslinking CD45 could either costimulate (Marvel et al. 1989) or inhibit T cell activation (Prickett et al. 1990; Ledbetter et al. 1988a). Furthermore, CD45 has tyrosine phosphotase activity in its cytoplasmic tail (Charbonneau et al. 1988; Fischer et al. 1991) and CD45 deficient cell lines were found to be defective in signal transduction, and this defect was restored by transfection of the cell line with wild type CD45 (Koretzky et al. 1991, 1990).

Alternative splicing of the exons 4, 5, and 6 (generally referred to as A, B and C) located at the distal region of the extracytoplasmic domain of CD45, can encode for at least 8 potential different isotypes (Streuli et al. 1987; Trowbridge et al. 1994; Janeway et al. 1992). Monoclonal antibodies that recognise epitopes dependent on the expression of the exons A, B, and C were generated. Thus for example, the mAbs SN130 (Munro et al. 1988) and 2H4 (Morimoto et al. 1985a; Streuli et al. 1987) recognise an epitope expressed on exon A of human CD45 and are consequently termed CD45RA antibodies. These mAbs recognise all isoforms expressing exon A which includes ABC, AB, AC and A isoforms. Similarly, the CD45RB antibodies will recognise the isoforms ABC, AB, BC and B, whilst the CD45RC antibodies will recognise ABC, BC, AC, and C isoforms. However the mAh UCHLl only recognises an epitope expressed on the human CD45 isoform with all the exons spliced out (Smith et al. 1986a; Terry et al. 1988). In humans the mAbs to CD45RA and CD45RO isoforms, revealed the existence of heterogeneous populations of cells of approximately equivalent proportions within both the CD4 and CD8 T cell subsets in PB (Smith et al. 1986a; Morimoto et al. 1985a). In other species there are many mAbs that recognise the CD45RA, RB and RC isoforms and are collectively referred to as CD45R mAbs, however there are no mAbs that recognise the CD45RO isoform. In mice, the most commonly used mAh 16A, identifies the CD45RB isoforms which can separate the CD4 T cells into high (65%) and low (35%) expressing cells (Bottomly et al. 1989; Dianzani et al. 1990). The murine CD45RA antibodies, such as RA3-2C2 (Coffman et al. 1981) however identifies only 10-30% of the CD4 subset (Marvel et

al. 1988, 1989). In the rat, the mAh (0X22) recognises high molecular weight isoforms (Woollett et al 1985), (which is likely to be through the recognition of the RC exon) and identifies 50-75% of the CD4 T cells (Powrie et al 1990). Thus in animal studies the CD45R mAbs isolates different cell populations to those isolated in the human studies with the CD45RO and/or CD45RA antibodies.

The association of CD45 splice variants with the functional state of the T cell was suggested by the finding that the CD45 subsets within CD4 T cell population differed in their ability to respond to soluble antigen and help B cells and were consequently referred to as helper/inducer or suppresser/inducer (Smith et al. 1986a; Morimoto et al. 1985). It was also shown that on activation of T cells, the high molecular weight isoforms were down regulated whilst the low molecular isoforms were upregulated (Akbar et al. 1988; Serra et al. 1998; Byrne et al. 1988; Sanders et al. 1988; Ledbetter et al. 1985). On re-exposure of these primed T cells to the primary stimulus, an increase in the rate of the proliferative response could be observed but only in the CD45RO population (Akbar et al. 1988). Furthermore the acquisition of the CD45RO phenotype correlated with an acquisition of the ability to help B cells produce antibodies (Clement et al. 1988).

However the association of the CD45RO phenotype with T cell memory was suggested by the finding that proliferative responses to previously encountered antigen such as IFZ A were predominantly in the CD45RO population, whereas responses to alloantigen were found to have similar precursor frequencies in both the CD45RO+ and CD45RO- T cells (Merkenschlager et al. 1988). In these earlier studies, because the expression of CD45RO was found to be more stable than the other known markers of activation such as MHC class n or the lL-2 receptor, it was concluded that CD45RO expression was also irreversible.

This theory was further substantiated by the finding that immunologically immature T cells isolated from human UCB were predominantly CD45RO- and only acquired CD45RO after activation (Sanders et al. 1988); UCBMC or CD45RA T cells from PB did not respond as well as the CD45RO phenotype from PB to superantigens (Horgan et al. 1990; Takahashi et al. 1995); CD45RA T cells isolated from cord blood were

suppressive for the B cell helper function of the minority CD45RO T cells present in cord blood, although activation of the T cells could inhibit this suppressive function (Clement et al. 1990); and the levels of T cells expressing CD45RA were found to decrease whilst CD45RO expressing cells increased with age and only reached adult levels after 20 years of age (Hayward et al. 1989).

From these very convincing experiments it was concluded that this unidirectional expression of CD45RO on T cells represented the loss of naive and acquisition of a memory phenotype. Following these studies, many other in vitro and in vivo examples in man, mice and other animals provided evidence to show that priming of T cells resulted in the loss of the high molecular weight isoforms and the gain of the low molecular weight isoforms. Thus despite the different populations isolated by the CD45R antibodies in the different species, T cells expressing the high molecular weight isoforms, in all species gave very poor responses to recall antigens and were ineffective at providing help for B cells. Conversely, cells expressing the low molecular weight isoforms, gave very good responses to recall antigens and provided help for B cells.

1.24b The expression of CD45RA mav not alwavs represent a naive phenotvpe.

There are however, a number of studies in both man and mouse in which the results are not consistent with the above theory and suggests that the expression of high molecular weight isoforms and low molecular weight isoforms may not always represent naive and memory phenotypes.

Although the activation of CD45RA T cells with mitogens does down regulate the expression of CD45RA, expression is not completely lost (Morimoto et al. 1986) even after 21 days in culture (Rothstein et al. 1990) and the retention of CD45RA correlated with an inability to help B cells (Rothstein et al. 1990). There has also been a report of transgenic unprimed mice bearing a percentage of cells with low molecular weight isoforms (Lightstone et al. 1993). Furthermore, priming of these mice resulted in an increase in percentage of the high molecular weight isoforms, which eventually decreased but again, were never lost.

The mathematical models used to analyse the human studies of the decay of cells with stable/unstable chromosomal dicentric lesions, also indicated that the CD45RO T cells could revert to CD45RA phenotype in vivo and the rate of reversion was estimated as 3.5 years (Michie et al 1992; Mclean et al 1995). In addition CD45RA T cells which were activated in vitro to express CD45RO, re-expressed CD45RA in a

subpopulation of the cells after 2-3 weeks. However T cells originally expressing CD45RO remained stable (Warren et al. 1991). In addition, the expression of CD45RA was found to vary in a cyclic manner after activation of CD45RA T cell lines with mitogens (Rothstein et al. 1991). Evidence for the instability of the CD45 isoforms was also found in rats, where adoptive transfer of either the high or low molecular weight isoforms to athymic nude recipients, resulted in the reversion to the reciprocal isotype in both subsets (Bell et al. 1990).

In general, the expression of the low molecular weight isoforms correlates with the expression of many activation markers, but co-expression of some of these markers on the high molecular weight isoforms has been noted in a few studies. Thus CD29 was found to be co-expressed with CD45RA-H population in both human neonates and in children but in adults was mainly found in the CD45RA- population (Pilarski et al.

1991). A subpopulation of CD45RA T cells were also found to co-express CD25 (the IL-2 receptor a-chain ) in neonates as well as in children and adults (Kanegane et al.

1991). These cells were also found to express mRNA for the cytokine IL-4, IL-5 and IFNy suggesting their recent activation. In addition, young mice were also found to co-express CD44 with CD45RA in the CD4 T cells (Lightstone et al. 1991).

Although the cells leaving the thymus are functionally naive, a subpopulation of thymocytes have been found to express the low molecular weight isoforms in humans (Pilarski et al. 1989), mice (Goff et al. 1990) and rats (Law et al. 1989). Furthermore thymectomy of mice led only to a reduction in CD45RA in the CD8 subset but not in the CD4 T cell subset (Lightstone et al. 1991).

Another paradox is that the expression of CD45RA was significantly higher on cells in the Gi/S/M phases compared to the Gq/Gi phases of activation in both T cell clones

activation of the PB T cells, an increase in the percentage of double positive cells in both CD45RA and CD45RO T cells was observed. These double positive cells can also be found in freshly isolated resting PB derived T cells (Wallace et al. 1990) and in a subpopulation (2-10%) of cells in all secondary lymphoid tissue (Picker et al

1993). Consistent with the above studies, it was found that after phytoheamoglutinin (PHA) activation of CD45RA^’®^ T cells, the transition from CD45RA to CD45RO phenotype occurs by an initial increase in expression in both CD45RA and CD45RO isoforms, followed by the downregulation in expression of CD45RA (Picker et al.

1993). This observation was further supported by the finding that the majority of T cells with CD45RA^'^^/R0^^^^ expression were blasts, in the S phase of cell cycle and expressing early activation antigens, whilst the CD45RO^‘^VCD45RA*°'^ T cells were predominantly small cells in G2/M phase of cell cycle and expressing persistant

activation antigens (Picker et al. 1993).

Despite the stringent activation requirements demonstrated in many experiments for CD45RA T cells, IL-2 alone was found to be able to activate these cells and induce the expression of CD45RO as well as other activation markers such as CD2, CD29 and GDI la (Roth et al. 1994). In addition, IL-2 in combination with anti-CD28 has been found to induce differentiation of PB derived CD45RA T cells into Th2 effector cells (Brinkmann et al. 1996). However in another study, IL-2 in combination with TN Fa and IL-6 could induce the proliferation of CD45RA T cells but could not induce expression of CD45RO or change the functional activity of these cells (Unutmaz et al. 1994).

Responses to recall antigens have also been demonstrated in PB derived CD45RA T cells when costimulated with CD28 (Pilling et al. 1996). However, the precursor frequency of CD45RA T cells to recall antigens remained below the precursor frequency obtained in the CD45RO population to the same antigen. In addition, CD28 costimulation could not induce activation of CD45RA T cells from PB in response to non-recall antigens or from UCB in response to recall antigens.

A possible explanation for at least some of these discrepancies was that the expression of CD45RO was merely a marker of activation and that the memory component of

these cells was due to their hyperresponsiveness and increased frequency (Mitchison et al. 1992). The ability of CD45RA T cells to respond to recall antigens could be explained if these cells were CD45RO T cells that had reverted to a CD45RA resting state but had retained the hyperresponsive state in some capacity.

It is possible that the functional differences that have been observed in freshly isolated T cells separated on the basis of CD45RA and CD45RO expression may have been observed under limiting costimulatory signals. Indeed when the necessary costimulatory signals are provided by antibodies or professional APC such as DC and activated B cells, the hyporesponsiveness of these cells can be overridden (see section 1.22). However in other cases, these differences in activation requirements between CD45RA and CD45RO T cells cannot be overcome even when costimulation is not limiting (Kuiper et al. 1994).

Taken together these results suggest that the expression of CD45RA on T cells does not always denote a naive status. In light of this evidence, one very pertinent question to this study is whether the use of CD45RO and CD45RA expression to identify and isolate memory and naive cells in vitro, denotes a true representation of these respective functional states. What is clear from the above literature is that on encounter with its specific antigen, the T cell does undergo many dramatic changes both biochemically, phenotypically and functionally. It is likely that at least some of these changes may be retained, but the above studies indicate that expression of CD45RO may eventually be lost and that there may be “re-expression” of CD45RA. Thus the distinction between memory and naive cells based on CD45RO and CD45RA expression respectively cannot suffice to identify these functional states, and this is particularly important where previous exposure to antigen is unknown. Thus, other phenotypic markers and functional capacity (e.g. B cell help or CTL function) should be considered in any attempt to define these two T cell functional states (see below ).

1.24c Other markers of activation/memory in humans

As a consequence of the ambiguity in using CD45RA as a marker of naive T cells, a number of recent studies in humans have used other phenotypic markers in conjunction with CD45RA and CD45RO to differentiate more specifically between the different states of activation. As in the mouse, CD45RB*°'^ expression in humans has also been shown to correlate to an activated phenotype (Matthews et al. 1993; Gordan et al. 1996). The relative expression of CD45RB on CD45RO T cells was used to distinguish between the differentiation states of the T cell (Horgan et al. 1994; Salmon et al. 1994). This was possible because unlike CD45RA, which is down regulated relatively rapidly, CD45RB was found to be lost slowly. The progression from CD45RB^'^^ to CD45RB^°'^ phenotype was shown to correlate with the susceptibility to apoptosis as suggested by the expression of bcl-2 and Fas antigen expression, as well as a change in cytokine profile from a predominantly EL-2 producing profile characteristic of naive cells, to an EL-4 producing phenotype of differentiated Th2 cells (Salmon et al. 1994). Furthermore on activation, the expression of CD45RB on CD45RO T cells could interconvert between an intermediate and a low level (Horgan et al. 1994).

CD31 (an adhesion molecule involved in platelet-endothelial cell interaction), has also been found to be restricted in expression to the CD45RA T cell subset (Morimoto et al. 1993; Ashman et al. 1991). Functional analysis revealed that these cells were unable to produce IL-4 and had poor or suppressive B cell helper function (Morimoto et al. 1993). In contrast CD31 negative cells provided help for IgG production and responded to recall antigens such as tetanus toxoid and mumps. However CD314- expression was not lost on activation of CD45RA T cells despite its absence on CD45RO T cells suggesting that it’s expression may only be lost after the T cells are fully activated or gain a memory phenotype.

Several studies have suggested that the molecule CD27 (a member of the nerve growth factor receptor family) may be differentially expressed on T cells. Thus CD27 expression was found at high levels on all CD45RA T cells but was either absent or expressed at a very low level on the CD45RO subset (Hintzen et al. 1993; Sugita et al. 1992). On activation with anti-CD3 or mitogens, a higher expression was observed on

the CD45RA subset and was maintained even after prolonged culture. In contrast activation of the CD45RO subset resulted in the eventual loss of CD27 expression, and which could not be induced after long term stimulation of these cells. Functional analysis showed that the production of IL-4 and B cell help was found to be predominantly from the CD45RA-, CD27- cells whilst the majority of CD27+ CD45RA4- expressing cells were unable to perform either of these functions (Morimoto et al. 1993; Sugita et al. 1992). The preferential ability to help B cell was confirmed in a more recent study where the loss of CD27 was shown to correlate with the down regulation of CD45RB expression and increase in B cell helper function (Tortorella et al. 1995). In addition, CD27- T cells showed a propensity to respond to recall antigens such as tetanus toxoid or to allergens in cells derived from atopic individuals (De Jong et al. 1992).

The molecule CD26 with dipeptidyl peptidase enzymatic activity was also found to be preferentially expressed on T cell responding to recall antigens and activated T cells and clones irrespective of cell cycle. Further distinction was made by the increased protein kinase C activity in the CD26+ population compared to the CD26- cells (Hafler et al. 1989; Hollsberg et al. 1993)

As discussed previously, many other molecules have been shown to vary with CD45RA/RO expression. This includes CD25 (Taga et al. 1991), MHC class

n,

LFA- 3, LFA-1, ICAM-1, CD44, CD29 (Sanders et al. 1988, 1989; Wallace et al. 1990; Okumura et al. 1993), L-selectin (Picker et al. 1993), bcl-2/Fas antigen expression (Akbar et al. 1993), and the expression of an IL-2 silencer (Mouzaki et al. 1993). In addition, LFA-1 was also found to follow the age related rise in expression in conjunction with CD45RO (Okumura et al. 1993). A number of studies have also shown that functional differences can be used to distinguish between CD45RA and CD45RO T cells independent of response to novel/recall antigen. This includes the production or expression of different cytokines (Salmon et al. 1989; Kristensson et al. 1992; Unutmaz et al. 1994; Semnani et al. 1994; {see also section 1.3}); provision of help for B cells (Clement et al. 1988; Smith et al. 1986a; Morimoto et al. 1985 Martensson et al. 1994); sensitivity to costimulatory signals (Horgan et al. 1990 Semnani et al. 1994; Van de Velde et al. 1993; Fischer et al. 1992; Byrne et al. 1988

Damle et al. 1992) and inhibitory signals (Akbar et al. 1990; Lecomte et al. 1992); and adhesive properties (Lecomte et al. 1992; Parra et al. 1993).

In document Análisis Crítico del Nivel de Implementación de los Procesos que forman parte de la Subgerencia de Finanzas de la Empresa Pública de Hidrocarburos Del Ecuador, EP PETROECUADOR (página 129-149)