5. Resultados y discusi´ on
5.5. Factor de transmisi´ on
5.6.1. Verificaci´ on de dosis en vivo
The proportion of CD3 T cells as a percentage of total lymphocytes was reduced in TB patients compared to healthy people (Fig. 6.1). CD3
is the main inarkei lo r T cells whereas CD 19 is the m ain m arker for B cells involved i.. L .\li c:.d HI. respectively. Therefore, the proportions o f T and B
lymphocytes might influence the predominance of CMI or humoral immunity. The decrease in the relative proportion of CD3 T cells is consistent with the impairment of CMI in TB patients.
The relative contribution of non-T cells to protective or undesirable immunity might be increased when the proportion of T cells is lowered. Some diseases with impaired CMI such as lepromatous leprosy are accompanied with an increased humoral response. A relative increase in CD 19 B cells has been reported in patients with elevated humoral responses, such as allergic rhinitis patients (Davidsson et al, 1994). However, the proportion of B cells was not increased in TB patients from this study. Possible changes in B cell function as a result of CD23 expression is discussed later in 6.3.5.
The ontogenic process of T cells takes them from the bone marrow to the thymus as well as other lymphoid organs where complex differentiation steps occur. The progenitor cells in the thymus cortex begin as double negative (CD4‘8 ) T cells which then acquire either CD4 or CDS markers and become singly positive. Thereafter, they become double positives (CD4^8"^) before they reach the medulla of the thymus, where they lose one or both of their markers and finally become CD4, CDS, CD4 8 and y ô -T cells.
Figure 6.1 % of CD3+ T and CD 19+ B lymphocytes in TB patients and healthy controls(±SD)
100 7 5 - 5 0 - 2 5 - p<0.025 T n Healthy (n= 20) 0 T B (n= 2 0 ) -r- T C D 3 C D 1 9 T and B cells
Figure 6.1 The proportion of circulating CD3+ lymphocytes is significantly reduced in tuberculosis patients (p<0.025, Mann-Whitney test ).
35% of TB patients had <53% CD3 T cells/ total lymphocytes (the lowest value of healthy people)
6.3.2 PROPORTIONS OF CD4. CD8 & yS-T ceUs
CD3 T cells include a(3-T cells w hich can be C D 4, C D 8 o r C D 4 CD8 and yô-T cells. T he a p - T cells have a p - T cell recep to rs w hereas the y5- T cells have yô-T cell receptors. T hese cells d iffer in recep to rs involved in antigen recognition as w ell as th eir e ffe cto r responses such as cytolysis and cytokine production. TB p atients fro m this study show ed a m ore m arked increase in their CD8 T cells than the red u ctio n o f th e ir CD4 T cells (Fig. 6.2). As a result, nine out o f tw en ty TB patients had a low er C D 4;C D 8 ratio <1.3 (the lo w est ratio in a h ealth y person).
ALTERED CD4:CD8 RATIO
TB patients are reported to have a reduction in the total T cell and CD4 counts and an increase in the CD8 count, with a concomitant reduction in the CD4:CD8 ratio (Singhal et al, 1989). TB patients with more severe disease such as those with chronic disease refractory to chemotherapy were found to have a lower CD4:CD8 ratio compared to newly diagnosed drug-sensitive TB patients (Bose et al, 1995). A fter successful treatment the CD4:CD8 ratio of TB patients was found to return to the normal levels found in the healthy controls.
The changes in the balance of CD4 and CD8 T cells could have occurred as a result of their differential synthesis, release, apoptosis or emigration into the sites of disease. The CD4:CD8 ratio has been reported to be altered in the pleural fluid and broncho-alveolar lavage samples compared to the peripheral blood of TB patients (Ainslie et al, 1992). The change in the proportions of CD4 and CD8 T cells has also been reported to occur as a result of external antigenic challenge. A
Figure 6.2 The % of CD3T cells which are CD4, CDS, CD4-8-, yô-T cells(±SD) 80
i
H +8
o 6 0 - 4 0 - 2 0 -J p<0.007 C D 4I
T
*i l
« _ * d ] H ealthy (n = 2 0 ) □ T B (n = 2 0 )I I
T
T
C D 8 C D 4 -8 - CD3+ T CELLSFigure 6.2 When the CD3+ T cells were analysed to calculte the proportions CD4, CDS, CD4-CDS-ap- andyÔ-T cells, TB patients showed a slightly decreased proportion of CD4 T cells and a significantly increased proportion of CDS T cells (p<0.007, Mann-Whitney test). As a result, the CD4:CDS ratio was reduced in TB patients (1.5±0.5) compared to the healthy controls (1.9±0.4) (p<0.01,
Mann-Whitney tes$.
40% TB patients had > 43% CDS T cells ( the highest value of healthy people). Only 10% TB patients had <40% CD4 T cells (the lowest value of healthy people).
study of asthmatic children showed that different subsets of lymphocytes were recruited into the peripheral blood under allergen challege resulting in the increased CD4:CD8 ratio (Schmitt et al, 1993).
No differences in pathology or symptoms could be found between the patients with different CD4:CD8 ratios. It is not known whether the reduction of CD4:CD8 ratio precedes pathogenesis of tuberculosis or if it is the result of the disease itself. HIV patients with lower CD4 T cell counts show an increased susceptibility to tuberculosis. The frequency of mycobacteraemia increases from 4% in HIV patients with >200 CD4 cells/|il to 49% in HIV patients with <100 CD4 T cells/jLll (Jones et al,
1993). The superimposition of mycobacterioses is also known to further accelerate the progression of Acquired Immune-deficiency Syndrome (AIDS). In fact, the T hl-Th2 cytokine imbalance has been implicated in the activation-induced apoptosis of CD4 T cells (Clerici and Shearer,
1996).
y5-T CELLS
My results showed that the proportion of yô-T cells as a percentage of CD3 T cells in the peripheral circulation was not changed in TB patients (Fig. 6.2). However, previous reports have found an increased proportion of y8-T cells in tuberculosis patients who were thought to mount a protective immune response such as patients with tubercular pleurisy disease (Barnes et al, 1992). In addition, they were found to be raised in patients who had positive skin-tests to tuberculin and less serious forms of disease compared to patients with more advanced pulmonary or miliary disease (Sanchez et al, 1994). The proportion of yS-T cells has also been found to be elevated in healthy people exposed to tuberculosis patients (Ueta et al, 1994).
6.3.3 PROPORTION OF CD8^ y8-T CELLS
The majority of peripheral yS-T cells in healthy individuals as well as TB patients are CD4 C D 8 . Tuberculosis patients showed higher levels of CDS expression on their yS-T cells compared to healthy controls (Fig. 6.3). It is not known how the expression of CD4 and CDS is regulated on yS-T cells compared to a p -T cells. The mean percentages of CDS^ aP -T cells / CD3 T cells and CDS^ y8-T cells / yS-T cells was similar in both TB patients and healthy people. Therefore, the expression of CDS on both a p - and yS-T cells could be under similar control. For example, an increase in the level of CDS^ yS-T cells might occur before or alongside an increase in the CDS^ a P -T cells. However, the expression of CDS on aP~ and yô-T cells did not show any correlation ie. individuals with raised levels of CDS^ a p -T cells did not necessarily have raised levels of CDS^ yÔ-T cells. It is possible that the different individuals could have had variable initial levels of CDS^ aP -T cells and CDS^ yÔ-T cells, but the changes in their levels might be similar.
The proportion of CDS^ yô-T cells is known to be increased amongst the intra-epithelial lymphocytes (lEL). The level of CDS^ yô-T cells has also been found to be increased in the peripheral circulation of patients infected with HIV (Margolick et al, 1991). Patients with colorectal cancer showed higher proportions of CDS^ yô-T cells amongst tumour- infiltating lymphocytes and normal intestinal tissue than in the peripheral circulation (Watanabe et al, 1995). Therefore, these CDS receptor molecules might be involved in the migration of yÔ-T cells to specific anatomical locations such as the mucosal epithelia.
Figure 6.3 The % of CD4-8- and CD8+yô-T cells in TB patients and healthy controls (±SD)
80 o 60 - 4 0 - 2 0- n H ealthy (n=20) □ TB (n=20) p<0.001
I
CD4-8- CD8+ y§-T cellsFigure 6.3 The majority ofyô-T cells are CD4-CD8- in healthy people and tuberculosis patients although this was reduced slightly in the latter. The proportion of CD8+yô-T cells was significantly increased in tuberculosis patients (p<0.001, Mann-Whitney test).
30% of TB patients had > 46% yô-T cells (highest value of healthy people).
CDS chains expressed on
y5-T
cells differ from their counterparts in CD8^ap-T
cells. CDS^ expression onyS-T
cells had lower fluorescence intensity than CDS^ap-T
cells. Similar types of CDS expression were also shown by CD3 CDS^ T cells, most of which are likely to be NK cells. CDS^yô-T
cells only havea
chains and lackp
chains found in CDS^ap-T
cells. NK cells and CDS^yÔ-T
cells both possess a chains which might be important in MHC-independent recognition of target cells. Cell lines from CDS^yô-T
cells of a patient with T-gamma lymphocytosis were also found to have strong cytotoxic activities against NK-sensitive target cells (Morikawa, 1990). These cells with potent/
cytotoxic abilities can kill mycobacteria-laden cells as well as causing tissue damage.