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In this latter part of the chapter, in order to improve on the isolation protocol in preparation for later studies analysing the various subsets involved in the HRV specific response, the E+ HD cells were compared with responder populations obtained using negative selection with magnetic beads. The following responder populations were analysed: HD cells; HD cells depleted of CD 14+ and CD 19+ cells; HD cells depleted of CD 14+, CD 19+ and MHC class n+ cells; and E+ HD cells. Various parameters (table 3) were then explored using these responder populations.

Table 3; Cell populations used in defining the parameters investigated

Parameter Responders Stimulators

Tim e course HD CD14, C D 19, MHC class II depleted H D E- LD D ose o f antigen HD E+HD E- LD CD14, C D 19, MHC class II depleted HD E- LD

T cell requirement E+HD E -L D

CD 14, CD 19, MHC class II depleted HD

E- LD

APC requirement E+HD E -L D

C D 14, CD 19 depleted HD E- LD C D 14, C D 19, MHC class II depleted HD a E - LD b. CD3, CD 14 depleted LD c. CDS, C D 14, C D 19 depleted LD d. DC enriched, CDS, C D 14, CD 19 depleted LD

3.61 Phenotypic analysis of the various responder populations.

The HD fraction contained approximately 50-70% CDS T cells. Further enrichment for T cells by either rosetting with SRBC (Figure 3.5), or depletion with antibodies to CD 14 and CD 19 (Figure 3.6), increased this population to between 80% and 90% of CD3+ cells. However, with both these cell preparations, a population of cells expressing CD 19, CD 14 or MHC class H could still be found (the high expression of CD 14 and CDS in figure 3.5 column 2, suggests that the anti-CD 14 mAh used in these experiments may not have been specific for CD14). In order to remove all APC, an antibody to MHC class n was included in the depleting cocktail. Figure 3.7 shows that under these conditions, effective depletion of all MHC class H-h cells from the responder population was achieved, with the CDS-k cells now comprising 97% of the total population.

3.62 Determination of the antigen concentration required to induce an optimal response to HRV.

The optimal dose of HRV to use in the proliferation assays was determined by culturing the HD cells in the presence or absence of HRV 15 and lA at concentrations ranging from 0.25-4)ig/ml for 7 days (figure 3.8). The dose response for both serotypes was very similar. There was a slight but progressive increase in the proliferation of HD cells with the dose of the antigen used. This was true for both serotypes. A maximal response was reached between 0.5-4 |ig/ml.

The dose response for HRV 15, 2 and lA using E-k HD cells was also examined over a concentration ranging from 0.1-6|Xg/ml, and a representative examples for each serotype is shown in figure 3.9a-c. These assays were performed using irradiated E- LD cells as a basic APC population. As observed above, the proliferative response increases with the dose of virus and a maximal response was obtained at the highest concentration used. As shown in figure 3.9, in some preparations E+ HD cells responded in the absence of exogenous APC (figure 3.9a), while in others the response was very low (figure 3.9b-c). In many cases, the addition of exogenous APC substantially increased background proliferation, and hence decreased the SI. Analysis of 34 tonsils showed that a SI greater than 2 could be obtained at 1 |ig/ml in 59% of the tonsils in the presence of APC and 74% in the absence of APC. At 4 |ig/ml, a response could be obtained in 87% in the presence of APC and 96% in the absence of APC. Thus, at 4|ig/ml a higher proportion of tonsils gave positive responses, both in the presence and absence of APC. Consequently 4|ig/ml was used as the standard dose in subsequent assays. The ability of the responder populations to respond better to HRV in the absence of additional APC suggested either that sufficient residual APC were present in the E+ HD cells or that the T cells were capable of presenting to each other. This residual APC population is examined in more detail in section 3.65.

The dose response pattern for HRV 15 and lA was confirmed with CD 14, CD 19, MHC class n depleted responders. These T cells were cultured in the presence or absence of HRV 15 and lA over a concentration ranging from 0.6-5pg/ml (figure 3.10). As for the

HD and E+ HD cells, the HRV specific response increases with the dose of virus and was maximal at the highest concentration used (5 |ig/ml).

3.63 The effect of altering the T cell number on the proliferative response to HRV.

In order to estimate the precursor frequency of the HRV specific T cells in human tonsils, the E+ HD cells were titrated in the range of 0.5-8x10^ cells/well in the presence of a fixed number of E- LD cells (figure 3.1 la-b). The antigen specific response increases with the number of T cells. A response was obtained even at 2x10^ cells/well (a and b). However when the cells were titrated to lower concentrations (figure 3.1 la), the HRV specific response dramatically decreases after 2x10^ cell/well.

The precursor frequency of the HRV specific T cells was confirmed using CD 14, CD 19, MHC class n depleted HD cells in figure 3.12. Thus the CD 14, CD 19, MHC class II depleted HD cells were titrated in the range of 0.5-4x10^ cells/well in the presence of a fixed number of E- LD cells at 10^ cells/well. The HRV specific response increases with the number of T cells and a minimum of 10^ T cells/well were required to observe an HRV specific response. These experiments suggested that HRV specific T cells were present at a minimum precursor frequency of 1 in 10^.

The minimum requirement for T cells observed above using triplicate wells for each condition was confirmed in small scale limiting dilution assays. Thus 96 well plates were set up using multiple wells for each dilution of CD 14, CD 19, MHC class II depleted HD cells, ranging from 0.44-4x10^ cells/well. All wells also contained E- LD as APC (4x10"^ cells/well) and HRV 15 or lA at 4 pg/ml (Figure 3.13). At 4x10^ cells/well, 7/8 wells have positive responses for HRV 15 and 4/8 wells for HRVIA. At 1.33x10^ cell/well, 6/8 well have positive responses for HRV 15 and 5/8 for HRVIA. However, at 0.44x10^ cells/well, nearly all the wells have no responses for both serotypes. Thus from these results it was possible to predict an estimate of the precursor frequency for HRV 15 and lA specific T cells to be between 1 in 1.33-4x10^ cells/well.

3.64 Determination of the time course of the HRV specific in vitro proliferative response in cells from human tonsils.

To determine the time course of the HRV response, multiple 96 well plates were set up under identical conditions. The time course, measured from 3-7 days, using unseparated HD cells as responders was first determined. The response to IFZ A was used as a model of a conventional recall response. The response to HRV 15, lA and IFZ A was observed to increase with time and a maximal response was obtained after 6-7 days in culture (figure 3.14). However, a response could also be observed after 3 days in culture for HRV 15 and 1 A.

The time course for the CD 14, CD 19, MHC class II depleted HD population was also determined. The T cells were cultured in the presence or absence of HRV 15, lA and IFZ A. Proliferation was measured after periods of 3-8 days. The proliferation of enriched T cells followed a similar pattern to the unseparated HD cells (figure 3.15). After 3 days in culture, responses to HRV 15, lA and IFZ A was observed and the highest response was again found at the latest time point of 8 days. As a response could be achieved after 7 days in culture for all the responders tested, this period was therefore used in subsequent experiments as the standard time point.

3.65 APC requirement

a Titration of B cell enriched populations

In order to determine the requirement for exogenous APC of the various responders used, the proliferative response of a fixed number of responder cells was measured at varying concentrations of E- LD cells as APC. Although an HRV specific response could be observed in the absence of exogenous APC when E-h HD cells were used, a small enhancement of the response could be observed at an APC concentration of 0.25x10^ E- LD cells (figure 3.16). Further addition of APC increased background proliferation but decreased the foreground proliferation. A similar inhibition of the SI was also observed when the APC requirement of CD 14, CD 19 depleted HD cells was examined (figure 3.17). Thus despite the substantial reduction in CD 19+ and MHC class

n+

n+

the CD 14, CD19 depleted responder population (figures 3.6), it appears that exogenous APC are not essential to respond to HRV. In contrast, the CD 14, CD 19, MHC class II depleted responder cells had an absolute requirement for exogenous APC and showed a maximal response on addition of only 0.11x10^ cells/well of E- LD cells (figure 3.18). As for the other responder populations, excess APC were inhibitory to the response, and sharply increased the “background” proliferation in the absence of virus.

b Comparison of B cell and DC enriched populations.

In order to examine the possibility that the APC population in the CD 14, CD 19 depleted stimulator population were of DC origin, the LD cells were depleted of B cells, monocytes and T cells and compared with a B cell enriched population. The LD cells were chosen over the HD cells to isolate DC since the MHC class II expression of the former cells were in excess of the CD 19+ population, suggesting the presence of DC. However, the HD cells had a lower percentage of MHC class II expression and therefore suggested a smaller population of DC.

The LD cells were depleted with antibodies to CD 19 and/or CD3 and CD 14 and compared with E- LD cells. The phenotypic profiles of these cell populations are shown in figure 3.19. There was no difference between the E- LD cells (column 1) and the CD3, CD 14 depleted LD cells (column 2) and therefore the latter population was used as the B cell population in functional assays comparing B cells and DC. The depletion of CD 19 from the LD fraction was not very effective at removing the CD 19+ population, and resulted in only a small reduction in CD 19+ cells as suggested by the small shift to the left of the curve for these cells (column 3).

The CD3,| CD14 depleted LD cells were then compared with the CD 19, CD3, CD 14 depleted LD cells for their ability to present HRV (figure 3.20). In these studies the CD 14, CD 19, MHC class II depleted HD cells were used, since this population was demonstrated to have an absolute requirement for APC. The CD 19, CD 14, MHC class II depleted HD cells were cultured in the presence or absence of either CD 19, CD3, CD 14 depleted LD cells, or CD3, CD14 depleted LD cells at concentrations ranging from 0.036x10^-10^ cells/well in the presence or absence of HRV 15 and lA. The lower background proliferation with the CD3, CD 14 depleted LD cells (figure 3.20b), gave a

higher HRV specific response for this population at all concentrations than observed for the CD3, CD 14, CD19 depleted LD cells (figure 3.20a). The capacity to stimulate high “background” proliferation (autologous MLR) is a characteristic feature of DC.

If the residual APC population were DC then a difference should be apparent when CD45RA enriched T cells (“naive” T cells) are used as responders. In order to further enrich for DC, the LD cells were subjected to a 2 hour adherence step on day 1, followed by depletion with antibodies to CD 19, CD3 and CD 14. The phenotypic profile of the DC enriched population is shown in figure 3.21. The inclusion of this step for the enrichment for DC, resulted in a substantial reduction in the percentage of CD 19+ cells but only a small reduction in MHC class n+ cells, suggesting the presence of another population of APC, such as DC. However, in nearly all of the 11 tonsils examined, the CD 19+ cells could not be completely removed even by including a second round of depletion of these cells with dynabeads.

The DC enriched, CD 19, CD3, CD 14 depleted population was compared with the CD3, CD 14 depleted population as stimulators for CD45RA T cells (the isolation and activation of CD45RA T cells are described in chapter 4). Thus CD45RO, CD 14, CD 19, MHC class n, depleted HD cells were cultured in the presence or absence of either DC enriched CD3, CD 14, CD 19 depleted LD cells or a CD 14, CD3 depleted LD cells, at concentrations ranging from 0.13-8x10"^ cells/well (Figure 3.22a-b). As before, although both populations were capable of presenting HRV, the high background proliferation of the DC enriched population (a) resulted in a lower HRV specific response than obtained for the B cell enriched population (b) at the higher APC numbers. However, at the lower APC concentration of 0.13x10"^ cells/well, the DC enriched population were better stimulators than the B cell enriched population. Similar results were observed in two of the other 12 tonsils examined, where the DC enriched population was able to stimulate

CD45RA enriched T cells when the B cell enriched population failed (at the lower APC concentrations). However, in the majority of tonsils, the B cell enriched population was equivalent or the better stimulator, at all concentrations tested.