**2.6.1 Methods to determine the CD277-counter receptor identity**

In spite of the weak expression of the counter-receptor in different cell lines (Compte *et al.*, 2004), C91 cells were selected because of the expression profile is higher than in other cell lines such as Raji, TF1, HUT78 or JA16. In order to obtain a cell population with a stable expression of the CD277 counter-receptor, C91 cells were stained with the CD277-Fc fusion protein as described above. The positive population was identified by comparing to unstained control and the 1% most positive population was selected and purified using a

CD277 an Immune Regulator of T Cell Function and Tumor Cell Recognition 421

From C91T2 cells stained with the CD277-Fc fusion protein we proceeded to clone from the 1% most positive cells (Figure 7). Cloned cells were followed to measure the CD277 counterreceptor expression and the most positive clone obtained was selected and named C91T3.3. As shown in figure 8, increasing expression levels of CD277 counter-receptor were reached with enrichment each step. However, after C91T3.3 no more clones with stable expression of CD277 counter-receptor were obtained. Thus, the highest stable expression of CD277

In the first approach to identify the CD277-counter receptor, our preliminary results shown only the homo-polymerization of CD277-Fc by the crosslinking agent BS3 and its detection by mass spectrometry (MALDI-TOF) led us to postulate that this approach is appropriate to determine the identity of CD277 counter-receptor. Nonetheless, the use of additional controls such as CTLA4-Fc is needed to further validate the results. Moreover, as can be seen in our results, it is possible that these homo-polymers mask the potential CD277-Fc - CD277 counter-receptor heterodimerization. Therefore they should be eliminated as collateral products of the reaction in order to select the appropriate band to be analyzed. Representative 4-12% PAGE-SDS and western-blot analysis are shown in figure 9. The selected bands were analyzed by MALDI-TOF, but only the polymerization of CD277-Fc

The use of the appropriated controls, negative and positive, is essential to validate the results. As a positive control, we used CTLA4-Fc fusion protein in C91 cells (positive by flow cytometry). However, the determination of the identity of CD277 counter-receptor is in

In both approaches used (crosslink and co-immunoprecipitation with post-nuclear membranes) the CD80/86 molecules were used to validate these methods. We hypothesized that if detecting CD80/86 was possible, this methodological approach would be useful for the CD277 counter-receptor identification. However, we were not able to detect CD80/86 by any of the strategies tested. Thus, even though this kind of approach has been used in similar circumstances, like the identification of the B7\_H6 counter-receptor (Brandt *et al.*, 2009), technical modifications are needed in order to have the validated conditions for the

Fig. 8. Enhanced expression of CD277 counter-receptor in C91, C91T, C91T2 and C91T3.3 cells after cell sorting and cloning. Expression was followed-up using CD277-Fc fusion

counter-receptor was that obtained in C91T3.3 cells.

fusion protein was identified.

detection of the CD277 counter-receptor.

protein by flow cytometry.

process.

FacsAria (BD Bioscience). These cells were then cultured in RPMI 1640 supplemented with 10% FCS and antibiotics. Expression of CD277 counter-receptor was followed by flow cytometry to corroborate that its surface expression was stable.

The cells obtained, were used in two different approaches to determine the identity of the CD277 counter-receptor. First, the use of cross-linking assays with Bis (sulfosuccinimidyl) suberate (BS3) a homobifunctional, water-soluble, non-cleavable and membrane impermeable crosslinker and; second, a classical co-immunoprecipitation assay from post-nuclear membranes.

In the first approach, C91T3.3 cells were incubated with 30 µg/mL of the CD277-Fc fusion protein and then the interaction was covalently stabilized by using 3 mM of BS3. After crosslink a standard co-immunoprecipitation protocol was carried out using anti-CD277 monoclonal antibodies and protein G. A 4-12% polyacrilamide gel electrophoresis-sodium dodecyl sulfate (PAGE-SDS) and western-blot were then used to detect the CD277-Fc crosslinked, bands were then cut from the PAGE-SDS gel and sent to be identified by MALDI (Matrix-Assisted Laser Desorption/Ionization)-TOF in a mass-spectrometry assay.

The second strategy to determine the identity of CD277 counter-receptor involves a procedure to obtain the postnuclear membranes. This procedure leads us to enrich the membrane proteins and eliminate the nuclear contaminants that can interfere with the interaction between CD277 and its counter-receptor. In this approach, the C91T3.3 cells were treated with 3 mM imidazole for cell membrane lysis but not the nuclear membrane. After centrifugation, membranes were recovered and used in a classical co-immunoprecipitation assay with CD277-Fc and CTLA4-Fc as a control. A 4-12% PAGE-SDS was carried out to cut the putative counter-receptors for CD277-Fc and CTLA4-Fc, compared with a control without recombinant proteins. Bands that are in the assay but not in the controls were selected to be identified by MALDI-TOF.

#### **2.6.2 Experimental results**

The procedure to enhance the expression of CD277 counter-receptor was done twice and cells obtained were named C91T and C91T2. The expression of CD277 counter-receptor in C91T2 was higher than it was in C91T (Figure 8). A third assay to enrich the cell population expressing CD277 counter-receptor by this method was not possible, because the expression after the procedure was not stable.

Fig. 7. Population selected from C91, C91T, or C91T2 cells. Flow cytometry was carried out to enhance the stable expression of the CD277 counter-receptor.

FacsAria (BD Bioscience). These cells were then cultured in RPMI 1640 supplemented with 10% FCS and antibiotics. Expression of CD277 counter-receptor was followed by flow

The cells obtained, were used in two different approaches to determine the identity of the CD277 counter-receptor. First, the use of cross-linking assays with Bis (sulfosuccinimidyl) suberate (BS3) a homobifunctional, water-soluble, non-cleavable and membrane impermeable crosslinker and; second, a classical co-immunoprecipitation assay from post-nuclear

In the first approach, C91T3.3 cells were incubated with 30 µg/mL of the CD277-Fc fusion protein and then the interaction was covalently stabilized by using 3 mM of BS3. After crosslink a standard co-immunoprecipitation protocol was carried out using anti-CD277 monoclonal antibodies and protein G. A 4-12% polyacrilamide gel electrophoresis-sodium dodecyl sulfate (PAGE-SDS) and western-blot were then used to detect the CD277-Fc crosslinked, bands were then cut from the PAGE-SDS gel and sent to be identified by MALDI (Matrix-Assisted Laser Desorption/Ionization)-TOF in a mass-spectrometry assay. The second strategy to determine the identity of CD277 counter-receptor involves a procedure to obtain the postnuclear membranes. This procedure leads us to enrich the membrane proteins and eliminate the nuclear contaminants that can interfere with the interaction between CD277 and its counter-receptor. In this approach, the C91T3.3 cells were treated with 3 mM imidazole for cell membrane lysis but not the nuclear membrane. After centrifugation, membranes were recovered and used in a classical co-immunoprecipitation assay with CD277-Fc and CTLA4-Fc as a control. A 4-12% PAGE-SDS was carried out to cut the putative counter-receptors for CD277-Fc and CTLA4-Fc, compared with a control without recombinant proteins. Bands that are in the assay but not in the controls were

The procedure to enhance the expression of CD277 counter-receptor was done twice and cells obtained were named C91T and C91T2. The expression of CD277 counter-receptor in C91T2 was higher than it was in C91T (Figure 8). A third assay to enrich the cell population expressing CD277 counter-receptor by this method was not possible, because the expression

Fig. 7. Population selected from C91, C91T, or C91T2 cells. Flow cytometry was carried out

to enhance the stable expression of the CD277 counter-receptor.

cytometry to corroborate that its surface expression was stable.

selected to be identified by MALDI-TOF.

**2.6.2 Experimental results** 

after the procedure was not stable.

membranes.

From C91T2 cells stained with the CD277-Fc fusion protein we proceeded to clone from the 1% most positive cells (Figure 7). Cloned cells were followed to measure the CD277 counterreceptor expression and the most positive clone obtained was selected and named C91T3.3. As shown in figure 8, increasing expression levels of CD277 counter-receptor were reached with enrichment each step. However, after C91T3.3 no more clones with stable expression of CD277 counter-receptor were obtained. Thus, the highest stable expression of CD277 counter-receptor was that obtained in C91T3.3 cells.

In the first approach to identify the CD277-counter receptor, our preliminary results shown only the homo-polymerization of CD277-Fc by the crosslinking agent BS3 and its detection by mass spectrometry (MALDI-TOF) led us to postulate that this approach is appropriate to determine the identity of CD277 counter-receptor. Nonetheless, the use of additional controls such as CTLA4-Fc is needed to further validate the results. Moreover, as can be seen in our results, it is possible that these homo-polymers mask the potential CD277-Fc - CD277 counter-receptor heterodimerization. Therefore they should be eliminated as collateral products of the reaction in order to select the appropriate band to be analyzed.

Representative 4-12% PAGE-SDS and western-blot analysis are shown in figure 9. The selected bands were analyzed by MALDI-TOF, but only the polymerization of CD277-Fc fusion protein was identified.

The use of the appropriated controls, negative and positive, is essential to validate the results. As a positive control, we used CTLA4-Fc fusion protein in C91 cells (positive by flow cytometry). However, the determination of the identity of CD277 counter-receptor is in process.

In both approaches used (crosslink and co-immunoprecipitation with post-nuclear membranes) the CD80/86 molecules were used to validate these methods. We hypothesized that if detecting CD80/86 was possible, this methodological approach would be useful for the CD277 counter-receptor identification. However, we were not able to detect CD80/86 by any of the strategies tested. Thus, even though this kind of approach has been used in similar circumstances, like the identification of the B7\_H6 counter-receptor (Brandt *et al.*, 2009), technical modifications are needed in order to have the validated conditions for the detection of the CD277 counter-receptor.

Fig. 8. Enhanced expression of CD277 counter-receptor in C91, C91T, C91T2 and C91T3.3 cells after cell sorting and cloning. Expression was followed-up using CD277-Fc fusion protein by flow cytometry.

CD277 an Immune Regulator of T Cell Function and Tumor Cell Recognition 423

The production and use of monoclonal antibodies is another attractive strategy to enhance our knowledge of CD277 counter-receptor. However, the identity of the receptor or a recombinant counter-receptor is not available yet. Thus, we decided to obtain monoclonal antibodies to recognize this counter-receptor using cells expressing it as the immunogenic stimulus. With this rationale we immunized rats with C91T3.3 cells, which as described above, enhanced expression of the CD277 counter-receptor. Spleen cells from these immunized rats were used for the production of hybridomas following standard protocols. Important to note is the fact that when using this type of approach, the screening systems are crucial to identify the relevant hybridomas to be clone and antibodies to be purified. Considering that the rat immune system will produce antibodies against many of the cell surface proteins of C91T3.3, a first screening was done using the CD277 counter-receptor expressing human erythroleukaemic cell line TF1. Hybridomas secreting antibodies against TF1 cells were selected for a second screening. In addition, since Raji cells stimulated with anti-CD277 mAbs show enhanced expression of the CD277 counter-receptor, these cells were used for a second screening of the clones selected in the first one. This second screening was done in parallel with an inhibitory test using C91T3.3 cells and the CD277-Fc.. With this system, hybridomas secreting antibodies against the Raji cells and with inhibitory

In figure 11 a representative figure is shown. The basal fluorescence of Raji cells is shown (11A). The percentage of positive non-stimulated Raji cells is shown in red and it is compared to the amount of positive stimulated Raji cells (blue) (11B). This supernatant was a potentially useful, thus it was tested for inhibitory activity on C91T3.3 cells (Figure 12). We can see that effectively the antibodies in this supernatant have an inhibitory effect on the CD277-Fc binding. Interestingly, we found that some supernatants enhance the CD277-Fc binding on C91T3.3. These clones were selected too, because they could recognize some

We selected a total of 6 clones with inhibitory activity on C91T3.3 cells. We also obtained one clone whose supernatant has the ability to enhance the CD277-Fc binding on C91T3.3

Fig. 11. Representative flow cytometry pattern of a selected clone. A) Basal fluorescence and B) fluorescence of supernatant tested on Raji non stimulated (red) and stimulated with anti-

CD277 (blue). The percentage of positive cells is indicated in each pattern.

cells. The identification of the proteins recognized by these antibodies is in progress.

**2.6.3 Monoclonal antibodies against the CD277 counter receptor** 

activity on C91T3.3 were selected for cloning.

accessory molecules for the counter-receptor.

Fig. 9. A typical PAGE-SDS and western-blot analysis for the identification of CD277 counter-receptor. A) 1.- control BSA 2.- molecular weight markers, 3.- assay without BS3 and 4.- with BS3. B) Western-blot line 1 with BS3, line 2 without BS3 and line 3 molecular weight markers. Numbers indicate the selected band to be analyzed by MALDI-TOF.

Fig. 10. A typical PAGE-SDS used in co-immunoprecipitation assays using the post-nuclear membranes. 1) Using CTLA4-Fc 2) Using CD277-Fc and 3) control without recombinant proteins. The arrow shows the band to be analyzed.

Fig. 9. A typical PAGE-SDS and western-blot analysis for the identification of CD277

markers. Numbers indicate the selected band to be analyzed by MALDI-TOF.

counter-receptor. A) 1.- control BSA 2.- molecular weight markers, 3.- assay without BS3 and 4.- with BS3. B) Western-blot line 1 with BS3, line 2 without BS3 and line 3 molecular weight

Fig. 10. A typical PAGE-SDS used in co-immunoprecipitation assays using the post-nuclear membranes. 1) Using CTLA4-Fc 2) Using CD277-Fc and 3) control without recombinant

proteins. The arrow shows the band to be analyzed.

#### **2.6.3 Monoclonal antibodies against the CD277 counter receptor**

The production and use of monoclonal antibodies is another attractive strategy to enhance our knowledge of CD277 counter-receptor. However, the identity of the receptor or a recombinant counter-receptor is not available yet. Thus, we decided to obtain monoclonal antibodies to recognize this counter-receptor using cells expressing it as the immunogenic stimulus. With this rationale we immunized rats with C91T3.3 cells, which as described above, enhanced expression of the CD277 counter-receptor. Spleen cells from these immunized rats were used for the production of hybridomas following standard protocols. Important to note is the fact that when using this type of approach, the screening systems are crucial to identify the relevant hybridomas to be clone and antibodies to be purified.

Considering that the rat immune system will produce antibodies against many of the cell surface proteins of C91T3.3, a first screening was done using the CD277 counter-receptor expressing human erythroleukaemic cell line TF1. Hybridomas secreting antibodies against TF1 cells were selected for a second screening. In addition, since Raji cells stimulated with anti-CD277 mAbs show enhanced expression of the CD277 counter-receptor, these cells were used for a second screening of the clones selected in the first one. This second screening was done in parallel with an inhibitory test using C91T3.3 cells and the CD277-Fc.. With this system, hybridomas secreting antibodies against the Raji cells and with inhibitory activity on C91T3.3 were selected for cloning.

In figure 11 a representative figure is shown. The basal fluorescence of Raji cells is shown (11A). The percentage of positive non-stimulated Raji cells is shown in red and it is compared to the amount of positive stimulated Raji cells (blue) (11B). This supernatant was a potentially useful, thus it was tested for inhibitory activity on C91T3.3 cells (Figure 12). We can see that effectively the antibodies in this supernatant have an inhibitory effect on the CD277-Fc binding. Interestingly, we found that some supernatants enhance the CD277-Fc binding on C91T3.3. These clones were selected too, because they could recognize some accessory molecules for the counter-receptor.

We selected a total of 6 clones with inhibitory activity on C91T3.3 cells. We also obtained one clone whose supernatant has the ability to enhance the CD277-Fc binding on C91T3.3 cells. The identification of the proteins recognized by these antibodies is in progress.

Fig. 11. Representative flow cytometry pattern of a selected clone. A) Basal fluorescence and B) fluorescence of supernatant tested on Raji non stimulated (red) and stimulated with anti-CD277 (blue). The percentage of positive cells is indicated in each pattern.

CD277 an Immune Regulator of T Cell Function and Tumor Cell Recognition 425

This work was supported by Institut National du Cancer, Institut Paoli Calmettes, Institut National de la Santé et de la Recherche Médicale. J. F Z-Z was a Post-doctoral fellow from

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**4. Acknowledgment** 

**5. References** 

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Fig. 12. Representative flow cytometry pattern of a selected clone tested on C91T3.3 cells. A) Basal fluorescence; B) Normal binding of CD277-Fc fusion protein; and C) Inhibitory test showing a reduction in the CD277-Fc binding. The percentage of positive cells is indicated in each pattern.

#### **2.7 Perspectives on the potential use of CD277 and its counter-receptor in cancer therapy**

The wide variety of function of CD277 described in this chapter shown the future potential of the use of mAbs anti-CD277 to regulate the immune response against tumor cells. The fact that stimulation of different cells by CD277 lead to the secretion of different cytokines, activation of different signalling pathways, enhance the proliferation of T cells, can be useful to modulate the immune response against tumor cells. Moreover, our results showing that Raji cells stimulated by CD277 are better killed by γ T cells are potentially useful for cancer therapy.

However, it is necessary to elucidate the mechanisms by which these molecules act and to identify the counter-receptor for CD277. The use of mAbs should be a tool to elucidate the mechanisms, but to be used no to mark the tumor cells, but to modulate the response that lead to kill them.

In the near future, regulation of the expression or activity of molecules such as CD277 or its counter-receptor can be useful for enhance the anti-tumoral immune response, however it is necessary the molecular and fully functional characterization of these molecules, and the mechanisms involved in the observed functions, to be used as modulators of immune response in cancer.
