COUPLING ASSAY FOR T CELL SPECIFICITY (CATS) AND METHOD OF ITS USE

Abstract

A technique, called the Coupling Assay for T-cell Specificity (CATS), to identify antigen-specific cells using cell lines expressing MHC II molecules with tethered peptides. CATS successfully identified antigen-specific T cells with a low-affinity peptide, while tetramer failed to identify cells with this same peptide. Increasing avidity on artificial antigen presenting cells can overcome low affinity TCR-pMHC interactions, can identify more responding endogenous populations, and may be specific for the MHCII.

Claims

1. A system for detecting activation of T cell receptor (TCR), comprising a plurality of cells expressing a major histocompatibility complex (MHC) molecule and a peptide, wherein the peptide is tethered to the MHC to form an MHC-peptide complex, the MHC-peptide complex being presented on surface of said cell, wherein the copy number of the MHC-peptide complex ranges between 10.sup.3 and 10.sup.7, or between 10.sup.4 and 10.sup.6 or about 10.sup.5 per cell.

2. The system of claim 1, wherein the MHC molecule is an MHC class II molecule (MHCII).

3. The system of claim 2, wherein the plurality of cells is derived from a cell line that is capable of perpetuating indefinitely.

4. The system of claim 3, wherein the plurality of cells is derived from a lymphoma cell line.

5. The system of claim 3, wherein the plurality of cells is derived from M12 cells.

6. The system of claim 3, wherein the plurality of cells is M12 cells expressing full-length I-E.sup.k MHC.

7. The system of claim 2, wherein the peptide is derived from a foreign pathogen.

8. The system of claim 1, wherein the peptide is derived from a tumor cell and the MHC is an MHC class I.

9. The system of claim 1, wherein the peptide is selected from a library comprising a plurality of peptides that are randomly synthesized.

10. The system of claim 1, wherein the peptide is 10-50 amino acids long, or 10-20amino acids long, or 12-18 amino acids long.

11. The system of claim 1, further comprising a second cell, the second cell comprising a TCR.

12. The system of claim 1, wherein the second cell is 58.sup..sup. cell or a primary CD4 T cell.

13. The system of claim 11, wherein the K.sub.D between the TCR and the peptide is greater than 10.sup.6M, or greater than 10.sup.5M.

14. The system of claim 1, wherein the peptide is tethered to the MHCII through a linker comprising an amino acid sequence of S-G-G-G-G-S.

15. A method for detecting activation of a T cell receptor (TCR), the method comprising (a) contacting a T cell comprising a TCR with a plurality of cells expressing an MHC molecule and a peptide, and (b) determining association between the T cell and the plurality of cells expressing the MHC molecule and the peptide, wherein the peptide is tethered to the MHC to form an MHC-peptide complex, the MHC-peptide complex being presented on surface of said cell, wherein the copy number of the MHC-peptide complex ranges between 10.sup.3 and 10.sup.7, or between 10.sup.4 and 10.sup.6 or about 10.sup.5 per cell.

16. The method of claim 15, wherein step (b) is performed by flow cytometry.

17. The method of claim 15, wherein the T cell and the plurality of cells expressing the MHC molecule and the peptide are labeled by different dyes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1A shows Representative flow plots showing dye-labeled 5c.c7 TCRG (GFP) TCR 58.sup..sup. cells coupled to specific APC dye-labeled pMHC+ M12 cells. Peplide-dependent coupling percentages and known Ko values are shown.

[0024] FIG. 2A shows Representative flow plot showing 5c.c7 CD4.sup.+ T cells coupled with tethered pMHC-II expressing M12 cells. FIG. 2B shows Coupling percentage of T cells to MCC M12 cells coupled 1:1:1 T cell:MCC:Hb at various time points with PP2 kinase inhibitor or DMSO control. FIG. 2C shows Same process as 2B with cells coupled 1:2:2 T cell:MCC:Hb. Statistical analysis was performed using multiple t-test comparison with Holm-Sidak post-test between the average of 3 experiments of PP2 and DMSO treated samples with SEM error bars shown. Significant p values are shown.

[0025] FIG. 3 shows results when 110.sup.5 dye-labeled 5c.c7 CD4.sup.+ T cells are adoptively transferred into a B1 O.A recipient mouse. After 24 hours, spleen and lymph nodes were collected and CATS or tetramer analysis was performed. (A) Representative flow plots showing 5c.c7 CD4+ T cells to specific pMHC-II+M12 cells. (B) Representative flow plots showing 5c.c7 CD4+ T cells stained with specific tetramer in two-color. (C) MCC or T102S cell coupling or tetramer staining percentage to total dye-labeled T cells. Statistical analysis was performed using an ordinary one-way ANOVA with Sidak post-test comparing each category to each other. Exact p values are shown. (n=8 mice).

[0026] FIG. 4A shows Representative flow plots showing dye-labeled B1 0.A CD4+ T cells coupled with tethered pMHC-II expressing M12 cells. 4B shows Representative flow plots showing the coupling in unblocked, stained with isotype control antibody, or blocked with aMHC-II antibody states. 4C shows Representative flow plot showing 20 g/mL 14-4-4S anti-I-E.sup.k antibody blocking MHCII I-E.sup.k epitopes. 4D shows Relative coupling rates of B10.A CD4+ T cells with tethered pMHC-II expressing M12 cells normalized to blocked states. Representative of 1 experiment.

DETAILED DESCRIPTION

[0027] T cells express TCRs that can interact with pMHC molecules in response to microbial infection. Detecting this interaction is important to understanding T cells. There are several techniques that can characterize a T cell response to infection, including antigen-specific T cell targeting. One such approach utilizes tetramers, a four-pronged pMHC molecule that has been used to target antigen-specific T cells. While extremely useful, tetramers possess limitations, as they are oftentimes difficult and costly to make.

[0028] CATS disclosed here offers a viable alternative to tetramer generation. Utilizing B or T cell lymphomas, cell lines are generated expressing pMHCII molecules with tethered peptide. These cell lines were used to target 58.sup..sup. cells expressing 5c.c7 TCR, 5c.c7 CD4+ T cells, and nave B10.A CD4+ T cells to better understand the capabilities and limitations of the CATS assay. Because tetramers were successful at identifying antigen-specific T cells with great specificity, we expected our M12 pMHC+ cell lines to be even more effective at detecting strong and weak TCR-pMHC interactions.

[0029] The data presented in FIG. 1 highlight the ability of CATS to identify TCR-pMHC interactions when 5c.c7 TCR+ 58.sup..sup. cells were coupled to pMHC+ M12 cells with tethered peptides of varying affinity for the 5c.c7 TCR. The trend we witness complies with our understanding of peptide affinity, with coupling percentage decreasing from MCC>T102S>T102G>Hb. Something to note is that we detected antigen-specific interactions data above background levels in the antagonist T102G, whose K.sub.D is too weak to measure via SPR. This suggests that CATS may overcome differences in affinity for peptide by increasing the avidity of the interaction.

[0030] Various parameters of CATS assay were altered to determine whether incubation time, ratio of T cell:specific APC:dump APC, or introduction of a kinase inhibitor influences coupling percentage. We concluded that incubating the cell couples at 37 C. ultimately resulted in a decreased percentage of cell couples when the kinase inhibitor PP2 was not present. In fact, when PP2 was present, the coupling percentage appeared to trend slightly upward over time. Next, we observed a negligible shift in coupling percentage between a 1:1:1 and 1:2:2 ratio, indicating that a 1:1:1 ratio is sufficient for the purposes of this experiment. It is also determined that 2 minutes of incubation time provided the highest rate of coupling and is sufficient for future experiments. This factor could greatly reduce the waiting period before cell couples can be analyzed, especially since co-cultures and tetramer staining can take hours until the sample is ready to by analyzed.

[0031] Beyond establishing CATS and optimizing the parameters within it, the primary goal of this study is to compare CATS to tetramer. To approach this question, we measured them head-to-head in FIG. 3 to determine how well each method identified 5c.c7 CD4+ T cells within a polyclonal B10.A mouse. CATS was performed as determined by the optimal methods described above, and tetramer staining was mimicked after CATS by including a dump tetramer. However, for added security, we created the tetramer in two-color to ensure that no cells were randomly sticking to tetramer or auto fluorescing. We discovered that the T102S tetramer does not stain 5c.c7 CD4+ T cells with much success at all. However, we did notice that T102S tetramer could stain the polyclonal B10.A population to a certain extent. Because of this quality, we are certain that the tetramers were not compromised or folding incorrectly prior to engagement. CATS also utilized T102S peptide, however, we noticed a significant shift in 5c.c7 CD4+ T cell identification from 0% with tetramer to around 70% with the cell line. This confirms our prediction that increasing the relative avidity of the TCR-pMHC interactions can result in a higher frequency of identified antigen-specific T cells.

[0032] It is disclosed here that CATS is a useful tool to identify antigen-specific T cells, particularly when confronted with the obstacles of low-affinity peptide interactions that tetramers face. However, our next set of questions address cell populations that include the 5c.c7 TCR as well as many others within the TCR repertoire that exist in a polyclonal population. For the purposes of our experiment, we collected B10.A spleens and lymphocytes and conducted CATS on its CD4+ T cells. This experiment was targeted to determine how much the interaction of MHC-II with these T cells affected cell coupling, and hence, identification of endogenous B10.A CD4+ T cells. We predicted coupling to decrease with the addition of clone 14-4-4S (anti-I-E.sup.k) as IL-2 production has previously been shown to decrease after MHC-II blocking of pMHC+ M12 cells prior to cell coupling (Parrish et al. 2016). In FIG. 4, we determined that blocking with the 14-4-4S clone was successful at inhibiting TCR-pMHC interactions, as relative coupling in the blocked states were lower by a factor of 3-5 compared to the uncoupled and isotype control states. Although this was a pilot experiment, it provides promising data that MHC-II may play a critical role in CATS, especially when engaging endogenous polyclonal populations.

[0033] Despite the success we have had with CATS, there are a few factors we believe are necessary to explore more in-depth. First, our optimization of CATS implements three factors that we deemed critical to explore. However, testing CATS at further ratios and more time points could offer a more robust procedure with higher rates of coupling. Another factor that warrants further exploration is leaving cells on ice and staining with antibody. Initially, we conducted this assay at room temperature and avoided staining with antibody to preserve cell couples. However, if we can stain with antibody on ice, this can open the door to any number of possibilities to employ with CATS.

[0034] Because nave CD4+ T cells possess a large repertoire of TCRs, we learned that our MHCII blocking experiment lacks a true dump cell line, as there are approximately as many TCRs that can recognize Hb as can recognize MCC. One way to approach this problem is by switching to 58.sup..sup. generated cell lines and use 58.sup..sup. parentals as a dump. This may produce a lower background than M12 parentals, as there is no endogenous class II MHC on 58.sup..sup. cells, while there is on M12 cells. Another approach to this question can utilize M12 cells expressing full-length I-E.sup.k with no tethered peptide. These cells express several peptides in the MHC-II binding groove, therefore decreasing the avidity of a specific peptide for an antigen-specific T cell. In either case, these controls can better address whether CATS performed on endogenous polyclonal populations is a product of peptide or MHC-II specificity.

[0035] The present disclosure is further illustrated by the following embodiments: [0036] Item 1. A system for detecting activation of T cell receptor (TCR), comprising a plurality of cells expressing a major histocompatibility complex (MHC) molecule and a peptide, wherein the peptide is tethered to the MHC to form an MHC-peptide complex, the MHC-peptide complex being presented on surface of said cell, wherein the copy number of the MHC-peptide complex ranges between 10.sup.3 and 10.sup.7, or between 10.sup.4 and 10.sup.6 or about 10.sup.5 per cell. [0037] Item 2. The system of Item 1, wherein the MHC molecule is an MHC class II molecule (MHCII). [0038] Item 3. The system of any preceding Items, wherein the plurality of cells is derived from a cell line that is capable of perpetuating indefinitely. [0039] Item 4. The system of any preceding Items, wherein the plurality of cells is derived from a lymphoma cell line. [0040] Item 5. The system of any preceding Items, wherein the plurality of cells is derived from M12 cells. [0041] Item 6. The system of any preceding Items, wherein the plurality of cells is M12 cells expressing full-length I-E.sup.k MHC. [0042] Item 7. The system of any of Items 2-6, wherein the peptide is derived from a foreign pathogen. [0043] Item 8. The system of Item 1, wherein the peptide is derived from a tumor cell and the MHC is an MHC class I. [0044] Item 9. The system of any preceding Items, wherein the peptide is selected from a library comprising a plurality of peptides that are randomly synthesized. [0045] Item 10. The system of any preceding Items, wherein the peptide is 10-50 amino acids long, or 10-20 amino acids long, or 12-18 amino acids long. [0046] Item 11. The system of any preceding Items, further comprising a second cell, the second cell comprising a TCR. [0047] Item 12. The system of any preceding Items, wherein the second cell is 58.sup..sup. cell or a primary CD4 T cell. [0048] Item 13. The system of any preceding Items, wherein the K.sub.D between the TCR and the peptide is greater than 10.sup.6M, or greater than 10.sup.5M. [0049] Item 14. The system of any preceding Items, wherein the peptide is tethered to the MHCII through a linker comprising the sequence of S-G-G-G-G-S. [0050] Item 15. A method for detecting activation of a T cell receptor (TCR), the method comprising (a) contacting a T cell comprising a TCR with a plurality of cells expressing an MHC molecule and a peptide, and (b) determining association between the T cell and the plurality of cells expressing the MHC molecule and the peptide, wherein the peptide is tethered to the MHC to form an MHC-peptide complex, the MHC-peptide complex being presented on surface of said cell, wherein the copy number of the MHC-peptide complex ranges between 10.sup.3 and 10.sup.7, or between 10.sup.4 and 10.sup.6 or about 10.sup.5 per cell. [0051] Item 16. The method of any Item 15, wherein step (b) is performed by flow cytometry. [0052] Item 17. The method of any of Items 15-16, wherein the T cell and the plurality of cells expressing the MHC molecule and the peptide are labeled by different dyes.

EXAMPLES

[0053] The disclosure will now be illustrated with working examples, which are intended to illustrate the working of disclosure and not intended to restrictively any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein.

Example 1 Materials and Methods

Mice

[0054] 6-to 8-week-old male and female 5c.c7 TCR Rag KO and B10.A mice were used for spleenocyte and lymphocyte cell coupling and tetramer staining. Mice were maintained under specific pathogen-free conditions in the University of Arizona animal facility. Experiments were conducted under the guidelines and approval of the University of Arizona Institutional Animal Care and Use Committee.

Cell Lines

[0055] 58.sup..sup. and M12 cells were generated by retroviral transduction using the MSCV-based retroviral expression vectors pP2 (IRES-puromycin resistance) and pZ4 (IRES-zeocin resistance) (Glassman et al., 2016; Lee et al., 2015; Parrishet al., 2016).

[0056] The 58.sup..sup. cell lines were retrovirally transduced to express 5c.c7 TCR, full-length CD3 subunits, and C-terminally truncated CD4 (CD4T aa:1-421) (Glassman et al., 2016). The C-terminus of the 5c.c7 a chain was fused to mEGFP via a long flexible linker (AAAGGGGSGGGGSGGGGS). The 5c.c7 B chain and CD4T were encoded by independent constructs and full-length CD3 subunits were encoded by a poly-cistronic construct as previously described (Glassman et al., 2016; Parrish et al., 2016).

[0057] M12 lines were generated by transducing M12 parental cells with full-length I-E.sup.k and full-length I-E.sup.k, fused at the N-terminus to a peptide as previously described (Parrish et al., 2016; Parrish et al., 2015). The peptides in this study include moth cytochrome c peptide (MCC) 88-103 (ANERADLIAYLKQATK), the altered peptide ligands of MCC, T102S and T102G, and the mouse hemoglobin d allele Hb 64-76 (GKKVITAFNEGLK).

[0058] Cell surface expression of CD4, TCR, TCR, and I-E.sup.k were determined by flow cytometry as previously shown (Glassman et al. 2016).

Lymph Node and Spleen Dissection and Dissociation

[0059] Inguinal, brachial, and axillary lymph nodes (LN) and spleens were collected from mice. They were dissociated using frosted glass cover slides and treated with Ack lysing buffer before being resuspended in RPMI.

CD4+ T-Cell Enrichment

[0060] T-cells were counted using the Hemavet instrument. Miltenyi CD4+ cell isolation kits were used in conjunction with Miltenyi LD columns and MACS magnetic separators to enrich the CD4+ T-cell population. Cells were spun down and resuspended in 40 L of complete RPMI per 10.sup.7 cells. Next, 10 L per 10.sup.7 cells of CD4+ antibody cocktail was added to the solution, mixed thoroughly, and left on ice for 5 minutes. In that time, 3 mL complete RPMI was flowed through the LD columns on the separators. 30 L per 10.sup.7 cells of complete RPMI was added to the tube after the 5-minute stain period. Finally, 20 L per 10.sup.7 cells of anti-biotin microbeads was added to the tube, mixed thoroughly, and left on ice for 10 minutes. After the waiting period, 3 mL of complete RPMI was added to the tube and the contents were transported into the MACS column. Flow through (CD4+ T cells) was collected. Pre- and post-enrichment analysis was conducted via Flow Cytometry.

Adoptive Transfer

[0061] 110.sup.5 Tag-it Violet stained 5c.c7 T cells in 100 L of PBS were retro-orbitally injected into a B10.A mouse. After 24 hours, LN and spleen were collected for analysis.

Cell Membrane Staining

[0062] Cells were counted, resuspended in 510.sup.6 cells/ml of 0.2% FBS PBS and cell surface stain dye. 1 L of 5 mM Tag it Violet, Cell Trace Far Red, or Cell Trace CFSE dye was added per 1 mL of 0.2% FBS PBS, for a final concentration of 5 M, as described by the manufacturer. Cells were mixed and incubated at 37 C. for 20 minutes. After the waiting period, 5 ml of complete RPMI was added to the sample to quench any remaining dye.

Cell Coupling Assay

[0063] Either TCR+ CD4+ T cell hybridomas or CD4+ T cells from mice were coupled with M12 cells expressing pMHC class II at a 1:1:1 ratio (T-cell:specific APC:dump APC), spun down for 5 minutes at 1500 RPM and incubated at 37 C. for 2 minutes. Cells were washed with 2% FBS PBS and immediately prepared to flow.

Tetramer Preparation

[0064] pMHC monomer was added to conjugated streptavidin at a ratio of 4:1 and 2% FBS PBS was added to achieve the final concentration of 4 M:1 M. The total concentration was further diluted into the cell population.

Tetramer Analysis

[0065] T cells expressing TCR were spun down and resuspended in 300 L 24.G2 FC Block (with 0.002% azide+2% mouse serum) and incubated for 20 minutes on ice. Cells were washed with 2% FBS PBS and resuspended in 190 L. 5L of tetramer was added to each tube for a total volume of 200 L. Cells were mixed thoroughly and allowed to stain overnight at 4 C. This results in a total tetramer concentration of 100 nM:25 nM monomer:streptavidin.

Flow Cytometry

[0066] BD LSR II and BD Fortessa measured cell engagement to CATS or tetramer

Example 2 Demonstration and Optimization of CATS

[0067] The first goal was to develop a working assay that can be used to demonstrate CATS' utility. Initially, CATS was performed by coupling 5c.c7 TCR+ 58.sup..sup. cells with Tag-it Violet labeled pMHC+ M12 cells at a ratio of 1:1 and incubated couples at 37 C. for time periods of 0 minutes, 2 minutes, 20 minutes, and 60 minutes. Flow cytometry was used to exclude single populations of 58.sup..sup. or M12 cells, gating on the double positive population (FIG. 1A). In this experiment, we used 4 distinct peptides tethered to MHCII molecules, all with affinity for 5c.c7 TCR as follows: MCC>T102S>T102G>Hb. MCC represents the cognate peptide for 5c.c7 TCR, while T102S is a weak agonist and T102G is an antagonist. Hb represents the null peptide, as the MHCII class is the same, but the only interactions between the two is due to nonspecific binding. The data collected demonstrates that cell coupling is both possible and dependent on the affinity of peptide for 5c.c7 TCR in 58.sup..sup. cells.

[0068] Although cell coupling is possible between 58.sup..sup. and M12 cells, we wanted to know if this assay can be applied to nave mouse cells. Following a similar process in our first experiments, we coupled CFSE labelled CD4+ T cells from a 5c.c7 Rag KO mouse with Tag-it Violet labeled MCC and Hb M12 cells under varying conditions. First, the cells were coupled at a ratio of 1:1:1 or 1:2:2 t-cell:specific APC:dump APC with MCC acting as the specific APC and Hb acting as the dump. A dump APC was used because dump APC should eliminate any nonspecific binding between MCC and the CD4+ T cells. The next variable we analyzed was again the incubation times of 0 minutes, 2 minutes, 20 minutes, and 60 minutes. The third was whether pMHC engagement caused TCR signaling, and consequently, downregulation of TCRs that would inhibit coupling. If the CD4+ cells are indeed downregulating their TCR's as a result of signaling, there should be less coupling observed using flow cytometry. Therefore, a kinase inhibitor, PP2, was introduced which prevents the TCRs from signaling or a DMSO vehicle control. Flow cytometry was used to determine MCC M12 cells coupled to 5c.c7 TCR CD4+ T cells, while excluding Hb-TCR or MCC-Hb-TCR double or triple positive events (FIG. 2A). MCC coupling drastically increased from 30% with the hybridomas to anywhere between 60%-85% with the nave T cells. The 1:1:1 (FIG. 2B) and 1:2:2 (FIG. 2C) ratio saw similar trends in the incubation time and +/PP2 coupling. With the DMSO vehicle control, cell coupling initially peaked by 2 minutes and slowly decreased until reaching its lowest % by 60 minutes. When PP2 was added, coupling slowly increased in the 1:1:1 ratio, but it increased up to 20 minutes and slightly decreased by the 60-minute mark in the 1:2:2 ratio. In both ratios, the addition of PP2 seems to eliminate TCR downregulation as coupling is statistically higher than with the DMSO control. Although there was not statistical significance between the different ratios +/PP2 from 0-20 minutes, coupling trended to its highest point by 2 minutes. Previous data suggests that TCR signaling begins quickly (Huse et al., 2007), so this data is consistent with that assertion. It is determined that the optimal parameters to perform CATS is at a 1:1:1 ratio for 2 minutes without PP2.

Example 3 CATS and Tetramer Analysis of Low Affinity TCR-pMHC Interactions

[0069] This example shows how well CATS can pull out antigen specific TCRs within a polyclonal population. Specifically, we wanted to know if the disclosed CATS can be used as or more effectively as tetramers, which represent another antigen-binding method of T cell identification. Tetramers are highly specific, but previous literature suggests that they are ineffective at identifying low affinity TCR-pMHC interactions. In order to determine if CATS could overcome low affinity challenges through its increased avidity, we compared both the cognate pMHCII, MCC:I-E.sup.K, M12 cells and MCC tetramer to the lower affinity pMHCII, T102S:I-E.sup.k, M12 cells and T102S tetramer. To address this comparison, 110.sup.5 dye-labeled 5c.c7 CD4+ T cells were transferred into a polyclonal B10.A mouse and spleen and lymph nodes were taken 24 hours post-transfer. Flow plots demonstrate how CD4+ T cells were identified when coupled 1:1:1 with MCC or T102S and the dump Hb M12 cells using CATS (FIG. 3A) or how the T cells were identified using MCC:I-E.sup.k or T102S:I-E.sup.k tetramer while keeping Hb:I-E.sup.k tetramer as a dump (FIG. 3B). For further confirmation that our tetramer staining was real, we stained our MCC:I-E.sup.k or T102S:I-E.sup.k tetramer in 2 colors and gated on the double positive events, excluding any nonspecific binding. We collected the entire sample of cells in order to collect as many dye-labeled T cells that remained in the mouse and compared the percentage of these T cells that were coupled to M12 cells or stained with tetramer (FIG. 3C). Cell coupling with MCC:I-E.sup.k and the lower affinity T102S:I-E.sup.k M12 cells identified a similar coupling percentage with the dye-labeled T cells (FIG. 3C). And while MCC:I-E.sup.k tetramer identified a high percentage of the dye-labeled T cells, T102S:I-E.sup.k tetramer failed to identify any weak TCR-pMHC interactions (FIG. 3C). This data demonstrates that CATS can be used to identify weak TCR-pMHC interactions that cannot be identified with tetramer.

Example 4 Functional Analysis of MHCII in CATS

[0070] Based on our results from the immunization experiments, we next asked whether the cell coupling we observed was dependent on the pMHCII molecules themselves or if it was a consequence of nonspecific binding. In this pilot study, spleen and LNs from a nave and B10.A mouse were taken, and CATS analysis was performed under conditions described below. We saturated the I-E.sup.k MHC epitope with 14-4-4S I-E.sup.k MHCII antibody, thus blocking any TCR interactions that are dependent on MHC in MCC:I-E.sup.k, T102S:I-E.sup.k, and Hb:I-E.sup.k M12 cells. Furthermore, if these interactions are indeed dependent on MHCII, we would expect coupling to decrease with the addition of the blocking antibody. So, prior to cell coupling, the M12 cells were either left unblocked, stained with an isotype control antibody IgG2a , which binds MHCII on epitopes that are different from the 14-4-4S epitope, or stained with MHCII I-E.sup.k antibody. 510.sup.5 CD4+ T cells were then coupled with specific APC and dump APC 1:1:1, and cell couples were determined based on flow gating (FIG. 4A). Since the MHCII antibodies are colorless, we wanted to determine that blocking with 20 g/mL fully saturated the M12 cells. To approach this question, M12 cells were stained with the unconjugated 14-4-4S antibody for 15 minutes at room temperature, excess antibody was washed out, and stained again this time with conjugated 14-4-4S antibody. If the MHCII epitopes of interest were saturated, we would expect to see no staining from the conjugated antibody, which is precisely what occurred (FIG. 4C). We then collected 10,000 events of T cells coupled to our specific APC and determined coupling based on flow gates (FIG. 4B). Cell coupling was then normalized to the blocked cell coupling by dividing the coupling percentage per cell line by its coupling percentage in its blocked state (FIG. 4D). We saw nearly a 4 decrease in coupling between the B10.A CD4+ T cells and MCC and Hb when blocked with the 14-4-4S antibody, and we observed a 3 decrease in coupling between the B10.A CD4+ T cells and T102S (FIG. 4E). These data suggest that the coupling we observe in endogenous polyclonal populations is likely not a consequence of random, nonspecific events. Rather, the data suggest that MHCII is integral to engagement of TCR's with respect to the CATS method, especially regarding the I-E.sup.k epitope.

REFERENCES

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