Methods and devices for detecting cellular responses

Abstract

Systems and methods for identifying cellular responses to candidate drugs, including treatment cycling, are described. These systems and methods utilize rapid response readout in combination with gentle removal and / or exchange of the analyte fluid. The systems and methods can be used, for example, in T-cell epitope mapping, T-cell diagnostics, and other cell-based high-throughput screening methods.

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 526,370, filed Jul. 12, 2023. The entire content of the above application is incorporated herein by reference.

Background Art

[0002] In ex vivo and in vitro biological tests, especially when working with primary cells from patients or experimental animals, and when screening treatment conditions (drug screening, receptor specificity studies) up to and exceeding 1000, the small sample volume and underrepresentation of rare cell types can be a substantial limitation. An example of such a limitation is the screening of the exact peptide specificity of the T cell receptor (TCR).

[0003] T cell activation is important for the initiation of the immune response and involves three stages. The first stage involves the processing and presentation of an antigen as an epitope on the surface of an antigen-presenting cell (APC) in a complex with a major histocompatibility complex (MHC) molecule. The second stage involves the binding between an antigen-specific T cell receptor (TCR) and the antigen-MHC complex. This results in the formation of an intercellular interaction between the T cell and the APC called an immunological synapse. The third stage involves intracellular signaling and the activation of the T cell "itself", leading to phenotypic changes, cytokine secretion, migration, and proliferation. ELISpot (enzyme-linked immunosorbent spot), and flow cytometry (fluorescence-activated cell sorting, FACS) combined with staining for intracellular cytokine staining (ICS) or activation-induced marker (AIM) are common research methods used to detect T cell activation. ELISpot typically uses specific capture and detection antibodies to detect cytokine spots generated around cytokine-secreting cells. Flow cytometry detects either cytokine-secreting T cells or T cells that begin to express specific activation-induced markers on their surface.

[0004] Antigens recognized by a given T cell are typically present at extremely low frequencies, e.g., one or fewer per 100,000 antigens. Furthermore, not all APCs presenting a given antigen encounter T cells with specific specificity for that APC. Therefore, the probability of a positive response is low. For example, using conventional research methods, the probability of a positive response is 0.01% or less under individual experimental conditions, and more than 99.99% of the sample is used to obtain a negative readout. Thus, conventional research methods are associated with practical limitations, including complex logistics and high costs.

[0005] In this field, there is still a need for high-throughput methods and devices for identifying such low-probability positive responses using small amounts of biological samples. [Overview of the project]

[0006] The present invention is at least in part based on the discovery of a method that enables multiple rapid cycles of testing different candidate drugs on the same small sample (e.g., the sample contains cells). The methods described herein can be used in T cell epitope mapping, T cell diagnostics, and other cell-based high-throughput screening methods. The systems and methods described employ rapid response readout (e.g., less than one hour) in combination with gentle removal, optional washing of the analyte fluid, and treatment exchange. The systems and methods provide automated methods and systems for high-throughput and / or screening of candidate drugs on small samples containing cells, in combination with desired features of fluorescence microscopy and / or label-free or label-based readout, in combination with gentle removal / washing or treatment exchange methods. The systems and methods described herein can also be used to identify cells and / or cell counts that respond to an analyte, such as an analyte library.

[0007] This invention encompasses a method for detecting cellular responses to candidate drugs, and this method is This involves contacting a sample containing ia cells with an analyte fluid, Here, the sample containing cells is on a solid support, The analyte fluid contains the candidate drug in a liquid culture medium, and contact is required. b. Detecting cellular responses by detecting optical signals in a sample on a solid support, c. A first cycle comprising removing the analyte fluid from the solid support, thereby removing the candidate drug while retaining the sample containing cells on the solid support, ii.a. Contacting a retained sample containing cells from a preceding cycle with a new analyte fluid, wherein the new analyte fluid contains a new candidate drug in a liquid medium. The new candidate drug differs from the candidate drugs in the previous cycle in that it requires contact. b. Detecting cellular responses by detecting optical signals in a sample held on a solid support, c. A subsequent cycle comprising removing the analyte fluid from the solid support, thereby removing the candidate drug while retaining the sample containing cells on the solid support, The process involves repeating the subsequent cycle at least N times, where N is a non-negative integer, and where each new analyte fluid in each subsequent cycle contains a different candidate drug than that of the preceding cycle. The removal step is carried out using the mild removal / washing methods and / or treatment exchanges described herein. In certain embodiments, the removal of the analyte fluid (e.g., by treatment exchange or mild washing) is a method that does not involve high-speed centrifugation, in which the cell viability and / or cell volume of the retained sample decreases by about 5% or less after a total of 2 to 10, or 10 or more total cycles. For example, the cell viability and / or cell volume of the retained sample decreases by about 5% or less after a total of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more total cycles. In some embodiments, the cells are non-adherent cells, and the cell viability and / or cell volume of the retained sample decreases by about 5% or less after a total of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more total cycles. As used herein, high-speed centrifugation is centrifugation with a centrifugal force greater than 250 × g. In certain additional embodiments, the removal of the analyte fluid is a method in which the cell viability and / or cell volume of the retained sample does not decrease by more than about 5% after a total of 10 cycles, and is a method that does not involve centrifugation. Non-limiting examples of cells that may be used in this method are immune cells. Immune cells include, for example, B cells, T cells, innate lymphoid cells, natural killer cells, natural killer T cells, gamma delta T cells, macrophages, monocytes, dendritic cells, neutrophils, myeloid suppressor cells, hematopoietic stem cells or mesenchymal stem cells, induced pluripotent stem cells (iPSCs), and iPSC-derived cells. In some embodiments, the population of immune cells is one or more of B cells, T cells, innate lymphoid cells, natural killer cells, natural killer T cells, gamma delta T cells, regulatory T cells, macrophages, monocytes, dendritic cells, neutrophils, and myeloid suppressor cells. Further examples of cells that may be used in this method include mixed cultures and suspensions of immune and non-immune cells, such as blood cells, peripheral blood mononuclear cells, lymph node cells, mucus samples, tumor cell suspensions, and tumor-infiltrating leukocytes (TILs).In certain embodiments, the sample consists of multiple samples, and optionally, the multiple samples are located on a multiwell plate. For example, the multiple samples (including cells) are located on the membrane of a transwell insert, and each insert is placed in a well of a multiwell plate.

[0008] This invention also includes a method for identifying candidate drugs, and this method is iii.a. Contacting a sample containing cells with an analyte fluid, wherein the sample containing cells is on a solid support and the analyte fluid is a liquid culture medium containing a candidate drug. b. Detecting the response by detecting the optical signal in the sample on the solid support, c. A first cycle comprising removing the analyte fluid from the solid support, thereby removing the candidate drug while retaining the sample containing cells on the solid support, iv.a. Contacting a retained sample containing cells from a preceding cycle with a new analyte fluid, wherein the new analyte fluid contains a new candidate drug in a liquid medium, and the new candidate drug is different from the candidate drug in the preceding cycle. b. Detecting the response by detecting the optical signal in the sample held on a solid support, c. A subsequent cycle comprising removing the analyte fluid from the solid support, thereby removing the candidate drug while retaining the sample containing cells on the solid support, The process involves repeating the subsequent cycle at least N times, where N is a non-negative integer, and where each new analyte fluid in each subsequent cycle contains a different candidate drug than that in the preceding cycle. The removal step is carried out using the gentle removal / washing method described herein. In certain embodiments, the removal of the analyte fluid is a method in which the cell viability and / or cell volume of the retained sample does not decrease by more than about 5% after a total of 10 cycles, and does not involve high-speed centrifugation. As used herein, high-speed centrifugation is centrifugation with a centrifugal force greater than 250 × g. In certain additional embodiments, the removal of the analyte fluid is a method in which the cell viability and / or cell volume of the retained sample does not decrease by more than about 5% after a total of 10 cycles, and does not involve centrifugation. Non-limiting examples of cells that may be used in this method are immune cells. Immune cells include, for example, B cells, T cells, innate lymphoid cells, natural killer cells, natural killer T cells, gamma delta T cells, macrophages, monocytes, dendritic cells, neutrophils, myeloid-derived suppressor cells, hematopoietic stem cells or mesenchymal stem cells, induced pluripotent stem cells (iPSCs), and iPSC-derived cells. In some embodiments, the population of immune cells is one or more of B cells, T cells, innate lymphoid cells, natural killer cells, natural killer T cells, gamma delta T cells, regulatory T cells, macrophages, monocytes, dendritic cells, neutrophils, and myeloid-derived suppressor cells. Further examples of cells that may be used in this method are mixed cultures and suspensions of immune and non-immune cells, such as hematopoiesis, peripheral blood mononuclear cells, lymph node cells, mucinous samples, tumor cell suspensions, and tumor-infiltrating leukocytes (TILs).

[0009] In certain embodiments of the method described herein, the cellular response is detected less than 6 hours, less than 4 hours, less than 2 hours, less than 1 hour, or less than 30 minutes, less than 15 minutes, or less than 15 minutes after the cells have been brought into contact with the analyte fluid.

[0010] In an additional embodiment, the method is a method for detecting T cell activation, and the method is The process involves contacting a sample containing iaT cells with an analyte fluid, where the T cell sample is on a solid support and the analyte fluid is a liquid culture medium containing a candidate drug. b. Detecting T cell activation by detecting optical signals in a sample on a solid support, c. A first cycle comprising removing the analyte fluid from the solid support, thereby removing the candidate drug while retaining the sample containing T cells on the solid support, ii.a. Contacting a retained sample containing T cells from a preceding cycle with a new analyte fluid, wherein the new analyte fluid contains a new candidate drug in a liquid medium, and the new candidate drug is different from the candidate drug in the preceding cycle. b. Detecting T cell activation by detecting optical signals in a sample held on a solid support, c. A subsequent cycle comprising removing the analyte fluid from the solid support, thereby removing the candidate drug while retaining the sample containing T cells on the solid support, The process involves repeating a subsequent cycle at least N times, where N is a non-negative integer, and where each new analyte fluid in each subsequent cycle contains a different candidate drug than that in the preceding cycle. The removal step is carried out using the gentle washing and / or treatment exchange method described herein. In certain embodiments, the removal of the analyte fluid is a method that does not involve high-speed centrifugation, in which the cell viability and / or cell volume of the retained sample decreases by about 5% or less after a total of 10 cycles. In certain additional embodiments, the removal of the analyte fluid is a method that does not involve centrifugation, in which the cell viability and / or cell volume of the retained sample decreases by more than about 5% after a total of 10 cycles. In certain embodiments, the sample is a plurality of samples, and optionally, the plurality of samples are on a multiwell plate. For example, the plurality of samples (containing cells) are on the membrane of a transwell insert, and each insert is placed in the wells of a multiwell plate.

[0011] In an additional embodiment, the method is a method for identifying candidate drugs that activate T cells, and the method is The process involves contacting a sample containing iaT cells with an analyte fluid, where the T cell sample is on a solid support and the analyte fluid is a liquid culture medium containing a candidate drug. b. Detecting T cell activation by detecting optical signals in a sample on a solid support, c. A first cycle comprising removing the analyte fluid from the solid support, thereby removing the candidate drug while retaining the sample containing T cells on the solid support, ii.a. Contacting a retained sample containing T cells from a preceding cycle with a new analyte fluid, wherein the new analyte fluid contains a new candidate drug in a liquid medium, and the new candidate drug is different from the candidate drug in the preceding cycle. b. Detecting T cell activation by detecting optical signals in a sample held on a solid support, c. A subsequent cycle comprising removing the analyte fluid from the solid support, thereby removing the candidate drug while retaining the sample containing T cells on the solid support, The process involves repeating the subsequent cycle at least N times, where N is a non-negative integer, and where each new analyte fluid in each subsequent cycle contains a different candidate drug than that in the preceding cycle. The removal step is carried out using the gentle washing / treatment exchange method described herein. In certain embodiments, the removal of the analyte fluid is a method in which the cell viability and / or cell volume of the retained sample does not decrease by more than about 5% after a total of 10 cycles, and does not involve high-speed centrifugation. In certain additional embodiments, the removal of the analyte fluid is a method in which the cell viability and / or cell volume of the retained sample does not decrease by more than about 5% after a total of 10 cycles, and does not involve centrifugation.

[0012] The method described herein enables the detection of multiple low-probability events within the same sample. For example, this method enables the detection of different subpopulations of cells (e.g., T cells) that respond specifically to different treatments (e.g., treatments containing their corresponding peptides). This method may be more sample-efficient and / or more sensitive for users who prefer not to reduce the sample volume. For some users, the method described herein may be high in both sample efficiency and sensitivity.

[0013] In some embodiments, cells are incubated for a sufficient time for the cells to respond (e.g., a sufficient time for T cells to be activated). In certain embodiments, the response (positive or negative readout) is detected less than about 6 hours, less than about 4 hours, less than about 2 hours, less than about 1 hour, or less than about 30 minutes, less than about 15 minutes, less than 10 minutes, or less than 5 minutes after the cells have been brought into contact with the analyte fluid. Positive and / or negative readouts of a sample during a particular cycle can be recorded. In certain embodiments, a cycle includes more than one response detection step (e.g., detection of optical signals at different time points and / or detection of different optical signals).

[0014] In certain embodiments, samples in which a response (e.g., T cell activation) is detected are further characterized using a downstream assay. In certain embodiments, the downstream assay is performed on or carried out on the same support as the treatment cycling. In further embodiments, the staining and / or imaging of the downstream assay is carried out by the same device used for treatment exchange. In some examples, the downstream assay is automated. One specific method recommended as a downstream assay is automated post-cycle staining of live or fixed cells with a dye or antibody (e.g., a labeled antibody) to confirm or enrich the cycle data, a method referred to herein as "AIMSpot". In certain embodiments, the downstream assay is an activation-inducing marker (AIM) assay.

[0015] AIMSpot is a method that combines treatment cycling with automated or semi-automated immunofluorescence staining or other imaging techniques to detect activation-inducing markers. AIMSpot is a research method that includes (a) a cell culture system that allows imaging and treatment exchange for live cell culture to track individual cells without cell loss and without significant changes in cell location, (b) a live cell culture observation system with detection of individual cell features and responses over time, and (c) a system or method for downstream (post-cycle) staining and characterization of further cellular features and markers in the same traceable cells or their corresponding locations. Post-cycle characterization can be performed using live or fixed cells. Cellular features may include phenotypic markers (e.g., CD4, CD8, and others), expressed cytokines or other secretory molecules trapped within cells (e.g., IFN-γ, IL-2, TNF, and others, perforin, granzyme B, and others), and expressed activation-inducing markers (collectively referred to as AIMs, e.g., CD69, 4-1BB, OX40, CD40L, and others).

[0016] "A method in which removal of the analyte fluid results in no more than about a 5% decrease in the cell viability and / or cell amount of the retained sample after 10 total cycles", or "A method in which removal of the analyte fluid would result in no more than about a 5% decrease in the cell viability and / or cell amount of the retained sample after 10 total cycles", even if described as such, does not mean that the method requires at least 10 total cycles (or that N is at least 8), but rather that the method of removing the analyte fluid is such that it would result in no more than about a 5% decrease if 10 total cycles were completed. Thus, in one example, N is 1 (e.g., there are 3 total cycles), and the removal of the analyte fluid is a method in which the cell viability and / or cell amount of the retained sample would not decrease by more than about 5% if 10 total cycles were completed, and is a method that does not include centrifugation. In another example, N is 15, and the removal of the analyte fluid is a method in which the cell viability and / or cell amount of the retained sample does not decrease by more than about 5% if 10 total cycles were completed, and is a method that does not include centrifugation.

[0017] In certain embodiments, N (the number of cycles in addition to the first and second cycles) is about 0 or more, 1 or more, 2 or more, about 5 or more, about 10 or more, about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 49 or more, about 50 or more, about 74 or more, about 75 or more, about 99 or more, about 100 or more, or about 200 or more. In further embodiments, the total number of cycles (N + 2) is about 2 or more, 3 or more, about 5 or more, about 10 or more, about 15 or more, about 20 or more, about 30 or more, about 40 or more, about 49 or more, about 50 or more, about 75 or more, about 100 or more, or about 200 or more. The number of cycles is adjusted according to the particular sample and the particular response being read. For example, in an automated system, the number of cycles can be programmed according to the particular sample being detected and the reading of the particular response.

[0018] The methods described herein can be automated and / or high-throughput.

[0019] The present invention further encompasses an automated system or device for performing the methods described herein.

[0020] In one embodiment, the present invention is an automated system or device for identifying a candidate agent that induces a response in a cell, The system comprises, a. a dispensing unit configured to dispense an analytical species fluid onto a solid support, wherein, for example, a sample containing cells is within a well of a multi-well plate or on a transwell insert disposed within a well of a multi-well plate, and the dispensing unit is configured to dispense the analytical species fluid into each well of the multi-well plate; b. an optical imaging unit configured to detect a cell response by detecting an optical signal; c. a washing unit configured to remove the analytical species fluid from the solid support (e.g., each well), thereby removing the candidate agent, and to retain the sample containing cells on the solid support. and comprises.

[0021] In certain aspects, the automated system or device includes a multi-well plate. The multi-well plate may include a transwell insert. In another example, the multi-well plate does not include a transwell insert. The multi-well plate is configured such that cells can be exposed to more than one treatment cycle and the relative positions of individual cells do not change over different treatment cycles. Each "unit" may be separate or may be combined with another unit; for example, the washing unit may be part of the dispensing unit, i.e., it is understood that the same element of the device dispenses and removes the analytical species. In a further aspect, the multi-well plate is configured such that cells can be exposed to more than one treatment cycle and the relative positions of individual cells do not significantly change over different treatment cycles.

[0022] In a particular embodiment, the present invention relates to an automated system or device for identifying T cell activation, wherein the system is a. A dispensing unit configured to dispense an analyte fluid onto a solid-phase support, wherein, for example, a sample containing T cells is located in a well of a multi-well plate or on a transwell insert placed in a well of a multi-well plate, and the dispensing unit is configured to dispense the analyte fluid into each well of the multi-well plate. b. An optical imaging unit configured to detect T cell activation responses by detecting optical signals, c. A solid-phase support, for example, a washing unit configured to remove the analyte fluid from each well, thereby removing the candidate drug, and to retain the sample containing T cells in the wells (which may be the same part as the dispensing unit). It is equipped with.

[0023] In a particular embodiment, the automated system or device includes a multiwell plate. The multiwell plate may include a Transwell insert. In another embodiment, the multiwell plate does not include a Transwell insert. The multiwell plate is configured such that cells can be exposed to one or more treatment cycles, and the position of individual cells does not change across different treatment cycles. Each “unit” may be separate or combined with another unit of the device; for example, a washing unit may be part of a dispensing unit. In a further embodiment, the multiwell plate is configured such that cells can be exposed to one or more treatment cycles, and the relative position of individual cells does not change significantly across different treatment cycles.

[0024] In certain embodiments, the system or device described herein further comprises a holder, platform, or stand for a plate. Optionally, the holder or stand includes a transport unit for transporting a multiwell plate to engage with one or more of a dispensing unit, an optical imaging unit, and a washing unit. In some embodiments, the holder, platform, or stand remains in a fixed position during cycling. In such embodiments, dispensing, imaging, and washing are performed while the plate remains in a fixed position. In other embodiments, the holder or stand moves to different modules or units during cycling.

[0025] The optical imaging units described herein may further include microscopes, such as fluorescence microscopes. In certain embodiments, the optical imaging unit (e.g., fluorescence microscope) may be positioned beneath a multiwell plate and configured to optionally move from well to well. In other examples, the optical imaging unit is positioned beneath a plate, and the plate moves so that different wells are above the optical imaging unit.

[0026] The dispensing unit is configured to dispense the analyte fluid onto a plate. For example, the dispensing unit may be configured to dispense different analyte fluids into each well. In some embodiments, the dispensing unit includes a nozzle and / or pipette tip.

[0027] The cleaning unit may include one or more aspirators configured to draw in the analyte fluid. The cleaning unit may be configured, for example, for laminar flow cleaning. In some examples, the holder or stand is configured to tilt (e.g., rotatably coupled to a pivot point), and the cleaning unit draws in or otherwise removes the fluid when the plate is tilted. As described above, the cleaning unit can be integrated into the dispensing unit.

[0028] In some embodiments, the dispensing and washing unit includes tubing, connectors, manifolds, pumps, syringes, a vacuum system, or connections to a vacuum system. In some embodiments, the dispensing and washing unit may be components of a single system or may have shared components.

[0029] The system or device may further include a processor that electronically communicates with an optical imaging unit, and a non-transient computer-readable medium accessible to the processor and storing instructions that, when executed by the processor, cause the processor to receive or record optical signals. In certain embodiments, the system or device includes one or more processors configured to dispense analyte fluids, detect optical signals, and / or remove candidate drugs. In further embodiments, the system includes a control module having a user interface that enables an operator to perform a method of screening cellular responses, e.g., the method described herein. The system or device may further include a system for storing and supplying new analytes in a screening library with optional temperature control, and one or more liquid handling devices that manage chips, liquids, move plates or other containers containing specimens or analytes, and are connected to all other system components and capable of processing one or more containers (e.g., plates) containing specimens.

[0030] The present invention also encompasses a non-temporary computer-readable medium that stores computer-readable instructions governing the methods described herein when executed by one or more processors.

[0031] The aforementioned and other objects, features and advantages of the present invention will become apparent from the following more detailed description of preferred embodiments of the invention, as shown in the accompanying drawings, where similar reference numerals in different figures refer to the same parts. The drawings are not necessarily to scale and instead focus on illustrating the principles of the invention. [Brief explanation of the drawing]

[0032] [Figure 1]This figure illustrates the principle of the treatment cycling method. A cell culture is shown, which may contain more than one cell species, with several cells possessing unique characteristics. Given the potential for low response probabilities, treatment cycling allows for screening of multiple treatments using the same sample. This method enables the study of cellular phenotypes (e.g., CD4 / CD8) and / or confirmation of long-term responses such as activation-inducing markers and cytokine expression by surface antibody staining of fixed cells using the same automated device (e.g., using labeled antibodies). Data analysis can be overlaid with long-term / final readouts (shown as small dark "X"s at the bottom of the figure) and rapid responses associated with specific cycles of the same cells (shown as white circles). More specifically, the figure shows that cellular responses to a library of treatments (analyte fluids) are being tested. The treatment exchange cycles indicate how many cells produce rapid photo(fluorescence) readouts associated with activation in each treatment cycle. After cycling, different downstream assays may be applied. In this example, AIMSpot is demonstrated. After cycling, cells are incubated to induce a long-term response and then stained via membrane in an automated manner with fluorescent antibodies that indicate either their activated phenotype (e.g., by detecting AIM markers such as CD69, 4-1BB, OX40, or CD40L, or combinations thereof) or cytokine production. The same procedure may be performed simultaneously or in one cycle in multiple wells of a multiwell plate or multiple alternative containers, or different procedures may be performed simultaneously or in one cycle in different wells. In another example, the same sample (or multiple samples) may be dispensed for different procedures, and / or the same procedure may be performed in different wells in a different order. Cells are shown as a monolayer. In different embodiments, they may form multiple layers or represent non-dissociated tissue. Layers may further be separated by a filtration mesh (as discussed elsewhere) or retained together by different means including cross-linked gels, bispecific antibodies, and fibrous proteins.Magnetic microbeads bound to cells can, in some embodiments, assist in layer formation and stabilization. In one embodiment, the sample is pre-treated to elicit fluorescence readout. However, a portion of the sample or mixed sample cells may be pre-isolated, differentiated, and pre-treated to obtain optimal readout. The pre-treatment scheme may include treatment with different biologics, including low molecules, antibodies, nucleic acids, and their analogues, along with the delivery vehicle, viruses, and pseudoviruses. In certain embodiments, these additional treatments may be performed with the analyte or between cycles in which the analyte is added. [Figure 2] This figure shows the principles for recording direct (A) and indirect (B, C, D) responses. An embodiment in which T cells become fluorescent after activation represents a situation of direct detection of activated cells (A). In other embodiments, T cell signals may be read indirectly through the formation of immunological synapses (C) or by detecting signals (B) originating from cells that present antigens and activate T cells. These signals may include, for example, the detection of cytoskeletal or mitochondrial network rearrangement, and several other events occurring in cells that present antigens and actively interact with T cells. Detection of these changes may be fluorescence or depend on other optical detection methods. Another indirect method of detecting T cell activation is (D) detecting changes in adjacent cells following T cell activation and downstream signaling. For example, some cells in a sample may respond to T cell cytokines and produce detectable readouts. In some examples, the readouts may depend on the responding cell. In additional examples, the readouts may be optical or based on other methods. [Figure 3]This figure shows alternative methods for downstream workflows after treatment cycling. This illustrates some potential downstream assay alternatives. In the case of T cell activation, it is important to note that researchers are typically interested in the T cell phenotype and type of activation. Downstream assays can provide these data. The cycling assay itself may involve a cycle of surface and intracellular staining with antibodies and cell fixation, providing sample preparation for downstream use. Downstream workflows may involve identifying cellular features related to the response. Downstream workflows are optional but can be helpful in cleaning and enhancing data. In one example, a downstream workflow may include one or more of the following optional steps, and others: 1. AIMSpot: Cells are stained for surface markers at their relative locations, fixed, and stained for intracellular proteins. 2. Cell cultures may be sorted for selected cell types / responses. 3. Cell cultures may be left for proliferation / death / other events. 4. Cell culture media may be taken for biological testing. 5. Cell cultures may be stained for flow cytometry or with barcoded antibodies / tetramers for downstream sequencing. 6. Cell cultures may be used for bulk or single-cell sequencing. 7. The relative positions of cells are fixed for spatial transcriptomics. For example, if the response is proliferation, the use of proliferated clones has several analytical advantages. [Figure 4]This figure illustrates the principle of spatial data superposition using AIMSpot (cycle and post-cycle). Dynamic data of the presence or absence of activation response can be potentially contaminated by false-positive readouts (derived from unrelated cell activation events, e.g., the initiation of apoptosis). Due to the maintenance of cell coordinates during multiple treatment exchange cycles, dynamic presence / absence response data can ultimately be superimposed (juxtaposed) with post-cycle data of in situ antibody staining of the same cell layer. True cell activation can be confirmed by AIM marker expression, cytokine production, or a combination thereof. More complex studies are possible when T cell clones are proliferated and spatial sequencing or TCR sequencing is applied. The figure shows that the position / coordinates of individual cells are tracked. Downstream analyses may include T cell clone proliferation and transcriptome analysis. T cell clones can be picked by an automated colony picker, for example, for T cell receptor (TCR) sequencing. This combination of methods can result in up to 1000-fold and more efficiency increases in sample use for TCR discovery. [Figure 5] This figure illustrates an example of fluid exchange applied substantially simultaneously in multiple wells. The figure shows cells in a Transwell insert. As shown in the figure, the fluid is transferred by a pump through a tube. Treatment exchange takes place beneath the membrane of the Transwell insert. The pump fluid transfer system may be connected to the plate by a tube. Each well contains a Transwell insert with a membrane at the bottom of the insert. The cells are a layer on the membrane. One fluid transfer system removes the previous treatment from beneath the membrane while new wash / analyte / stain solution is added. [Figure 6]This is a schematic diagram illustrating a second example of fluid exchange in a single representative well of a multiwell plate. In this example of treatment exchange, cells are perfused on the membrane and stacked on the membrane of a transwell insert. The treatment contains a peptide and / or peptide pool corresponding to the antigen. The readout is fluorescence of activated T cells by fluorescence microscopy. For treatment exchange, the fluid can be slowly added above the perfused cells inside the transwell insert and collected from the bottom of the chamber. Separation principle: mesh filtration, risk of disturbance exists. Signal detection: from bottom to top relative to the cells, in the same unit or separate units. [Figure 7] This is a schematic diagram illustrating a third example of fluid exchange in a single representative well of a multiwell plate. In this example of treatment exchange, laminar flow is present over immobilized cells, and the cells are immobilized at the bottom of a flattened well. The treatment is a peptide library corresponding to a common vaccine. Readout is immunosynapse formation by fluorescence / optical microscopy. Treatment exchange is by laminar flow. Separation principle: gel or adhesion, non-disturbing shear flow. Signal detection: from below or above the cells, in the same unit or separate units. [Figure 8] This is a schematic diagram illustrating a fourth example of fluid exchange in a single representative well of a multiwell plate. In this example of treatment exchange, a flow is present on the membrane above the cells, and the cells are adherent cells with a reporter system for pathway activation. The treatment is a large drug library with a low probability of positive response, and all drugs can be tested at escalating concentrations. Readout is fluorescence / luminescence / death of activated cells, etc. Treatment exchange is performed on the membrane without disturbing the cells. Separation principle: Mesh shielding with cells at the bottom of the well. Signal detection: From below or above the cells, in the same unit or a separate unit. [Figure 9]This figure shows a fifth example of fluid exchange in a single representative well of a multiwell plate. In this example of treatment exchange, cells are located inside a microwell / microgrid array, and the cells are stacked on flat-bottomed microwells / grids within the well. The treatment is a peptide or peptide pool corresponding to an antigen. Readout may be immunosynapse formation by fluorescence / optical microscopy. For treatment exchange, layers of fluid are exchanged on the microwells. Treatment exchange can be performed by pipetting, a fluid system, or a combination thereof. Separation principle: Cells are protected from flow by the walls of the microwell / grid. Signal detection: From below or above the cells, in the same unit or in separate units. [Figure 10] This figure shows one example of a treatment cycling device in which the container containing the sample has a fixed position during treatment cycling. This approach is preferred because it is important to track the position / coordinates of the cells throughout the assay. The principle of liquid transfer is solely pipetting. Liquid transfer can be performed solely by pipetting (e.g., by a liquid handler robot). In this embodiment, the cells do not change their position until they are removed. [Figure 11]This figure illustrates an example of another treatment cycling device in which, during treatment cycling, the container containing the sample has a fixed position, or the container on the stage of the microscope / imaging device can be moved for imaging (allowing for a stable optical system). This approach may be less desirable in certain situations because tracking the position / coordinates of cells throughout the assay is important. However, if the movement is slow, they can be managed. The plate must remain in place throughout all cycles. The principle of fluid transfer is hybrid and includes both a pipetting system and a fluid system. The fluid system may be based on a combination of multiple peristaltic pumps, diaphragm pumps, vacuum pumps, or other pumps. If the connecting tubing is flexible, the stage holding the cell container (e.g., plate) can be moved. Special plate designs with fluid inlets and outlets can simplify the tubing connections. In this embodiment, the cells do not change their position until removed. The detection module may be stable, and the plate may be moved if the tubing is flexible. Pipetting may be performed to an intermediate transfer plate or directly to the test plate. [Figure 12] This figure illustrates yet another example of a treatment cycling device in which the container containing the specimen has a fixed position during treatment cycling. The principle of liquid transfer is solely a fluid system (no pipetting). The liquid exchange system may be configured under or around the wells to allow optical detection from the top of the plate and from below the plate. Not all possible embodiments are shown. The substantial difference in this example is the absence of pipetting by liquid handling robotics, which is replaced by a flow manifold driven by a peristaltic pump, diaphragm pump, vacuum pump, or other pump. In this example, the cells do not change their position until they are removed. [Figure 13]This figure illustrates an example of a further treatment cycling device in which a container containing a sample is automatically moved between modules (based on multiple separate units) during treatment cycling. This approach allows for the construction and programming of automated sets of commercially available third-party instruments to create treatment cycling workflows. Although the plate is moving in this embodiment, this approach also allows for tracking the position or coordinates of specific cells throughout the assay by providing smooth plate transfer, cell fixation by attachment to a plate or membrane, or cell embedding in a gel. The cells are stable in the plate as it moves between units. In this example, the cells in the plate are transferred between instrument units. If the cells are attached or gel-embedded, plate transfer can be improved. [Figure 14] This figure illustrates the potential uses of the methods described herein for studying vaccination status and vaccine effectiveness, studying the spread of infection, and studying the immunogenicity of cell and gene therapies that introduce potential T cell antigens into the organism. The advantages are minimal sample volume and high sensitivity compared to conventional methods. Researchers can use smaller sample volumes to detect responses, or they can use the same sample volume but obtain more data from the sample (epitope mapping or multiple responses). This figure illustrates the process for vaccine / infection protection status. The sample volume required is minimal for the same amount of data. [Figure 15] This figure illustrates the potential uses of the methods described herein for studying vaccination status and vaccine effectiveness, studying the spread of infection, and studying the immunogenicity of cell and gene therapies that introduce potential T cell antigens into the organism. It shows the process for pandemic response preparation. The time after infection for test effectiveness is minimal due to high sensitivity. The earliest potential time is 2-3 days after contact. The cost of the test is much lower than analogues based on T cell receptor sequencing. [Figure 16]This figure shows the potential uses of the method described herein for studying vaccination status and vaccine effectiveness, studying the spread of infection, and studying the immunogenicity of cell and gene therapies that introduce potential T cell antigens into the organism. It illustrates the process regarding the non-immunogenicity of cell and gene therapies. The time after treatment for test effectiveness is minimal due to high sensitivity. The earliest time is 2-3 days after treatment. The cost of the test is significantly lower than that of sequencing-based analogues. Further applications: testing the effects of tolerance and treatment, cancer vaccination, and numerous other applications. [Figure 17] This figure shows fluorescence microscopy images of cells in human PBMCs demonstrating Ca2+ flux. The image on the left is a field of view of cells in a human PBMC monolayer showing Ca2+ flux after two cycles of treatment (no treatment, Flu peptide pool), with n=62 activated cells (cells brighter than a preset threshold are counted using QUpath). The image on the ring is a field of view showing the number of cells in a human PBMC monolayer showing Ca2+ flux after three cycles of treatment (first two cycles + Covid peptide pool), with n=183 activated cells. Previously activated cells are not excluded from the analysis. [Figure 18] This figure shows fluorescence microscope images. The top two images: T cell markers can be stained 24 hours after Ca++ signaling data was collected (Ca2+ sensor signals photobleached before staining). Human PBMCs were stimulated with a Flu peptide pool, and the cellular phenotype was studied; staining was performed via a Transwell membrane. Images can be overlaid for data analysis. Bottom image: Intracellular staining of IFN-γ "spots" to confirm T cell activation, readout of negative control (NC) vs. positive control (PC). Cell density is shown in transmitted light images. [Modes for carrying out the invention]

[0033] As used herein, the words "a" and "an" shall, unless otherwise specified, encompass one or more.

[0034] As used herein in relation to numerical values ​​or ranges, the term “about” allows for some degree of variability in the value or range, for example, within 20%, 10%, 5%, 4%, or 2% of the value or range, depending on the context.

[0035] As used herein, “specimen” or “biological sample” refers to a biological specimen or sample obtained from an experimental animal, human, in vitro cell line, or other source. An essential characteristic of specimens described herein is that they contain viable, functional cells capable of responding to a variety of treatments. Non-limiting examples of biological samples include tissues, body fluids, and other specimens taken directly from a subject, as well as specimens resulting from one or more processing steps such as separation, centrifugation, genetic engineering (e.g., transduction with a viral vector encoding a recombinant chimeric receptor), washing, and / or incubation. A biological sample may be a specimen obtained directly from a biological source or a processed specimen. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, saliva, sweat, and tissue and organ specimens (e.g., specimens from tissues or organs including tumors), and processed specimens derived therefrom. In some embodiments, a biological sample is a biological fluid specimen or a biological tissue specimen. In some embodiments, a biological sample is a biological tissue. In some embodiments, the biological sample from which immune cells are derived or isolated is blood or a blood-derived sample, or derived from apheresis or leukocyte apheresis products. When blood is used as the biological sample, an anticoagulant, such as heparin, citrate, or ethylenediaminetetraacetic acid, may be added to the blood as needed. A fraction containing T cells and antigen-presenting cells, prepared from blood by conventional methods, may be used as the biological sample.

[0036] "Cell culture" is the proliferation of living cells under controlled conditions. The term "primary cell culture" means that the cell culture originates from a living organism, as opposed to immortalized cell lines, which are also studied in vitro. In a further aspect, a biological sample is a dissociated tissue culture, a primary cell culture, a cell line, or a mixture thereof. In a particular aspect, a biological sample is a mixed culture of T cells and antigen-presenting cells. Cell cultures can be, for example, adherent, partially adherent, non-adherent, or suspension cultures. Biological samples or cell cultures can be preconditioned by a variety of methods, including, but not limited to, mechanical or enzymatic dissociation, centrifugation, magnetic or other tools for specific cell types, isolation, separation, concentration or depletion, freeze / thaw, pretreatment or incubation under different culture conditions, transfection, and other genetic modifications.

[0037] As used herein, the term “sample” may be used to refer to a specimen on a solid-phase support, a biological specimen, or a specific portion of a cell culture. For example, if the support is a 6-well plate, the plate may be described as having a sample in each well. The samples in each well may be derived from the same biological specimen or from different biological specimens. In certain embodiments, the sample is a cell culture.

[0038] The terms “analyte fluid,” “analyte composition,” “analyte liquid,” or “analyte solution” are used interchangeably herein to mean a liquid composition containing a candidate drug. The candidate drug may be, for example, a small molecule or a biological formulation. A non-limiting example of an analyte fluid is a peptide diluted with DMSO and further diluted with an optimized T cell culture medium.

[0039] A "candidate drug" is a drug being tested or screened for a biological response. Non-limiting examples of candidate drugs include small molecule drug candidates, peptides, antibodies, and libraries of any of these. A "candidate library" or "candidate drug library" is a mixture of two or more (usually up to several hundred) individual candidate drugs. A "candidate drug" may be, for example, a potentially activating drug and / or one added from outside the cell.

[0040] As used herein, “single-cell sensitivity” means that a method or system can distinguish a response from the background, even if only one single cell responds to the treatment. In certain embodiments, the treatment cycling systems and methods described herein have single-cell sensitivity.

[0041] The terms “Transwell system,” “well insert,” “Transwell insert,” and “culture insert” are used interchangeably herein to refer to products that are semipermeable to air and liquids and are placed inside the regular wells or “outer wells” of a multiwell cell culture plate or other container. These outer wells may have a design that allows for easy addition, removal, or replacement of culture medium / liquid. A “well insert” may be referred to as a “top chamber” or “upper chamber,” and a multiwell plate or other support, or its wells or reservoir, may be referred to as a “bottom chamber.” In some embodiments, these inserts allow cells to move through a semipermeable membrane or mesh at the bottom of the insert. In other embodiments, the membrane or mesh at the bottom of the insert is not permeable to cells.

[0042] As used herein, “mesh membrane” or “mesh” refers to a semi-permeable or permeable membrane having an open weave with openings through which a liquid can pass, and is formed as a woven material, a perforated material, etc. In certain embodiments, the mesh is a nylon mesh. The openings or pores of the mesh may be 30 μm to 1 mm. In certain embodiments, the openings or pores of the mesh may be about 0.1 μm to 5 μm in size.

[0043] As used herein, “laminar flow cell washing” is a method of replacing the liquid components of a cell suspension or culture monolayer by creating a liquid shear flow that does not affect cell adhesion to the bottom of the cell culture vessel.

[0044] As used herein, “downstream workflow,” “downstream assay,” “downstream method,” or similar terms refer to methods applied after treatment cycling.

[0045] "Direct readout" involves the direct detection of cellular responses, such as changes in their optical properties.

[0046] "Indirect readout" is the indirect detection of cellular responses, such as their interactions with neighboring cells or the responses of neighboring cells.

[0047] A positive readout indicates the detection of a cellular response (e.g., T cell activation).

[0048] Negative readout (e.g., lack of T cell activation) indicates that no cellular response was detected.

[0049] The systems and methods described herein utilize treatment cycling. “Treatment cycling” is a method described herein that allows repeated treatment of the same sample. Repeated treatment allows for the detection of one or more low-probability events (different subpopulations of cells responding to a particular treatment) within the same sample, and thus makes the process either more sample-efficient and / or more sensitive, for example, for users who do not prefer to reduce the sample volume. For some users, the methods described herein may be higher in both sample efficiency and sensitivity simultaneously. Alternatively, this process may be considered as recycling or reusing the same sample in a number of experimental treatments. This process may also be described as repeating an experiment seeking a positive response until it appears. In some embodiments, a positive response may be obtained in multiple cells and / or samples in different treatment cycles. In certain embodiments, if a sample is positive for a response (e.g., T cell activation) in a cycle, that sample is not subjected to subsequent cycles of different treatments. In yet another embodiment, if a sample is either positive or negative for a cellular response (e.g., T cell activation) in a cycle, the sample may still be subjected to one or more subsequent cycles of different treatments. A positive response may be, for example, fluorescence, luminescence, or other signals from cells above the background, cellular interactions with other cells in the sample, or their metabolites, and / or changes in the phenotypic characteristics of one or more target cells, or one or more cells interacting with the target cells or their metabolites, as determined by optical imaging. Optical signals can be detected by microscopy, for example, fluorescence microscopy.

[0050] Regarding standard T cell epitope mapping methods, most T cell immunologists seek answers to several questions: “Are T cells activated?”, and if yes, “How are T cells activated?”, as well as “What cytokines do T cells produce after activation?” or “What surface markers do T cells express after activation?”. The methods available in the art aim to answer these questions in a single experiment. The systems and methods described herein can answer the question “Are T cells activated?” more effectively with fewer samples and multiple times (depending on the number of cycles). After treatment cycling, downstream assays, e.g., AIMSpot, AIM assay, and / or other methods described herein or known in the art, may then be used downstream of treatment cycling to further characterize the cellular response or T cell activation, e.g., the type of T cell activation and sequencing (e.g., nucleic acid sequencing of the TCR receptor).

[0051] The systems and methods described herein combine rapid readout with gentle washing / analyte exchange and / or treatment exchange. The gentle washing / analyte exchange and / or treatment exchange methods described herein avoid the use of high-speed centrifugation. In certain embodiments, the use of centrifugation is avoided in the gentle washing / analyte exchange and / or treatment exchange methods described herein. Centrifugation is almost universally used to wash cells. Centrifugation is difficult to incorporate into automated systems and methods and can adversely affect cell viability and volume, especially when centrifugation is performed at normal to high speeds for cell culture (>250 × g) and repeated multiple times, which can lead to the formation of cell aggregates and degrade cell performance in assays. Therefore, the present invention utilizes methods and techniques that can be easily automated and do not result in cell loss or damage. Treatment cycling can also utilize methods and techniques that maintain the relative spatial position or coordinates of cells on a support, for example, in wells or on a membrane.

[0052] The systems and methods described herein include cell culture systems in which imaging and treatment exchange are performed while enabling tracking of individual cells without cell loss and without significant changes in the relative cell position (e.g., the location or position of cells relative to other cells in the culture), and / or cell culture systems that enable the detection of individual cell features and responses over time. Optionally, the systems and methods described herein also include downstream (post-cycling) staining and characterization of additional cell features and markers in the same traceable cells and / or at their corresponding locations and / or on the same support as the treatment cycling. Post-cycling characterization may be performed using live or fixed cells. For example, a device including a multiwell plate configured such that the relative positions of individual cells do not change significantly across different treatment cycles is described herein. In some examples of the methods and devices described herein, the relative positions of cells do not change significantly if at least about 85%, 90%, or 95% of the cells in a sample retain their relative positions after two, three, four, five, six, seven, eight, nine, or ten or more total cycles. In certain embodiments, the cells are non-adherent cells, and the relative positions of cells do not change significantly if at least about 85%, 90%, or 95% of the cells in a sample retain their relative positions after two, three, four, five, six, seven, eight, nine, or ten or more total cycles. Because the treatment cycling described herein retains the relative positions of cells, individual cells can be tracked or monitored using imaging over multiple cycles.

[0053] The treatment cycling technique ensures that cells are not exposed to a culture medium-free environment during the cycling process. Therefore, the systems and methods described herein can provide cell culture conditions over several hours or days. In some examples, the analyte fluid may differ for each cycle, and multiple cycles are possible, e.g., 5, 10, 20, 50, 75, or 100 cycles. In additional examples, the treatment schedule (e.g., analyte fluid) may differ for different wells. The treatment exchange cycles may be automated and / or standardized, for example, for multiple wells, and / or coordinated with downstream assays, e.g., imaging. In some examples, at least 1 million cells are present on the support.

[0054] In one specific example, the cells of the sample form a layer on the semipermeable membrane of the Transwell insert, and the Transwell insert is placed in the wells of a multiwell plate. The membrane restricts or prevents the movement of cells from the Transwell insert (e.g., into the bottom of the wells of the multiwell plate). Treatment from a preceding cycle can be removed from the wells of the multiwell plate without disturbing the position of the cells and without exposing the cells to an environment without culture medium. By adding new analyte fluid to the wells of a microwell plate, it can diffuse and reach the cells in the insert. Removal of the analyte fluid in any cycle can be carried out, for example, by pipette and / or tubing connected to a pump with adjustable flow rate. Other methods for treatment exchange of new analyte fluid or separating cells from the flow are described, for example, in the figure. In certain embodiments, the Transwell insert is fixed or immobilized when placed in the wells of a multiwell plate. In additional examples, the openings of the wells and / or the Transwell insert are covered to prevent or reduce evaporation.

[0055] When the treatment cycling approach is combined with spatial transcriptomics, i.e., multiple signals per container / well / microchip for cycle position barcoding (CLB), the effectiveness of sample use for determining TCR sequences and activation types can be further increased by up to 10-fold or more. Applying treatment cycling and CLB together can increase assay effectiveness by up to 1000-fold or more.

[0056] As described herein, each treatment cycle includes a response readout step (detection of a cellular response or detection of T cell activation) and a removal step in which the analyte fluid is removed. Analyte fluid removal removes all or part of the analyte fluid from a solid support (e.g., a multiwell plate) and thus removes candidate (potentially activating) agents, while retaining the sample containing cells on the solid support (e.g., a cell culture). As used herein, “gentle analyte removal,” treatment exchange, “gentle analyte removal / treatment exchange,” and similar terms refer to a set of methods that allow the analyte fluid to be removed from a sample without causing extreme stress or damage to living cells, preferably while maintaining their ability for functional response and / or their relative position on the support or the bottom of the well or membrane. In certain embodiments, gentle analyte removal and / or treatment exchange maintains the ability of cells for functional response and their relative position. In certain embodiments, the removal and / or treatment exchange of the analyte fluid is sufficiently gentle that the cell viability and / or cell volume of the retained sample does not decrease by more than approximately 5% after a total of 10 cycles. The method used for removal and / or treatment exchange does not involve high-speed centrifugation. In certain embodiments, the method used for removal and / or treatment exchange does not involve centrifugation. Standard centrifugation at high centrifugal force is not considered gentle cell washing as it can result in cell loss, death, or dysfunction. In certain embodiments, gentle analyte removal and / or treatment exchange may include analyte exchange in which a previous potentially T-cell activating analyte solution is removed and a subsequent potentially T-cell activating analyte solution is added. In further embodiments, the removal step may include gentle washing, which is a washing step in which, for example, a washing buffer that does not contain any cell-activating components is introduced into the sample. An example of a washing buffer for T-cell activation cycling is T-cell medium. Gentle washing and / or treatment exchange can be performed at an optimal physiological temperature, ideally +37°C, but temperature variations should be possible without affecting the assay. In a particular embodiment, the temperature is between +20°C and +40°C.Ideally, gentle washing and / or treatment changes will maintain the cells in the same 2D orientation within the well. In some assay embodiments, these stable cell coordinates are important.

[0057] As described above, the present invention encompasses the detection of cellular responses to candidate drugs (e.g., potentially activating drugs), and this method is This involves contacting a sample containing ia cells with an analyte fluid, Here, the sample containing cells is on a solid support, The analyte fluid contains the candidate drug in a liquid culture medium, and contact is required. b. Detecting cellular responses by detecting optical signals in a sample on a solid support, c. A first cycle comprising removing the analyte fluid from the solid support, thereby removing the candidate drug while retaining the sample containing cells on the solid support, ii.a. Contacting a retained sample containing cells from a preceding cycle with a new analyte fluid, wherein the new analyte fluid contains a new candidate drug in a liquid medium. The new candidate drug differs from the candidate drugs in the previous cycle in that it requires contact. b. Detecting the response by detecting the optical signal in the sample held on a solid support, c. A subsequent cycle comprising removing the analyte fluid from the solid support, thereby removing the candidate drug while retaining the sample containing cells on the solid support, The process involves repeating the subsequent cycle at least N times, where N is a non-negative integer, and where each new analyte fluid in each subsequent cycle contains a different candidate drug than that in the preceding cycle. The analyte fluid is removed by a mild analyte removal or treatment exchange method described herein. In certain embodiments, the removal of the analyte fluid or treatment exchange is a method that does not reduce the cell viability and / or cell volume of the retained sample by more than about 5% after a total of 10 cycles, and does not involve centrifugation. In certain examples, the detection of optical signals in a cycle includes more than one round of detection before analyte removal or treatment exchange and subsequent cycles. For example, a cycle may include multiple imaging or detection steps performed over time, e.g., separated by a few seconds, a few minutes, or a few hours.

[0058] A solid-phase support can be any support on which cells can be cultured, and this includes, but is not limited to, cuvettes, tubes, capsules, microtiter plates having microtiter wells (also referred herein as multiwell or multiwell plates), e.g., 6-well plates, 12-well plates, 24-well plates, 96-well plates, 192-well plates, 384-well plates, 1536-well plates, or the membrane surface of transwell inserts within these multiwell plates. Another example of a solid-phase support is the “wallless” plate sold by Curiox and / or described, for example, in U.S. Patent Application Publication No. 20140235468(A1), the contents of which are expressly incorporated herein by reference. Further examples of solid-phase supports are plates having a bottom of wells containing microwells or microgrid arrays for cell culture (similar to those described in U.S. Patent No. 9,068,155 (B2), U.S. Patent No. 1,1969,702 (B2), and U.S. Patent Application Publication No. 20230407223 (A1)), where the microwells or microchambers prevent the flow of liquid close to the cells at the bottom, but allow for treatment exchange within the microwells / microchambers via diffusion if the liquid is exchanged across a larger well or container. Solid-phase supports can be formed from any suitable material, including but not limited to polystyrene or polyvinyl, or derivatives thereof. Solid-phase supports can also be microfluidic devices.

[0059] A treatment cycling consists of a first cycle and at least one subsequent cycle. N is the number of cycles after the second cycle. Therefore, if N is 0, there are 2 total cycles. In another example, if the total number of cycles is 10, this means there is a first cycle and 9 subsequent cycles (or, in other words, N is 8). In another example, if the total number of cycles is 100, this means there is a first cycle and 99 subsequent cycles. Each subsequent cycle comes into contact with the sample from the preceding cycle from which the analyte fluid has been removed. The preceding cycle is the cycle immediately preceding the referenced cycle; for example, if the total number of cycles is 3, the first cycle is the “preceding cycle” of the second cycle, and the second cycle is the “preceding cycle” of the third cycle. In another example, if the solid-phase support is a 96-well plate, where each well contains a sample, and the total number of cycles is 3, the analyte fluid may be removed from each well at the end of the first cycle (while retaining the sample containing cells in the well), and the retained sample is treated with a new candidate drug in the second cycle. The analyte fluid may then be removed from each well at the end of the second cycle (while retaining the sample containing cells in the well), and the retained sample is treated with a new candidate drug (different from the candidate drug in the second cycle) in the third cycle.

[0060] The analyte fluid can be removed from the solid-phase support (e.g., a multi-well plate) by any method that removes all or part of the analyte fluid. For example, the analyte fluid can be aspirated or otherwise removed so that cells are retained in the wells with minimal loss of viability and / or function (such methods may include tilting the plate and / or using a mesh to cover the cells). The analyte fluid can be removed, for example, using a pipette and / or tubing. In a further method, a fluid (e.g., a buffer, other washing fluid, or new analyte fluid) can be added to the solid-phase support to remove the analyte fluid by elimination as waste (treatment change without washing). New analyte fluid can be added to the solid-phase support to remove the analyte fluid from the previous cycle, for example, using a pipette and / or tubing connected to a pump with adjustable flow rate.

[0061] In some examples, the solid-phase support is a Transwell insert (upper chamber) used with a multiwell plate (lower chamber), and the sample containing T cells is located in each well of the insert. When such a Transwell insert is used, the analyte fluid can be removed or excluded from the wells of the multiwell plate. Such a method is shown, for example, in Figure 4. In yet another example, a mesh is placed above the cells, and the analyte fluid is removed from the top of the plate. Such a method is shown, for example, in Figure 6. In yet another example, the cells are located above the mesh, or sandwiched between two layers of mesh, and the analyte fluid is removed from below the mesh. Such a method is shown, for example, in Figure 7.

[0062] In a further example, the analyte fluid is removed by laminar flow washing. Laminar flow washing devices are sold by Curiox (curiox.com) and are described, for example, in International Publication No. 2020028406(A1) and U.S. Patent No. 9557318(B2), the contents of which are expressly incorporated herein by reference. In laminar flow washing, the buffer or other washing fluid is dispensed and aspirated at a precise rate at opposing positions, thereby resulting in a laminar flow with a high flow velocity at the top of the well and a low or static flow velocity where cells are present. Laminar flow washing is shown, for example, in Figure 5.

[0063] In a particular embodiment, the present invention relates to a method for identifying or detecting T cell activation, wherein the method is The process involves contacting a sample containing iaT cells with an analyte fluid, where the T cell sample is on a solid support and the analyte fluid is a liquid culture medium containing a candidate drug. b. Detecting T cell activation by detecting optical signals in a sample on a solid support, c. A first cycle comprising removing the analyte fluid from the solid support, thereby removing the candidate drug while retaining the sample containing T cells on the solid support, ii.a. Contacting a retained sample containing T cells from a preceding cycle with a new analyte fluid, wherein the new analyte fluid contains a new candidate drug in a liquid medium, and the new candidate drug is different from the candidate drug in the preceding cycle. b. Detecting T cell activation by detecting optical signals in a sample held on a solid support, c. A subsequent cycle comprising removing the analyte fluid from the solid support, thereby removing the candidate drug while retaining the sample containing T cells on the solid support, The process involves repeating a subsequent cycle at least N times, where N is a non-negative integer, and where each new analyte fluid in each subsequent cycle contains a different candidate drug than that in the preceding cycle. The removal step is carried out using the gentle removal, washing methods and / or treatment exchanges described herein. In certain embodiments, the removal of the analyte fluid (e.g., by treatment exchange or gentle washing) is a method that does not involve high-speed centrifugation, resulting in a reduction of no more than approximately 5% of the cell viability and / or cell volume of the retained sample after 2 to 10, or 10 or more total cycles. In certain embodiments, the removal of the analyte fluid is a method that does not involve centrifugation, resulting in a reduction of no more than approximately 5% of the cell viability and / or cell volume of the retained sample after 10 total cycles. In certain examples, the detection of optical signals in a cycle includes the removal of the analyte and / or treatment exchange, and the detection of multiple (e.g., at least two) different optical signals, or multiple detections of the same optical signal, prior to the subsequent cycle. For example, if the method involves multiple detections of the same optical signal, the cycle may include multiple imaging or detection steps performed over time (e.g., separated by a few seconds, minutes, or hours).

[0064] If the solid-phase support is a multi-well plate, a microtiter plate, or any support containing multiple separate samples, each sample on the support may be in contact with a different candidate drug in one or all cycles, for example, if all samples originate from the same biological sample. In another example, each sample on the support may be in contact with the same candidate drug in one or all cycles, for example, if some or all samples originate from different biological samples. In certain embodiments, one or more cycles, or all cycles, may include a positive control (a drug known to induce a response in cells, e.g., a drug known to activate T cells). In certain further embodiments, one or more cycles, or all cycles, may include a negative control (a drug known not to induce a response in cells, e.g., a drug known not to activate T cells).

[0065] Non-limiting examples of cells that may be used in the systems and methods described herein include cells of multicellular organisms, e.g., cells of invertebrates and vertebrates, e.g., myoblasts, neutrophils, erythrocytes, osteoblasts, chondrocytes, basophils, eosinophils, adipocytes, invertebrate neurons (e.g., Helix aspersa). Aspersa), vertebrate neurons, mammalian neurons, adrenal medullary cells, melanocytes, epithelial cells, and endothelial cells; all types of tumor cells (e.g., melanoma, myeloid leukemia, carcinomas of the lung, breast, ovary, colon, kidney, prostate, pancreas, and testis); cardiomyocytes, endothelial cells, lymphocytes (e.g., T cells and B cells), mast cells, vascular intima cells, hepatocytes, leukocytes including mononuclear leukocytes; stem cells, e.g., hematopoietic stem cells, neural stem cells, skin stem cells, lung stem cells, kidney stem cells, liver stem cells, and myocyte stem cells; osteoclasts, connective tissue cells, keratinocytes, melanocytes, hepatocytes, and kidney cells; induced stem cells and stem cell-derived cells. Appropriate cells also include known cell lines, including but not limited to Jurkat T cells, NIH3T3 cells, CHO, COS, etc.

[0066] In certain embodiments, the cells or culture include immune cells, including but not limited to T cells, B cells, and NK cells. In further embodiments, the cells or culture include peripheral blood mononuclear cells (PBMCs). In further embodiments, the cells or culture include T cells. Such T cells include, for example, CD4 + CD8 + This includes T cells and regulatory T cells. A preferred example is Jurkat cells. Jurkat cells are immortalized T lymphocytes that first originate in the peripheral blood of children with T-cell leukemia (Schneider et al., 1977, Int J Cancer 19(5):621-6). The sample may contain T cells and APCs such as professional APCs. Such professional APCs may be selected from the group consisting of dendritic cells, macrophages, monocytes, and B cells.

[0067] In certain embodiments, candidate agents are peptides, peptide libraries, or disease-related antigens (e.g., bacterial antigens, viral antigens, or cancer antigens). Candidate agents may be those whose effects on T-cell activation and / or differentiation and / or modulation of other cellular responses are unknown. Examples of candidate agents (or libraries thereof) include oligopeptides, polypeptides, proteins, antibodies, (peptide-)mimiles, and small molecules. In some examples, candidate agents are screened for their ability to activate immune cells. Candidate agents may be, for example, infection-related candidate epitopes, autoimmune disease-related candidate epitopes, or tumor-related candidate epitopes. An epitope refers to the portion of an antigen recognized by B cells or T cells, and the portion of the antigen to which an antibody binds. More specifically, an epitope refers to the portion of an antigen recognized by B cells or T cells, and the portion of the antigen to which an antibody binds. Epitopes are typically small peptides consisting of two or more amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids). Class I epitopes are typically about 8–15 amino acid long, more typically about 8–12 amino acid long. Class II epitopes are typically about 8–24 amino acid long, more typically about 15 amino acid long.

[0068] Biological samples, or samples containing T cells, can be stored in a suitable solution so that the T cells and, optionally, antigen-presenting cells remain viable. Such liquids include, for example, culture media commonly used for culturing immune cells in vitro. Such media are well known and include, for example, MEM, DMEM, RPMI-1640, and CTL-Test medium (Cellular Technology Limited). If necessary, the medium may be mixed with additives such as fetal bovine serum (FBS) or L-glutamine. In a preferred embodiment, the biological sample or specimen is stored in the medium under conditions in which immune cells can be cultured. Such conditions are well known and include, for example, conditions at 37°C in a 5% CO2 atmosphere.

[0069] The solid-phase support may be a microfluidic device. The candidate drug may be added to the microfluidic device containing cells, or the candidate drug may already be present on the microfluidic device (e.g., pre-loaded) when cells (e.g., as part of a patient sample) are added. The microfluidic device may be made of, for example, glass, polymer or other suitable material. The flow of the analyte fluid (fluid containing the candidate drug) over the cells does not alter the position of the cells. The analyte fluid may be removed using the gentle analyte removal or treatment exchange method described herein. In certain embodiments, the method includes a gentle washing step. In other embodiments, the method does not include a washing step. After gentle analyte removal and / or gentle washing or treatment exchange, the cells are retained on the microfluidic device, and then the cells may be treated with a new analyte fluid (new treatment cycle). In certain embodiments, T cell activation treatment cycling is performed using a microfluidic device containing T cells.

[0070] The systems and methods described herein detect cellular responses (e.g., T cell activation) by detecting optical signals in a sample on a support, for example, by fluorescence microscopy. In certain embodiments, the optical signal indicates T cell activation. For T cell activation, the optical signal may indicate, for example, calcium flux, cytokine release, or the formation of a complex containing a T cell and an antigen-presenting cell (APC). In certain embodiments, the optical signal is immunological synapse formation, for example, a T cell and an APC complex. In further additional examples, the optical signal indicates calcium flux.

[0071] In certain embodiments, the optical signal indicates a calcium flux. An increase in calcium concentration in T cells is a rapid and highly sensitive measure of cell activation. Therefore, the optical signal may be provided by a calcium-binding moiety (or calcium probe) containing a detectable label (e.g., a fluorescent label). Calcium-binding moieties with fluorescent labels may be referred to as "fluorescent calcium indicators" or "fluorescent probes," and may contain fluorescent dyes conjugated to calcium chelators, and various products are commercially available. Examples of such fluorescent calcium indicators include Fura2, Fluo3, Fluo4, Indo1, and Rhod2. Fluorescent probes may be protected with acetoxymethyl (AM) groups. Protection with AM groups confers cell permeability to the fluorescent probe. Non-limiting examples of labeled calcium probes / indicators that may be used in accordance with the methods described herein include x-Rhod-1 (red fluorescent calcium indicator), Calbryte® 630 AM (AAT Bioquest), Calbryte® 520 AM (AAT Bioquest), and Fluo-4, Fluo-Gold, Fluo-3, and Fluo-2, available from Ion Biosciences. Labeled calcium probes may be added before, after, or simultaneously with the analyte fluid. For example, the calcium probe may be added to the cells before the analyte, incubated for about 1 hour, and then washed.

[0072] In additional embodiments, the optical signal indicates cytokine release. Examples of cytokines secreted by activated T cells include interferon-gamma (IFN-γ) and interleukin-2 (IL-2). Therefore, in some embodiments, the optical signal may be provided by a cytokine-binding labeling moiety. Examples of such labeling moieties are fluorescently labeled anti-cytokine antibodies, such as anti-IFN-γ antibodies or anti-IL-2 antibodies. The cytokine-binding labeling moiety may be added before, after, or simultaneously with the analyte fluid.

[0073] Furthermore, in a further embodiment, the optical signal is the formation of an immunological synapse, in other words, a complex of T cells and APCs. Non-limiting examples for detecting immunological synapses are described, for example, in U.S. Patent Application Publication No. 20210356454(A1) and Calvo et al. (2018), Front Immunol 9:684, the contents of which are expressly incorporated herein by reference.

[0074] Representative labels suitable for optical detection (e.g., associated with or bound to anti-cytokine antibodies or calcium probes) include radioisotopes, phosphors, chemiluminescent materials, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, and metal sols. In some embodiments, the labels are fluorescent. Representative examples of non-proteinogenic fluorescent labels include allophycocyanin (trade name XL665), as well as luminescent organic molecules such as rhodamine, cyanine (e.g., Cy5), squaline, coumarin, proflavin, acridine, fluorescein, boron-dipyrromethene derivatives (commercially available under the trade name "BODIPY"), fluorophores known as "Atto", fluorophores known as "DY", compounds known as "Alexa", and nitrobenzoxadiazole. "Alexa" compounds are commercially available, for example, from Invitrogen; "Atto" compounds from Atto-tec; "DY" compounds from Dyomics; and "Cy" compounds from Amersham Biosciences. Fluorescently labeled anti-cytokine antibodies are commercially available. Protein-based fluorescent labeling may also be useful. Representative examples of fluorescent polypeptides include yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), GFP, mRFP, RFP(tdimer2), and HCRED. Biotin-based labeling may also be useful. Biotinylation of target molecules, including antibodies, is well known in the art. Biotinylated anti-cytokine antibodies can be detected by binding to a detectably labeled biotin-binding partner such as avidin or streptavidin.

[0075] In a further embodiment, cells express a heterologous reporter gene, the expression of which provides an optical signal. Gene reporters are widely used in pharmaceutical and biomedical research as indicators for studying gene expression and cellular events linked to gene expression. Typically, the reporter gene encoding the reporter is cloned into an expression vector, which is then transferred into cells. Expression vectors may include viral vectors, plasmids, mRNA, and others. After transfer, cells are assayed for the presence of the reporter by directly measuring the reporter protein itself or the enzymatic activity of the reporter protein. Preferred reporters are those that can be readily identified and quantitatively measured when expressed in effector T cells. Many suitable examples are known to those skilled in the art, and these include fluorescent and luminescent reporters. Optionally, the reporter is a bioluminescent reporter, e.g., luciferase.

[0076] In some cases, a sample showing a positive readout, or cells within a sample, can be identified as indicating a cellular response, such as T cell activation, without the need for downstream assays. For example, if the optical signal is immunosynapse formation, downstream assays may not be required to confirm or characterize T cell activation.

[0077] Samples exhibiting positive readout in one or more cycles may be further analyzed or characterized using downstream assays. Such downstream workflows may include, for example, incubating cells for several days (e.g., five days or more) and evaluating cellular responses (e.g., cell death, proliferation, cytokine production), collecting cell culture media for analysis at different time points, sorting the obtained cells of a sample by cell type, activation state, or other characteristics using magnetic separation or other cell sorting methods, performing staining for flow cytometry or barcode antibody staining for sequencing, determining the coordinates of the responsive cells, and optionally applying a series of methods to perform spatial transcriptomics on two-dimensional samples retained attached to the bottom of a container or a Transwell mesh, or between two layers of mesh, performing colony picking with third-party automated equipment, and / or performing bulk or single-cell sequencing of DNA or RNA, such as sequencing of T cell receptor (TCR) mRNA to find their sequences in parallel with peptide specificity, if the treatment involved peptides from a peptide pool.

[0078] Downstream assays can, for example, determine the T cell subtype and function of positive samples. T cell type and function can be determined by antibody-based methods, such as flow cytometry, immunohistochemistry, or immunofluorescence, or by transgenic fluorophor expression, based on the expression of surface proteins (e.g., CD3, CD4, CD8, CD45RA, etc.) and cytokine production (e.g., interferon-gamma (IFN-γ), transforming growth factor beta (TGF-β), interleukin (IL)-2, IL-4, IL-17, etc.). Downstream assays may include, for example, repeated cell activation or use in subsequent experiments, either alone or in the presence of other cell or tissue culture samples.

[0079] In certain embodiments, the downstream assay may be performed on the same support as the treatment cycling. In additional embodiments, the downstream assay is performed by the same device that removes the analyte fluid.

[0080] As described above, downstream assays may include staining and / or characterization of further cellular features and markers in the same traceable cells or at their corresponding locations. Such post-cycling characterization may be performed using live or fixed cells. Cellular features include phenotypic markers (e.g., CD4, CD8, and others), expressed cytokines or other secretory molecules trapped within cells (e.g., IFN-γ, IL-2, TNF, and others, perforin, granzyme B, and others), and expressed activation-inducing markers (collectively referred to as AIMs, e.g., CD69, 4-1BB, OX40, CD40L, and others). The use of FACS for the detection of activation-inducing markers is described, for example, in Lemieux, Audree, et al., "Enhanced detection of antigen-specific T cells by a multiplexed AIM assay," Cell Reports Methods 4.1 (2024), the contents of which are expressly incorporated herein by reference. Modern methods of serial staining using sets of fluorescent antibodies allow for further multiplexing of the final readout. The main advantages of AIMSpot over ELISpot include the potential for cell phenotyping analysis and high-resolution data redundancy regarding activated cells. The main advantages over FACS-ICS / AIM are the high potential for workflow automation and the availability of high-resolution data regarding antigen specificity (activation cycling).

[0081] In certain embodiments, the downstream assay is an activation-inducing marker (AIM) assay, or a method for detecting an activation-inducing marker (or its expression). An "activation-inducing marker" (AIM) is a marker that is expressed after T cell activation, or whose expression is upregulated. An AIM assay can detect any marker upregulated by T cells upon T cell activation. In certain embodiments, the assay includes the use of a labeled antibody that binds to the AIM, such as a fluorescently labeled antibody. Non-limiting examples of activation-inducing markers include, for example, CD137 / 4-1BB, CD107, IFNγ, PD-1, CD40L, OX40, CD25, CD69, CD28, HLA-DR, CX3CR1, TIM3, LAG3, TIGIT, or any combination thereof. In further embodiments, the AIM is CD69, 4-1BB, OX40, and CD40L, or a combination thereof. In certain embodiments, the downstream assay is a flow cytometry activation-inducing marker (AIM) assay. In certain embodiments, a downstream assay is a method for detecting an activation-inducing marker, wherein the optical signal (detectable during cycling) indicates a calcium flux, for example, by adding a calcium-binding moiety or calcium probe containing a detectable label to the assay.

[0082] In some embodiments, downstream assays are, for example, imaging of cells using dyes or labeled antibodies. One specific method of a downstream (post-cycling) assay is the staining or imaging of live or fixed cells using dyes or antibodies (e.g., fluorescently labeled antibodies, or antibodies containing fluorescent or dye labeling) to confirm or provide further characterization of data or readouts obtained during treatment cycling. In some embodiments, staining or imaging is automated. Such methods are referred to herein as "AIMSpot". A non-limiting example of the AIMSpot method is the treatment of live human PBMCs for one or more cycles using a candidate compound that potentially activates T cells. In this example, the solid support is a Transwell insert. All treatments of cells in this example are performed without changing the cell position, and all treatments are carried out under the Transwell insert membrane while the cells are cultured on the membrane (e.g., treatments using brefeldin A, fixation / permeabilization buffer, blocking solution, staining solution, washing solution, cell preservation solution and imaging solution). After treatment, cells are given 2 hours to upregulate cytokine IFN-γ expression. Next, IFN-γ secretion is blocked by treating the cells with brefeldin A, followed by a 4-hour incubation. The cells are then fixed in a buffer containing paraformaldehyde, which can permeate the cell membrane. Subsequently, the cells are treated with a blocking solution containing serum and unstained antibodies that block the FcG receptor. The cells are then stained with a fluorescent antibody against human IFN-γ. Excess antibody is washed away after treatment. The cells are imaged using fluorescence microscopy. Small, bright "spots" correspond to cells producing IFN-γ, and the antigen specificity of these cells can be determined by the number of cycles at which Ca++ flux is detected in them.

[0083] As described herein, one area of ​​research where the treatment cycling approach can be very advantageous is T cell research, specifically the study of T cell receptors, the peptide specificity of TCRs. The genetic diversity of TCRs is enormous. A single person has up to 10 TCRs corresponding to individual T cells or their clones. 8 TCRs can have individual variants with distinct characteristics. TCRs specifically interact with complexes of HLA molecules and peptides (typically short amino acid sequences produced during normal protein metabolism in living cells) (although they exhibit some levels of cross-specificity). In an inflammatory environment, TCRs can recognize corresponding non-self peptides presented as HLA-peptide complexes on the surface of antigen-presenting cells. The consequences of this interaction may include T cell activation, phenotypic changes, proliferation, and cytokine secretion. Activated T cells help induce antigen-associated antibody responses, participate in antigen-specific signaling, and kill target cells exposing non-self peptides on their surface. Often, it is sufficient to determine whether any T cells in a sample can respond to any random portion of one or more antigens associated with some pathogen or gene therapy vector. However, studying the precise peptide specificity of TCRs and corresponding T cells can be useful for optimizing responses. For example, in simplified terms, a strong T cell response would be beneficial in the development of vaccines, cancer, and chronic infection immunotherapies. Demonstrating "low or no" T-cell responses will be important in the development of cell and gene therapies, autoimmune treatments, and allergy desensitization immunotherapies. Measuring T-cell responses can be of high importance in both preclinical and clinical studies, as well as as a diagnostic tool in clinical practice.

[0084] TCR(10 8Given the diversity of both HLA-peptide and peptide lengths associated with HLA-TCR interactions, the probability of activating even a single T cell in a sample using a single peptide corresponding to the antigen (protein) of interest may be so small that it is negligible. In such cases, not only is the probability of activation very low, but there is also a significant waste of sample if the response is negative (and for the patient, this sample is usually blood collected for research). Therefore, the treatment cycling approach described herein may be useful.

[0085] Another area where a treatment cycling approach may be advantageous is the study of the activation of rare primary cell types with libraries of compounds less likely to induce a response of interest. Mixed primary human cell cultures are perhaps the most relevant ex vivo and in vitro research models that can capture the features of human-specific effects and the diversity of human genetic background (as opposed to the use of experimental animals or animal and human immortalized cell lines). The main drawbacks of these models include a lack of standardization and questions regarding the representativeness of the samples compared to the general population. Another issue is the logistical access to material from many donors and the cost of such material, in parallel with generally limited access to primary cells. The problem is further complicated when the study must be conducted in rare cell types that constitute only a small proportion of the total cell population in the tissue. Attempts to screen extensive libraries of drug-like molecules (up to 1000 or more analytes) to determine whether these molecules activate rare cell types can be highly wasteful if the cell culture is treated once and the sample is discarded after a negative response is recorded (which can happen more than 99.99% of the time).

[0086] If 100 cycles are permissible by the model, the treatment cycling approach described herein can facilitate the use of rare cells and primary cells with a 100-fold improvement in sample use efficiency, or a 100-fold improved sensitivity, or a combined gain in sample use efficiency and sensitivity. If these rare cells have highly genetically diverse receptors of interest, a single sample may contain a mixture of cells from different patients (with immune responses between them blocked, e.g., immune cells and antibodies removed), and then response features / cycles can be correlated with genetic signatures. Such a mixture of primary cells from 10 patients may result in a further 10-fold increase in bioassay efficacy, and cycling may result in a 1000-fold increase in sample use efficacy.

[0087] The present invention includes a device and method for cycling the testing of sample reactivity to multiple analytes in order to increase the frequency of detecting low-probability positive responses. The device or set of devices may have the following functions: (a) enabling the addition of a treatment to a suspension of cells of interest; (b) measuring the cell response readout as negative or positive if one or more cells are activated; (c) removing excess treatment from the cell suspension and optionally replenishing with washing solution; and (d) excluding samples or wells with positive responses from subsequent cycles by repeating the cycle of steps (a) to (c) and optionally programming individual controls of treatment exchange in this sample or well. As a result, repeated treatment cycles substantially reduce the need to use large quantities of biological samples for screening, where the probability of activating any single cell during a single treatment cycle is low. The device and method can advantageously operate with limited sample supplies, particularly with a limited proportion of responding cells of interest in the sample, where their responses are to be screened against multiple treatments with a low probability of positive response to any given treatment.

[0088] The present invention encompasses (1) a treatment cycling approach that allows for repeated use of the same sample compared to the industry's conventional approach of one treatment per experimental sample / well / container to evaluate the effect of the treatment; (2) the use of rapid readouts to enable cycling compared to the industry's conventional readouts that are typically recorded at least several hours after treatment; (3) the use of various rapid cell washes or treatment exchanges without centrifugation at high centrifugal force compared to the industry's conventional approach; and (4) reliance on the use of single-cell generation responses and colonies related to specific activation treatment conditions (number of cycles or coordinates or responsive cells) for sequencing their characteristics, compared to the industry's conventional alternative of single-cell sequencing or bulk sequencing performed on unknown proliferating colonies without tracking individual treatment responses.

[0089] The present invention allows for the use of limited sample supply (which is restricted by cost, logistics, ethical reasons, etc.), particularly a limited proportion of response cells of interest within a sample, where these responses are screened against multiple treatments with a low probability of positive response to any given treatment. An increase in bioassay effectiveness of up to 100 to 1000 times can be expected compared to conventional approaches. This increase in effectiveness can (a) simplify research logistics and reduce costs, thereby enabling research that was previously impossible; (b) reduce the amount of sample required, making research procedures less harmful or painful for experimental animals or patients; (c) make research procedures less wasteful, improving the ecological outcomes of research; and (d) stimulate new areas of research, such as the study of T cells and B cells, the study of rare cell types, and compounds (drugs, peptides), etc., that interact with these cells and their receptors in the context of high biological and genetic diversity.

[0090] The present invention will be explained by the following non-limiting examples.

[0091] example Example 1: Multiple trials for T-cell reactivation due to widespread infection Human frozen PBMCs from three donors, pre-tested by ELISpot for a high T-cell response to influenza virus antigen (over 100 spots per 400,000 cells), will be purchased (Cellular Technology Limited, OH, USA). PBMCs collected from June 2022 onwards will be used to expect a high T-cell response to Covid antigen (post-vaccination and post-infection). Cells will be thawed according to a general PBMC thawing protocol. Briefly, 9 mL of T-cell medium (with the addition of OpTmizer T Cell Expansion SFM, Gibco, 1:100 GlutaMAX, Gibco) preheated to +37°C is prepared. Frozen cell vials are warmed in a +37°C water bath until 2 / 3 of the contents are liquid. Vial contents are quickly transferred to a tube containing warm medium and centrifuged at 300xg for 5 minutes at +20°C. The pelleted cells are resuspended in T cell medium and immediately used for assays, or seeded overnight in a CO2 incubator at +37°C at 1-2 M / mL in 3 mL / well of a 6-well plate.

[0092] Cells are isolated at 200 μl in 200 K in T cell medium for staining with green or red fluorescent dyes (Calbrite 520 AM and Calbrite 630 AM, ATT Bioquest, CA, USA), i.e., markers of calcium flux (a rapid event that occurs after T cell activation). These dyes diffuse and remain within the cells, and are not fluorescent unless T cells are activated and the concentration of Ca++ ions in the cytoplasm increases. Staining is performed at +37°C for 1 hour according to the manufacturer's protocol. Cells are then washed by centrifugation at 300 x g for 5 minutes at +20°C and resuspended in T cell medium. Unstained cells can be used as an additional negative control to demonstrate potential autofluorescence before and after T cell activation.

[0093] Cells in 100 µl of T cell medium are added to a Transwell insert (permeable cell culture insert, 0.4 µm pore, Celltreat, USA). The Transwell insert is placed in a droplet of 10–20 µl of T cell medium (hereinafter referred to as the droplet) at the bottom of a 6-well plate. The plate is centrifuged at 250 x g for 5 minutes at +20°C to allow for cell concentration at the bottom of the insert.

[0094] Imaging is performed using an EVOS M7000 imager and a fluorescent green or red channel capable of recording the corresponding dye signal. Fluorescence settings are established using a positive control sample treated with a positive control at +37°C for 10 minutes. Two positive controls are used: CytoStim, human (Miltenyi Biotek, USA), and a cell stimulation cocktail (eBioscience®, USA).

[0095] In different cycles, the droplets under the Transwell insert are removed by pipetting. Then, either (1) a negative control, T cell medium, (2) a Flu antigenic peptide pool (PepTivator® Influenza A(H1N1)HA, Miltenyi Biotek, USA), or (3) a Covid antigenic peptide pool (PepTivator® SARS-CoV-2 Prot_S1, Miltenyi Biotek, USA), or one of the two positive controls, is added under the Transwell insert in the same volume as the initial droplet, at the recommended concentration or up to 5 times higher.

[0096] In this example, gentle wash / treatment exchange is the exchange of liquid in droplets while the cells remain in T cell medium within the Transwell insert. The insert has pores of 0.4 μm and shows no signs of leakage when removed for droplet exchange. In this example, the process is manual and not automated. If the process were automated, different methods of gentle wash / treatment exchange may be used. Treatment cycles may include or omit washing in T cell medium between activation cycles. Cycles may include adding new analytes to the analytes from the previous cycle and mixing them, instead of removing the previous analytes.

[0097] The stimulating peptide or positive control interacts with the cells via the Transwell membrane. After each stimulation, the cells are returned to a CO2 incubator or placed on a heating pad at +37°C for 5–10 minutes. Alternatively, a plate heating system, i.e., an on-stage incubator, may be used to maintain the plate at +37°C for most of the time. A green or red signal is then recorded. If multiple wells are used, the signal is obtained by programmed scanning of a selected area of ​​the plate using the corresponding green or red channel. At least some wells may be scanned in less than one minute.

[0098] The number of activated cells after treatment with one or two consecutive antigenic peptide pools, or any type of positive control, should not vary by more than 5% based on treatment cycles of 10 or more. For consistent counting, a cell count cutoff value, as well as consistent fluorescence imaging conditions, should be selected.

[0099] In some cases, treatment cycling may be stopped if a positive result is obtained (e.g., treatment with stoppage). For example, after a cell response to one of the peptide pools is obtained, the data is recorded and the sample is not used for further cycling. This approach can be used to compare the consistency of the number of cells responding to the same peptide pool across different treatment cycles, e.g., cycle #1 and cycle #10. In the case of single peptide use, and if only a small number of cells respond to the treatment, the cells may be proliferated (optionally) and isolated for T cell receptor (TCR) sequencing.

[0100] In a further example, treatment cycling may be continued if a positive result is obtained (treatment without stopping). In this example, the cycle is continued even after some cells have responded to one peptide (pool) to see if more cells respond to other peptides (pool). In the “treatment without stopping” approach, in the simplest circumstances, the number of cells newly responded to the last cycle treatment is represented by the number of fluorescent cells in the current readout cycle minus the number of fluorescent cells in the previous readout cycle (because the decrease in fluorescence after induction is a slow process and can take several hours). Since Ca++ signaling spikes degrade over time, a more sophisticated data analysis workflow should be implemented for multiple cycles, where every individual cell is tracked, and only newly occurring activation events are counted for each cycle, while signals from previously activated cells are ignored. This approach makes it possible to test the T cell response of a patient sample to multiple antigens within the same sample. Quality control in the final cycle may involve the addition of a positive control.

[0101] Furthermore, one or more strategies may be employed to improve the specificity of the method. A significant limitation when using Ca++ flux as a T cell activation readout is the non-specific nature of this event in cellular signaling. This event is present when apoptosis or phagocytosis is initiated, or when different other cell types are activated. Users of the method may improve the specificity of the method by applying additional sensors for apoptosis and other non-specific events and excluding these events from the count. Users may screen or pre-label cell types to avoid counting non-specific events originating from irrelevant cell types. Users may select more specific methods for identifying T cell activation, such as immunological synapse formation. Even if all T cell activation events captured during cycling are true, there may be T cell populations that do not secrete cytokines upon activation, and this should be taken into consideration.

[0102] Example 2: Treatment cycling using downstream ELISpot Treatment cycling can be an assay performed using cells prior to single-cytokine and multi-cytokine ELISpot assays of the FLUOROSpot method for detecting T cells that secrete cytokines after activation. Immediately after periodic treatment, data are recorded within 20 hours of the start of treatment, and the treated cell suspension can be transferred to an ELISPOT membrane coated with one or more antibodies. Cells in this assay are activated at different time points before placement on the membrane (pre-incubation), and the dynamics of cytokine secretion may vary based on this factor. One strategy to avoid inconsistent timing of T cell stimulation before adding them to the ELISpot membrane is to use two cycling rounds. The first round is performed using a portion of the sample to detect the most immunogenic peptide (e.g., 10 out of 100), and the second round involves fewer cycles before using the cells in ELISpot. Thus, the pre-incubation time difference is minimized.

[0103] Another alternative is to detect the most immunogenic peptides and use their pool exclusively to activate cells before ELISpot. In this case, the pre-incubation time lag is eliminated, and epitope mapping data is still obtained.

[0104] Another alternative is to simply perform treatment cycling and ELISpot in parallel. The total number of activated T cells per million PBMCs should be consistent or at least well correlated. One assay provides epitope mapping data, and the other provides information about the type of stimulation.

[0105] More complex alternative methods allow for the simultaneous detection of specific stimulating peptides and types of T cell activation. However, these differ significantly from ELISpot and will not be discussed in this section.

[0106] A 20-hour pre-incubation may be considered too long and represent a non-standard approach. However, it is known that for optimal ELISpot results, cells should be pre-incubated after stimulation to detect some cytokines, such as IL-2. Furthermore, in cytokine secretion assays, 48 ​​and 72 hours after T cell stimulation are preferred time points compared to 24 hours, as higher cytokine secretion activity is observed after 24 hours. Despite the differences in pre-incubation periods for cells activated in different cycles, cycling-ELISpot with 100 different peptides and regular ELISpot with a peptide pool of 100 peptides are expected to correlate well (with proper exclusion of false-positive activation events or more accurate inclusion of true T cell activation events). Moreover, the results of cycling-ELISpot are supported by the expected spot volume after cycling (total of all cells stimulated in all cycles). Additionally, cycling-ELISpot provides higher data resolution and T cell epitope mapping data compared to regular ELISpot, which only gives the overall result of T cell stimulation with the peptide mixture. Both approaches require at least one well containing a negative control and one well containing a positive control.

[0107] Example 3: Treatment cycling by downstream ICS-FACS Treatment cycling can be an assay performed using cells prior to single-cytokine and multi-cytokine ICS-FACS to detect T cells that secrete cytokines after activation. ICS-FACS is a flow cytometry-based method for detecting intracellular cytokines, typically for detecting antigen-specific T cells. In some embodiments, it requires T cell stimulation and a 2-hour pre-incubation, after which a Golgi blocking reagent is added to the cells. Four hours after the use of the blocking reagent, the cells are stained for viability, surface markers, and intracellular cytokines using fluorescent dyes and fluorescently labeled antibodies. In this assay, the standard 2-hour pre-incubation can be replaced with a 20-hour cycling without the Golgi blocking agent, and a further 2-hour pre-incubation without cycling to allow cells stimulated in the most recent cycle to upregulate cytokine expression (as in the standard approach). The outcome of cytokine secretion is partially affected by the time difference in T cell stimulation (actual pre-incubation times will differ for cells stimulated in different cycles). Strategies to overcome this limitation are discussed in the section on the combination of cycling-ELISpot methods. A similar strategy may be employed to detect cells with up-controlled activation-inducing markers (AIMs) by using a longer post-cycling incubation timing prior to cell staining and analysis (FACS-AIM).

[0108] Example 4: Treatment cycling with downstream cytokine secretion assays Treatment cycling can be an assay performed using cells before detecting cytokines secreted by T cells after activation. This combination is perhaps the simplest. After all cycles have been performed, the cells are placed in a CO2 incubator for an additional 24–48 hours. Many multiple cytokine detection methods, such as LEGENDplex, Luminex, and MSD (Meso Scale Discovery), can detect the secreted cytokines. If cycling is performed so that only one cell per well is activated and cycling stops, then a longer incubation time should be recommended, and a more sensitive method such as MSD should be prioritized.

[0109] Example 5: Treatment cycling with downstream cell proliferation and TCR sequencing Treatment cycling can be performed before T cells are proliferated after stimulation. Proliferation is one of the oldest methods for detecting T cell activation. This typically requires 5-7 days or more. Current instruments allow for automated colony counting and picking as needed. Bulk RNA sequencing can make it possible to find TCR sequences if only one colony is proliferating per well (especially if the wells used are very small). Even if several colonies are generated per well from cells activated in a known cycle, in silico predictive methods can make it possible to find out which activating peptide corresponds to which TCR. Pre-staining of cells with oligonucleotide barcoded antibodies and MHC / HLA multimers preloaded with antigenic peptides (which may also be barcoded) can make it possible to pre-select cells, control cell phenotypes, and perform single-cell sequencing.

[0110] Example 6: Testing a recurrent T cell activation protocol using green dye staining. The cell source was a vial of human frozen PBMCs from one donor, pre-tested by ELISpot (Cellular Technology Limited, OH, USA) for a high T-cell response to influenza virus antigen (over 100 spots per 400,000 cells). PBMCs collected from June 2022 onward were used to anticipate a high T-cell response to Covid antigen (post-vaccination or post-infection). Cells were thawed according to a general PBMC thawing protocol. Briefly, 9 mL of T-cell medium (with the addition of OpTmizer T Cell Expansion SFM, Gibco, 1:100 GlutaMAX, Gibco) preheated to +37°C was prepared. The frozen cell vial was warmed in a +37°C water bath until 2 / 3 of its contents were liquid. The vial contents were quickly transferred to a tube containing warm medium and centrifuged at 300 × g for 5 minutes at +20°C. The cells were resuspended in T cell medium and seeded overnight in a CO2 incubator at +37°C at a concentration of 1–2 M / mL, 3 mL / well in a 6-well plate. The cells were then separated into 200 μl of T cell medium at 200 K for staining with the green fluorescent dye Calbrite 520 AM (ATT Bioquest, CA, USA) in a CO2 incubator at +37°C for 1 hour. The cells were then washed by centrifugation at +20°C at 300xg for 5 minutes and resuspended in T cell medium.

[0111] Cells (100K in 100 μL of T cell medium) were added to a Transwell insert (permeable cell culture insert, 0.4 μm pore, Celltreat, USA). The bottom of the Transwell insert was placed in a 20 μL droplet of T cell medium (hereinafter referred to as the droplet) at the bottom of a well in a 6-well plate. Imaging was performed using an EVOS M7000 imager with a green fluorescence channel. In different cycles, the droplet below the Transwell insert was removed by pipetting. Next, (1) a negative control, T cell medium, (2) a Flu antigenic peptide pool (PepTivator® Influenza A(H1N1)HA, Miltenyi Biotek, USA), or (3) a Covid antigenic peptide pool (PepTivator® SARS-CoV-2 Prot_S1, Miltenyi Biotek, USA), or one of the two positive controls, is added under the Transwell insert in the same volume as the initial droplet, but at a 5-fold higher concentration. Two positive controls are used: CytoStim, human (Miltenyi Biotek, USA), and a cell stimulation cocktail (eBioscience®, USA). For treatment cycling, the liquid in the droplet was replaced with the next treatment type while the cells remained in the T cell medium within the Transwell insert (which was manually removed to replace the droplet). The insert had pores of 0.4 μm and showed no signs of leakage when removed for droplet exchange. No washing was performed between treatment cycles in this experiment. The stimulating peptide or positive control interacted with the cells via the Transwell membrane. After each stimulation, the cells were returned to a CO2 incubator at +37°C for 8 minutes. They were then imaged, and a green signal was observed. Five treatment cycles were performed, and a stepwise increase in the number of fluorescent spots (cells) was recorded. The T-cell activation function immediately after staining and the suitability of the green dye in T-cell activation experiments were confirmed. Similar experiments were performed using the red dye Calbrite 630 AM (Example 7).

[0112] Example 7: Medium exchange protocol and red dye staining test The cell source was a vial of human frozen PBMCs from one donor, pre-tested by ELISpot (Cellular Technology Limited, OH, USA) for a high T-cell response to influenza virus antigen (over 100 spots per 400,000 cells). PBMCs collected from June 2022 onward were used to anticipate a high T-cell response to Covid antigen (post-vaccination or post-infection). Cells were thawed according to a general PBMC thawing protocol. Briefly, 9 mL of T-cell medium (with the addition of OpTmizer T Cell Expansion SFM, Gibco, 1:100 GlutaMAX, Gibco) preheated to +37°C was prepared. The frozen cell vial was warmed in a +37°C water bath until 2 / 3 of its contents were liquid. The vial contents were quickly transferred to a tube containing warm medium and centrifuged at 300 × g for 5 minutes at +20°C. The cells were resuspended in T cell medium and seeded overnight in a CO2 incubator at +37°C in a 6-well plate at a concentration of 1–2 M / mL.

[0113] Cells were separated into 200 μl of T cell medium at 200 K for staining with the red fluorescent dye Calbrite 630 AM (ATT Bioquest, CA, USA) in a CO2 incubator at +37°C for 1 hour. The cells were then washed by centrifugation at 300xg for 5 minutes at +20°C and resuspended in T cell medium. The cells were placed in 2 mL capped tubes with caps slightly loosened for aeration and incubated in a CO2 incubator at +37°C for 24 hours.

[0114] Cells (100K in 100 μL of T cell medium) were added to a Transwell insert (permeable cell culture insert, 0.4 μm pore, Celltreat, USA). The bottom of the Transwell insert was immersed in 200 μL of T cell medium at the bottom of the wells of a 24-well plate. Imaging was performed using an EVOS M7000 imager with a red fluorescence channel.

[0115] In different cycles, the medium beneath the Transwell insert was removed by pipetting. Then, either (1) negative control, T cell medium, (2) Flu antigenic peptide pool (PepTivator® Influenza A(H1N1)HA, Miltenyi Biotek, USA), or (3) Covid antigenic peptide pool (PepTivator® SARS-CoV-2 Prot_S1, Miltenyi Biotek, USA), or one of the two types of positive controls, was added beneath the Transwell insert in the same volume and at the recommended concentration. Two types of positive controls were used: CytoStim, human (Miltenyi Biotek, USA), and cell stimulation cocktail (eBioscience®, USA). For treatment cycling, the liquid beneath the insert was replaced with the next treatment type while the cells remained in the T cell medium within the Transwell insert (which remained inserted in the plate). In this experiment, there was no washing between treatment cycles. The stimulating peptide or positive control interacts with the cells via the Transwell membrane. After each stimulation, the cells are returned to a CO2 incubator at +37°C for 8 minutes. They are then imaged, and a red signal is observed. Five treatment cycles are performed, and a stepwise increase in the number of fluorescent spots (cells) is recorded. The T cell activation function 24 hours after staining, and the suitability of the red dye in T cell activation experiments requiring more than 24 hours, are confirmed. Representative images showing the increase in activated T cells in response to Flu and Covid peptide treatment between two treatment cycles are shown in Figure 17. Activated cells are outlined and counted using the free software package QuPath. These proof-of-concept data have not been cleaned for potential false-positive events, and methods for data cleaning are discussed elsewhere.

[0116] Example 8: Testing of a fully automated system for analyte fluid exchange and imaging. Automated "new treatment" transfer between the analyte fluid library and the transfer plate was implemented as fluid handling by an OT-2 robot (Opentrons, USA) programmed via a PC. Automated scheduled analyte fluid transfer in a 12-channel format between the transfer plate and the test plate containing the biological sample was implemented by multiple tubes and multiple diaphragm pumps switched on and off by relays and microcontrollers programmed via a PC. Automated scheduled removal of analyte fluid from the test plate was implemented by multiple tubes connected to a vacuum pump switched on and off by relays and microcontrollers programmed via a PC. Automated scanning of pre-selected areas of pre-selected wells in a multi-well plate was implemented using a microscope with an automated stage (BZ-X800, Keyence, Japan) programmed via a PC, or an automated optical system moving beneath the plate (Celloger Mini Plus, Curiosis, Korea). The implemented automated instrumentation set is illustrated by the scheme shown in Figure 11.

[0117] Example 9: Testing the AIMspot method for staining AIM markers or intracellular cytokines through the membrane of a Transwell insert. AIMspot is a method that enables the combination of treatment cycling and automated or semi-automated immunofluorescence staining or other imaging techniques to show the final results of activation cycling. Spatial data superposition allows for the elimination of false-positive activation events and confirmation of true positivity. For true positive events, the cycling data indicates how many cells were activated (yes / no) by any particular analyte fluid, and the downstream portion of AIMspot provides data on cellular phenotype and activation outcomes.

[0118] In this example, the cell source was a vial of human frozen PBMCs derived from one donor. The cells were thawed according to a general PBMC thawing protocol. Briefly, 9 mL of T cell medium (OpTmizer T Cell Expansion SFM, Gibco, with GlutaMAX and glutamine added, Gibco) was prepared preheated to +37°C. The frozen cell vial was warmed in a water bath at +37°C until 2 / 3 of its contents were liquid. The vial contents were quickly transferred to a tube containing the warm medium and centrifuged at +20°C for 5 minutes at 300 × g. The cells were resuspended in T cell medium and plated in a transwell insert with a volume of 50–100 μl per insert at 100–150 K, with 300 μl of cell culture medium below the insert. In the test wells, the medium was replaced with medium containing a pool of Flu peptide (as described in the previous example). In the positive control wells, the culture medium was replaced with a medium containing a cell stimulation cocktail (eBioscience®, USA).

[0119] For the study of phenotypic and AIM markers, cells were pre-loaded with Ca++ sensors and imaged after activation (as discussed in previous examples; in this example, only one cycle of treatment and Ca++ readout were performed), and plates were left in a CO2 incubator at +37°C for 24 hours post-stimulation (AIM markers typically appear on the surface of activated cells 24–48 hours after antigen-specific stimulation). Residual fluorescence of the Ca++ sensor in the red channel was removed by photobleaching for 30 minutes under LED light. Replacing the fluid beneath the cells with a Ca-free solution also helped to completely remove the Ca++ sensor signal.

[0120] Staining with fluorescent antibodies was performed by changing the medium under the Transwell insert without centrifugation. Blocking before antibody staining was performed at room temperature for 20 minutes using blocking buffer A containing 2% FBS and Fc receptor blocking solution (Human TruStain FcX, Biolegend, USA) in Ca-free PBS, at a rate of 300 μl per well. Antibody staining was performed using 1× to 3× of the manufacturer's recommended antibody concentration in the blocking buffer. In this particular example, the phenotypic marker was CD4, and the AIM markers included CD69 and 4-1BB. The antibodies used for staining included anti-human CD4 Alexa Fluor 488-labeled antibody, anti-human CD69 PE-labeled antibody, and anti-human 4-1BB PE-labeled antibody. Staining was performed at room temperature for 40 minutes. The samples were washed three times at room temperature for 20 minutes each by changing the fluid under the Transwell with PBS. Imaging was performed using a microscope BZ-X800 (Keyence, Japan). Green CD4+ and red (CD69 / 4-1BB)+ cells can be distinguished (Figure 18, top two images).

[0121] For the study of intracellular protein expression (IFN-γ), only the downstream portion of the AIMSpot protocol was tested in this example (not Ca++ signaling imaging, although methods for performing such imaging are described above). The same cells without Ca++ sensor preloading were plated into the same Transwell insert and treated only with a positive control treatment, or left untreated as a negative control. Plates were left at +37°C for 2–12 hours. The medium in the lower chamber of the Transwell was replaced with medium containing brefeldin A in a semi-automated format and left for an additional 4 hours of incubation in a CO2 incubator at +37°C. Next, cell fixation and permeabilization were carried out using the eBioscience® Foxp3 / transcription factor staining buffer set according to the manufacturer's protocol, with major modifications: (1) the centrifugation and cell resuspension step was replaced by semi-automatic fluid exchange under the Transwell membrane with an additional incubation time to allow diffusion; (2) cell resuspension in permeabilization buffer was replaced by a 20-minute incubation at room temperature with this buffer under the Transwell insert (Perm wash step); (3) blocking was carried out with blocking buffer B containing 2% FBS and Fc receptor blocking solution (Human TruStain FcX, Biolegend, USA) in permeabilization buffer, which was added under the Transwell insert for a 20-minute incubation at room temperature; and (4) staining was performed after blocking by adding primary mouse IgG antibody against human IFN-γ under the Transwell insert for 2 hours at room temperature, two additional Perm wash steps, an additional blocking step, and Alexa Fluor under the Transwell insert. This was carried out by adding a 647-labeled secondary anti-mouse IgG antibody at room temperature for 1 hour. Cells were imaged after three additional Perm washes and a final wash in which the fluid under the Transwell insert was replaced with PBS. Cells were imaged using a Nikon Ti2 inverted fluorescence microscope.The positive control exhibits numerous intracellular fluorescent INF-γ spots (Figure 18, bottom four images; transmitted light image demonstrates cell density above the Transwell membrane).

[0122] All references, papers, patent applications, patent publications, and patents are incorporated herein by reference in their entirety. While the present invention has been specifically shown and described with reference to its preferred embodiments, it will be understood by those skilled in the art that various modifications in form and detail can be made without departing from the scope of the invention as encompassed in the appended claims.

Claims

1. A method for detecting T cell activation, wherein the method is i. a. A step of bringing a sample containing T cells into contact with an analyte fluid, wherein the sample containing T cells is on a solid support, and the analyte fluid contains a candidate drug in a liquid culture medium. b. A step of detecting T cell activation by detecting an optical signal in the sample on the solid support, c. A step of removing the analyte fluid from the solid support, thereby removing the candidate drug while retaining the sample containing the T cells on the solid support, The first cycle includes, ii. a. A step of contacting the retained sample containing the T cells from the preceding cycle with a new analyte fluid, wherein the new analyte fluid contains a new candidate drug in a liquid culture medium, and the new candidate drug is different from the candidate drug from the preceding cycle. b. A step of detecting T cell activation by detecting an optical signal in the sample held on the solid support, c. A step of removing the analyte fluid from the solid support, thereby removing the candidate drug while retaining the sample containing the T cells on the solid support, Subsequent cycles including, Includes, The subsequent cycle is repeated at least N times, where N is an integer greater than or equal to 0, and each new analyte fluid in each subsequent cycle contains a different candidate drug than that in the preceding cycle. A method, which does not involve centrifugation, in which the removal of the analyte fluid is such that the cell viability and / or cell volume of the retained sample does not decrease by more than about 5% after a total of 10 cycles.

2. The sample containing T cells is a plurality of samples containing T cells, The solid support is a multi-well plate, and the plurality of samples containing T cells are in the plurality of wells of the plate, or The method according to claim 1, wherein the solid phase support is a transwell insert, the plurality of transwell inserts are arranged in the wells of a multiwell plate, the plurality of samples containing T cells are placed on the plurality of transwell inserts, and the analyte fluid is added to the wells of the multiwell plate.

3. The method according to claim 2, wherein the sample in at least two of the wells or at least two of the inserts is brought into contact with different candidate drugs in the first cycle and / or subsequent cycles.

4. The method according to claim 3, wherein the sample in each of the wells or each of the inserts is brought into contact with a different candidate drug in the first cycle and / or subsequent cycles.

5. The method according to any one of claims 1 to 4, wherein N is 5 or more.

6. The method according to claim 5, wherein N is 10 or more.

7. The method according to claim 6, wherein N is 25 or more.

8. The method according to claim 7, wherein N is 50 or more.

9. The method according to claim 8, wherein N is 100 or more.

10. The method according to claim 3 or 4, wherein at least one candidate drug is a positive control.

11. The method according to claim 3 or 4, wherein at least one candidate drug is a negative control.

12. The method according to any one of claims 1 and 2, wherein the candidate drug is a library of candidate drugs.

13. The method according to any one of claims 1 and 2, wherein the candidate drug is selected from the group consisting of small molecules or biological preparations.

14. The method according to claim 13, wherein the biological preparation is an antibody or a peptide.

15. The method according to claim 13, wherein the candidate drug is a peptide.

16. The method according to claim 15, wherein the candidate drug is a library of candidate peptides.

17. The method according to any one of claims 1 and 2, wherein the candidate drug is a bacterial antigen, a viral antigen, or a cancer antigen.

18. The method according to claim 15, wherein the candidate drug is an infectious disease-related candidate epitope, an autoimmune disease-related candidate epitope, or a tumor-related candidate epitope.

19. The method according to claim 15, wherein the candidate drug is a peptide with a length of 8 to 24 amino acids, a peptide with a length of 8 to 15 amino acids, or a peptide with a length of 8 to 12 amino acids.

20. The method according to any one of claims 1 and 2, wherein the optical signal indicates calcium flux, cytokine release, or the formation of a complex comprising T cells and antigen-presenting cells (APCs).

21. The method according to claim 20, wherein the optical signal indicates calcium flux, and the signal is fluorescence of a calcium-binding portion including a fluorescent label.

22. The method according to claim 20, wherein the optical signal indicates the formation of a complex between a T cell and an APC, and the signal is a change in cell shape.

23. The method according to any one of claims 1 and 2, wherein the optical signal is detected less than six hours after the sample is brought into contact with the analyte fluid.

24. The method according to claim 23, wherein the optical signal is detected less than two hours after the sample is brought into contact with the analyte fluid.

25. The method according to claim 24, wherein the optical signal is detected less than one hour after the sample is brought into contact with the analyte fluid.

26. The method according to claim 25, wherein the optical signal is detected less than 30 minutes after the sample is brought into contact with the analyte fluid.

27. The method according to any one of claims 1 and 2, wherein the method or a part thereof is automated.

28. The method according to any one of claims 1 and 2, wherein the method is high throughput.

29. The method according to any one of claims 1 and 2, wherein the sample containing T cells also contains PBMCs.

30. The method according to claim 29, wherein the PBMC is obtained from a mammalian subject.

31. The method according to any one of claims 1 and 2, wherein the sample containing T cells further contains antigen-presenting cells.

32. The method according to claim 31, wherein the antigen-presenting cells are professional APCs.

33. The method according to claim 32, wherein the professional APC is selected from the group consisting of dendritic cells, macrophages, monocytes, and B cells.

34. The method according to any one of claims 1 and 2, wherein the T cells include CD8+ T cells.

35. The method according to any one of claims 1 and 2, wherein the T cells include CD4+ T cells.

36. The method according to any one of claims 1 and 2, wherein in a cycle, a sample positive for T cell activation is not subjected to a subsequent cycle.

37. The method according to any one of claims 1 and 2, wherein in a cycle, a sample positive for T cell activation is used in a subsequent cycle.

38. The method according to any one of claims 1 and 2, wherein the removal of the analyte fluid is performed by laminar flow cell washing.

39. The method according to any one of claims 2 to 38, wherein the solid-phase support is a transwell insert, the plurality of transwell inserts are added to the wells of a multiwell plate, the plurality of samples containing T cells are placed on the plurality of transwell inserts, and the analyte fluid is added to the wells of the multiwell plate.

40. The method according to any one of claims 1 and 2, wherein the sample is washed after the removal of the analyte fluid and before contact with the new analyte fluid.

41. The method according to any one of claims 1 and 2, wherein the sample is not washed after the removal of the analyte fluid and before contact with the new analyte fluid.

42. The method according to any one of claims 1 and 2, wherein in any given cycle, samples that are positive for T cell activation are further analyzed by a downstream assay.

43. The method according to claim 42, wherein the downstream assay is ELISPOT.

44. The method according to claim 42, wherein the downstream assay is flow cytometry.

45. The method according to claim 42, wherein the downstream assay comprises staining or imaging the sample, and the staining or imaging comprises a dye or labeled antibody, optionally a fluorescently labeled antibody.

46. The method according to claim 42, wherein the downstream assay detects an activation-inducing marker.

47. The method according to claim 46, wherein the optical signal indicates calcium flux, and the signal is fluorescence of a calcium-binding portion including a fluorescent label.

48. The method according to claim 42, wherein the downstream assay is sequencing.

49. A method for identifying cellular responses to candidate drugs, wherein the method is iii. a. A step of bringing a sample containing cells into contact with an analyte fluid, wherein the sample containing cells is on a solid support and the analyte fluid contains a candidate drug in a liquid culture medium. b. A step of detecting a response by detecting an optical signal in the sample on the solid support, c. A step of removing the analyte fluid from the solid support, thereby removing the candidate drug while retaining the sample containing the cells on the solid support, The first cycle includes, iv. a. A step of contacting the retained sample, which contains cells from the preceding cycle, with a new analyte fluid, wherein the new analyte fluid contains a new candidate drug in a liquid culture medium, and the new candidate drug is different from the candidate drug from the preceding cycle. b. A step of detecting a response by detecting an optical signal in the held sample on the solid phase support, c. A step of removing the analyte fluid from the solid support, thereby removing the candidate drug while retaining the sample containing the cells on the solid support, Subsequent cycles including, Includes, The subsequent cycle is repeated at least N times, where N is an integer greater than or equal to 0, and each new analyte fluid in each subsequent cycle contains a different candidate drug than that in the preceding cycle. A method for removing the analyte fluid in which the cell viability and / or cell volume of the retained sample does not decrease by more than about 5% after a total of 10 cycles, and which does not involve centrifugation.

50. The method according to claim 49, wherein the cellular response is selected from the group consisting of activation or cell death.

51. A non-temporary computer-readable medium that, when executed by one or more processors, stores computer-readable instructions for managing automated cycling operations of the method according to any one of claims 1 and 2.

52. A non-temporary computer-readable medium that, when executed by one or more processors, stores the computer-readable instructions that govern the automated cycling operation of the method according to claim 49.

53. An automated system or device for performing the method according to any one of claims 1 to 42, wherein the solid-phase support is a well of a multiwell plate or a transwell insert disposed in a well of a multiwell plate, and the system a. A dispensing unit configured to dispense the analyte fluid into each well of the multiwell plate, b. An optical imaging unit configured to detect the activated T cells by detecting an optical signal, c. An automated system or device comprising: a washing unit configured to remove the analyte fluid from each well, thereby removing the candidate drug, and retaining the sample containing the T cells in the wells.

54. The system according to claim 53, further comprising at least one processor configured to execute instructions causing the at least one process to perform the method according to claim 1.

55. An automated system for detecting T cell activation, wherein the sample containing T cells is located in the wells of a multiwell plate or on a transwell insert placed in the wells of a multiwell plate. a. A dispensing unit configured to dispense an analyte fluid into each well of the multiwell plate, wherein the analyte fluid includes a candidate drug and a liquid culture medium, b. An optical imaging unit configured to detect activated T cells by detecting an optical signal, c. A system comprising a washing unit configured to remove the analyte fluid from each well, thereby removing the candidate drug, and to retain the sample containing the T cells in the wells.

56. The system according to any one of claims 53 and 55, wherein the multiwell plate is optionally configured such that cells can be exposed to one or more treatment cycles, and the relative positions of individual cells do not change significantly across different treatment cycles.

57. The system according to any one of claims 53 and 55, further comprising a holder or platform for holding the multiwell plate.

58. The system according to any one of claims 53 and 55, wherein the holder is configured to hold the multiwell plate in a fixed position.

59. The system according to claim 58, wherein the holder is configured to transport the multiwell plate so as to engage with the dispensing unit, the optical imaging unit, and the washing unit.

60. The system according to any one of claims 53 and 55, wherein the optical imaging unit is located below the multiwell plate.

61. The system according to any one of claims 53 and 55, wherein the optical imaging unit is configured to move from well to well.

62. The system according to any one of claims 53 and 55, wherein the dispensing unit is configured to dispense different analyte fluids into each well.

63. The system according to any one of claims 53 and 55, wherein the dispensing unit is configured to dispense a new analyte fluid into each well for each cycle.

64. The system according to any one of claims 53 and 55, wherein the optical imaging unit is a fluorescence imaging microscope.

65. The system according to any one of claims 53 and 55, wherein the cleaning unit comprises one or more suction devices configured to aspirate the analyte fluid.

66. The method according to any one of claims 53 and 55, wherein the cleaning unit is configured for laminar flow cleaning.

67. The method according to claim 57, wherein the holder is configured to be inclined.

68. The method according to claim 67, wherein the holder is rotatably coupled to the pivot point.

69. The system according to any one of claims 53 and 55, further comprising a processor that electronically communicates with the optical imaging unit, and a non-temporary computer-readable medium that is accessible to the processor and stores instructions that, when executed by the processor, cause the processor to receive or record the optical signals.

70. The system according to any one of claims 53 and 55, further comprising one or more processors configured to dispense the analyte fluid, detect an optical signal, and / or remove the candidate drug.

71. The system according to any one of claims 53 and 55, further comprising a control module having a user interface on which an operator can perform a method for screening T cell activation.

72. The system according to any one of claims 53 and 55, wherein the method is the method described in claim 1.

73. The system according to any one of claims 53 and 55, further comprising a system for storing and supplying new analytes from the screening library, with optional temperature control.

74. The system according to any one of claims 53 and 55, further comprising a liquid handling device capable of managing tips and liquids, moving the plate or other container containing the sample or analyte, and being connected to all other system components and capable of processing one or more containers (e.g., plates) containing the sample.

75. The system according to any one of claims 53 and 55, wherein the liquid handling device comprises a liquid handling robot integrated into the system to operate an electronic pipette, or an automated fluid system comprising tubing and a pump, or a combination thereof, and in some embodiments, the liquid transfer system is capable of moving up and down relative to the sample plate.

Images