Non-invasive method for enhancing immune and t cell function using low intensity vibration

Abstract

The disclosure is to a method for non-invasively augmenting T cell function in a subject in need thereof comprising a step of administering a high frequency, low magnitude physical stimulus, e.g. vibration, to the subject for a time duration effective to augment T cell function in the subject. Multiple administering steps can be delivered with refractory or rest periods therebetween. The method increases T cell function, enhances T cell function, stimulates an immune system in the subject, enhances CAR-T therapy, and slows the loss of T cell function. The enhanced CAR-T therapy can be an adjunct to treatment of cancer in the subject. The augmentation provided by the method improves an immune system response in the subject, including a subject in a non-disease state to, e.g. improve longevity in the subject, or both.

Description

NON-INVASTVE METHOD FOR ENHANCING IMMUNE AND T CELL FUNCTION USING LOW INTENSITY VIBRATIONGOVERNMENT SUPPORT

[0001] This invention was made with government support under HL127522 awarded by the National Institutes of Health. The government has certain rights in the invention.PRIORITY

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 675,575, filed July 25, 2024, the entire contents of which are incorporated herein by reference.FIELD

[0003] The disclosure concerns a non-invasive method for augmenting T cell function in subject, such as a human.BACKGROUND

[0004] Immunotherapy refers to the treatment of disease by inducing, enhancing, or suppressing an immune response. A variety of approaches are known in this regal’d, including the use of monoclonal antibodies, checkpoint inhibitors, and adoptive cell transfer therapies. These therapies aim to harness the body's immune system to fight diseases such as cancer, autoimmune disorders, and infectious diseases. The mechanisms of immunotherapy involve modulating the activity of immune cells, including T cells, to enhance their ability to recognize and destroy diseased cells. Applications of immunotherapy are broad, ranging from cancer treatment to managing chronic infections and autoimmune diseases.

[0005] One goal is to enhance the activation and proliferation of T cells, which play a crucial role in the immune response. T cells arc responsible for identifying and eliminating infected or malignant cells. Effective T cell activation requires the engagement of the T cell receptor (TCR) with an antigen, along with co- stimulatory signals provided by molecules such as CD28. Additionally, the production of cytokines like interleukin-2 (IL-2) is essential for T cell proliferation and function. Enhancing T cell activation and proliferation can improve the efficacy of immunotherapies, leading to better clinical outcomes for patients.

[0006] Achieving robust T cell activation and proliferation faces several obstacles. One significant challenge is the phenomenon of T cell exhaustion, which occurs when T cells are chronically stimulated, leading to a loss of function and the up-regulation of inhibitory receptors such as PD-1 and LAG-3. This exhaustion limits the effectiveness of T cell-based therapies. Another challenge is the immunosuppressive tumor microenvironment, which can inhibit T cell activity and proliferation. Additionally, the ex vivo expansion of T cells for adoptive cell transfer therapies is time-consuming and can result in a heterogeneous population of cells with varying levels of functionality.

[0007] Various methods have been explored to enhance T cell activation and proliferation. These include the use of cytokines, such as IL-2, to stimulate T cell growth and the application of checkpoint inhibitors to block inhibitory signals and restore T cell function. However, these approaches have limitations. Cytokine therapies can cause severe side effects, including cytokine release syndrome, and may not be effective in all patients. Checkpoint inhibitors, while effective in some cases, can also lead to immune-related adverse events and are not universally successful in overcoming the immunosuppressive tumor microenvironment.

[0008] Additionally, in Chimeric Antigen Receptor cell (CAR-T), used in treating various cancers, therapy involves the genetic modification of a patient's T cells to express a chimeric antigen receptor (CAR) that specifically targets cancer cells. The process begins with the extraction of T cells from the patient, followed by their genetic engineering to express the CAR, which combines an antigen recognition domain with T cell activation domains. These modified Tcells are then expanded in vitro and reinfused into the patient, where they seek out and destroy cancer cells expressing the target antigen.

[0009] CAR-T therapy has been used in treating hematologic malignancies, particularly B-cell malignancies such as acute lymphoblastic leukemia (ALL) and certain types of non-Hodgkin lymphoma. Its efficacy is attributed to the CAR's ability to direct T cells to recognize and kill cancer cells with high specificity. However, the therapy also faces several challenges, including the risk of severe side effects such as cytokine release syndrome (CRS) and neurotoxicity. Additionally, the manufacturing process for CAR-T cells is complex and time-consuming, often taking several weeks to produce a sufficient number of functional CAR-T cells for infusion.

[0010] One of the challenges in CAR-T therapy is the phenomenon of T cell exhaustion, where T cells lose their functionality due to chronic stimulation in the tumor microenvironment. This exhaustion is characterized by the upregulation of inhibitory receptors such as PD-1 and LAG-3, which dampen the T cells' anti-tumor activity. Overcoming T cell exhaustion is for improving the durability and efficacy of CAR-T therapy. Strategies to enhance T cell activation and proliferation, while reducing exhaustion, are actively being explored to optimize CAR-T therapy outcomes.

[0011] There is thus a need for a non-invasive method to augment T cell function, e.g., to enhance T cell activation and proliferation in a subject such as a human that can overcome the limitations of current approaches. The method should be capable of stimulating T cell function in a subject without causing significant side effects or requiring complex and costly treatments. Additionally, it should be applicable to a broad range of patients, including those with compromised immune systems due to aging, chronic diseases, or immunosuppressive therapies.

[0012] While various in vitro techniques exist to enhance the action of various types of cells which are then re-infused or otherwise reintroduced into a subject, applying such techniques directly to a human subject in vivo can be fraught with unpredictability. For example, in the context of in vivo T cells, various pertaining and impending cascade mechanisms and otherimmune system or bodily processes exist — the existence and effects of which are not encountered with isolated cells in vitro — can appear and / or fade to unexpected degrees.SUMMARY

[0013] In one aspect, the disclosure is directed to a method for non-invasively augmenting T cell function in a subject in need thereof comprising a step of administering a high frequency, low magnitude physical stimulus to the subject, e.g. a human, for a time duration effective to augment T cell function in the subject. In one embodiment, the physical stimulus is delivered by vibration.

[0014] In another embodiment, more than one administering step is delivered to the subject; and the respective frequency, magnitude, and time duration for each administering step can individually be the same or different. In one practice, a refractory period is provided between the delivery of at least two consecutive administering step to the subject. In another instance, a refractory period is provided between the delivery of each administering step to the subject, and wherein each refractory period can individually be the same or different.

[0015] In another embodiment, the augmentation of T cell function provided by the disclosed method includes increasing T cell function in the subject, enhancing T cell function in the subject, stimulating an immune system in the subject, enhancing CAR-T therapy for the subject, and slowing the loss of T cell function in the subject. In one practice, the enhanced CAR-T therapy is an adjunct to treatment of cancer in the subject. In another embodiment, the method of the disclosure is useful as a standalone cancer immunotherapy or as an adjunctive treatment with any other pharmacologic or cell-based cancer immunotherapy such as without limitation checkpoint blockade therapy, bi-specific T cell engagers, T cell receptor (TCR) therapy etc. In another embodiment, the augmentation of T cell function provided by the disclosed method improves an immune system response in the subject, improves longevity in the subject, including a non-disease state subject, or both.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 shows the effects of an embodiment of the method of the disclosure (denoted LIV) on CD62L expression in T cells from mice, including a histogram of CD62L expression, a bar graph of CD62L percent positive cells, and a bar graph of CD62L median fluorescence intensity (MFI).

[0017] FIG. 2 shows the effects of an embodiment of the method of the disclosure (denoted LIV) on CD69 expression in T cells from mice, including a histogram of CD69 expression, a bar graph of CD69 percent positive cells, and a bar graph of CD69 MFI.

[0018] FIG. 3 shows the effects of an embodiment of the method of the disclosure (denoted LIV) on CD25 expression in T cells from mice, including a histogram of CD25 expression, a bar graph of CD25 percent positive cells, and a bar graph of CD25 MFI.

[0019] FIG. 4 shows the effects of an embodiment of the method of the disclosure (denoted LIV) on PD-1 expression in T cells from mice.

[0020] FIG. 5 shows the effects of an embodiment of the method of the disclosure (denoted LIV) on LAG-3 expression in T cells from mice.

[0021] FIG. 6 shows the effects of an embodiment of the method of the disclosure (denoted LIV) on the expression of CD25, CD69, CD62L, LAG3, and PD1 on aged mice.

[0022] FIG. 7 shows the effects of an embodiment of the method of the disclosure (denoted LIV) on the immune response to an influenza viral infection in aged mice.DETAILED DESCRIPTION

[0023] The contents of U.S. Published Patent Application No. 2023 / 0203421 and U.S. Patent No. 10,029,089 are incorporated herein by reference in their entirely for any purpose.

[0024] The present disclosure will now be described in greater detail by referring to the following discussion. In the following description, numerous specific details are set forth, such as particular structures, components, materials, dimensions, processing steps and techniques, in order to provide an understanding of the various embodiments of the present disclosure.However, it will be appreciated by one of ordinary skill in the art that the various embodiments of the present disclosure may be practiced without these specific details.

[0025] As used herein, the term “about” indicates that the value listed may be somewhat altered, as long as the alteration does not result in nonconformance of the method or system herein described. For example, the term “about” can refer to a variation of ±0.1% to ±10%, or any point therebetween.

[0026] When a range is expressed in the present disclosure as being from one number to another number (e.g., 20 to 40), the present disclose contemplates any numerical value that is within the range (i.e., 22, 24, 26, 28.5, 31, 33.5, 35, 37.7, 39 or 40) and includes endpoints, fractional numbers, and whole numbers, and includes any amount that is bounded by any of the two values that can be present within the range (e.g., the range 20-40 includes 28.5-35).

[0027] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present disclosure. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and / or “comprising”, when used in this disclosure, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition ofone or more other features, integers, steps, operations, elements, components, and / or groups thereof

[0028] As used herein, the term “augmenting T cell function” and derivative terms such as “augment” or “augmentation” and the like representatively intends the improvement of T cell function, the slowing of loss of T cell function, the increasing of T cell function, the enhancement of T cell activity and activation, the stimulation of an immune system, the enhancing of CAR-T therapy enhancement, preservation of T cell function in a subject.

[0029] As used herein, the term a “physical stimulus” comprises an external force or signal applied to a subject's body to elicit a physiological response, including without limitation, mechanical stimulus, heat, light, electric fields, or combinations of any of the foregoing. In the context of the representative embodiments of the method described herein, “physical stimulus” refers to a high frequency, low magnitude mechanical signal, such as vibration, administered toward a subject in need thereof. In one practice, the physical stimulation is delivered by vibration. Devices that can be used to deliver vibration in this regard arc known in the art, see e.g. U.S. Patent No. 10,029,089.

[0030] As used herein, the term “refractory period” is a time interval following the administration of a physical stimulus during which the subject is free of the physical stimulus (e.g. not subject to the vibration), allowing the subject’s cells to recover and respond optimally before the next session of stimulus administration. In the context of the embodiments of the method described herein, the refractory period can be up to about 3 hours, e.g. it can be between 1 second to 3 hours.

[0031] As used herein, the term “subject” includes mammals, such as humans, dogs, cats, horses, mice, etc.

[0032] Various embodiments of the disclosure described herein provide a non-invasive method for enhancing immune and T cell function using low intensity vibration. The method leveragesthe mechanosensitivity of T cells to enhance their activation and proliferation without the need for pharmacological interventions. By administering a high frequency, low magnitude physical stimulus in a controlled manner, the method aims to improve immune function in subjects in need thereof, offering a novel and safer alternative to existing treatments. Administering a high frequency, low magnitude physical stimulus to a subject enhances T cell function by leveraging the mechanosensitivity of T cells. This method stimulates T cell activation and proliferation without the need for pharmacological interventions, providing a non-invasive alternative to existing treatments. The physical stimulus, characterized by specific frequency and magnitude parameters, induces mechanical signals that enhance immune function, potentially improving health outcomes for individuals with compromised immune systems. The disclosed method involves administering the physical stimulus in controlled sessions with refractory periods, ensuring that the stimulus is delivered effectively while allowing the subject's cells to recover and respond optimally. This approach mimics the mechanical benefits of exercise, which is known to enhance immune function, but does so in a controlled and repeatable manner that can be applied to individuals who may not be able to engage in regular physical activity.

[0033] Compared to traditional pharmacological treatments, the disclosed method reduces the risk of adverse side effects and is suitable for a broader range of patients, including those with contraindications to certain medications. The non-invasive nature of the method also makes it more accessible and easier to administer, potentially increasing patient compliance and overall effectiveness.

[0034] Specifically, the physical stimulus enhances T cell activation by increasing the expression of activation markers such as CD69 and CD25, while simultaneously decreasing the expression of inhibitory receptors like PD-1 and LAG-3. This dual effect not only promotes T cell activation but also reduces the likelihood of T cell exhaustion, a common issue in chronic activation scenarios. The mechanical signals generated by the physical stimulus activate downstream elements of the CD28 co-stimulatory pathway, including the phosphorylation of key proteins such as AKT and GSK3p. This activation leads to the nuclear translocation of transcription factors like NFAT, which are crucial for T cell activation and function.

[0035] By enhancing T cell activation, the method improves the body's ability to mount an effective immune response, which is particularly beneficial for individuals undergoing immunotherapy, such as CAR-T therapy, or those with weakened immune systems due to aging or chronic diseases. The non-invasive nature of the method also makes it more accessible and easier to administer, potentially increasing patient compliance and overall effectiveness.

[0036] In addition to enhancing T cell activation, the method also increases the production of pro-inflammatory cytokines such as IL-2 and TNF-a, which play essential roles in immune regulation and response. By boosting cytokine production, the method further stimulates the immune system, enhancing its ability to respond to infections and other immune challenges.

[0037] The method also reduces the expression of inhibitory receptors like PD-1 and LAG-3, which are associated with T cell exhaustion and immune suppression. By decreasing the expression of these inhibitory receptors, the method helps maintain T cell functionality and prevents the immune system from becoming over-exhausted, which is particularly important in chronic disease conditions and during prolonged immune responses.

[0038] By stimulating the immune system, the method improves the body's ability to mount an effective immune response, which is particularly beneficial for individuals undergoing immunotherapy, such as CAR-T therapy, or those with weakened immune systems due to aging or chronic diseases. The non-invasive nature of the method also makes it more accessible and easier to administer, potentially increasing patient compliance and overall effectiveness.

[0039] In one aspect, the disclosure is to a method for non-invasively augmenting T cell function in a subject in need thereof comprising a step of administering a high frequency, low magnitude physical stimulus to the subject for a time duration effective to augment T cell function in the subject. In one embodiment, the physical stimulus is delivered to the subject by vibration. The physical stimulus, e.g. vibration, can be delivered to any part of the body of the subject. For example, the physical stimulus can be delivered to one or both feet, or to any one both arms orand / or legs of the subject; or the subject can lie down on or against the device providing the physical stimulus. In another practice, the physical stimulus is administered toward the subject using a device configured to deliver the physical stimulus to the entire body of the subject.

[0040] In different embodiments, the device is a platform on which the subject stands, ensuring that the mechanical signals are transmitted effectively through the feet and up into the body. In one practice, the device is equipped with adjustable settings to control the frequency and / or magnitude of the vibrations, allowing for customization based on the subject's specific needs and conditions. In another embodiment, the physical stimulus is administered using a full-body vibration device, where the subject either stands or lies down on a platform that delivers the mechanical signals uniformly across the entire body. This full-body approach ensures that the stimulus reaches a broader range of tissues and cells, potentially enhancing the overall immune response. In one practice, the device comprises ergonomic features to ensure comfort during the sessions, and may include safety mechanisms to prevent overexposure to the vibrations. In other embodiments, the device is portable, allowing for use in various settings such as clinics, homes, or fitness centers. In yet another embodiment, the device is a wearable device that delivers localized vibrations to specific parts of the body, such as the legs or arms. In one aspect, this wearable device is in the form of straps or pads that are secured around the target area, providing a more focused and controlled delivery of the physical stimulus. In one practice, the wearable device is battery-operated and include a user interface for adjusting the vibration parameters, making the wearable device convenient for daily use. Each of these embodiments ensures that the physical stimulus is administered in a controlled manner, with sessions comprising about one second to one hour, followed by a refractory period of one second to three hours, and then a second session of similar duration, thereby optimizing the stimulation of T cell function. Devices that can be used to deliver vibration are known in the ail, see e.g. U.S. Patent No. 10,029,089.

[0041] In one embodiment wherein the physical stimulus is delivered by vibration, there is no upper limit on frequency of the vibration, except that frequencies so high that damage is done to the subject are not used. In one instance, the high frequency is up to about 1000 hertz. In one practice, the frequency is up to about 200 hertz. In other instances the frequency is betweenabout 5 hertz to about 500 hertz, between about 10 hertz to about 200 hertz, between about 5 to about 100 hertz, and between about 20 to about 90 hertz. The magnitude of the low vibration should not be greater than 2.0 g which is an order of magnitude that causes damage to the subject, e.g. cause unwanted inflammation in the subject. Thus in one embodiment, the magnitude of the low vibration is less than 2.0 g. In another embodiment, the magnitude is less than 1.0 g. In other instances, the magnitude is between about 0.1 g to about 1.5 g, between about 0.4 g to about 1.0 g, between about 0.3 to about 0.5 g. The high frequency, low magnitude physical stimulus is administered to the subject for a time duration that is effective to augment T cell function in the subject.

[0042] In one embodiment, the time duration is up to about 2 hours. In another embodiment, the time duration is about 2 minutes to about 1 hour. In another embodiment, the about 5 minutes and about 20 minutes. In one practice, the administering step occurs at least once a day for up to about 6 months. In various instances, the administering step is at least once a day for up to about 3 months, at least once a day for up to about 8 weeks, at least once a day for up to about 4 weeks, at least once a day for about 1 week.

[0043] In one embodiment, more than one administering step is delivered to the subject; in this embodiment, the respective frequency, magnitude, and time duration for each administering step can each individually be the same or different. In one practice of this embodiment, up to 10 administering steps per day are delivered to the subject. In another practice, 2 to 4 administering steps are delivered to the subject per day. In one embodiment, a refractory period is provided between the delivery of at least two consecutive administering step to the subject. In one practice, a refractory period is provided between the delivery of each administering step to the subject; in this practice, each refractory period can each individually be the same or different. In one instance, the refractory period is up to about 12 hours. In other practices, the refractory period is up to about 3 hours, between about 5 minutes to about 30 minutes, between about 10 minutes to about 20 minutes. In one practice, the refractory period is provided immediately after the completion of an administration step.

[0044] In one practice of the method, the frequency is about 20 hertz to about 90 hertz; the magnitude is about 0.3 g to about 0.5 g; and the time duration is about 10 minutes to about 20 minutes; and wherein 2 to 4 administering steps are delivered to the subject per day; and a refractory period of about 15 minutes to about 30 minutes is provided between the delivery of each administering step to the subject; and wherein the respective frequency, the magnitude, the time duration, and the refractory period can each individually be the same or different .

[0045] In one embodiment, the augmentation of T cell function provided by the disclosed method includes increasing T cell function in the subject, enhancing T cell function in the subject, stimulating an immune system in the subject, enhancing CAR-T therapy for the subject, and / or slowing the loss of T cell function in the subject. In one instance, the enhanced CAR-T therapy is an adjunct to treatment of cancer in the subject. In another practice, the augmentation of T cell function provided by the method is applied to a subject in a non-disease state to improve an immune system response in that subject, improve longevity in that subject, or both. In one embodiment, the method of the disclosure leverages the mechanosensitivity of T cells to enhance their activation and proliferation thereby improving their anti-tumor efficacy. The practice of the disclosed method increases the expression of activation markers such as CD69 and CD25, while reducing the expression of inhibitory receptors like PD-1 and LAG-3. This dual effect not only promotes T cell activation but also mitigates T cell exhaustion, thereby enhancing the overall functionality of CAR-T cells.

[0046] In one embodiment, the method of the disclosure is integrated into a CAR-T therapy protocol. Doing so enhances T cell activation and reduces T cell exhaustion, and moreover improve the expansion and functionality of CAR-T cells, which can lead to improved clinical outcomes. Additionally, the non-invasive nature of the method of the disclosure makes it a safer and more accessible adjunct to CAR-T therapy than known adjuncts, thereby reducing the risk of adverse side effects associated with pharmacological interventions and can expand the use of CAR-T therapy to a broader range of patients, including those with compromised immune systems or those who are unable to tolerate conventional treatments.

[0047] EXAMPLES

[0048] In the following pilot study and Examples FIGS 1-5, the subjects were wild type (WT) mice and Duchenne Muscular Dystrophy (DMD) mice. The WT mice represent a standard model, while the DMD mice provide a model for Duchenne Muscular Dystrophy, allowing for the assessment of the effects of the method of the disclosure across different genetic backgrounds. The mice were exposed in vivo to the method of the disclosure using vibration at the parameters identified below. Mice that were subjected to the method of the disclosure are identified in the Figures as LIV (low intensity vibration).

[0049] The vibration device was configured to deliver high frequency, low magnitude physical stimulus to the mice. The device administered the physical stimulus non-invasively by having the mice placed on a platform that vibrated in controlled sessions using the parameters identified below ensuring that the stimulus was delivered effectively while allowing the mice's cells to recover and respond optimally. The T cells were collected from the mice's blood and analyzed via flow cytometry. This analysis involved measuring changes in positive and negative population distribution and median fluorescence intensity (MFI) of various markers.

[0050] Specifically:

[0051] In example FIG. 1, the T cells were analyzed for changes in the expression of activation marker CD62L. Flow cytometry provided detailed information on the distribution and intensity of CD62L, allowing for the assessment of T cell activation and exhaustion levels.

[0052] In example FIG. 2, the T cells were analyzed for changes in the expression of activation marker CD69. Flow cytometry provided detailed information on the distribution and intensity of CD69, allowing for the assessment of T cell activation and exhaustion levels.

[0053] In example FIG. 3, the T cells were analyzed for changes in the expression of activation marker CD25. Flow cytometry provided detailed information on the distribution and intensity of CD25, allowing for the assessment of T cell activation and exhaustion levels.

[0054] In example FIG. 4, the T cells were analyzed for changes in the expression inhibitory receptors PD- 1. Flow cytometry provided detailed information on the distribution and intensity of PD-1, allowing for the assessment of T cell activation and exhaustion levels.

[0055] In example FIG. 5, the T cells were analyzed for changes in the expression of inhibitory receptor LAG-3. Flow cytometry provided detailed information on the distribution and intensity of these markers, allowing for the assessment of T cell activation and exhaustion levels.

[0056] In example FIG. 6, the T cells were analyzed for changes in the activation markers CD25, CD69, and CD62L, and in the inhibitory receptors LAG3 and PD1 in aged mice.

[0057] In example FIG. 7, the weight loss of aged mice challenged with a PR* influenza virus strain was measured over 12 days (D0-D12). The weight loss was an indicator of overall health of the challenged mice over time.

[0058] Pilot Study:

[0059] A pilot study was conducted using a mix of 3 pairs of wild type (WT) mice, and 2 pairs of Duchenne Muscular Dystrophy (DMD) mice. In this pilot study, 5 mice were exposed to the method of the disclosure using vibration at the following conditions: magnitude was 0.4 g, frequency was 90 hertz, time duration of administration was 10 minutes, twice a day for 4 weeks with a 30 minute refractory period between administrations. Following the fourth week the mice were sacrificed and T cells were analyzed via flow cytometry from the mouse whole blood. T cells were analyzed both for changes in positive / negative population distribution and median fluorescence intensity (MFI) where changes in MFI are used to determine changes in receptor expression. Changes in expression of activation markers CD62L, CD69, and CD25 as well asinhibitory receptors PD-1 and LAG-3 which are closely linked with T cell exhaustion were measured. It was found that there was a 22.6% increase in the CD62L negative population and a 16.1% decrease in the MFI of CD62L. It was also found that there was a 7.8% reduction in PD-1 expression and a 9.6% reduction in LAG-3 expression. The pilot study showed that the method of the disclosure mechanically stimulated the immune system and that vibrated mice had higher numbers of CD62L (negative population) cells and lower expression of CD62L overall. Loss of CD62L is known to correlated with higher levels of activation in T cells. Moreover, the decreases in both PD- 1 and LAG-3 expression indicates that T cell activation was enhanced while expression of inhibitory receptors and protecting against T cell exhaustion was reduced.

[0060] Examples FIGS. 1-5:

[0061] FIGS.1-5 illustrate experimental results of applying the method of the disclosure to a mix of WT and DMD mice under the following conditions of vibration: the mice were exposed to vibration at a magnitude of 0.3 g, a frequency of 60 Hertz, a time duration of administration was 10 minutes, twice a day for 4 weeks with a 30 minute refractory period between administrations. The mice were sacrificed and T cell activation status was analyzed from collected whole blood. T cells were analyzed for both changes in positive and negative population distribution, and median fluorescence intensity. Representative histograms in FIGS. 1-5 demonstrate positive and negative gating. CD62L (negative population) increased by 22% and the MFI decreased by 16%. CD69 and CD25 showed no significant changes, PD-1 MFI decreased by 7.8% and LAG-3 MFI decreased by 9.6% (n=5 data presented as mean + / - SD).

[0062] FIG. 1 shows the effects of the method on T cell activation in mice. The figure includes a histogram of CD62L expression, a bar graph of CD62L percent positive cells, and a bar graph of CD62L median fluorescence intensity (MFI). The histogram compares the CD62L expression between control and mice treated with the method of the disclosure (LIV). The bar graphs quantify the changes in CD62L percent positive cells and CD62L MFI between the two groups. The histogram in FIG. 1 illustrates the distribution of CD62L expression in T cells from control and mice treated with the method of the disclosure. The control group shows a distinct peak forCD62L positive cells, while mice treated with the method of the disclosure showed a shift in the distribution, indicating a change in CD62L expression. The bar graph of CD62L percent positive cells in FIG. 1 shows that mice treated with the method of the disclosure resulted in a significant increase in the percentage of CD62L negative cells compared to the control group. This indicates that mice treated with the method of the disclosure enhanced T cell activation, as CD62L negative cells are associated with higher levels of activation. The bar graph of CD62L MFI in FIG. 1 shows that mice treated with the method of the disclosure resulted in a decrease in the median fluorescence intensity of CD62L compared to the control group. This indicates that mice treated with the method of the disclosure had enhanced T cell activation, as lower CD62L expression is correlated with higher activation levels.

[0063] The flow cytometry analysis showed that treatment with the method of the disclosure resulted in a 22% increase in the CD62L negative population and a 16% decrease in the MFI of CD62L. Additionally, there was a 7.8% reduction in PD-1 expression and a 9.6% reduction in LAG-3 expression. These changes indicate that mice treated with the method of the disclosure enhanced T cell activation while reducing the expression of inhibitory receptors associated with T cell exhaustion. The representative histograms and bar graphs in Figure 1 illustrate these changes, showing the distribution of CD62L expression and quantifying the differences between control and mice treated with the method of the disclosure. The data provided in FIG. 1 further demonstrates the effects of the method of the disclosure on T cell expression marker CD62L and indicates that administering a high frequency, low magnitude physical stimulus enhanced T cell function, enhanced T cell activation, and stimulated the immune system, in a subject in need thereof.

[0064] FIG. 2 shows the effects of the method of the disclosure on CD69 expression in T cells from the mice. The figure includes a histogram of CD69 expression, a bar graph of CD69 percent positive cells, and a bar graph of CD69 median fluorescence intensity (MFI). The histogram compares the CD69 expression between control and mice treated with the method of the disclosure (LIV). The bar graphs quantify the changes in CD69 percent positive cells and CD69 MFI between the two groups. The histogram in FIG. 2 illustrates the distribution of CD69expression in T cells from control and mice treated with the method of the disclosure. The control group showed a distinct peak for CD69 positive cells, while the mice treated with the method of the disclosure showed a shift in the distribution, indicating a change in CD69 expression. The bar graph of CD69 percent positive cells in FIG. 2 shows that mice treated with the method of the disclosure resulted in a significant increase in the percentage of CD69 positive cells compared to the control group. This indicates that mice treated with the method of the disclosure had enhanced T cell activation, as CD69 positive cells are associated with higher levels of activation. The bar graph of CD69 MFI in FIG. 2 shows that mice treated with the method of the disclosure resulted in a decrease in the median fluorescence intensity of CD69 compared to the control group. This further supports the finding that mice treated with the method of the disclosure had enhanced T cell activation, inasmuch as a lower CD69 expression is correlated with higher activation levels. The flow cytometry analysis revealed that mice treated with the method of the disclosure resulted in a significant increase in the percentage of CD69 positive cells and a decrease in the median fluorescence intensity of CD69. These changes indicate that mice treated with the method of the disclosure had enhanced T cell activation. The representative histograms and bar graphs in FIG. 2 illustrate these changes, showing the distribution of CD69 expression and quantifying the differences between control and mice treated with the method of the disclosure. The data provided in FIG. 2 further demonstrates the effects of the method of the disclosure on T cell expression marker CD69 and indicates that administering a high frequency, low magnitude physical stimulus enhanced T cell function, enhanced T cell activation, and stimulated the immune system, in a subject in need thereof.

[0065] FIG. 3 shows the effects of the method of the disclosure on CD25 expression in T cells from mice. The figure includes a histogram of CD25 expression, a bar graph of CD25 percent positive cells, and a bar graph of CD25 median fluorescence intensity (MFI). The histogram compares the CD25 expression between control and mice treated with the method of the disclosure. The bar graphs quantify the changes in CD25 percent positive cells and CD25 MFI between the two groups. The histogram in FIG. 3 illustrates the distribution of CD25 expression in T cells from control and mice treated with the method of the disclosure (LIV). The control group showed a distinct peak for CD25 positive cells, while the mice treated with the method ofthe disclosure showed a shift in the distribution, indicating a change in CD25 expression. The bar graph of CD25 percent positive cells in FIG. 3 showed that mice treated with the method of the disclosure resulted in an increase in the percentage of CD25 positive cells compared to the control group. This indicates that mice treated with the method of the disclosure had enhanced T cell activation, as CD25 positive cells are associated with higher levels of activation. The bar graph of CD25 MFI in FIG. 3 shows that mice treated with the method of the disclosure resulted in an increase in the median fluorescence intensity of CD25 compared to the control group indicating that the mice treated with the method of the disclosure had enhanced T cell activation, as higher CD25 expression is correlated with higher activation levels. The flow cytometry analysis revealed that mice treated with the method of the disclosure resulted in an increase in the percentage of CD25 positive cells and an increase in the median fluorescence intensity of CD25. These changes indicate that mice treated with the method of the disclosure enhanced T cell activation. The representative histograms and bar graphs in FIG. 3 illustrate these changes, showing the distribution of CD25 expression and quantifying the differences between control and mice treated with the method of the disclosure. The data provided in FIG. 3 further demonstrates the effects of the method of the disclosure on T cell expression marker CD25 and indicates that administering a high frequency, low magnitude physical stimulus enhances T cell function, enhances T cell activation, and stimulates the immune system, in a subject in need thereof.

[0066] FIG. 4 shows the effects of the method of the disclosure on PD-1 expression in T cells from mice. The figure includes a histogram of PD-1 expression, a bar graph of PD-1 percent positive cells, and a bar graph of PD-1 median fluorescence intensity (MFI). The histogram compares the PD-1 expression between control and mice treated with the method of the disclosure (LIV). The bar graphs quantify the changes in PD-1 percent positive cells and PD-1 MFI between the two groups. The histogram in FIG. 4 illustrates the distribution of PD-1 expression in T cells from control and mice treated with the method of the disclosure. The control group shows a distinct peak for PD-1 positive cells, while the mice treated with the method of the disclosure showed a shift in the distribution, indicating a change in PD-1 expression. The bar graph of PD-1 percent positive cells in FIG. 4 shows that mice treated withthe method of the disclosure resulted in a decrease in the percentage of PD-1 positive cells compared to the control group. This indicated that mice treated with the method of the disclosure had reduced the expression of inhibitory receptors, which is associated with lower levels of T cell exhaustion. The bar graph of PD-1 MFI in FIG. 4 shows that mice treated with the method of the disclosure resulted in a decrease in the median fluorescence intensity of PD- 1 compared to the control group. This further supports the finding that mice treated with the method of the disclosure had reduced expression of inhibitory receptors, enhancing T cell functionality. The flow cytometry analysis revealed that mice treated with the method of the disclosure resulted in a decrease in the percentage of PD-1 positive cells and a decrease in the median fluorescence intensity of PD-1. These changes indicate that mice treated with the method of the disclosure had reduced expression of inhibitory receptors associated with T cell exhaustion. The representative histograms and bar graphs in FIG. 4 illustrate these changes, showing the distribution of PD-1 expression and quantifying the differences between control and mice treated with the method of the disclosure. The data provided in FIG. 4 further demonstrates the effects of the method of the disclosure on PD-1 expression in T cells, and indicates a high frequency, low magnitude physical stimulus reduces the expression of inhibitory receptors and enhance T cell function in a subject in need thereof.

[0067] FIG. 5 shows the effects of the method of the disclosure on LAG-3 expression in T cells from mice. The figure includes a histogram of LAG-3 expression, a bar graph of LAG-3 percent positive cells, and a bar graph of LAG-3 median fluorescence intensity (MFI). The histogram compares the LAG-3 expression between control and mice treated with the method of the disclosure (LIV). The bar graphs quantify the changes in LAG-3 percent positive cells and LAG- 3 MFI between the two groups. The histogram in FIG. 5 illustrates the distribution of LAG-3 expression in T cells from control and mice treated with the method of the disclosure. The control group showed a distinct peak for LAG-3 positive cells, while the mice treated with the method of the disclosure showed a shift in the distribution, indicating a change in LAG-3 expression. The bar graph of LAG-3 percent positive cells in FIG. 5 shows that mice treated with the method of the disclosure resulted in a decrease in the percentage of LAG-3 positive cells compared to the control group. This indicates that mice treated with the method of the disclosurehad reduced expression of inhibitory receptors, which is associated with lower levels of T cell exhaustion. The bar graph of LAG-3 MFI in FIG. 5 shows that mice treated with the method of the disclosure resulted in a decrease in the median fluorescence intensity of LAG-3 compared to the control group. This indicates that mice treated with the method of the disclosure had reduced expression of inhibitory receptors, which enhances T cell functionality. The flow cytometry analysis revealed that mice treated with the method of the disclosure resulted in a decrease in the percentage of LAG-3 positive cells and a decrease in the median fluorescence intensity of LAG- 3. These changes indicate that mice treated with the method of the disclosure had reduced expression of inhibitory receptors associated with T cell exhaustion. The representative histograms and bar graphs in FIG. 5 illustrate these changes, showing the distribution of LAG-3 expression and quantifying the differences between control and mice treated with the method of the disclosure. The data provided in FIG. 5 further demonstrates the effects of the method of the disclosure on LAG-3 expression in T cells, and indicates that administering a high frequency, low magnitude physical stimulus reduces the expression of inhibitory receptors and enhance T cell function in a subject in need thereof.

[0068] Examples FIGS. 6-7:

[0069] FIG. 6 shows effects of another embodiment of the method of the disclosure on CD25, CD69, CD62L, LAG3 and PD1 expression in mice. Aged mice (18 months old) were treated with an embodiment of the method of the disclosure using the following vibration parameters: magnitude was 0.4 g, frequency was 30 hertz, time of duration was 10 minutes, twice a day for 4 weeks with a 30 minute refractory period between administrations. Blood samples were taken and measured as in examples 1-5. As seen in FIG. 6, the method of the disclosure increased CD25 expression by +102% (* = p<0.05, n=8), increase CD69 expression by +44.2% (* = p<0.05, n-7). and increased LAG-3 expression by +8.7% (* = p<0.05, n-8), while CD62L expression decreased by -30.9% (* = p<0.05, n=8). In vivo application of the method of the disclosure had no significant effect on PD-1 (ns = p>0.05, n=8). All data was normalized to average of controls and presented as median + / - IQR. From these results, it is seen that the method of the disclosure increased the T cell activation in the aged mice.

[0070] FIG. 7 shows the effects of an embodiment of the method of the disclosure (denoted LIV) on the immune response to an influenza viral infection in aged mice using weight loss as an indicator of immune response. Aged mice (18 months old) were treated with an embodiment of the method of the disclosure using the following vibration parameters: magnitude was 0.4 g, frequency was 30 hertz, time of duration was 10 minutes, twice a day for 4 weeks with a 30 minute refractory period between administrations. At 4 weeks, the mice were challenged with 0.3LD50 of an influenza A virus strain, PR8. Weight loss was measured daily (D0-D12) as a measure of overall health and disease severity during the period the mice were being treated with the vibration. As seen in FIG. 7, at 12 days post infection (D12), the treated mice had lost 18.2% less weight than the sham handled controls (* = p<0.05, n=9). All data presented as median + / - IQR. From these results, it is seen that the method of the disclosure enhanced the immune response to a viral infection, increased the T cell activation in the aged mice.

[0071] The following advantages accrue in the certain embodiments of the method of the disclosure manifests:

[0072] Non-Invasive Stimulation: Unlike traditional methods that rely on pharmacological treatments, embodiments of the disclosure use a non-invasive physical stimulus to enhance immune function. This reduces the risk of adverse side effects and makes the method suitable for a broader range of patients, including those with contraindications to certain medications.

[0073] Controlled Sessions with Refractory Periods: Embodiments of the disclosure include administering the physical stimulus in controlled sessions with refractory periods, ensuring that the stimulus is delivered effectively while allowing the subject's cells to recover and respond optimally. This approach mimics the mechanical benefits of exercise in a controlled and repeatable manner.

[0074] Dual Effect on T Cell Activation and Exhaustion: The physical stimulus enhances T cell activation by increasing the expression of activation markers such as CD62L, CD69 and CD25,while simultaneously decreasing the expression of inhibitory receptors like PD-1 and LAG-3.This dual effect not only promotes T cell activation but also reduces the likelihood of T cell exhaustion, a common issue in chronic activation scenarios.

[0075] Activation of CD28 Co-Stimulatory Pathway: The mechanical signals generated by the physical stimulus activate downstream elements of the CD28 co-stimulatory pathway, including the phosphorylation of key proteins such as AKT and GSK3p. This activation leads to the nuclear translocation of transcription factors like NF AT, which are crucial for T cell activation and function.

[0076] Broad Applicability: The method is applicable to a wide range of patients, including those with compromised immune systems due to aging, chronic diseases, or immunosuppressive therapies. It can also be integrated into existing immunotherapy protocols, such as CAR-T therapy, to enhance treatment outcomes.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method for non-invasively augmenting T cell function in a subject in need thereof comprising a step of administering a high frequency, low magnitude physical stimulus to the subject for a time duration effective to augment T cell function in the subject.

2. The method of Claim 1 wherein the physical stimulus is delivered by vibration.

3. The method of Claim 2 wherein the frequency is (i) up to about 1000 hertz,(ii) up to about 500 hertz, (iii) about 5 hertz to about 100 hertz, (iv) about 10 hertz to about 200 hertz, or (v) about 20 hertz to about 90 hertz.

4. The method of Claim 2 wherein the magnitude is (i) no greater that 2.0 g, (ii) up to about 1.8 g, (iii) about 0.1 g to about 1.5 g, (iv) about 0.4 g to about 1.0 g, or (v) about 0.3 g to about 0.5 g.

5. The method of Claim 2 wherein the time duration is (i) up to about 2 hours, (ii) about 2 minutes to about 1 hour, or (iii) about 5 minutes and about 20 minutes.

6. The method of Claim 2 wherein the administering step occurs (i) at least once a day for up to about 6 months, (ii) at least once a day for up to about 8 weeks, (iii) at least once a day for up to about 4 weeks, or (iv) at least once a day for up to about 2 weeks, or (v) at least once a day for up to about 1 week, or (vi) at least once a day for less than 1 week.

7. The method of Claim 2 wherein more than one administering step is delivered to the subject; and wherein the respective frequency, magnitude, and time duration for each administering step can individually be the same or different.

8. The method of Claim 7 wherein up to 10 administering steps per day are delivered to the subject.

9. The method of Claim 8 wherein 2 to 4 administering steps are delivered to the subject per day.

10. The method of Claim 7 wherein a refractory period is provided between the delivery of at least two consecutive administering steps to the subject.

11. The method of Claim 10 wherein the refractory period is provided immediately after the first of the at least two consecutive administering steps to the subject.

12. The method of Claim 7 wherein a refractory period is provided between the delivery of each administering step to the subject, and wherein each refractory period can individually be the same or different.

13. The method of Claim 7 wherein the refractory period is up to about 3 hours.

14. The method of Claim 13 wherein the refractory period is about 5 minutes to about 30 minutes.

15. The method of Claim 14 wherein the refractory period is about 10 minutes to about 20 minutes.

16. The method of Claim 2 wherein the frequency is about 20 hertz to about 90 hertz; the magnitude is about 0.3 g to about 0.5 g; and the time duration is about 10 minutes to about 20 minutes; and wherein 2 to 4 administering steps are delivered to the subject per day; and a refractory period of about 15 minutes to about 30 minutes is provided between the delivery of each administering step to the subject; and wherein the respective frequency, the magnitude, the time duration, and the refractory period can each individually be the same or different .

17. The method of Claim 1 wherein the augmentation of T cell function includes one or more of the following: increasing T cell function in the subject, enhancing T cell function in the subject, stimulating an immune system in the subject, enhancing CAR-T therapy for the subject, enhancing TCR-T cell therapy for a subject, and slowing the loss of T cell function in the subject.

18. The method of Claim 17 wherein the enhanced CAR-T therapy is an adjunct to treatment of cancer in the subject.

19. The method of Claim 18 wherein the enhanced CAR-T therapy is an adjunct with one or more pharmacologic cancer immunotherapies, one or more of a cell -based cancer immunotherapies, or both.

20. The method of Claim 17 wherein augmentation improves an immune system response in the subject, improves longevity in the subject, or both.

21. The method of Claim 1 wherein the physical stimulus is delivered to the feet of the subject.

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