In addition to the readily detectible humoral immune response in the context of neutralizing antibodies in convalescent patients who had recovered from COVID, these studies have collectively shown strong SARS-CoVspecific memory T cells are frequently observed 11 , Furthermore, significantly larger T cell responses appear to correlate with severity of the disease, underscoring the importance of T cells above and beyond the humoral antibody response for combating infection This is an important consideration for a novel therapeutic, as most prophylactic vaccines currently in development for COVID are designed to focus on eliciting antibody responses to the spike protein of SARS-CoV-2 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , In addition, the antibody response in recovered COVID patients has been shown to decline several months after infection, raising concerns that therapeutics or vaccines designed to elicit primarily neutralizing antibody responses may not be sufficient to engender the cellular immunity required for long-term duration of protection or to protect from potential repeat infections Thus, the a priori T cell repertoire, both quantity and quality, may portend COVID disease prognosis and may influence the outcome between mild or severe disease.
The transfer of neutralizing antibodies reflects a more passive serological immune engagement versus the more active cellular immune response that SARS-CoVspecific T cells would provide.
Given the crucial importance of a strong virus-specific T cell response for patients with severe disease, especially during the critical days where respiratory distress is common, the adoptive transfer of a quantity of expanded, activated, effector memory T cells capable of mounting a robust virus-specific response may be important to reduce viral load and improve patient outcomes. Furthermore, these expanded VIL are enriched in virus antigen-specificity, show polyfunctional cytokine responses and acquire a T effector memory phenotype, making them highly suited for participating in an active cellular immune response when adoptively transferred back to patients after this minimal ex vivo expansion time.
There are unfortunately limited therapeutic options for the treatment of COVID that have demonstrated robust clinical relevance amidst the ongoing pandemic. Considerable, contemporaneous actual experience in the critical care of COVID patients dating from the first quarter of in New York City, NY, USA, including observations of the clinical time course, hallmark clinical features, and treatment from presentation through critical illness to ultimate resolution or mortality, provides guidance as to the need for a vein-to-vein time of 7-days in order to intercede timely.
A protective virus-specific T cell therapy may be an important novel modality for the treatment of SARS-CoV-2 and other pandemic viral pathogens. To develop a platform for robust and rapid ex vivo expansion of viral antigen-specific T cells from patients exposed to viral pathogens, we generated a micro-aAPC capable of providing an immunogenic viral peptide in the context of MHC Class I or MHC Class II molecules in combination with anti-CD28 stimulation molecules Fig.
By comparison, donor T cells cultured in cytokine alone in the absence of VIPR particles showed a minimal enrichment in antigen-specific VIL proportions, even though the T cells proliferated robustly. Next we sought to optimize the design of the VIPR particle aAPCs to further enhance the expansion of antigen-specific T cells within the rapid 7-day stimulation culture. A dose-dependent enrichment was seen with lower doses of particles and higher numbers of T cells, such that an optimal enrichment was observed with a ratio of T cells to VIPR particles Fig.
These culture plates enable gas exchange from the base of the culture well, allowing cells to be cultured with a large ratio of media per surface area and abundant access to nutrients, and have been shown to facilitate a large and rapid expansion of primary human T cells This proliferative expansion coupled with the enrichment of the VIL population resulted in an average of 2.
Collectively these data demonstrate the ability of the VIPR particle expansion protocol to rapidly enrich and expand VIL from low numbers in CMV-positive individuals and near undetectable numbers in COVID convalescent individuals, to significantly large numbers of virus-specific T cells. Analysis of expanded antigen-specific VIL that co-express PD-1 in combination with either TIM-3, LAG-3 or TIGIT, revealed a detectible population of cells after 7-days at similar frequencies observed in the non-antigen-specific population, suggesting that after 7-days these TCR stimulated cells have not acquired an extensive exhaustive T cell phenotype that would be seen by expression of multiple co-inhibitory markers Supplementary Fig.
The same profile of activation marker and checkpoint gene expression was observed when CMV-specific VIL were stimulated after 7-day rapid expansion with VIPR particles, with a similarly observed variability between different CMV-positive individuals, indicating this culture platform is effective at rapid T cells expansion and activation with multiple viral antigens Fig. When analyzed together we see an elevated proportion of cells expressing 1, 2 or all 3 cytokines in combination when compared to the non-SARS-CoVspecific T cells within the expanded culture Fig.
Taken together, these analyses demonstrate that elevated numbers of virus-specific VIL can be rapidly expanded in 7-days by VIPR particle culture and form robust activated, polyfunctional effector memory T cells. Given the paucity of therapeutic options for the treatment of COVID and the provocative data suggesting the importance of the immune T cell response to viral infections, we investigate a novel potential therapeutic modality to augment the anti-viral T cell response by providing a therapeutic immune boost of virus-specific T cells VIL.
Unlike T cell immuno-oncology in which T cell expansion and subsequent efficacy requires substantial critical mass of quantity, and thus time, VIL serve a catalytic immune booster function and can be isolated and expanded ex vivo both autologously and also MHC typed for allogeneic delivery in a 7-day vein-to-vein time which is clinically practical and relevant as depicted in Fig.
These antigen-specific VIL expand at an average of fold prior to adoptive transfer back to HLA-matched patients to mediate a T cell immune response to support the eradication of the SARS-CoV-2 virus and to engender protective immunity against repeat infection. In the setting of COVID pathogenesis, studies have found individuals suffering from a more severe presentation of the disease typically require a duration of hospitalization ranging from 5 to 29 days 33 , Developments in Rapid Expansion Protocols REP for T cell and TIL expansion in the setting of clinical oncology have enabled methods for the robust and exponential ex vivo expansion of unenriched, as well as neoantigen-enriched, T cells for the autologous treatment of solid tumors 35 , 36 , 37 , In fact, large quantities of antigen-specific TIL can be expanded in just 22 days using well established protocols This study demonstrates the antigen-specific expansion of SARS-CoV-2 T cells from convalescent COVID patients to demonstrate applicability for this viral pathogen, yet one important consideration is that these individuals have very low numbers of recirculating SARS-CoV-2 specific T cells in their blood due to the timeframe since their infection and recovery Fig.
Hospitalized, symptomatic COVID patients with a severe form of the disease will be undergoing a significant cellular immune response whereby the numbers of virus-specific T cells may have expanded, even if overall T cell numbers may be reduced in some individuals While this T cell response needs therapeutically boosting to potentiate viral clearance and ensure positive disease outcome, a potentially more robust VIL expansion may be achievable when T cells are acquired from suffering COVID patients as opposed to recovered convalescent individuals, although this would need to be empirically assessed in the clinic.
When considering the translation of this platform into the clinic as a potential cell therapy for hospitalized COVID patients, based on the level of enrichment and expansion demonstrated in this study, and a prediction of the number of SARS-COV-2 cells in the blood of COVID individuals, we calculate an estimated capability to expand and deliver an average of approximately 3.
The expansion of VIL in G-REX plates and flasks also improves overall T cell proliferation by supporting more effective gaseous exchange and nutrient availability, further adding to the rapidity by which significant numbers of virus-specific VIL can be generated Fig. While studies have also shown that T cells can indeed be expanded, form memory, and protect form viral infections in animal models in the absence of co-stimulation 43 , 44 , it was found that very high levels of TCR stimulation were needed to overcome the need for co-stimulation 45 , Thus, if a strong, shared, immunodominant and well validated peptide antigen is known and available for a viral pathogen, large numbers of VIL could potentially be expanded ex vivo by soluble peptide in the absence of an aAPC or co-stimulation.
However, this situation is clinically irrelevant for most pathogens whereby multiple immunogenic epitopes typically exist and the comparative affinity for their cognate TCRs may not be known as in an emerging pandemic. Thus, VIL expansion platforms for newly emerged pathogens, as well as seasonal variants of existing viruses, will benefit from aAPC-mediated approaches, where the presence of CD28 co-stimulation should lower the T cell activation threshold and provide robust co-stimulation to ensure strong proliferation and effector memory T cell formation.
Dendritic cells are typically generated from autologous monocytes through a series of ex vivo maturation steps with different cytokines which includes approximately 6 days of culture in IL-4 and GM-CSF to generate immature DCs before further maturation Therefore, we believe the VIPR platform based on artificial APCs offers a clinically meaningful and practical alternative to this approach.
When the optimized VIPR particles were combined with other elements of the protocol, including high cytokine concentrations and G-REX flasks, we thus observed not only a significant expansion in virus-specific VIL cell numbers Fig. This was crucial to establish a pool of virus-reactive T cells that have the potential to not only mount a primary immune response against the virus when adoptively transferred to the patient, but may also engender immunological memory to ensure the duration of the response is long lasting.
This theory remains to be tested and will require in vivo assessment in the setting of a viral infection. Thus, the expanding virus-specific VIL that can subsequently be transferred back to the patient are primed to mount a functional cellular response in the context of cytokine release and recruitment of other immune cells.
Thus, this technology can be used to expand both T Helper and Cytolytic virus-specific VIL to provide a modality to tune a specific cell therapy product to treat different viral diseases. The enrichment and expansion of CMV-specific T cells demonstrates the flexibility of the VIPR particle system for addressing cell therapies for diverse pathogens where immunogenic epitopes are known. VIPR particles are off-the-shelf reagents that can be rapidly utilized for ex vivo T cell expansion in the clinic without any additional manufacturing lead time.
We expect that this technology would provide a modality for expansion of all clinically relevant virally-mediated diseases where an immune boosting dose of virus-specific T cells would aid in viral clearance and disease outcome. Furthermore, due to the immune catalytic nature of T cells and their ability to expand further in vivo and recruit additional cells of the immune system, we believe that an efficacious therapeutic dose of virus-specific T is likely to be well within the numbers achievable within short durations of expansion.
In instances whereby such a short duration of T cell expansion is not as critical as required in diseases like COVID, this technology can also enable the expansion and cryopreservation of virus-specific VIL. One drawback of this rapid antigen-specific T cell expansion platform is the requirement for the validated immunodominant epitope of the viral pathogen to be known and available to generate the peptide conjugated VIPR particle.
For an emerging pathogen, such as SARS-CoV-2, this will thus involve the scientific community to first study the pathogen and its disease and identify the antigenic peptide sequence and MHC restriction. While this process was effective in rapidly identifying robust SARS-CoV-2 epitopes in the early phase of the global pandemic, this may not always be as rapid or successful for other viral pathogens as they emerge.
This could also be a concern for pandemics like Covid whereby additional variants emerge via rapid mutation and lead to subsequent waves of infection that could become established before the dominant epitopes have been identified and reagents made available.
Additionally, the collection, isolation and ex vivo expansion of patient T cells requires suitable clinical facilities and expertise that adds to the complexity of delivering such a personalized T cell therapy and may limit the utility of this approach in hard-to-reach or resource limited areas and communities.
To be broadly applicable as a pragmatic modality that is scalable and addresses some of these limitations on cost and accessibility, we have MHC typed donor pools of SARS-CoVspecific VIL for use as an allogeneic off-the-shelf therapy at scale. In this setting, these T cells are tissue matched to COVID patients and provided as an allogeneic cell therapy product to combat an active infection.
Furthermore, cryopreservation of other VIL, such as MHC -typed CMV-specific VIL, could, as an example, be used to immunize bone marrow transplant recipients or other immunocompromised individuals against adventitious pathogens In summary we demonstrate a novel technology platform for the robust and rapid expansion of virus-specific T cells as a potential cell therapy for COVID and other viral pathogens. Heretofore, the promising antigen-specific therapeutic approaches to date, including polyclonal antibody cocktails and monoclonal antibodies, and current prophylactic vaccine approaches to COVID, all have been focused on neutralizing antibodies.
However, complete immune protection is likely also a function of the long-lasting central memory T cell response to provide both cellular immunity, and potentiate humoral immunity, and thus prolong the duration of protection 8 , Validated therapies are few and largely supportive or non-specific, such as the use of dexamethasone to delay mechanical ventilation in COVID induced pulmonary failure.
Thus, there is an urgent need for specificity and efficacy in the clinic to mitigate disease progression and severity.
Methods were approved by Harvard University and informed consent was obtained from all subjects providing PBMC samples. Purity and viability of isolated T cells was assessed using flow cytometry prior to cryopreservation of isolated T cells in CryoStore CS10 cryopreservation media Stem Cell Technologies at a density of 1—1. In addition, a sample of the T cells was also analyzed by flow cytometry at Day-0 to measure the starting proportion of antigen-specific T cells see methods below.
For some cultures the media was replaced every 2—3 days with fresh complete media including cytokines and NAC but no extra addition of VIPR particles and media in G-REX cultures was left unchanged for the duration of 7-days in some experiments to promote cell expansion.
The proportion of expanded antigen-specific T cells was assessed at Day-7 by flow cytometry. After 1-h of peptide stimulation, GolgiStop solution was added containing Monensin protein transport inhibitor to block intracellular protein transport BD Bioscience. Cells were then stained for surface markers followed by intracellular cytokines using antibodies specific for IFN-y 4S.
Flow analysis was carried out on a Fortessa flow cytometer BD Bioscience and data analyzed and flow cytometry figures generated using FlowJo 10 software BD Biosciences. All graphs were generated using Prism Software version 8 GraphPad. P values are included in the figures where statistical analyses have been carried out.
Intima Bioscience has patents filed based on the findings described herein. Stevanovic, S. Complete regression of metastatic cervical cancer after treatment with human papillomavirus-targeted tumor-infiltrating T cells. Tran, E. Science , — Zacharakis, N. Table 2 displays these findings. Thumbnail Table 1 Median and mean for T subpopulations cells. T lymphocytes values according to lifestyle: We didn't find any difference for the T lymphocytes absolute values when stratifying individuals by alcohol intake and stress level.
Lymphocyte values and geographical origin: We observed a differential pattern for absolute lymphocytes count according to the city of origin of blood donors. Table 4 summarizes the values found for these T-cells subsets count. The comparison of T lymphocytes in healthy and blood donors from distinct regions of Brazil, demonstrate that there are significant differences in these parameters, depending on the cell subsets evaluated, and the geographical origin of individuals.
In the present work we compared these parameters for blood donors from cities of other different regions, and included children in two of them 8 8. The immune response usually is modulated according to different factors, like nutritional aspects, ethnicity, age, gender and others 11 Lymphocyte subsets in human immunodeficiency virus type 1-infected and uninfected children in Nairobi.
Pediatr Infect Dis J. This may impact some specific aspects of the response against infections, for instance 5 5. T-lymphocyte subsets in West African children: impact of age, sex, and season.
J Pediatr. In addition, some available evidence suggest that nutritional factors like the amount of 3-omega, arginin, glutamine, proteins supplement, and nucleic acids in the food may help the immune system to produce a higher level of cytokines and other important factors for cell function 4 4.
Nutritional supplementation may enhance immune functions even for healthy individuals 22 Hematological parameters and T lymphocytes subsets are presented differently in literature, according to geographical area, economic level, age and nutritional status. Lower platelets and leukocytes values were reported in some studies realized with blood donors from Ethiopia, in comparison with standards from other regions 12 European Collaborative Study.
Are there gender and race differences in cellular immunity patterns over age in infected and uninfected children born to HIV-infected women? J Acquir Immune Defic Syndr. In Italy, CD3 T absolute values showed a variation 3. Lysine fortification of wheat flour improves selected indices of the nutritional status of predominantly cereal-eating families in Pakistan. Food Nutr Bull. Age- and sex-related changes in lymphocyte subpopulations of healthy Asian subjects: from birth to adulthood.
Although these variations are not wide, they can be significant when we use such parameters for taking clinical decisions. Regarding reference levels for children, we found a wide variation in lymphocytes count and its subsets in this population. These findings are in conformity with the previous reports on hematological parameters in children 1 1.
This is in agreement with some studies in Asian and European children 15 Low platelet counts in Zambians. Reference values for peripheral blood lymphocyte phenotypes applicable to the healthy adult population in Switzerland. Eur J Haematol. Chandra RK. Nutrition and immunity: lessons from the past and new insights into the future. The reasons for this disparity are not clear but both genetic and environmental factors may play a role 31 Telles E. The Brazilian population has a wide diversity per geographic region with unique nutritional, ethnics, and behavior characteristics.
For instance, the African heritage in Salvador is very strong, and some costumes were well preserved, like dietary, music, and others. Most of Salvador's population J Trop Pediatr. The South and North regions have less African influence, but a strong European and Amerindian presence, respectively 17 Brasil: anos de povoamento. Brasilia: IBGE; Most of the Indian population are localized at North and Mid-West regions from Brazil, while in South and Southeast predominates the European influence.
These findings can be associated with the different results found by T lymphocytes subsets among Brazilian cities in our study, and reinforce the importance of defining the regional variations for these parameters 24 Niger Postgrad Med J. Others variables can also influence the immune response. Immunization introduces antigens or weakened pathogens to a person in such a way that the individual does not become sick but still produces antibodies. Because the body saves copies of the antibodies, it is protected if the threat should reappear later in life.
Because the immune system is so complex, there are many potential ways in which it can go wrong. Types of immune disorder fall into three categories:. These arise when one or more parts of the immune system do not function. Immunodeficiencies can be caused in a number of ways, including age, obesity , and alcoholism.
In developing countries, malnutrition is a common cause. AIDS is an example of an acquired immunodeficiency. In some cases, immunodeficiencies can be inherited, for instance, in chronic granulomatous disease where phagocytes do not function properly. In autoimmune conditions, the immune system mistakenly targets healthy cells, rather than foreign pathogens or faulty cells. In this scenario, they cannot distinguish self from non-self. With hypersensitivity, the immune system overreacts in a way that damages healthy tissue.
An example is anaphylactic shock where the body responds to an allergen so strongly that it can be life-threatening. The immune system is incredibly complicated and utterly vital for our survival. Several different systems and cell types work in perfect synchrony most of the time throughout the body to fight off pathogens and clear up dead cells.
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Researchers shed light on how dark chocolate can offer benefits for brain health and…. How the immune system works. Medically reviewed by Daniel Murrell, M. White blood cells The immune response Immunity Immune system disorders Our immune system is essential for our survival. White blood cells. Share on Pinterest A white blood cell yellow , attacking anthrax bacteria orange. The white line at the bottom is 5 micrometers long. Image credit: Volker Brinkmann. How an immune response works.
Share on Pinterest B lymphocytes secrete antibodies pictured that lock onto antigens.
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