June 27, 2022

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Immunotherapy: What’s Treg source and mechanism?

Immunotherapy: What’s Treg source and mechanism? CAR-Treg is Treg’s Future?

 

Immunotherapy: What’s Treg source and mechanism? Regulatory T cells (Treg) are a small part of immune cells that are specifically used to suppress excessive immune activation and maintain immune homeostasis.

In the past two decades, the development of chimeric antigen receptors (CAR) and advances in genome editing have promoted the optimization of T cell therapy. These technologies are now being used to enhance the specificity and functionality of Treg cells. Basic autoimmune and transplantation treatments are being developed rapidly.

Immunotherapy: What's Treg source and mechanism?
Source Reference 2

 



Treg source and mechanism


Regulatory T cells are a subset of T cells, which have the function of maintaining homeostasis and preventing autoimmunity.

Tregs account for 5-10% of the total number of CD4+ T cells, and are characterized by the co-expression of CD4, CD25, FOXP3 and low-level CD127. High levels of FOXP3 and demethylation of the specific demethylation region (TSDR) are notable features of Treg, and TSDR is a conserved region in the FOXP3 gene.

Tregs are divided into thymus-derived Tregs (tTregs) and peripheral-derived Tregs (pTregs). During the development of T cells, primitive CD4+ T cells receiving intermediate TCR signals are driven to differentiate into Treg cells. The difference in signal intensity determines whether primitive T cells differentiate into regular T cells or regulatory T cells.

In addition, the conventional T cells of FOXP3- will also be transformed into Treg cells when they are repeatedly stimulated by non-self antigens or exposed to IL-10 and TGF-β.

Treg suppresses the immune system through different mechanisms, including contact-dependent mechanisms, such as through CTLA-4, and non-contact-dependent mechanisms, such as the release of cytokines, such as IL-35 or IL-10. In view of the role of Tregs in preventing autoimmune diseases, Tregs have obvious potential and good application prospects in promoting immune tolerance.

 



Adoptive Treg-from polyclonal to antigen specificity


The initial clinical trials of adoptive Treg mainly used polyclonal or in vitro expanded Tregs. Although polyclonal Tregs have achieved a certain degree of encouraging results, the number of cells required for infusion is quite large. In addition, there is a risk of non-specific immunosuppression. In fact, the virus reactivation after infusion of polyclonal Tregs has been There are reports.

These shortcomings can be overcome by using antigen-specific Tregs. Compared with polyclonal Tregs, antigen-specific Tregs require fewer cells to perform more local and targeted inhibition. In addition, many studies have proved that in animal models, Tregs specific for the desired antigen are functionally superior to polyclonal or unmodified Tregs.

The traditional method of generating antigen-specific Treg mainly relies on the use of APCs and specific antigen amplification, or the use of T cell receptors (TCRs) engineered Treg. However, amplification of Treg with APCs is inefficient, and Tregs constructed with TCR (TCR-Tregs) are still limited by MHC, which limits the modular application for different patients.



 

Engineering CAR-Treg


Another way to give Tregs specificity is to transduce these cells with chimeric antigen receptors (CAR). Compared with TCR-Treg, CAR has some unique advantages: these CARs expressing T cells bypass HLA restriction when activated, the activation of co-receptor signals increases specificity, and the targeting flexibility of CARs (any soluble or Surface multivalent antigens can be used as targets).

In 2009, the first study using CAR to redirect human Treg cells was conducted. In immunodeficiency mouse models, human CEA-CAR-Treg cell-mediated inhibition was observed.

The most direct application of CAR-Treg cells is GvHD and organ transplant rejection. Unlike most autoimmune diseases, there is a very clear target in transplantation, namely HLA molecules.

In 2016, HLA-A2 CAR Treg cells were reported for the first time. Studies have shown that HLA-A2-CAR-Treg cells inhibit the proliferation of Teff cells and prevent HLA-A2+PBMC-mediated GvHD in an immunodeficient NSG mouse model.

Immunotherapy: What's Treg source and mechanism?
Source Reference 3

New applications of CAR-Treg cells are still emerging, and B cell targeting antibody receptor (BAR) Treg cells has been a hot research topic in recent years.

Human factor VIII (FVIII) injection is used to treat patients with hemophilia A, and over time, the development of anti-FVIII neutralizing antibodies will lead to an increase in morbidity and mortality. The BAR containing the FVIII immunodominant domain (A2 or C2) as the extracellular domain is designed to target FVIII-specific B cells, and the intracellular signal domain is still CD28-CD3ζ. Strikingly, in vitro, human BAR-Treg cells displaying A2 domain or C2 domain inhibited the production of anti-FVIII antibodies in mice immunized with recombinant FVIII.

 

Optimization of engineered CAR-Treg

At present, most studies on preclinical disease models focus on monospecific CAR-Treg. Increasing the specificity of CAR-Treg can improve its therapeutic effect, and at the same time indirectly exert the functional advantages of Treg through bystander inhibition.

The first option is the combination of CAR-Treg. This method has been tested on CAR-T, including the combination of CD19 and CD123 for B-ALL, and the combination of HER2 and IL-13Rα2 for glioblastoma. However, this is logically challenging because the expansion of autologous CAT-Tregs for different target antigens will be limited by the number of available autologous Tregs and the number of highly expressed target antigens.

 

 

Therefore, using CAR-T cells transfected with two different specific antigens and signal domains, dual CAR-T cells have been developed. Dual CAR-T cells are more effective than CAR-T cell combination to prevent the escape of antigens and show improved anti-tumor effects. In addition, bispecific CARs for two different antigens can also be used.

Immunotherapy: What's Treg source and mechanism?

The development of modular or universal CAR (UniCAR) is another strategy. This strategy can be customized to control Treg activities, because the activation of general CAR-Tregs strictly depends on the target module, and the general CAR is open to the target module.

Immunotherapy: What's Treg source and mechanism?

 

 

Next-generation engineered Treg

Synthetic biology

The development of Treg cells as drugs for the treatment of autoimmune diseases is not limited to the application of TCRs and CARs. Synthetic immunology has produced many artificial receptors and systems, which are being tested in Treg cells.

These systems include T cell antigen coupling agents that recruit endogenous TCR complexes to non-MHC targets through linked single-chain antibodies, CARs that can be bound and activated by soluble ligands, and separable, universal, and programmable CAR (SUPRA).

Utilize cytokines

There is no doubt that cytokines play a key role in the immune response. Treg cells constitutively express the high-affinity chain CD25 of IL-2 receptor, effectively depriving Teff cells of IL-2. In addition, through the transformation of engineered CAR-Treg cells, pro-inflammatory cytokine signals can be converted into IL-2 or IL-10 signals to increase the inhibition of inflammation.

Gene editing

Some preclinical studies using CRISPR-Cas9 to edit human T cell genes have been published. These include knocking out the CCR5 gene in CD4+ T cells to produce T cells resistant to HIV infection; knocking out the CD7 gene in CD7-CAR T cells , Because T cells express CD7, thereby preventing cannibalism; and knocking out the PD1 gene in CD19-CAR T cells, thereby improving the tumor clearance rate of the humanized mouse model.

Improve the delivery system

Currently, the manufacture of CAR T cells uses retrovirus and lentiviral transduction to deliver and integrate genetic material into T cells. However, these methods are time-consuming, expensive, and suffer from safety issues.

 

Some non-viral delivery methods are under development, such as CRISPR-RNPs co-electrotransformation, which can knock more than 1 kb of DNA into specific genomic sites of human T cells. This method is simple and safe, and double-stranded DNA templates are non-toxic. Using this method to correct the pathogenic CD25 mutations in the cells of patients with single-gene autoimmune diseases has proved its potential application value, but this method is also limited by the size of the insert.

Design Treg cells

One obstacle to Treg cell therapy is to ensure the survival of cells after infusion. Using gene editing to knock out the JNK1 gene in Treg cells can make them resistant to apoptosis. JNK1-deficient Treg cells secrete higher levels of IL-10 and TGFβ. It can protect the transplanted islets from rejection by 100 days longer than the control.

In addition, Treg cells may become unstable in an inflammatory environment and transform into pathogenic TH17 cells. By knocking out the PRKCQ gene, the tendency of Treg cells to differentiate into TH17 cells can be reduced while maintaining the inhibitory function of Treg cells.

Finally, obtaining the latest generation of low immunogenic human pluripotent stem cells through gene editing, coupled with continuous efforts to differentiate stem cells into Treg cells, may completely change the means of engineered Treg cell therapy.

 

 

Summary

At present, the use of Treg cells to design and treat autoimmune diseases is in the ascendant. Engineered CAR-Treg shows unique advantages in this regard and has great potential. However, there are still many problems in the field of Treg cell biology and Treg cell therapy.

In the next ten years, as these unresolved issues are resolved, we look forward to seeing continuous improvements in Treg cell manufacturing, as well as the wide application of synthetic biology and gene editing, which will make Treg cell therapy more and more important The role of.

 

 

(source:internet, reference only)


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