Regulation of T Cell Activation in Immunotherapy

Introduction

Immune checkpoint blockade attenuates T cell activation by removing inhibitory signals to enable tumor-reactive T cells to overcome regulatory mechanisms and mount an effective antitumor response. These regulatory mechanisms are normally utilized to protect the host from autoimmunity, however, malignant tumors can take advantage of these immune suppressive and tolerance mechanisms to avoid immune clearance1. The development of immune checkpoint blockade therapy relies on previously performed basic research that established key regulatory mechanisms of T cell activation. This review discusses the regulatory mechanisms of two popular checkpoint blockade targets, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1), and how their potential to induce tumor rejection. The clinical progress of immune checkpoint blockade therapy is also outlined along with future checkpoint inhibitory targets and combination treatment options.

CTLA-4 Negative Costimulation Mechanics

CTLA-4 is a protein receptor that is constitutively expressed on regulatory T cells and also becomes upregulated in conventional T cells upon T cell receptor (TCR) engagement to peptide:MHC (pMHC) complexes2,3. CTLA-4 expression acts as a negative costimulation for T cell activation both intrinsically and extrinsically. Intrinsically, the upregulated CTLA-4 on activated T cells act as a competitive inhibitor with CD28 for B7-1 and B7-2 ligands expressed on antigen presenting cells (APCs) as shown in Figure 1. CTLA-4 has a higher avidity and affinity for the B7 ligands to outcompete CD28 binding, thus attenuating T cell activation4,5.

CD28 binding with B7-1 and B7-2 provide positive costimulation for T cell activation. Without CD28 costimulation, an activated T cell with only TCR and pMHC binding results in an anergic T cell. The lack of costimulation also limits all of the CD28 downstream signaling including PI3K and AKT, which are responsible for certain transcription factor activation, cytoskeletal reorganization, and cell survival signals6. Inhibiting CD28 will also decrease IL-2 production, which is responsible for cell proliferation and survival7. Another source of CTLA-4 due to T cell activation comes from rapidly trafficking intracellular vesicles to the immunologic synapse.

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At the immunologic synapse, the CTLA-4 will outcompete CD28 binding to B7 ligands in the central supramolecular activation cluster (cSMAC)8. CTLA-4 inhibition primarily occurs at sites of T cell priming, such as lymph nodes and the spleen, but it can also attenuate T cell activation in peripheral tissues because B7 ligands are constitutively expressed by APCs. This negative costimulation of CTLA-4 is critical for normal immunologic homeostasis, as proven by an experiment where genetic deletion of Ctla4 led to a massive lymphoproliferation9,10.

Extrinsically, CTLA-4 suppression is mediated through T regulator cells (Tregs)11. CTLA-4 derived by Tregs has been shown to be essential for immunologic tolerance because loss of CTLA-4 in Tregs will induce irregular T cell activation and give rise to autoimmunity12. Treg derived CTLA-4 contributes extrinsically by limiting the availability of B7 ligands through competing in trans or through transendocytosis of the B7 ligands [image: ]from APCs13,14. However, the level of contribution to T cell tolerance of these extrinsic pathways is still unknown, particularly in context of tumor immunity. The Treg transcription factor, Foxp3, promotes expression of CTLA-4 and inhibits expression of IL-2.

Tregs rely heavily on CTLA-4 to elicit regulatory function, but not exclusively15. Recent studies have shown that despite genetic loss of CTLA-4 in Tregs during adulthood led to resistance from experimental autoimmune encephalomyelitis (EAE)16. This study shows potential implications that a combination of Treg depletion along with CTLA-4 blockade may lead to enhanced efficacy of anti-CTLA-4 therapy. Figure 1. Schematic representation of mechanisms behind attenuation of T cell activation by CTLA-4 and PD-1. Some known downstream signaling of different aspects of T cell activation is noted along with potential downstream mechanisms of both coinhibitory molecules27.

PD-1 Mediated Attenuation of T Cell Activity Mechanics

As opposed to CTLA-4 which primarily acts at sites of T cell priming, PD-1-mediated attenuation primarily occurs in inflamed peripheral tissue. That is, PD-1 predominantly regulates effector T cell activity within tissue and tumors, whereas CTLA-4 predominantly regulates T cell activation17. Similar to CTLA-4, PD-1 is highly expressed on Tregs where its presence may enhance their proliferation. However, PD-1 is also more broadly expressed on B cells and natural killer (NK) cells, suggesting that anti-PD-1 therapy may also enhance NK cell activity and antibody production18,19.

PD-1 is expressed upon activation of T and B lymphocytes and interact with widely expressed PD-L1 and PD-L2. Both PD-L1 and PD-L2 are also part of the B7 family and studies have shown that they might not bind exclusively to PD-1 and could bind to CD80 or other costimulatory receptors20. PD-L1 expression is induced in response to inflammatory cytokines such as IFNγ, indicating that PD-1 regulation occurs in response to CD8 or TH1 CD4 T cells. PD-L2 is predominantly expressed on APCs, whereas PD-L1 can be expressed on a variety of cell types including cells in the immune system, epithelial cells and endothelial cells21.

Recent studies have shown that PD-1 can directly target the TCR or CD28 to induce attenuation of T cell signaling. PD-1 is thought to provide negative costimulation by dephosphorylating proximal signaling elements through tyrosine phosphatase SHP222 as shown in Figure 1. Similar to CTLA-4, the dephosphorylation of kinases due to PD-1 ligation will affect downstream signaling of TCR and CD28 including PI3k, AKT, RAS, ERK, VAV, and PLCγ leading to decreased T cell activation, proliferation, survival, cytokine production, and metabolism. PD-1 ligation also induces metabolic restriction by attenuating glycolysis and promoting fatty-acid oxidation and lipid catabolism23.

PD-1 is essential for homeostasis in peripheral tolerance as evidenced by autoimmune pathologies upon genetic deletion of Pdcd124. However, a recent study has shown that SHP2 is not essential for T cell exhaustion or anti-PD-1 therapy response25. T cell exhaustion is a state of T cell dysfunction defined by poor effector function, sustained expression of inhibitory receptors, and a transcriptional state distinct from that of functional effector or memory T cells. T cell exhaustion is caused by chronic antigen stimulation due to conditions such as chronic viral infection or cancer leading to high levels of persistent PD-1 expression26. Currently, PD-1 is used often as a marker for T cell exhaustion, but other coinhibitory molecules are expressed including LAG3 and TIM3.

CTLA-4 and PD-1/PD-L1 Blockade Impact on Tumor Rejection

The contrast of function between CTLA-4 and PD-1 acting primarily at T cell priming and periphery tissue respectively reflect their different mechanisms of inducing tumor rejection. CTLA-4 inhibits through steric inhibition of B7 interactions, however, tumor cells do not express B7 ligands. Thus, CTLA-4 inhibition occurs mostly in tumor-draining lymph nodes where tumor antigens can be cross-presented by APCs to prime tumor targeting T cells28. CTLA-4 blockade results in a specific expansion of tumor neoantigen-specific CD8 T cells and a PD-1+ICOS+TBET+ TH1-like CD4 effector T cell in the tumor microenvironment (TME)29. This unique population of TH1 T cells suggest that anti-CTLA-4 may also affect T cell differentiation. Another impact of anti-CTLA-4 therapy is the depletion of Treg populations in intratumoral populations, but not peripheral populations. Both of these mechanisms indicate that effective tumor rejection will be most effective through the blockade of both effector and regulatory T cell CTLA-430.

On the other hand, PD-1 blockade is able to induce tumor rejection through restoration of activity in some exhausted CD8 effectors depending on a threshold level of exhaustion. A recent study has shown that some CXCR5+PD-1+CD8 T cells are responsible for immediate proliferative expansion after PD-1 blockade31. Furthermore, we know that CD4 T cells are required for effective responses, but specific are still unclear. Some speculate that CD4 helper T cells help CD8 T cells and antibodies enter into the peripheral tissue sites32. Evidence has also shown that anti-PD-1 and anti-PD-L1 therapy are not mechanistically equivalent because anti-PD-L1 therapy may also derive some of its efficacy from ADCC or TH1 skewed responses against tumors33.

Clinical Progress of Checkpoint Blockade Therapy

The first clinical tests using checkpoint blockades started in 2000 with two fully humanized CTLA-4 antibodies, ipilimumab (Bristol-Myers Squibb, Princeton, NJ) and tremelimumab (MedImmune/AstraZeneca, Wilmington, DE), for the treatment of metastatic melanoma34. Both antibodies produced durable clinical responses in some patients, but also exhibited some immune-related toxicities in various tissue sites. Unfortunately, phase III for tremelimumab did not show statically significant improvement in overall survival for patients with advanced melanoma.

Ipilimumab, however, performed significantly better by improving overall survival in two phase III studies. The mean survival benefit was increased by 3.5 months and survival rate after 2 years was 18% compared to 5% of the control group35. Ipilimumab was a fully human IgG1 antibody and tremelimumab was a fully human IgG2 antibody. This may have been a primary reason for the success of ipilimumab over tremelimumab because antibody-dependent cellular cytotoxicity (ADCC) – mediated killing of Tregs is more effectively mediated by IgG136. This success in preclinical trials led to an FDA approval of ipilimumab in 2011. Table 1. Summary of the FDA approved immune checkpoint blockade therapeutic agents, target coinhibitory molecules, target tumor type, and FDA approval year37.

Since then, 6 additional checkpoint blockade therapies targeting PD-1/PD-L1 axis have been approved for the treatment of various tumor types. These include Nivolumab (Bristol-Myers Squibb, Princeton, NJ), Pembrolizumab (Merck, Whitehouse Station, NJ), Atezolizumab (Genentech, San Francisco, CA), Avelumab (Merck and Pfizer), Durvalumab (MedImmune/AstraZeneca, Wilmington, DE), and Cemiplimab (Regeneron, Tarrytown, NY). Nivolumab, pembrolizumab, and cemiplimab are anti-PD-1 antibodies and atezolizumab, avelumab, and durvalumab are anti-PD-L1 antibodies.

Furthermore, these antibody treatments have expanded beyond treatment of metastatic melanoma to a variety of tumor types including non-small cell lung cancer (lung), renal cell carcinoma (kidney), urothelial carcinoma (bladder), Hodgkin lymphoma (lymphocyte), merkel cell carcinoma (skin), and cutaneous squamous cell carcinoma (skin)37 based on Table 1. The list of checkpoint blockade therapy antibodies in preclinical or clinical trials is constantly expanding to other types of cancers and other coinhibitory/costimulatory molecules such as LAG3, TIM3, TIGIT, VISTA, ICOS, OX40, CD40, GITR, 4-1BB, and CD27.

LAG3 is furthest along in clinical development and has shown promising results for patients with renal cell carcinoma27. However, all of these immune checkpoint inhibitor treatments share a common concern that a reinvigorated immune system could attack some healthy organs in the body leading to some serious side effects. These side effects include fatigue, nausea, skin rash, and more serious ones such as inflammation of the lung, intestines, kidney, heart, or neurologic problems. Some of these side effects can be managed using immunosuppressive agents such as corticosteroids, which do not appear to interfere with the clinical benefits of checkpoint blockade therapy38.

Combination Treatment

Monotherapies have shown remarkable progress in cancer treatment, but there still remains a large margin of improvement that needs to be achieved in treatment efficacy across various tumor types. A better understanding of the responses in the TME to the immune checkpoint blockade therapy will lead to more effective treatments. Studies have shown that tumor heterogeneity will often upregulate a separate inhibitor molecule in response to the therapy, thus limiting the therapeutic efficacy of monotherapy approaches. For example, an increased expression of PD-L1 has been found in many anti-CTLA-4 patients.

For this reason, many researchers are looking into combination treatments for higher therapeutic efficacy39. In 2015, FDA approved a combination treatment of anti-CTLA-4 and anti-PD-1 for melanoma that showed a 57% 3 year overall survival and produced a complete response in 36% of patients. Predictions indicate that over half of the patients treated with combination therapy will achieve long term responses lasting 10 years or more40. However, before we start combining treatments together, a lot of the mechanisms behind the enhanced efficacy of combination therapy is still unclear.

Conclusion

This review has covered our current understanding of the biological mechanisms behind two important coinhibitory molecules, CTLA-4 and PD-1. Both of these molecules have been shown clinically to be able to induce tumor rejection in a variety of cancer types. However, much more research on these coinhibitory molecules is necessary before we have a thorough understanding of all the aspects regarding attenuation of T cell activation the role they play to induce tumor regression separately and in combination. This deeper level of mechanistic insight is essential for continued improvement and development of immunotherapeutic strategies. Immunotherapy has brought new light to cancer treatment and this review highlights some of the reasons for researchers showing excitement for the field.

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