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Soluble Factors and Immune Checkpoint in STS

Soluble Factors: Cytokines, Chemokines and Growth Factors

Cytokines, chemokines are different types of proteins that are made by certain immune and non-immune cells and have various effect on the immune system.

Some cytokines stimulate the immune system and others slow it down. Examples of cytokines are interleukins (such as IL-6, IL-8 and IL-10), interferons, and colony-stimulating factors.

Chemokines stimulate the movement of certain types of white blood cells and attract them to areas of inflammation. They also help keep the immune system working the way it should.

Growth factor is a substance made by the body that functions to regulate cell division and cell survival. For example, Vascular endothelial growth factor (VEGF) is a substance made by cells that stimulates new blood vessel formation.

The network of pro- and anti-inflammatory cytokines, chemokines and growth factors orchestrates the immune cell signaling and function and, as such, largely contributes to the complexity of the tumor micro-environment (TME). They have been studied in a broad range of tumors, and their involvement in cancer development, progression, and recurrence has been suggested. Moreover, their profile might be a prognostic factor for clinical outcome.

Soluble Factors in STS

Preliminary studies have found an elevated serum level of some cytokines, growth factors, and immune-related soluble receptors in patients with STS. Higher serum level of VEGF has been reported. It promotes angiogenesis, facilitating the tumor’s growth and increased metastatic spread. Furthermore, VEGF also promotes the proliferation of immunosuppressive cells and T cell exhaustion, contributing largely to immune escape and cancer development. In addition, increased serum levels of interleukin 6 (IL-6), receptors for TNF (TNF-RI and TNF-RII), interleukin 2 receptor α (IL-2Rα), interleukin 10 (IL-10), macrophage-colony stimulating factor (M-CSF), and interleukin-8 (IL-8) were also found in STS patients.

Rutkowski et al. analyzed the serum levels of 13 cytokines and soluble receptors in STS patients before treatment. The results confirmed the elevated levels of VEGF, FGF, IL-6, TNF RI, TNF RII, IL-2Rα, IL-10, M-CSF, and IL-8 stated above. Furthermore, they tried to correlate the serum levels of these cytokines with clinic-pathological features. IL-2Rα, TNF RI, M-CSF, and VEGF correlated with tumor size, IL-8 was associated with tumor grade, and IL-6 appeared to be correlated with tumor size, grade, and metastases. Additionally, it was proved that IL-6 and IL-8 were correlated with decreased survival.

Immune checkpoint

Immune checkpoints are regulators of the immune system. These pathways are crucial for self-protection against infections or cancers, as well as self-tolerance, which prevents the immune system from attacking cells indiscriminately.

• Stimulatory checkpoint molecules

At least five stimulatory checkpoint molecules are members of the tumor necrosis factor receptor superfamily (TNFRSF) —CD27, CD40, OX40, GITR and CD137. Some other stimulatory checkpoint molecules belong to the B7-CD28 superfamily—CD28 itself and ICOS.

CD27: This molecule supports antigen-specific expansion of naïve T cells and is vital for the generation of T cell memory. CD27 is also a memory marker of B cells. CD27's activity is governed by the transient availability of its ligand, CD70, on lymphocytes and dendritic cells.

CD28: This molecule is constitutively expressed on almost all human CD4+ T cells and on around half of all CD8 T cells. Binding with its two ligands are CD80 and CD86, expressed on dendritic cells, prompts T cell expansion.

CD40: This molecule, found on a variety of immune system cells including antigen presenting cells has CD40L, otherwise known as CD154 and transiently expressed on the surface of activated CD4+ T cells, as its ligand. CD40 signaling is known to ‘license’ dendritic cells to mature and thereby trigger T-cell activation and differentiation.

CD137: When this molecule, also called 4-1BB, is bound by CD137 ligand, the result is T-cell proliferation. CD137-mediated signaling is also known to protect T cells, and in particular, CD8+ T cells from activation-induced cell death.

OX40: This molecule, also called CD134, has OX40L, or CD252, as its ligand. Like CD27, OX40 promotes the expansion of effector and memory T cells, however it is also noted for its ability to suppress the differentiation and activity of T-regulatory cells, and also for its regulation of cytokine production.

GITR: short for Glucocorticoid-Induced TNFR family Related gene, prompts T cell expansion, including Treg expansion. The ligand for GITR is mainly expressed on antigen presenting cells.

ICOS: This molecule, short for Inducible T-cell costimulator, and also called CD278, is expressed on activated T cells. Its ligand is ICOSL, expressed mainly on B cells and dendritic cells. The molecule seems to be important in T cell effector function.[26] The American biotechnology company Jounce Therapeutics is developing an ICOS agonist.

• Inhibitory checkpoint molecules

B7-H3: also called CD276, was originally understood to be a co-stimulatory molecule but is now regarded as co-inhibitory.

CTLA-4: short for Cytotoxic T-Lymphocyte-Associated protein 4 and also called CD152. Expression of CTLA-4 on Treg cells serves to control T cell proliferation.

LAG3: short for Lymphocyte Activation Gene-3, works to suppress an immune response by action to Tregs as well as direct effects on CD8+ T cells.

PD-1: short for Programmed Death 1 (PD-1) receptor, has two ligands, PD-L1 and PD-L2. An advantage of targeting PD-1 is that it can restore immune function in the tumor microenvironment.

TIM-3: short for T-cell Immunoglobulin domain and Mucin domain 3, expresses on activated human CD4+ T cells and regulates Th1 and Th17 cytokines. TIM-3 acts as a negative regulator of Th1/Tc1 function by triggering cell death upon interaction with its ligand, galectin-9.

Immune checkpoints in STS

Immune checkpoints are essential in regulating the immune response. In cancer, they can be dysregulated, working as an immune resistance mechanism.

In 2013, the impact of the immune checkpoints PD-1 and PD-L1 in STS was evaluated for the first time. The result from immunohistochemistry showed an intratumoral infiltration of PD-1 positive lymphocytes and the expression of PD-L1 in most STS samples. Additionally, PD-1 positivity, PD-L1 positivity, and the combined PD-1/PD-L1 pattern were independent prognostic indicators of OS and event-free survival. Furthermore, more studies have evaluated these immune checkpoints, the majority by immunohistochemistry, and confirmed the presence of PD-1 and PD-L1, and their association with a negative prognosis. However, in some studies, PD-1 and PD-L1 expression appear to be low or absent, and the PD-L1 expression has not been associated with the outcome in STS. Wunder et al. showed recently that the PD-1 and PD-L1 expression depended on the STS subtype and the prognostic value of PD-L1, justifying the discrepancies between studies with different subtypes of STS. In addition, these discrepancies may also be due to the use of different methods of expression assessment, cutoff values, antibody clones, and tissue samples analyzed before and after therapeutical interventions.

PD-1 and PD-L1 expression levels have also been correlated in some studies with T-cell infiltration, and PD-L1 expression has been associated with more PD-1 positive TILs.

Other immune checkpoints have been studied in several tumors, but there are only a few reports for STS. A recent study showed a high expression of lymphocyte-activation gene 3 (LAG3) on CD8 TILs. Other studies analyzed the expression of LAG3 by immunohistochemistry. They confirmed its overexpression on TILs and found a significant association of LAG3 expression with a poor clinical outcome. The expression of B7-H3 has been evaluated in metastatic gastrointestinal stromal tumors and rhabdomyosarcoma, respectively. In both studies, the expression of this molecule was associated with a worse prognosis.

Summary

Although the presence and prognostic value of these immune checkpoints has been controversial and underexplored in STS, they might still have a role in predicting the prognosis of STS patients. Furthermore, the expression of these immune checkpoints may also indicate the patients who will benefit from PD-1 therapies. In 2020, a study concluded that STS patients who responded to pembrolizumab, an anti-PD-1 monotherapy, exhibited more PD-L1-expressing macrophages than non-responders. Similarly, it is urgently worth studying the expression of stimulatory checkpoint molecules in STS, as well as the interactions between those stimulatory and inhibitory checkpoint molecules. The network of checkpoints together with soluble factors could have important and diagnostic and prognostic value for STS treatment proposal and follow-up surveillance.

Exemplary TNFRSFR stimulatory checkpoint molecules

Ref: Sousa, L.M.; Almeida, J.S.; Fortes-Andrade, T.; Santos-Rosa, M.; Freitas-Tavares, P.; Casanova, J.M.; Rodrigues-Santos, P. Tumor and Peripheral Immune Status in Soft Tissue Sarcoma: Implications for Immunotherapy. Cancers 2021, 13, 3885. https://doi.org/10.3390/ cancers13153885

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