**8.2 DC and CTLA4**

The co-inhibitory immune checkpoint CTLA4 (CD152) is constitutively expressed by regulatory T cells (Treg) and by effector T cells upon activation. CTLA4 is highly homologous to the co-stimulatory receptor CD28 and binds the same ligands CD80 and CD86 (B7.2), however with a much higher affinity. As such, CTLA4 outcompetes CD28 for ligand binding and reduces CD28-mediated co-stimulation of T cell functions. CTLA4 blockade promotes anti-tumour immunity by increasing the activation of effector T cells and by depleting Treg in the TME. The CTLA4 blocking mAb Ipilimumab and Tremelimumab have been approved for the treatment of metastatic melanoma, renal cell carcinoma and colorectal cancer [118].

CTLA4 on T cells directly alters DC functions by removing the CTLA4 ligands (CD80/86) from their cell surfaces. When human moDC are co-cultured with CTLA4<sup>+</sup> T cells, CD80/86 levels on DC decrease rapidly in a CTLA4 dependent manner. This mechanism, named trans-endocytosis, involves the physical capture of CTLA4 ligands by the receptor and their degradation. This process is upregulated by TCR engagement [139, 140]. Mouse *in vivo* studies show that trans-endocytosis is primarily carried out by regulatory T cells and impacts the migratory cDC1 and cDC2 [141]. In addition, CTLA4 interaction with CD80/CD86 on DC induces immunosuppression through reverse signalling. MoDC stimulated with soluble CTLA4 or agonistic anti-CD80/86 Ab produced indoleamine 2,3-dioxygenase (IDO), which is able to inhibit allogenic T cell activation [142]. IDO is expressed by human pDC [143], hence similar immunosuppressive pathways are likely to be induced downstream of CD80/86 in this subset, as reported in mouse pDC [144].

Besides their regulation through CTLA4-CD80/86 interaction, moDC also express the CTLA4 molecule upon activation by TLR stimuli. Treatment of these cells with an agonistic anti-CTLA4 Ab induced increased production of IL-10, reduced expression of IL-8 and IL-12 and decreased T cell stimulation capacity [145]. MoDC are also able to secrete CTLA4 in extracellular microvesicles. Microvesicular CTLA4 has been shown to downregulate CD80 and CD86 on moDC [146].

Combinatorial approaches of anti-CTLA4 mAb with cancer vaccines have been tested in clinics and have yielded mixed results. In melanoma patients, peptide vaccines, in combination with anti-CTLA4 Ipilimumab did not show better clinical outcomes compared to Ipilimumab alone [127, 147, 148]. However, other strategies using DC vaccines have provided promising results. For instance, the co-administration to melanoma patients of autologous moDC that have been pulsed with tumour peptide, together with Tremelimumab, resulted in objective and durable tumour responses [149]. Furthermore, a phase II study using Ipilimumab and moDC electroporated with synthetic mRNA (TriMixDC-MEL) has been tested in advanced melanoma patients and has shown an encouraging rate of highly durable tumour response [150].

#### **8.3 DC and TIM-3**

T cell immunoglobulin mucin-3 (TIM-3) is a co-inhibitory immune checkpoint receptor expressed by all T cell populations as well as B cells and a large variety of myeloid cells. Four TIM-3 ligands have been identified, including Galectin-9, CEACAM-1, HMGB1 and phosphatidylserine. Engagement of TIM-3 on

**113**

*Dendritic Cells and Their Roles in Anti-Tumour Immunity*

blocking in these cancers could potentially enhance CD8+

tumour-infiltrating T cells induces exhaustion and suppresses tumour immunity. Preclinical studies have reported high therapeutic activities of blocking anti-TIM-3 antibodies against various types of malignancies and clinical trials with TIM-3

High TIM-3 expression has been reported on cDC1 and cDC2 from peripheral

T cell infiltration. These data suggest that TIM-3

T cell recruitment to the

pDC are also found in the

blood [151–153] and on tumour-associated cDC1 and cDC2 from mammary tumour biopsies [152]. Mouse models indicated that blocking TIM-3 on cDC1 leads to an increase in the T cell chemoattractant CXCL9. Moreover, cDC1 expressing TIM-3 correlated with CXCL9 expression in human breast cancer biopsies and was

Lymphocyte activation gene-3 (LAG-3) is a co-inhibitory immune checkpoint receptor expressed on activated T cells and NK cells that recognise MHCII molecules on APCs as a ligand. LAG3 negatively regulates T cell activation and is frequently co-expressed with PD-1 on exhausted T cells in the TME. Several LAG-3-targeting cancer immunotherapies are currently in different phases of clinical

The interaction between MHCII and LAG-3 not only has effects in T cells, but also induces reverse signalling in DCs that is stimulatory. This was shown using the soluble LAG-3-Ig fusion protein that activates moDC, as indicated by the upregulation of co-stimulatory molecules, the production of several proinflammatory cytokines and chemokines and increased allogenic T cell activation. However, Ab-mediated MHCII ligation does not activate moDC, thus showing that the MHCII: LAG-3 interaction is required in this process [155–157]. Soluble LAG-3-Ig fusion protein in combination with the chemotherapy drug Paclitaxel has demonstrated elevated clinical activity in metastatic breast carcinoma during a phase I/II trial. This treatment also strongly stimulated the patients' APC, as evidenced by the increase in the number and activation of monocytes, pDC and

Notably, LAG-3 itself has been found expressed by DC, specifically by a sub-

tumour lesions and in the tumour-draining lymph nodes of melanoma patients and are thought to contribute to the immunosuppressive environment. Engagement of LAG-3 on pDC provides an activating signal, independent of TLR signalling, inducing low IFN-α and high IL-6 expression [159]. Hence, LAG-3-specific mAb in cancer immunotherapies may enhance the anti-tumour immune response by

Inducible T cell costimulatory (ICOS) belongs to the co-stimulatory immune checkpoint receptor family and similarly to CD28, enhances the proliferation and effector functions of T cells. ICOS is expressed on activated T cells and constitutively on a subpopulation of Treg [160] while ICOS-L is present at the surface of APC. High ICOS expression on T cells has been particularly observed during anti-CTLA4 therapies and the co-administration of agonistic ICOS-specific mAb further

population of circulating pDC in healthy donors. LAG-3<sup>+</sup>

inhibiting LAG-3 signalling in both T cells and DC.

improves the efficacy to CTLA4 blockade [161].

*DOI: http://dx.doi.org/10.5772/intechopen.91692*

inhibitors are currently underway [128].

positively associated with CD8+

TME [152].

**8.4 DC and LAG-3**

development [154].

cDCs [158].

**8.5 DC and ICOS**

*Dendritic Cells and Their Roles in Anti-Tumour Immunity DOI: http://dx.doi.org/10.5772/intechopen.91692*

tumour-infiltrating T cells induces exhaustion and suppresses tumour immunity. Preclinical studies have reported high therapeutic activities of blocking anti-TIM-3 antibodies against various types of malignancies and clinical trials with TIM-3 inhibitors are currently underway [128].

High TIM-3 expression has been reported on cDC1 and cDC2 from peripheral blood [151–153] and on tumour-associated cDC1 and cDC2 from mammary tumour biopsies [152]. Mouse models indicated that blocking TIM-3 on cDC1 leads to an increase in the T cell chemoattractant CXCL9. Moreover, cDC1 expressing TIM-3 correlated with CXCL9 expression in human breast cancer biopsies and was positively associated with CD8<sup>+</sup> T cell infiltration. These data suggest that TIM-3 blocking in these cancers could potentially enhance CD8+ T cell recruitment to the TME [152].

### **8.4 DC and LAG-3**

*Current Cancer Treatment*

**8.2 DC and CTLA4**

colorectal cancer [118].

subset, as reported in mouse pDC [144].

with CTLA4<sup>+</sup>

moDC [146].

tumour response [150].

**8.3 DC and TIM-3**

The co-inhibitory immune checkpoint CTLA4 (CD152) is constitutively expressed by regulatory T cells (Treg) and by effector T cells upon activation. CTLA4 is highly homologous to the co-stimulatory receptor CD28 and binds the same ligands CD80 and CD86 (B7.2), however with a much higher affinity. As such, CTLA4 outcompetes CD28 for ligand binding and reduces CD28-mediated co-stimulation of T cell functions. CTLA4 blockade promotes anti-tumour immunity by increasing the activation of effector T cells and by depleting Treg in the TME. The CTLA4 blocking mAb Ipilimumab and Tremelimumab have been approved for the treatment of metastatic melanoma, renal cell carcinoma and

CTLA4 on T cells directly alters DC functions by removing the CTLA4 ligands (CD80/86) from their cell surfaces. When human moDC are co-cultured

dependent manner. This mechanism, named trans-endocytosis, involves the physical capture of CTLA4 ligands by the receptor and their degradation. This process is upregulated by TCR engagement [139, 140]. Mouse *in vivo* studies show that trans-endocytosis is primarily carried out by regulatory T cells and impacts the migratory cDC1 and cDC2 [141]. In addition, CTLA4 interaction with CD80/CD86 on DC induces immunosuppression through reverse signalling. MoDC stimulated with soluble CTLA4 or agonistic anti-CD80/86 Ab produced indoleamine 2,3-dioxygenase (IDO), which is able to inhibit allogenic T cell activation [142]. IDO is expressed by human pDC [143], hence similar immunosuppressive pathways are likely to be induced downstream of CD80/86 in this

Besides their regulation through CTLA4-CD80/86 interaction, moDC also express the CTLA4 molecule upon activation by TLR stimuli. Treatment of these cells with an agonistic anti-CTLA4 Ab induced increased production of IL-10, reduced expression of IL-8 and IL-12 and decreased T cell stimulation capacity [145]. MoDC are also able to secrete CTLA4 in extracellular microvesicles. Microvesicular CTLA4 has been shown to downregulate CD80 and CD86 on

Combinatorial approaches of anti-CTLA4 mAb with cancer vaccines have been tested in clinics and have yielded mixed results. In melanoma patients, peptide vaccines, in combination with anti-CTLA4 Ipilimumab did not show better clinical outcomes compared to Ipilimumab alone [127, 147, 148]. However, other strategies using DC vaccines have provided promising results. For instance, the co-administration to melanoma patients of autologous moDC that have been pulsed with tumour peptide, together with Tremelimumab, resulted in objective and durable tumour responses [149]. Furthermore, a phase II study using Ipilimumab and moDC electroporated with synthetic mRNA (TriMixDC-MEL) has been tested in advanced melanoma patients and has shown an encouraging rate of highly durable

T cell immunoglobulin mucin-3 (TIM-3) is a co-inhibitory immune checkpoint receptor expressed by all T cell populations as well as B cells and a large variety of myeloid cells. Four TIM-3 ligands have been identified, including

Galectin-9, CEACAM-1, HMGB1 and phosphatidylserine. Engagement of TIM-3 on

T cells, CD80/86 levels on DC decrease rapidly in a CTLA4-

**112**

Lymphocyte activation gene-3 (LAG-3) is a co-inhibitory immune checkpoint receptor expressed on activated T cells and NK cells that recognise MHCII molecules on APCs as a ligand. LAG3 negatively regulates T cell activation and is frequently co-expressed with PD-1 on exhausted T cells in the TME. Several LAG-3-targeting cancer immunotherapies are currently in different phases of clinical development [154].

The interaction between MHCII and LAG-3 not only has effects in T cells, but also induces reverse signalling in DCs that is stimulatory. This was shown using the soluble LAG-3-Ig fusion protein that activates moDC, as indicated by the upregulation of co-stimulatory molecules, the production of several proinflammatory cytokines and chemokines and increased allogenic T cell activation. However, Ab-mediated MHCII ligation does not activate moDC, thus showing that the MHCII: LAG-3 interaction is required in this process [155–157]. Soluble LAG-3-Ig fusion protein in combination with the chemotherapy drug Paclitaxel has demonstrated elevated clinical activity in metastatic breast carcinoma during a phase I/II trial. This treatment also strongly stimulated the patients' APC, as evidenced by the increase in the number and activation of monocytes, pDC and cDCs [158].

Notably, LAG-3 itself has been found expressed by DC, specifically by a subpopulation of circulating pDC in healthy donors. LAG-3<sup>+</sup> pDC are also found in the tumour lesions and in the tumour-draining lymph nodes of melanoma patients and are thought to contribute to the immunosuppressive environment. Engagement of LAG-3 on pDC provides an activating signal, independent of TLR signalling, inducing low IFN-α and high IL-6 expression [159]. Hence, LAG-3-specific mAb in cancer immunotherapies may enhance the anti-tumour immune response by inhibiting LAG-3 signalling in both T cells and DC.

#### **8.5 DC and ICOS**

Inducible T cell costimulatory (ICOS) belongs to the co-stimulatory immune checkpoint receptor family and similarly to CD28, enhances the proliferation and effector functions of T cells. ICOS is expressed on activated T cells and constitutively on a subpopulation of Treg [160] while ICOS-L is present at the surface of APC. High ICOS expression on T cells has been particularly observed during anti-CTLA4 therapies and the co-administration of agonistic ICOS-specific mAb further improves the efficacy to CTLA4 blockade [161].

pDC are able to induce immunosuppression though ICOS stimulation. ICOS-L is strongly upregulated by human blood pDC, but not CD11c<sup>+</sup> cDC, in response to TLR stimuli or IL-3 [162]. Co-cultures of pDC with allogenic T cells induced IL-10 expression through a mechanism mediated by ICOS-L-ICOS interaction [162] and similar observations were reported with pDC isolated from ovarian carcinoma [163]. Furthermore, pDC are able to induce Treg proliferation though ICOS stimulation [160] and this mechanism likely explains the dramatic accumulation of ICOS+ Treg in ovarian, breast, liver and gastric tumour tissues, in close proximity with ICOS-L+ pDC [101, 164–166].
