Decreased frequency of CD4+ T cells in metastatic lymph nodes
The lymph node is a secondary lymphoid organ [14] that represents a pivotal meeting point of various immune cell types for adaptive immune responses [15]. To identify the immune cell types in metastatic lymph nodes, LN + and paired LN- collected from each OSCC patient were analyzed via flow cytometry. The clinical parameters of the 11 OSCC patients are shown in Table 1. The percentage of T cells was significantly lower in LN + than in LN- (p = 0.0028) (Fig. 2A). However, there was no significant difference between the percentage of B cells (p = 0.9825), dendritic cells (p = 0.7674), or macrophages (p = 0.1625) in LN + and paired LN- (Fig. 2A). In a further analysis, CD4+ T cells dramatically decreased in LN + compared with LN- (p < 0.0001) (Fig. 2B), whereas CD8+ T cells did not change in LN+ (Fig. 2B). This result indicated that the decreased frequency of CD4+ T cells was closely associated with metastatic lymph nodes in OSCC.
Table 1. The clinical parameter of OSCC patients were used.
NO.
|
Age
|
Gender
|
T classification
|
Lymph node
status
|
Tumor site
|
Perineural invasion
|
Prior Radiotherapy/ Chemosensitivety
|
Prior surgical treatment
|
1
2
3
4
5
6
7
8
9
10
11
|
78
49
51
59
71
68
85
45
52
66
58
|
Female
Female
Male
Male
Male
Male
Male
Female
Male
Male
Female
|
T2
T3
T3
T3
T2
T2
T4
T3
T2
T4
T3
|
pN positive
pN positive
pN positive
pN positive
pN positive
pN positive
pN positive
pN positive
pN positive
pN positive
pN positive
|
Tongue
Buccal mucosa
Tongue
Tongue
Tongue
Tongue
Buccal mucosa
Oral floor
Tongue
Tongue
Gingiva
|
No
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
|
No
No
No
No
No
No
No
No
No
No
No
|
Yes
Yes
Yes
Yes
Yes
No
Yes
No
No
Yes
No
|
pN: pathology lymph nodes status
Increased frequency of PD1 in CD4+ T cells occurred in metastatic lymph nodes
Immune checkpoint receptors on T cells can negatively determine their expansion, activation, and effector functions via inhibitory signals generated through binding to the receptors [6]. The interaction of PDL1 with its cognate ligand PD1 on activated T cells inhibits anti-tumor immunity by counteracting T cell-activating signals [16]. To detail the expression of immune checkpoint receptors PD1, PDL1, and CTLA4 in metastatic lymph nodes, immune checkpoint receptor transcriptional levels were detected using RT-PCR. The LN + and paired LN- from each OSCC patient were collected, and the clinical parameters of 11 OSCC patients are shown in Table 1. Only the PD1 expression level of CD4+ T cells was notably upregulated in LN + compared to that in LN- (p = 0.0158) (Fig. 3A). To further determine changes in immune checkpoints in metastatic lymph nodes, PD1, PDL1, and CTLA4 protein levels were detected using flow cytometry. As expected, the PD1 protein level of CD4+ T cells was significantly upregulated in LN + compared to LN- (p < 0.0001) (Fig. 3B and Fig. 3C, respectively). However, there was no significant difference in PDL1 and CTLA4 between LN + and LN- (Fig. 3B and Fig. 3C, respectively). Immunofluorescence analysis showed that PD1 was predominantly expressed in CD4+ T cells and markedly upregulated in LN + compared to LN- cells (Fig. 3D). Our findings revealed that the increased PD1 of CD4+ T cells in LN + was related to lymph node metastasis progression.
Elevated glycolysis related enzymes levels in CD4+ T cells from metastatic lymph nodes
T cells depend on dramatic increases in glucose metabolism as fuel to support the growth, function, survival, and differentiation of activated T cells [9, 17]. To determine whether glycolysis-related enzymes contribute to CD4+ T cells in metastatic lymph nodes, the mRNA expression levels of Glut1, Hk2, Hk3, Tpi1, Gpi1, Eno1, PKM, LDHa, and MCT4 in CD4+ T cells were detected via RT-PCR. The mRNA expression levels of Glut1, Hk2, Tpi1, Gpi1, Eno1, and LDHa in CD4+ T cells dramatically increased in LN + compared to LN- (Fig. 4). Although there was no statistical difference between Hk3, PKM, and MCT4 expression levels in LN + and LN-, the average values of Hk3, PKM, and MCT4 expression levels in LN + were higher than those in LN- (Fig. 4). These results suggest that an increase in PD1 of CD4+ T cells is linked to glucose metabolism and aerobic glycolysis.
PD1 and Hk2 expressions of CD4 + T cells in metastatic lymph nodes of OSCC patients with prior surgical treatments compared to those without
LN + in OSCC patients with a surgical treatment history (i.e., underwent neck lymph node dissection) was defined as P-LN+ (n = 7), and LN + in OSCC patients without a prior surgical treatment history was defined as N-LN+ (n = 4). The PD1 expression level of CD4+ T cells was markedly upregulated in P-LN + compared to N-LN+ (p = 0.0286), whereas there was no statistical difference in the PDL1 and CTLA4 expressions between P-LN + and N-LN+ (Fig. 5A, 5B and 5C, respectively). To determine whether glycolysis-related enzymes contributed to the upregulation PD1 of in CD4+ T cells in P-LN+, the mRNA expression levels of Glut1, Hk2, Hk3, Tpi1, Gpi1, Eno1, PKM, LDHa, and MCT4 in CD4+ T cells were analyzed according to the patients’ surgical treatment history. Only the Hk2 expression levels of CD4+ T cells dramatically increased in P-LN + compared to N-LN+ (p = 0.0061) (Fig. 5D). These data suggest that the increase in PD1 of CD4+ T cells in P-LN + was associated with elevated Hk2.