Poor survival in glioblastoma patients is associated with early signs of immunosenescence in the CD4 T-cell compartment after surgery

Figures & data

Figure 1. Levels of CD3⁺ T cells, γδ T cells, and CD4⁺CD28⁻ T cells are lower after surgery in GBM patients than in healthy controls (HC). T-cell phenotype was analyzed in PBMCs from GBM patients before and 3, 12, and 24 weeks after surgery. (A) CD3⁺ T cells were less abundant than in controls at all-time points (B and C) Levels of CD4⁺ and CD8⁺ T cells did not differ. (D) Levels of γδ T cells were higher in GBM patients than controls at all-time points. (E) Levels of CD4⁺CD28⁻ T cells were higher before and 3 and 12 weeks after surgery. (F) Levels of CD8⁺CD28⁻ T cells did not differ.

Table 1. Characteristics of glioblastoma patient cohort

Patient nr	Age at diagnosis	TTP (Months)	Treatment at tumor recurrance	Overall survival (Months)	Preoperative steroid intake (days)	Post-operative steroid discontimuation (days)	Surgical tumor reduction more than 90% (y/n)
1	64	6	none	14	10	12	y
2	58	3	none	17	1	10	y
3	64	12	Surgery	16	6	12	y
4	55	73	Temodal	75	3	8	y
5	74	16	Temodal	18	5	7	y
6	68	12	Surgery + Lomustine	30	18	8	y
7	59	3	none	8	13	9	y
8	68	3	Surgery + Radiotherapy	77	12	21	y
9	59	11	Lomustine	18	4	17	y
10	71	12	Lomustine	17	5	8	y
11	75	6	Lomustine	18	14	60	y
12	61	68	Surgery	70	8	9	y
13	52	6	Gammaknife	41	4	6	y
14	60	6	Radiotherapy	17	5	8	n (50% to 90% tumor reduction)
15	75	52	Gammaknife	59	2	long term (> 6 mo)	y
16	64	6	Temodal	14	11	19	y
17	73	3	none	8	16	9	y
18	62	3	Gammaknife	18	6	9	y
19	60	3	Gammaknife	55	10	8	y
20	49	3	none	5	2	long-term (> 6 mo)	y
21	34	3	Gammaknife + Avastin	24	3	9	y
22	65	3	Temodal + Avastin	19	7	16	y
23	64	3	Lomustine	10	14	11	y
24	51	3	Lomustine	12	3	12	y
25	77	3	Temodal	10	14	25	y
26	37	6	none	Lost to follow-up	9	8	y
27	60	3	Lomustine	19	13	10	y
28	52	4	Lomustine	13	4	60	y
29	59	9	Gammaknife	22	1	42	y
30	60	3	none	10	21	long term (> 6 mo)	y
31	33	3	Surgery + Gammaknife + Lomustine	8	3	9	y
32	57	12	none	15	2	30	y
33	65	24	Temodal + Avastin	38	20	14	y
34	69	6	Gammaknife	22	1	9	y
35	46	9	Surgery + Lomustine	18	5	9	y
36	46	3	Temodal + Avastin	16	1	26	y
37	49	6	Gammaknife	18	2	12	y
38	68	17	Gammaknife	31	1	8	y
39	64	6	Gammaknife	68	7	9	y
40	61	1	Gammaknife	25	7	9	y
41	66	1	none	4	3	9	y
42	65	6	Gammaknife	24	6	8	y
Median	61	6		18	5.5	9

Table 2. Summary of the analysis of immunological cell types (%) in blood in glioblastoma patients after surgery (weeks). Data shown in the table: median (significant p values)

	0 weeks	3 weeks	12 weeks	24 weeks	Healthy controls
CD3 cells	37.1**** (p < 0.0001)	44.6 *** (p = 0.0001)	40.9 ****(p < 0.0001)	43****(p < 0.0001)	62
CD4^+ cells	64	64.5	60.5	65	67
CD4CD28null cells	9** (p = 0.0044)	7* (p = 0.0141)	7* (p = 0.0203)	5	3
CD4CD57 cells	7.7 * (0.0106)	7.15 * (p = 0.0265)	8.1 * (p = 0.0132)	7* (p = 0 .0375)	4.5
CD4CD57CD28 cells	6.2 **** (p < 0.0001)	5 **** (p < 0.0001)	6 **** (p < 0.0001)	5.7 **** (p < 0.0001)	2
CD4CD57CD28null cells	0.5	0.9	1.35	0.9	1
CD4CD25 cells	1.3* (p = 0.0193)	1.8 * (p = 0.0355)	2.3	2.5	4.5
CD8^+ cells	26	27.5	28.5	28	25
CD8CD28null cells	36	35	40	29.5	43.5
CD8CD57 cells	33.4	26.5	27	30.8	30.5
CD8CD57CD28 cells	7.7	6.5	8.9	7.6	5
CD8CD57CD28null cells	21.1	14.4	19	17.9	27
CD8CD25 cells	1.7	1.2	3.1	1.1 * (p = 0 .02)	4
gd T cells	11.6****(p < 0.0001)	12.1***(p = 0.0003)	10.2***(p = 0.0002)	11.8***(p = 0.0006)	3.6

Figure 2. Levels of CD4⁺CD57⁺, CD4⁺CD57⁺CD28⁺, CD4⁺CD25⁺ and CD8⁺CD25⁺ T cells are higher after surgery in GBM patients than in healthy controls (HC). T-cell phenotype was analyzed in PBMCs from GBM patients before and 3, 12, and 24 weeks after surgery. (A) Levels of CD4⁺CD57⁺ T cells were higher in GBM patients at all-time points compared to HC. (B–D) Levels of CD8⁺CD57⁺, CD4⁺CD57⁺CD28⁻, and CD8⁺CD57⁺CD28⁻T cells did not differ. (E) Levels of CD4⁺CD57⁺CD28⁺ T cells were higher in GBM patients than in controls at all-time points. (F) Levels of CD8⁺CD57⁺CD28⁺ T did not differ. (G) Levels of CD4⁺CD25⁺T cells were lower at baseline and 3 weeks after surgery. (H) Levels of CD8⁺CD25⁺T cells were lower at 24 weeks after surgery.

Figure 3. GBM patients with longer survival time (≥20 mo) have higher levels of CD3⁺ but not CD4⁺ and CD8⁺ T cells. Survival was analyzed with respect to CD3, CD4⁺, and CD8⁺ expression (median values were used as a cut-off for Kaplan–Meier graphs). (A) Patients who survived longer had higher levels of CD3⁺ T cells at 3 and 12 weeks after surgery. (B and C) Survival did not differ in patients with high vs. low levels of CD3⁺ cells before and 3 weeks after surgery (D and G). No significant differences in CD4⁺ and CD8⁺ T-cell levels were observed in GBM patients with long-term survival. (E–H) Survival did not differ in patients with high vs. low levels of CD4⁺ cells at baseline and 3 weeks after surgery and low levels CD8⁺ cells at baseline. (I) GBM patients with higher levels of CD8⁺ T cells have shorter survival than patients with lower levels of these cells.

Figure 4. GBM patients with shorter overall survival have higher levels of CD4⁺CD28⁻ cells. (Median values were used as a cut-off for Kaplan–Meier analysis). Long-term GBM survivors had lower levels of CD4⁺CD28⁻ at 3 weeks after surgery. (A) γδ T-cell levels did not differ in GBM patients with longer or shorter survival. (B and C) Survival did not differ in patients with high vs. low levels of γδ T cells before and at 3 weeks after surgery. (D) Among GBM patients, short-term survivors had significantly higher levels CD4⁺CD28⁻ cells 3 weeks after surgery than long-term survivors. (E) Survival did not differ in patients with high vs. low levels of CD4⁺CD28⁻ T cell at baseline. (F) GBM patients with higher levels of CD4⁺CD28⁻ cells have shorter overall survival than those with lower levels of these T cells. (G) CD8⁺CD28⁻ T-cell levels did not differ in GBM patients with longer vs. shorter survival. (H and I) Survival did not differ in patients with high vs. low levels of CD8⁺CD28⁻ cells at baseline and 3 weeks after surgery.

Figure 5 (See previous page). Higher levels of CD4⁺CD57⁺ and CD4⁺CD57⁺CD28⁻ T cells are associated with poor survival in GBM patients using median values as a cut-off for Kaplan–Meier analysis. Direct correlation between expression of CD57 and loss of CD28 on T cells was observed. (A) GBM patients with short-term survival time had significantly higher levels of CD4⁺CD57⁺ cells at 3 and 24 weeks after surgery than long-term survivors. (B) Survival did not differ in patients with high vs. low levels of CD4⁺CD57⁺ T cell at baseline (C). GBM patients with higher levels of CD4⁺CD57⁺ T cells have shorter overall survival than those with lower levels of these cells. (D) Among GBM patients, short-term survivors had significantly higher levels of CD8⁺CD57⁺ cells at 24 weeks after surgery than long-term survivors. (E and F) Survival did not differ in patients with high vs. low levels of CD8⁺CD57⁺ cells before and 3 weeks after surgery. (G) GBM patients with shorter survival times (<20 mo) had significantly higher levels CD4⁺CD57⁺CD28⁻ cells 3 weeks after surgery than those with long-term survival. (H) Survival did not differ in patients with high vs. low levels of CD4⁺CD57⁺CD28⁻ cells at baseline. (I) GBM patients with higher levels of CD4⁺CD57⁺CD28⁻ cells have shorter overall survival than those with lower levels of these cells. (J) Linear regression analysis of direct correlation between expression of CD57 and loss of CD28 on CD4⁺ cells.

Figure 6. In GBM patients, levels of CD8⁺CD57⁺CD28⁻, CD4⁺CD57⁺CD28⁺ and CD8⁺CD57⁺CD28⁺ T cells do not correlate with survival (median values were used as a cut-off for Kaplan–Meier analysis) and did not differ in long-term vs. short-term survivors. (A–C) Survival did not differ in patients with high vs. low levels of CD8⁺CD57⁺CD28⁻ T-cells as well as in short-term vs. long-term GBM survivors. (D–I) Survival did not differ in patients with high vs. low CD4⁺CD57⁺CD28⁺ or CD8⁺CD57⁺CD28⁺ T-cell levels as well as in short-term vs. long-term GBM survivors.

Figure 7. (A–C) Levels of CD4⁺CD25⁺ T cells did not correlate with GBM patient survival.

Table 3. Linear regression analysis of patient cohorts with steroid treatment longer then 3 weeks versus shorter than 3 weeks post surgery

CD3 cells	Regression coefficient	p value
0 weeks	−0.2345511	0.1099
3 weeks	−0.4374638	0.1845
12 weeks	−0.3675055	0.2356
24 weeks	0.1472976	0.6469
CD4^+ cells
0 weeks	0.2300614	0.05
3 weeks	–0.0851683	0.4607
12 weeks	0.0847497	0.4321
24 weeks	−0.0672685	0.5645
CD8^+ cells
0 weeks	−0.1520693	0.3401
3 weeks	0.077754	0.736
12 weeks	−0.0734131	0.6772
24 weeks	−0.1384975	0.5219
γδ T cells
0 weeks	0.1478178	0.652
3 weeks	−0.0623596	0.8675
12 weeks	0.3344783	0.2845
24 weeks	0.2441888	0.4758
CD4/CD57 cells
0 weeks	0.1852333	0.5615
3 weeks	0.0553747	0.88
12 weeks	0.4497213	0.1782
24 weeks	−0.182609	0.5779
CD8/CD57 cells
0 weeks	−0.0922303	0.7747
3 weeks	0.0430214	0.8816
12 weeks	−0.0638975	0.8847
24 weeks	−0.1975558	0.5477
CD4/CD28null cells
0 weeks	0.4659717	0.2652
3 weeks	−0.5119411	0.2396
12 weeks	0.4751103	0.251
24 weeks	−0.5901584	0.0863
CD8/CD28null cells
0 weeks	0.3325712	0.3999
3 weeks	−0.8171889	0.0453
12 weeks	0.0879745	0.7117
24 weeks	−0.8251942	0.0114
CD4/ CD57/CD28null cells
0 weeks	−0.1180299	0.8643
3 weeks	0.9674673	0.1709
12 weeks	0.0219018	0.9694
24 weeks	0.4697549	0.5238
CD8/CD57/CD28null cells
0 weeks	−0.0862766	0.8732
3 weeks	−0.3726477	0.3934
12 weeks	0.0554057	0.8842
24 weeks	0.7204616	0.1374
CD4/CD25 cells
0 weeks	−0.6599094	0.1663
3 weeks	0.2641433	0.6468
12 weeks	0.2000327	0.6008
24 weeks	0.3657016	0.2899
CD8/CD25 cells
0 weeks	−0.9189226	0.0857
3 weeks	0.4819171	0.356
12 weeks	0.3546721	0.6073
24 weeks	0.1461828	0.7551

Table 4. Linear regression analysis comparing patient cohorts with Valcyte treatment versus placebo

CD 3 cells	Regression coefficient	p value
0 weeks	−0.1510336	0.2173
3 weeks	0.139136	0.4
12 weeks	−0.1315498	0.3092
24 weeks	−0.0630347	0.8129
CD 4^+ cells
0 weeks	−0.0385455	0.6983
3 weeks	−0.0523832	0.5933
12 weeks	0.0612635	0.5189
24 weeks	−0.0246153	0.7993
CD 8^+ cells
0 weeks	0.1092555	0.4089
3 weeks	0.3792077	0.0462
12 weeks	0.00146	0.9925
24 weeks	0.205801	0.2462
GD T cells
0 weeks	−0.4969697	0.0891
3 weeks	−0.3266768	0.3045
12 weeks	0.0294959	0.9175
24 weeks	−0.0437197	0.8812
CD 4/CD 57 cells
0 weeks	−0.2710945	0.3597
3 weeks	−0.1967002	0.5725
12 weeks	0.2718693	0.3706
24 weeks	−0.4021137	0.1652
CD 8/CD 57 cells
0 weeks	0.1518069	0.6128
3 weeks	−0.1124162	0.6826
12 weeks	−0.0695953	0.8608
24 weeks	−0.2129092	0.4692
CD 4/CD 28null cells
0 weeks	0.312091	0.3743
3 weeks	−0.5047389	0.1493
12 weeks	−0.0089941	0.9803
24 weeks	−0.4274857	0.1648
CD 8/CD 28null cells
0 weeks	0.1306234	0.6972
3 weeks	0.0858196	0.8044
12 weeks	−0.0895841	0.6666
24 weeks	−0.4305437	0.1522
CD 4/ CD57/CD 28null cells
0 weeks	1.110387	0.0673
3 weeks	1.203756	0.058
12 weeks	0.1420544	0.7847
24 weeks	0.5643537	0.3595
CD 8/ CD57/CD 28null cells
0 weeks	−0.0155809	0.9756
3 weeks	−0.6119404	0.137
12 weeks	0.3224287	0.3278
24 weeks	−0.6235189	0.1541
CD 4/CD 25 cells
0 weeks	−0.21233	0.6179
3 weeks	0.7711318	0.0947
12 weeks	0.4433576	0.1565
24 weeks	0.1041078	0.7244
CD 8/CD 25 cells
0 weeks	0.1311385	0.7944
3 weeks	0.2672356	0.5469
12 weeks	0.6383185	0.2732
24 weeks	0.0276734	0.9453