Deregulated Expression of Immune Checkpoints on Circulating CD4 T Cells May Complicate Clinical Outcome and Response to Treatment with Checkpoint Inhibitors in Multiple Myeloma Patients
Abstract
:1. Introduction
2. Results
2.1. CD4 T Cells from PB of MM Patients Are Maximally Stimulated In Vivo and Possess Strong Potential for Inhibition of the Immune Response
2.2. Expression of Immune Checkpoints in Myeloma CD4 T Cell Subsets Is Clearly Impaired, Especially in Newly Diagnosed Patients
2.3. Dysfunctional Characteristics of PB CD4 T Cells Depend on Myeloma Stage
2.4. Expansion of PB Treg Cells Is Most Pronounced at Less Advanced MM, Which May Create Conditions Promoting Disease Development
2.5. Markers of T Cell Exhaustion Are Associated with Adverse MM Clinical Behavior
3. Materials and Methods
3.1. Samples and Patient Characteristics
3.2. Cell Isolation from Peripheral Blood
3.3. Secretory Effector Function, Immunofluorescence Staining, and Flow Cytometric Analysis
3.4. Statistical Analysis
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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T-Cell Subset (%) | At Diagnosis (NDMM) (n = 26) | At Relapse (RRMM) (n = 14) | HC (n = 20) | p Value |
---|---|---|---|---|
CD3+CD4+PD-1+ | 15.33 (12.62–18.52) | 17.72 (11.75–19.86) | 12.55 (8.37–15.29) | (a) ns (b) 0.02 (c) 0.04 |
CD4+CD127+PD-1+ | 23.53 (16.42–29.99) | 25.00 (12.96–31.13) | 16.87 (10.52–21.75) | (a) ns (b) 0.088 * (c) 0.090 * |
CD4+CD127−PD-1+ | 9.70 (6.78–11.94) | 14.79 (5.31–26.50) | 6.14 (3.90–7.62) | (a) 0.037 (b) 0.041 (c) 0.0001 |
CD3+CD4+BTLA+ | 28.64 (20.04–43.21) | 23.85 (15.06–30.73) | 19.03 (13.25–37.52) | (a) ns (b) 0.018 (c) ns |
CD4+CD127+BTLA+ | 38.87 (26.26–50.23) | 40.30 (26.08–47.58) | 23.62 (10.85–40.79) | (a) ns (b) ns (c) ns |
CD4+CD127−BTLA+ | 3.98 (2.54–6.29) | 7.70 (5.21–11.35) | 2.47 (1.24–5.15) | (a) ns (b) ns (c) 0.010 |
CD3+CD4+CTLA-4+ | 1.91 (0.78–3.37) | 1.28 (0.89–1.88) | 1.27 (0.78–2.00) | (a) ns (b) ns (c) ns |
CD4+CD127+CTLA4+ | 3.23 (2.21–7.66) | 3.42 (1.68–4.36) | 3.55 (1.64–4.22) | (a) ns (b) ns (c) ns |
CD4+CD127−CTLA4+ | 1.93 (0.47–2.67) | 1.68 (1.45–2.19) | 1.15 (0.57–1.38) | (a) ns (b) ns (c) 0.031 |
CD3+CD4+CD69+ | 0.58 (0.23–1.09) | 0.70 (0.36–0.95) | 0.34 (0.22–0.44) | (a) ns (b) 0.060 * (c) 0.027 |
Fluorescence Intensity | At Diagnosis (NDMM) (n = 26) | At Relapse (RRMM) (n = 14) | HC (n = 20) | p Value |
---|---|---|---|---|
PD-1 in CD4+ | 72.99 (67.04–86.64) | 79.41 (56.39–111.33) | 83.04 (71.73–105.09) | (a) ns (b) 0.063 * (c) ns |
PD-1 in CD4+CD127+ | 67.19 (52.34–84.45) | 85.34 (57.28–112.96) | 90.27 (60.38–105.84) | (a) ns (b) 0.085 * (c) ns |
PD-1 in CD4+CD127− | 68.47 (54.77–93.55) | 73.92 (59.29–113.76) | 102.13 (79.79–127.27) | (a) ns (b) 0.016 (c) ns |
BTLA in CD4+ | 169.60 (146.40–184.92) | 184.12 (109.50–196.04) | 215.18 (192.79–289.32) | (a) ns (b) 0.0002 (c) 0.002 |
BTLA in CD4+CD127+ | 150.75 (142.37–167.89) | 186.19 (149.70–241.82) | 240.20 (166.25–304.14) | (a) ns (b) 0.001 (c) ns |
BTLA in CD4+CD127− | 147.00 (120.49–168.15) | 192.21 (154.61–235.12) | 240.60 (167.13–292.31) | (a) 0.033 (b) 0.001 (c) ns |
CTLA-4 in CD4+ | 44.95 (38.52–63.20) | 76.21 (63.09–129.84) | 65.40 (60.10–111.23) | (a) 0.002 (b) 0.005 (c) ns |
CTLA-4 in CD4+CD127+ | 36.22 (33.40–41.87) | 55.58 (49.57–95.97) | 80.96 (51.93–90.08) | (a) 0.0002 (b) 0.0005 (c) ns |
CTLA-4 in CD4+CD127− | 34.13 (30.84–40.13) | 53.69 (43.86–66.96) | 62.97 (41.04–74.55) | (a) 0.008 (b) 0.0008 (c) ns |
CD69 in CD4+ | 53.64 (47.87–58.82) | 75.69 (49.52–94.75) | 70.81 (55.55–123.46) | (a) ns (b) 0.017 (c) ns |
T-Cell Subset (%) | At Diagnosis (NDMM) (n = 26) | At Relapse (RRMM) (n = 14) | HC (n = 20) | p Value |
---|---|---|---|---|
CD3+CD8−IFN-γ+ | 4.79 (3.34–7.03) | 3.91 (3.01–6.40) | 9.00 (7.66–10.07) | (a) ns (b) 0.005 (c) 0.0002 |
CD3+CD8−IL-17+ | 0.51 (0.24–0.59) | 0.56 (0.29–1.08) | 0.32 (0.22–0.45) | (a) ns (b) 0.048 (c) 0.045 |
CD4+CD25+CD127− | 5.02 (4.40–6.02) | 6.61 (5.70–8.98) | 3.69 (2.72–3.90) | (a) 0.076 * (b) 0.0001 (c) 0.0001 |
CD4+CD25+FOXP3+ | 4.30 (3.43–5.34) | 4.58 (3.88–5.60) | 2.51 (1.66–3.19) | (a) ns (b) 0.0001 (c) 0.001 |
CD4+ FOXP3+CD127− | 4.52 (3.79–5.35) | 4.35 (3.42–6.17) | 2.34 (1.49–2.67) | (a) ns (b) 0.0001 (c) 0.001 |
Cell Subsets | Clinical Features | Median (IQ Range) | p Value |
---|---|---|---|
CD4+CD127+PD-1+ (%) | <3.5 mg/L of β2M | 18.66 (13.76–24.29) | |
≥3.5 mg/L of β2M | 23.99 (16.42–32.32) | 0.06 * | |
PD-1 in CD4+ (MFI) | ≤3.5 g/dL of albumin | 84.42 (72.42–129.93) | |
>3.5 g/dL of albumin | 69.05 (55.08–94.03) | 0.05 | |
PD-1 in CD4+CD127+ (MFI) | IgA | 107.59 (99.75–121.15) | |
IgG | 63.85 (52.34–84.45) | 0.01 | |
PD-1 in CD4+CD127− (MFI) | IgA | 123.41 (104.10–149.45) | |
IgG | 62.38 (54.77–85.70) | 0.01 | |
PD-1 in CD4+CD127− (MFI) | Stage ≤ 2 | 59.49 (53.62–85.70) | |
Stage > 2 | 96.70 (69.07–113.76) | 0.02 | |
CTLA-4 in CD4+ (MFI) | <10 mg/dL of serum calcium | 44.48 (39.87–62.12) | |
≥10 mg/dL of serum calcium | 72.71 (45.56–98.68) | 0.01 | |
CTLA-4 in CD4+CD127+ (MFI) | <10 mg/dL of serum calcium | 35.87 (32.94–39.74) | |
≥10 mg/dL of serum calcium | 51.21 (44.45–72.31) | 0.0006 | |
CTLA-4 in CD4+CD127− (MFI) | <10 mg/dL of serum calcium | 33.79 (27.96–35.29) | |
≥10 mg/dL of serum calcium | 53.69 (40.72–66.97) | 0.002 | |
CD4+CD69+ (%) | ≤12 g/dL of hemoglobin | 0.69 (0.56–1.09) | |
>12 g/dL of hemoglobin | 0.37 (0.21–0.91) | 0.08 * | |
CD4+CD69+ (%) | <3.5 mg/L of β2M | 0.30 (0.19–0.68) | |
≥3.5 mg/L of β2M | 0.69 (0.37–1.09) | 0.02 |
Prognostic Factors | Univariate | Multivariate | |
---|---|---|---|
p Value | HR (95% CI) | p Value | |
Age > 65 years | 0.10 * | 9.74 (0.94–100.33) | 0.05 |
ISS stage > 2 | 0.11 * | ||
Albumin < 3.5 g/dL | 0.005 * | 0.04 (0.01–0.29) | 0.001 |
β2-microglobulin ≥ 3.5 mg/L | 0.05 * | 21.65 (0.65–714.02) | 0.08 |
Creatinine ≥ 2.0 mg/dL | 0.04 * | ||
Serum calcium ≥ 10 mg/dL | 0.91 | ||
LDH > 190 U/L | 0.96 | ||
Hemoglobin ≤ 12 g/dL | 0.08 * | 0.16 (0.02–1.04) | 0.05 |
Platelets < 100,000/mm3 | 0.43 | ||
Plasmocytes > 5 % | 0.69 | ||
CD4+CD127+PD-1+ > median (%) | 0.10 * | ||
PD-1 in CD4+ > median (MFI) | 0.56 | ||
PD-1 in CD4+CD127+ > median (MFI) | 0.38 | ||
PD-1 in CD4+CD127− > median (MFI) | 0.34 | ||
CTLA-4 in CD4+ > median (MFI) | 0.11 * | ||
CTLA-4 in CD4+CD127+ > median (MFI) | 0.65 | ||
CTLA-4 in CD4+CD127− > median (MFI) | 0.58 | ||
CD4+CD69+ (%) > median (MFI) | 0.20 |
Characteristic | Newly Diagnosed (NDMM) | Relapsed/Refractory (RRMM) | Total |
---|---|---|---|
Number of patients, n (%) | 26 (65%) | 14 (35%) | 40 (100%) |
Sex (female) | 17 (65.5%) | 4 (28.5%) | 21 (52.5 %) |
Age of sampling (median, range) | 66 (50–76) | 72 (65–75) | 69 (59–76) |
ISS | |||
I | 5 (19%) | 1 (7%) | 6 (15.0 %) |
II | 10 (38.5%) | 6 (43%) | 16 (40.0 %) |
III | 11 (42.5%) | 7 (50%) | 18 (45.0 %) |
Myeloma isotype | |||
IgG | 18 (69%) | 9 (64%) | 27 (67.5 %) |
IgA | 3 (11.5%) | 3 (21.5%) | 6 (15.0 %) |
Light chain only | 5 (19.5%) | 2 (14.5%) | 7 (17.5 %) |
Type of Ig light chain (serum) | |||
Kappa | 16 (61.5%) | 7 (50%) | 23 (57.5 %) |
Lambda | 9 (34.5%) | 7 (50%) | 16 (40.0 %) |
None | 1 (4%) | 0 (0%) | 1 (2.5 %) |
Osteolytic bone lesion/s, n (%) | 15 (60.0%) | 13 (92.8%) | 28 (70.0 %) |
Laboratory values | |||
β2-microglobulin ≥ 3.5 mg/L | 18 (69%) | 9 (64%) | 27 (67.5 %) |
Creatinine ≥ 2.0 mg/dL | 9 (34.5%) | 4 (28.5%) | 13 (32.5 %) |
LDH > 190 U/L | 6 (23%) | 2 (14%) | 8 (20.0 %) |
Serum calcium ≥ 10 mg/dL | 9 (34.5%) | 11 (78.5%) | 20 (50.0 %) |
Hemoglobin ≤ 12 g/dL | 23 (88.5%) | 8 (57%) | 31 (80.0 %) |
Platelets < 100,000/mm3 | 2 (7.5%) | 1 (7%) | 3 (7.5 %) |
Prior treatment | |||
1–3 therapy lines | 0 (0%) | 8 (57%) | 8 (20.0 %) |
≥4 therapy lines | 0 (0%) | 6 (43%) | 6 (15.0 %) |
BTZ-based therapy | 0 (0%) | 12 (85.5%) | 12 (30.0 %) |
IMiD therapy | 0 (0%) | 11 (78.5%) | 11 (27.5 %) |
No therapy | 26 (100%) | 0 (0%) | 26 (65.0 %) |
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Kulikowska de Nałęcz, A.; Ciszak, L.; Usnarska-Zubkiewicz, L.; Frydecka, I.; Pawlak, E.; Szmyrka, M.; Kosmaczewska, A. Deregulated Expression of Immune Checkpoints on Circulating CD4 T Cells May Complicate Clinical Outcome and Response to Treatment with Checkpoint Inhibitors in Multiple Myeloma Patients. Int. J. Mol. Sci. 2021, 22, 9298. https://doi.org/10.3390/ijms22179298
Kulikowska de Nałęcz A, Ciszak L, Usnarska-Zubkiewicz L, Frydecka I, Pawlak E, Szmyrka M, Kosmaczewska A. Deregulated Expression of Immune Checkpoints on Circulating CD4 T Cells May Complicate Clinical Outcome and Response to Treatment with Checkpoint Inhibitors in Multiple Myeloma Patients. International Journal of Molecular Sciences. 2021; 22(17):9298. https://doi.org/10.3390/ijms22179298
Chicago/Turabian StyleKulikowska de Nałęcz, Anna, Lidia Ciszak, Lidia Usnarska-Zubkiewicz, Irena Frydecka, Edyta Pawlak, Magdalena Szmyrka, and Agata Kosmaczewska. 2021. "Deregulated Expression of Immune Checkpoints on Circulating CD4 T Cells May Complicate Clinical Outcome and Response to Treatment with Checkpoint Inhibitors in Multiple Myeloma Patients" International Journal of Molecular Sciences 22, no. 17: 9298. https://doi.org/10.3390/ijms22179298