No Recovery Of Vα7.2 MAIT Cells After Therapeutic Immunisation … · 2020. 4. 19. · No Recovery...
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No Recovery Of Vα7.2 MAIT Cells After Therapeutic Immunisation Plus IL-2/GM-CSF/rhGH Louise Greathead, Alexander Cocker, Anna Herasimtschuk, Sundhiya Mandalia, Peter Kelleher, Nesrina Imami Centre of Immunology & Vaccinology, Chelsea and Westminster Campus, Imperial College London, [email protected] INTRODUCTION Mucosal-associated invariant T (MAIT) cells are a recently described T cell subset enriched in mucosal tissue (liver, lung, intestinal epithelium) and involved in the defence against bacteria at mucosal surfaces 1 . They express the invariant TCRα chain (Vα7.2-Jα 33/12/20) and variable but restricted TCRβ chains and recognise small molecule vitamin B metabolites presented by the MHC-class I-like molecule MR1 2 . In HIV infection blood MAIT cells are reduced compared to healthy controls and do not recover on antiretroviral therapy (ART) 3 . Any remaining MAIT cells from HIV patients have impaired function (IFNγ and TNF production in response to bacteria, reduced granzyme A, B, CD107a and perforin) 3 . The loss of MAIT cells from mucosal tissue in HIV patients may contribute to systemic immune activation 4 . Therapeutic immunisation (GTU-MultiHIV Clade B DNA vaccine) together with interleukin-2 (IL-2), granulocyte-macrophage colony-stimulating factor (GM-CSF) and recombinant human growth hormone (rhGH), has been shown to reduce immune activation and improve HIV-1-specific T- cell responses in ART-treated HIV-1 infected individuals 5 . In this study we assessed the ability of this regimen to reconstitute MAIT cell populations. METHODS RESULTS 1. Mucosal-associated invariant T (MAIT) cells: an evolutionarily conserved T cell subset. E.Treiner et al. Microbes and Infection, 7(3):552-59, 2005. 2. MR1 presents microbial vitamin B metabolites to MAIT cells. Kjer- Nielsen et al. Nature, 491:717-23, 2012. 3. The role of mucosal-associated invariant T cells in infectious diseases. E Wong et al. Immunology, 150(1):45-54, 2017. 4. CD8+/CD161++ mucosal-associated invariant T-cell levels in the colon are restored on long-term antiretroviral therapy and correlate with CD8+ T-cell immune activation. L Greathead et al. AIDS, 17;28(11):1690-2, 2014. 5. Therapeutic immunisation plus cytokine and hormone therapy improves CD4 T cell counts, restores anti-HIV-1 responses and reduces immune activation in treated chronic HIV-1 infection. A Herasimtschuk et al. Vaccine, 5;32(51):7005-13, 2014. The immunotherapy trial was funded by the MRC (Grant number G0501957). This work was funded by the Westminster Medical School Research Trust and St Stephen’s AIDS Trust. We are grateful to all the patients that took part in the study and to the staff at Chelsea and Westminster Hospital. References Acknowledgements Immunotherapy trial treatment schedule overview: Patients were eligible to participate if they were over 18 years of age, chronically infected with HIV-1, on a stable ART regimen for ≥6 months, with CD4 T-cell counts >400 cells/mm 3 blood, and plasma HIV-1 RNA <50 copies/ml. Subjects could not be receiving or have received immunomodulatory drugs or immunisation. Patients who met the eligibility criteria were randomised into one of three arms of the trial: Arm 1) to receive the GTU®-MultiHIV DNA Clade B vaccine (FIT Biotech Plc, Tampere, Finland) at baseline, followed by administration of IL-2 (Aldesleukin, Proleukin; Novartis, Camberley, UK) and GM-CSF (Sargramostim, Leukine™; Berlex, Seattle, WA) for five days during week 1, and rhGH (Somatropin, Saizen™; Merck-Serono International, Geneva, Switzerland) for five days during week 2, with vaccine further administered at weeks 6 and 12; Arm 2) to receive vaccine alone; or Arm 3) to receive cytokines and rhGH alone, at the aforementioned time points. Vaccine was administered at 1mg/ml as 10 intradermal injections (five 100μl injections per arm); IL-2 given twice daily, 5x10 6 Units, administered by subcutaneous injection, 8h apart; GM-CSF, 150μg, was administered subcutaneously once daily, 4h from the IL-2; and rhGH self-administered subcutaneously daily at 4mg/day. Cryopreserved PBMCs from week 0, 2 and 48 were used for the current study. ART Sample collection 0 1 2 4 6 8 12 Week 16 24 48 Randomisation Therapeutic immunisation IL-2 and GM-CSF rhGH Therapeutic immunisation IL-2 and GM-CSF rhGH • Plasma HIV-1 RNA < 50 copies/ml • CD4 T-cell counts > 400 cells/mm 3 blood • Age > 18 years • On ART ≥ 6 months Baseline characteristics Arm 1: n=3 Arm 2: n=4 Arm 3: n=5 Figure 1. Immunotherapy trial overview • For trial design and eligibility see Figure 1. From 93 patient referrals and 21 screen visits, 12 patients meeting eligibility criteria were enrolled onto the trial. • Flow cytometry was performed on cryopreserved peripheral blood mononuclear cells (PBMCs) taken from patients at baseline, week 2 and week 48. MAIT cells were identified as live CD3+CD45RO+ T cells co- expressing the invariant T cell receptor Vα7.2 together with the C-type lectin CD161 (Figure 2). CD4 and CD8 memory T cell expression of CD38 was used as a marker of T-cell activation. • Wilcoxon matched-pairs signed rank test, and random intercept model using MIXED procedure in SAS with 95% confidence interval, were used to evaluate changes from baseline to week 2 and week 48. Differences between groups were assessed with Kruskall Wallis and Dunn’s post test. Correlations were performed using Spearman’s Rho. Patient short code Group Graph symbol Age (years) Gender Clade of infection Length of time since diagnosis (months) Duration of ART (months) ART regimen CD4 T-cell count (cells/mm 3 ) Plasma viral load at baseline (copies/ml ) Nadir Baseline R771 1 64 M B 160.13 139.05 FTC+TFV+EFV 391 884 <50 B784 1 40 M B 99.25 98.95 FTC+TFV+EFV 80 1332 <50 G739 1 43 M B 141.61 113.15 FTC+TFV+EFV 227 534 <50 P087 2 50 M B 172.95 162.75 FTC+TFV+EFV 210 731 <50 C789 2 29 M B 20.75 12.20 FTC+TFV+EFV 309 535 <50 L043 2 47 M B 90.69 45.87 FTC+TFV+EFV 303 782 <50 C319 2 48 M B 229.05 80.03 FTC+TFV+ETV 93 1077 <50 F810 3 ▲ 50 M B 233.41 193.54 FTC+TFV+NVP 166 582 <50 O523 3 ▲ 53 F C 84.10 62.89 FTC+TFV+ETR 284 892 <50 S648 3 ▲ 52 M B 300.39 158.43 TFV+DRV+RTV 227 578 <50 C241 3 ▲ 47 M B 144.23 87.18 FTC+TFV+DRV+RT V 180 466 <50 P054 3 ▲ 33 M B 40.80 28.80 FTC+TFV+EFV 200 840 <50 Mean 48 142.92 93.07 219 757 IQR 42-51 89.04- 186.98 58.63- 143.89 177- 289 567- 886 Table 1. Baseline characteristics of patients enrolled on the trial (n=12) IQR – interquartile range; FTC – emtricitabine; TFV – tenofovir; EFV – efavirenz; NVP – nevirapine; ETR – etravirine; DRV – darunavir; RTV – ritonavir. Figure 2. MAIT cell identification on cryopreserved PBMC -8 -6 -4 -2 0 2 4 6 Change in MAIT %CD3 from baseline Arm 1 Arm 2 Arm 3 Week 2 Week 48 Example MAIT cell gating (C789). After doublet discrimination and dead cell exclusion MAIT cells were identified as Va7.2+CD161+ dual positive memory (CD45RO) CD3 T cells. The majority of MAIT cells expressed CD8. Figure 3. MAIT cell frequencies, nadir CD4, duration of ART and T cell activation Baseline characteristics of the 12 patients recruited to the study are shown in Table 1. Median CD3 MAIT cell frequency at baseline was 1.49% (0.49-2.96%). The majority of MAIT cells were CD8 positive (Figure 2). There was no correlation between MAIT cell frequency and either T cell activation, nadir CD4 count or length of time on ART (Figure 3). Median CD8 T cell activation at baseline was 4.22% (2.06- 6.79). The ratio of CD4/CD8 memory T cells rose significantly at week 2 (p=0.002) and was maintained at week 48 (p=0.005) compared to baseline in the combined group of patients (Figure 4). CD4 and CD8 T cell activation (CD38+) also significantly rose at week 2. There was a significant fall of CD8 T cell activation at week 48 compared to baseline (p=0.021) in the combined group. MAIT cell frequency fell to 0.85% (0.46-1.71, p=0.021) at week 2 before rising to 1.32% (0.57-2.64) at week 48 in the combined group. At week 48 only patients in group 1, randomised to receive IL-2/GM-CSF and rhGH with vaccine, achieved significantly elevated CD4 counts compared to baseline (281 cells/μl, (78-484), p=0.008) 5 . CD38 expression on CD4 T cells was significantly reduced in group 1 (p=0.019) and group 3 (p=0.025) at week 48 compared to baseline. However only group 3 had significantly reduced CD8 activation by week 48 (p=0.014) 5 . We observed no significant change in CD3 or CD8 MAIT cell frequency in patients treated with IL- 2/GM-CSF and rhGH, either with (group 1), or without (group 3) vaccine (Figure 5) and we conclude that while this regimen improves some clinical parameters it fails to restore MAIT cell populations. Baseline MAIT cell frequency was not associated with Nadir CD4 count, duration of ART or CD8 T cell activation (CD38+). Figure 5. No change in MAIT cell frequency at week 48 -15 -10 -5 0 5 10 15 Change in MAIT %CD8 from baseline Arm 1 Arm 2 Arm 3 Week 2 Week 48 Figure 4. Change in CD4/CD8 ratio, T cell activation and MAIT cell frequency from baseline to week 2 and week 48 Error bars are 95% CI, Point estimates derived using MIXED procedure in SAS. A statistically significant difference compared to baseline can be observed where the 95% CI does not cross the line of origin.
Transcript of No Recovery Of Vα7.2 MAIT Cells After Therapeutic Immunisation … · 2020. 4. 19. · No Recovery...
No Recovery Of Vα7.2 MAIT Cells After Therapeutic Immunisation Plus IL-2/GM-CSF/rhGH
Louise Greathead, Alexander Cocker, Anna Herasimtschuk, Sundhiya Mandalia, Peter Kelleher, Nesrina Imami Centre of Immunology & Vaccinology, Chelsea and Westminster Campus, Imperial College London, [email protected]
INTRODUCTION Mucosal-associated invariant T (MAIT) cells are a recently described T cell subset enriched in mucosal tissue (liver, lung, intestinal epithelium) and involved in the defence against bacteria at mucosal surfaces1. They express the invariant TCRα chain (Vα7.2-Jα 33/12/20) and variable but restricted TCRβ chains and recognise small molecule vitamin B metabolites presented by the MHC-class I-like molecule MR12. In HIV infection blood MAIT cells are reduced compared to healthy controls and do not recover on antiretroviral therapy (ART)3. Any remaining MAIT cells from HIV patients have impaired function (IFNγ and TNF production in response to bacteria, reduced granzyme A, B, CD107a and perforin)3. The loss of MAIT cells from mucosal tissue in HIV patients may contribute to systemic immune activation4. Therapeutic immunisation (GTU-MultiHIV Clade B DNA vaccine) together with interleukin-2 (IL-2), granulocyte-macrophage colony-stimulating factor (GM-CSF) and recombinant human growth hormone (rhGH), has been shown to reduce immune activation and improve HIV-1-specific T-cell responses in ART-treated HIV-1 infected individuals5. In this study we assessed the ability of this regimen to reconstitute MAIT cell populations.
METHODS
RESULTS
1. Mucosal-associated invariant T (MAIT) cells: an evolutionarily conserved T cell subset. E.Treiner et al. Microbes and Infection, 7(3):552-59, 2005.
2. MR1 presents microbial vitamin B metabolites to MAIT cells. Kjer-Nielsen et al. Nature, 491:717-23, 2012.
3. The role of mucosal-associated invariant T cells in infectious diseases. E Wong et al. Immunology, 150(1):45-54, 2017.
4. CD8+/CD161++ mucosal-associated invariant T-cell levels in the colon are restored on long-term antiretroviral therapy and correlate with CD8+ T-cell immune activation. L Greathead et al. AIDS, 17;28(11):1690-2, 2014.
5. Therapeutic immunisation plus cytokine and hormone therapy improves CD4 T cell counts, restores anti-HIV-1 responses and reduces immune activation in treated chronic HIV-1 infection. A Herasimtschuk et al. Vaccine, 5;32(51):7005-13, 2014.
The immunotherapy trial was funded by the MRC (Grant number G0501957). This work was funded by the Westminster Medical School Research Trust and St Stephen’s AIDS Trust. We are grateful to all the patients that took part in the study and to the staff at Chelsea and Westminster Hospital.
References
Acknowledgements
Immunotherapy trial treatment schedule overview: Patients were eligible to participate if they were over 18 years of age, chronically infected with HIV-1, on a stable ART regimen for ≥6 months, with CD4 T-cell counts >400 cells/mm3 blood, and plasma HIV-1 RNA <50 copies/ml. Subjects could not be receiving or have received immunomodulatory drugs or immunisation. Patients who met the eligibility criteria were randomised into one of three arms of the trial: Arm 1) to receive the GTU®-MultiHIV DNA Clade B vaccine (FIT Biotech Plc, Tampere, Finland) at baseline, followed by administration of IL-2 (Aldesleukin, Proleukin; Novartis, Camberley, UK) and GM-CSF (Sargramostim, Leukine™; Berlex, Seattle, WA) for five days during week 1, and rhGH (Somatropin, Saizen™; Merck-Serono International, Geneva, Switzerland) for five days during week 2, with vaccine further administered at weeks 6 and 12; Arm 2) to receive vaccine alone; or Arm 3) to receive cytokines and rhGH alone, at the aforementioned time points. Vaccine was administered at 1mg/ml as 10 intradermal injections (five 100µl injections per arm); IL-2 given twice daily, 5x106 Units, administered by subcutaneous injection, 8h apart; GM-CSF, 150µg, was administered subcutaneously once daily, 4h from the IL-2; and rhGH self-administered subcutaneously daily at 4mg/day. Cryopreserved PBMCs from week 0, 2 and 48 were used for the current study.
ART
Sample collection
0 1 2 4 6 8 12 Week 16 24 48
Randomisation
Therapeutic immunisation
IL-2 and GM-CSF
rhGH
Therapeutic immunisation
IL-2 and GM-CSF
rhGH
• Plasma HIV-1 RNA
< 50 copies/ml • CD4 T-cell counts
> 400 cells/mm3 blood • Age > 18 years • On ART ≥ 6 months
Baseline characteristics
Arm 1: n=3
Arm 2: n=4
Arm 3: n=5
Figure 1. Immunotherapy trial overview
• For trial design and eligibility see Figure 1. From 93 patient referrals and 21 screen visits, 12 patients meeting eligibility criteria were enrolled onto the trial.
• Flow cytometry was performed on cryopreserved peripheral blood mononuclear cells (PBMCs) taken from patients at baseline, week 2 and week 48. MAIT cells were identified as live CD3+CD45RO+ T cells co-expressing the invariant T cell receptor Vα7.2 together with the C-type lectin CD161 (Figure 2). CD4 and CD8 memory T cell expression of CD38 was used as a marker of T-cell activation.
• Wilcoxon matched-pairs signed rank test, and random intercept model using MIXED procedure in SAS with 95% confidence interval, were used to evaluate changes from baseline to week 2 and week 48. Differences between groups were assessed with Kruskall Wallis and Dunn’s post test. Correlations were performed using Spearman’s Rho.
Patient short code
Group Graph
symbol Age
(years) Gender
Clade of infection
Length of time since
diagnosis(months)
Duration of ART
(months) ART regimen
CD4 T-cell count (cells/mm3)
Plasma viral load
at baseline (copies/ml
) Nadir Baseline
R771 1 64 M B 160.13 139.05 FTC+TFV+EFV 391 884 <50
B784 1 40 M B 99.25 98.95 FTC+TFV+EFV 80 1332 <50
G739 1 43 M B 141.61 113.15 FTC+TFV+EFV 227 534 <50
P087 2 50 M B 172.95 162.75 FTC+TFV+EFV 210 731 <50
C789 2 29 M B 20.75 12.20 FTC+TFV+EFV 309 535 <50
L043 2 47 M B 90.69 45.87 FTC+TFV+EFV 303 782 <50
C319 2 48 M B 229.05 80.03 FTC+TFV+ETV 93 1077 <50
F810 3 ▲ 50 M B 233.41 193.54 FTC+TFV+NVP 166 582 <50
O523 3 ▲ 53 F C 84.10 62.89 FTC+TFV+ETR 284 892 <50
S648 3 ▲ 52 M B 300.39 158.43 TFV+DRV+RTV 227 578 <50
C241 3 ▲ 47 M B 144.23 87.18 FTC+TFV+DRV+RT
V 180 466 <50
P054 3 ▲ 33 M B 40.80 28.80 FTC+TFV+EFV 200 840 <50
Mean 48 142.92 93.07 219 757
IQR 42-51 89.04- 186.98
58.63- 143.89
177- 289
567- 886
Table 1. Baseline characteristics of patients enrolled on the trial (n=12)
IQR – interquartile range; FTC – emtricitabine; TFV – tenofovir; EFV – efavirenz; NVP – nevirapine; ETR – etravirine; DRV – darunavir; RTV – ritonavir.
Figure 2. MAIT cell identification on cryopreserved PBMC
-8
-6
-4
-2
0
2
4
6
Ch
ange
in M
AIT
%C
D3
fro
m b
ase
line
Arm 1
Arm 2
Arm 3
Week 2 Week 48
Example MAIT cell gating (C789). After doublet discrimination and dead cell exclusion MAIT cells were identified as Va7.2+CD161+ dual positive memory (CD45RO) CD3 T cells. The majority of MAIT cells expressed CD8.
Figure 3. MAIT cell frequencies, nadir CD4, duration of ART and T cell activation
Baseline characteristics of the 12 patients recruited to the study are shown in Table 1. Median CD3 MAIT cell frequency at baseline was 1.49% (0.49-2.96%). The majority of MAIT cells were CD8 positive (Figure 2). There was no correlation between MAIT cell frequency and either T cell activation, nadir CD4 count or length of time on ART (Figure 3). Median CD8 T cell activation at baseline was 4.22% (2.06-6.79). The ratio of CD4/CD8 memory T cells rose significantly at week 2 (p=0.002) and was maintained at week 48 (p=0.005) compared to baseline in the combined group of patients (Figure 4). CD4 and CD8 T cell activation (CD38+) also significantly rose at week 2. There was a significant fall of CD8 T cell activation at week 48 compared to baseline (p=0.021) in the combined group. MAIT cell frequency fell to 0.85% (0.46-1.71, p=0.021) at week 2 before rising to 1.32% (0.57-2.64) at week 48 in the combined group. At week 48 only patients in group 1, randomised to receive IL-2/GM-CSF and rhGH with vaccine, achieved significantly elevated CD4 counts compared to baseline (281 cells/μl, (78-484), p=0.008)5. CD38 expression on CD4 T cells was significantly reduced in group 1 (p=0.019) and group 3 (p=0.025) at week 48 compared to baseline. However only group 3 had significantly reduced CD8 activation by week 48 (p=0.014)5. We observed no significant change in CD3 or CD8 MAIT cell frequency in patients treated with IL-2/GM-CSF and rhGH, either with (group 1), or without (group 3) vaccine (Figure 5) and we conclude that while this regimen improves some clinical parameters it fails to restore MAIT cell populations.
Baseline MAIT cell frequency was not associated with Nadir CD4 count, duration of ART or CD8 T cell activation (CD38+).
Figure 5. No change in MAIT cell frequency at week 48
-15
-10
-5
0
5
10
15
Ch
ange
in M
AIT
%C
D8
fro
m b
ase
line
Arm 1
Arm 2
Arm 3
Week 2 Week 48
Figure 4. Change in CD4/CD8 ratio, T cell activation and MAIT cell frequency from baseline to week 2 and week 48
Error bars are 95% CI, Point estimates derived using MIXED procedure in SAS. A statistically significant difference compared to baseline can be observed where the 95% CI does not cross the line of origin.
