The Impact of Immune Checkpoint Therapy on the Latent reservoir in HIV Infected Individuals with Cancer on Antiretroviral Therapy.

Abstract

Objective:

To quantify HIV specific immunological and virological changes in people living with HIV (PLWH) on antiretroviral therapy (ART) with malignancy who received immune checkpoint blockade (ICB).

Design:

Observational cohort study

Methods:

Blood samples were collected before and after four cycles of ICB in HIV positive adults on ART. Virological assessments performed on CD4+ T cells included: cell associated (CA) unspliced (US) HIV RNA, cell associated (CA) HIV DNA, Tat/rev Induced Limiting Dilution Assay (TILDA), and plasma HIV RNA using a single copy assay (SCA). Flow cytometry was used to assess the frequency of precursor exhausted T cells (Tpex) and exhausted T cells (Tex), and Gag-specific CD4+ and CD8+ T cells positive for IFN-γ, TNF-α, or CD107a by intracellular cytokine staining (ICS).

Results:

Participant (P)1 received avelumab (anti-PD-L1) for Merkel cell carcinoma. P2 and P3 received ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) for metastatic melanoma.

An increase in CA-US RNA following each infusion was noted in all 3 participants. There were no consistent changes in HIV DNA or the proportion of cells with inducible MS HIV RNA. P2 demonstrated a striking increase in the frequency of gag-specific central and effector memory CD8+ T cells producing IFN-γ, TNF-α, and CD107a following anti-PD1 and anti-CTLA-4. The frequency of CD8+ Tpex cells pre-ICB was also highest in this participant.

Conclusions:

In three PLWH with cancer on ART, we found that ICB activated latent HIV and enhanced HIV-specific T cell function but with considerable variation.

Introduction

People living with HIV (PLWH) require life-long antiretroviral therapy (ART) due to HIV persistence in long-lived CD4+ T cells. Immune checkpoints such as programmed death (PD)-1 and cytotoxic T lymphocyte-associated protein 4 (CTLA-4), are inhibitory receptors that limit T cell function and also play a role in the establishment and maintenance of HIV latency ^[1]. One strategy to eliminate latent infection is activating HIV expression in latently infected cells to promote virus-induced cytolysis or immune-mediated killing.

In people living with HIV on ART, latent HIV is enriched in CD4+ T cells expressing immune checkpoints, including PD1 and CTLA-4 ^[2]. Blocking these pathways can activate latent virus in in vitro and ex vivo HIV models ^[3]. In addition, there is increased expression of PD1 in total and HIV-specific T cells in PLWH on ART leading to decreased function ^[4], but antibodies to these proteins can increase HIV-specific T cell function when administered ex vivo to peripheral blood mononuclear cells (PBMCs) collected from PLWH ^[5]. In a monkey model of simian immunodeficiency virus (SIV) on ART, the administration of anti-PD1 together with anti-CTLA-4 resulted in greater latency reversal compared to either antibody alone, but did not enhance SIV-specific T cell function or delay viral rebound upon stopping ART ^[6].

Antibodies to PD1, the ligand for PD1 (PDL1) and CTLA-4 are licensed for the management of multiple malignancies. Although they appear to have a similar safety and anti-tumour profile in PLWH ^[7], there is limited knowledge on their effect on HIV latency and HIV-specific immunity. Here, we describe the effects of immune checkpoint blockade (ICB) on HIV latency and HIV-specific T cell function in a prospective study of PLWH on ART who received antibodies to either PD1, PDL1 and/or CTLA-4 for cancer.

Methods

Study participants

We conducted a prospective study of PLWH on suppressive ART, who initiated anti-PD1, anti-PDL1 and/or anti-CTLA-4 for cancer at tertiary hospitals in Melbourne, Australia. Sampling was performed before, 24 hours after, and 7 days after the first four infusions and 8 weeks after the 4th infusion of immune checkpoint therapy. The study was approved by the Alfred Health Human Research and Ethics Committee; informed consent was obtained from all participants.

Virology and immunology assays

Cell-associated unspliced HIV RNA (CA-US HIV RNA) and HIV DNA were quantified by real time polymerase chain reaction and plasma HIV RNA measured using a single copy assay (SCA) as previously described^{[8, 9]}(see also supplementary methods). Tat/rev Induced Limiting Dilution Assay (TILDA) was used to estimate the proportion of cells containing inducible multiply-spliced (MS) HIV RNA as a measure of the inducible HIV reservoir^[10] (supplementary methods).

HIV-specific T cell responses were quantified following incubation with overlapping gag and nef peptides together with intracellular cytokine staining (ICS) for IFN-γ, TNF-α, and CD107a (supplementary methods, supplementary figure 1). T cell proliferation was measured by CD4+ and CD8+ T cell expression of Ki67 using flow cytometry, (Supplementary Figure 2, supplementary methods). The frequencies of precursor exhausted T cells (Tpex) and exhausted T cells (Tex) amongst the pool of proliferating CD8 T cells were measured using flow cytometry (supplementary methods). Tpex were defined as PD-1+, CXCR5+ cells expressing transcriptional regulator T cell factor 1 (TCF-1), and Tex as PD-1+ and Tim-3+ cells respectively.

Cytokines, chemokines and soluble cytokine receptors were measured in plasma using multiplex panels from Milliplex (supplementary methods). Due to the rarity of the population being studied, which limited our sample size of this study, a descriptive analysis was performed without formal statistical tests.

Results

Clinical cases

Participant 1 (P1) was a 76-year old man with HIV diagnosed in 2007 and plasma HIV-RNA <20 copies/ml for 9 years on tenofovir disoproxil fumarate (TDF), emtricitabine (FTC) and efavirenz (EFV). Malignant Merkel cell carcinoma of the chest wall, axilla and liver was diagnosed in 2016 and treated with 5 cycles of carboplatin and etoposide. Due to disease progression he received avelumab (anti-PDL1) 10mg/kg every two weeks. After initial clinical response, he had disease progression and died 8 months after commencing avelumab.

Participant 2 (P2) was a 56-year old man diagnosed with HIV in 2001 and plasma HIV-RNA <20 copies/ml for 11 years on TDF/FTC/EFV. Malignant melanoma on his upper back was diagnosed in 2012 and managed with local excision. Widespread metastases were diagnosed in 2017, and he received dabrafenib (BRAF-inhibitor) and trametinib (Mitogen-Activated Protein Kinase 2 inhibitor), followed by ipilumumab (anti-CTLA-4) 1mg/kg and nivolumab (anti-PD1) 3mg/kg every three weeks. There was no clear response after four cycles, and he died within four months of commencing anti-PD1 and anti-CTLA-4.

Participant 3 (P3) was a 68-year-old man diagnosed with HIV in 2003, had plasma HIV-RNA <20 copies/ml for 10 years and was receiving elvitegravir (EVG), cobicistat (COBI), tenofovir alafenamide (TAF), and FTC at enrolment. Malignant melanoma was diagnosed in 2018 and managed with local excision. Brain metastases were diagnosed 7 months later. He was treated with radiotherapy, then ipilumumab 1mg/kg and nivolumab 3mg/kg every three weeks. P3 responded to anti-PD1 and anti-CTLA-4 and is maintained on nivolumab 3mg/kg every two weeks.

No immune related adverse events were reported in any of the participants. P1 and P3 continued to receive immune checkpoint therapy at the end-of-study visit. P2 died before the end-of-study visit. Patient demographics and clinical results are summarized in Table 1.

Table 1.

Participant demographics, baseline data and clinical outcomes.

	P1	P2	P3
Age/sex	76/m	56/m	68/m
Malignancy	Merkel Cell Carcinoma	Metastatic Melanoma	Metastatic Melanoma
Previous therapy	Carboplatin and etoposide	Dabrafenib and trametinib	Radiotherapy
Immune checkpoint blockade	Avelumab (anti-PDL1) 10mg/kg 2 weekly	Nivolumab (anti-PD1) 3mg/kg + Ipilimumab (anti-CTLA4) 1mg/kg 3 weekly	Nivolumab 3mg/kg + Ipilimumab 1mg/kg 3 weekly
Year of HIV Diagnosis	2007	2001	2003
ART at enrolment	TDF/3TC/EFV	TDF/3TC/EFV	EVG /COBI/TAF/FTC
CD4 nadir	53 cells/uL	210 cells/uL	125 cells/uL
Baseline CD4	323 cells/uL	468 cells/uL	265 cells/uL
Progress	Partial response Died 4 months after ICB commenced	Disease progression Died 2 months after ICB commenced	Disease responsive Maintenance nivolumab

P, participant; VL, viral load; BL, baseline; TDF, tenofovir disoproxil fumarate; 3TC, lamivudine; FTC, emtricitabine; EFV, efavirenz; EVG, elvitegravir; COBI, cobicistat; TAF, tenofovir alafenamide; ICB, immune checkpoint blockade.

Increases in CA-US HIV RNA after each infusion but no consistent changes in HIV DNA

To address whether immune checkpoint blockade reversed HIV latency, we quantified the level of CA-US RNA and found an increase following each infusion in all three participants (Fig 1A), with a mean fold-change from baseline to 24 hours post-infusion of 1.3 (range 1.08–2.57) in cycle one, 3.1 (1.45–5.46) in cycle two, 6.8 (1.95–13.69) in cycle 3 and 8.6 (1.00–16.06) in cycle four (Fig 1B). Plasma HIV RNA did not increase in P1 or P2, but in P3 rose from 4 copies/mL at baseline to 16 and 8 copies/mL post-infusion in cycle two and three, respectively (Fig 1C).

Figure 1. Virological and immunological effects of immune checkpoint blockade.

Levels of cell-associated unspliced HIV RNA around each infusion of immune checkpoint antibodies in Log-10 scale (a) with corresponding fold-changes from baseline to 24 h post each ICB infusion (b). Levels of plasma HIV RNA measured by single copy assay (c), levels of cell-associated total HIV DNA (d) and the proportion of cells with inducible MS RNA as measured by TILDA (e). The frequency CD8+ effector memory cells expressing CD107a, IFNg and TNFa following stimulation with gag peptides is shown for participant 2 in (f), the frequency of triple-positive polyfunctional CD8+ T cells for all three participants in (g) and changes in Ki67+CD8+ and total CD3+ T cells in (h). The gating strategy for detecting Tpex and Tex cells is shown in (i) and the frequencies of Tpex and Tex cells during ICB in (j).

BL, baseline; EOS, end of study; ICB, immune checkpoint blockade; TILDA, tat/rev induced limiting dilution assay; IFNg, interferon gamma; TNFa, tumor necrosis factor alpha; Tpex, precursor exhausted T cells; Tex, exhausted T cells.

Overall there were no consistent changes in HIV DNA or the proportion of cells with inducible MS RNA as measured by TILDA during immune checkpoint therapy (Fig 1D–E). For P1 and P2 specifically, inducible MS RNA was below the level of quantification both at baseline and at end of study (Fig. 1E). In P3 we observed a 55% reduction in HIV DNA and a 33% reduction in the proportion of cells with inducible MS RNA as measured by TILDA from baseline to the final study bleed after the fourth infusion, however these changes could be due to assay or sampling variation and may not represent a treatment effect on the HIV reservoir.

HIV-specific immune responses, T cell proliferation and Tpex/Tex dynamics

In participant 2 (P2), we detected a striking increase during combination anti-PD1/anti-CTLA-4 in the frequency of central and effector memory CD8+ T cells producing IFN-γ, TNF-α, and CD107a in response to gag-stimulation (Fig. 1f and Supplementary Figure 3A–C), as well as an increase in the proportion of polyfunctional HIV specific CD8+ T cells during therapy (Fig. 1g), which was not seen in P1 and P3. In P2, we also found an increase in effector memory CD4+ T cells (memory T cell subsets defined on the basis of CD45RA and CCR7, (supplementary methods) producing IFN-g and TNF-a (Supplementary Figure 3D).

Participant 2 displayed a sustained increase in CD8+ T cells expressing Ki67 (3.44% to 7.16%) from baseline to end of study (Fig 1H). We assessed frequencies of Tpex and Tex cells during ICB to explore the potential role of these cells in the HIV-specific T cell response in PLWH, given recent findings that Tpex is a key CD8+ T-cell subset responsible for the proliferative burst and increased effector functions after blocking PD-1 in malignancy ^[11]. We found that the frequency of CD8+ Ki67+ Tpex cells pre-ICB was highest in P2 (Fig. 1i, j), the same participant who had a dramatic increase in HIV-specific T cells following anti-PD1 and anti-CTLA-4. In addition, the frequency of CD8+ Ki67+ Tex cells increased during immune checkpoint therapy for all three participants (Fig. 1j). We noted no consistent change in total cell counts of CD3+, CD8+, Ki67+ CD8+ T cells, Tpex or Tex cells (Supplementary figure 4), but some caution is needed in interpreting these findings, given these numbers are influenced by sampling volumes. Finally, we did not see any consistent changes in plasma cytokines during immune checkpoint therapy, whereby these data clustered more strongly by participant, than by timepoint relative to therapy (Supplementary Figure 5).

Discussion

In this case-series of PLWH on ART receiving immune checkpoint therapy, we report reversal of HIV-latency evidenced by increased CA-US RNA in all 3 participants following anti-PDL1 (P1) or combination anti-PD1 and anti-CTLA-4 (P2 and P3). We found no consistent effect on the HIV reservoir but did note moderate decreases in HIV DNA and TILDA (estimates of the HIV reservoir) in one individual. In another participant we found a substantial increase in HIV-specific CD8+ T cell responses during anti-PD1 and anti-CTLA-4. Our study thus illustrates that immune checkpoint therapy can reverse HIV latency and that combination anti-PD1 and anti-CTLA-4 can enhance HIV-specific T cell function, but significant variation was noted between individuals.

Previous studies have shown an increase in CA-US HIV RNA following anti-CTLA-4 ^[12] or anti-PD1 ^{[1, 13]} in PLWH on ART but this has not been consistently assessed or observed^[13]. Similarly, a sustained reduction in HIV DNA has only been reported in one individual^[14]. Despite substantial relative increases in US RNA in P1 and P2, this did not lead to increased levels of plasma HIV RNA. This may reflect that cell-associated HIV RNA is a more sensitive measure of latency reversal, but also that induced US RNA may contain read-through transcripts, incomplete transcripts and/or defective sequences that don’t give rise to viral particles, as we have recently demonstrated ex vivo ^[15].

Concomitant administration of anti-PD1 and anti-CTLA-4 in this population has not been previously reported. A study of ART-treated SIV-infected monkeys found that the combination of anti-PD1 and anti-CTLA-4 reversed latency more potently than either antibody alone, but did not have an impact on SIV specific T cell responses ^[6]. Increased HIV specific immune responses during anti-PD1 (nivolumab) have been reported in two individuals ^{[14, 16]}. Also, in a study of anti-PDL1 in PLWH without cancer, a single low-dose increased Gag-specific CD8+ T cells in two of six participants but did not reverse HIV-latency ^[17].

Tpex were recently identified as a CD8+ T cell subset responsible for the proliferative burst and increased effector functions after blocking PD1 ^[11]. Our finding of a higher Tpex frequency in the pool of cells with proliferative potential (Ki67+) pre-ICB in the same individual with enhanced HIV-specific T cell function might indicate a similar relationship for anti-HIV immunity, however this was not reflected in a reduction of HIV DNA. We note, though, that this individual did not have a favourable antitumour response to immune checkpoint therapy. The ratio of Tex cell reinvigoration to tumour burden is associated with response to anti-PD1 in cancer patients ^[18]. Amongst proliferating cells we observed an increase in Tex cell frequencies during ICB in all participants, but noted varied clinical responses.

While the small number of participants limits our ability to draw generalisable conclusions from our findings, this reflects the rarity of this cohort of PLWH receiving immune checkpoint therapy for cancer. It also underlines the importance of studying the impact of immune checkpoint blockade on HIV specific immunological and virological responses in this population. Furthermore, NK cells are an emerging target for immune checkpoint inhibition, and blockade of CTLA-4, PD-1 and PD-L1 has been associated with antibody-dependent cell-mediated cytotoxicity (ADCC) or killing of malignantly transformed and virally infected cells ^[19]. It was not possible to measure NK cell activation and ADCC in this study due to limited available samples.

In summary, our results demonstrate that blocking PD1 and/or CTLA-4 can activate latent HIV and in some individuals enhance HIV-specific immune response. Further research and larger controlled studies are needed to fully understand the impact of ICB on HIV-specific T cell function, on elimination of HIV-infected cells and to identify biomarkers of an effective response.