key: cord-1014141-e65old40 authors: Kreuger, J.; Santinon, F.; Kazanova, A.; Issa, M.; Larrivee, B.; Milhalcioiu, C.; Rudd, C. E. title: Hydroxychloroquine (HCQ) reverses anti-PD-1 immune murine checkpoint blockade: TCF1 as a marker in humans for COVID-19 and HCQ therapy date: 2020-09-30 journal: nan DOI: 10.1101/2020.09.29.20193110 sha: 688b55199107fc2aa53c13f34c9b5b4f628d6d29 doc_id: 1014141 cord_uid: e65old40 Coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a serious threat to global public health. Hydroxychloroquine (HCQ) and the antibiotic azithromycin (AZ) are still being used by thousands and numerous hospitals to treat COVID-19. In a related context, immunotherapy using checkpoint blockade (ICB) with antibodies such as anti-PD-1 has revolutionised cancer therapy. Given that cancer patients on ICB continue to be infected with SARS-CoV-2, an understanding of the effects of HCQ and AZ on the elimination of tumors by anti-PD-1 ICB is urgently needed. In this study, we report that HCQ alone, or in combination with AZ, at doses used to treat COVID-19 patients, reverses the therapeutic benefit of anti-PD-1 in controlling B16 melanoma tumor growth in mice. No deleterious effect was seen on untreated tumors, or in using AZ alone in anti-PD-1 immunotherapy. Mechanistically, HCQ and HCQ/AZ inhibited PD-L1 expression on tumor cells, while specifically targeting the anti-PD-1 induced increase in progenitor CD8+CD44+PD-1+TCF1+ tumor-infiltrating T-cells (TILs) and the generation of CD8+CD44+PD-1+ effectors. Surprisingly, it also blocked the appearance of a subset of terminally exhausted CD8+ TILs. No effect was seen on the presence of CD4+ T-cells, FoxP3+ Tregs, thymic subsets, B-cells, antibody production, myeloid cells, or the vasculature of mice. Lastly, we identified TCF-1 expression in peripheral CD8+ T-cells from cancer or non-cancer human patients infected with SARs CoV2 as a marker for the effects of COVID-19 and HCQ on the immune system. This study indicates for the first time that HCQ and HCQ/AZ negatively impact the ability of anti-PD-1 checkpoint blockade to promote tumor rejection. The outbreak of coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/2019-nCoV) poses a serious threat to global public health. The anti-malarial 4-aminoquinoline drugs chloroquine (CQ) and hydroxychloroquine (HCQ) as well as the antibiotic azithromycin (AZ) have gained much attention potential as potential therapies. The parent compound chloroquine (CQ) was originally reported to inhibit SARS-Cov1 coronavirus infection (Vincent et al., 2005) and in vitro studies have shown activity against SARS-CoV-2 Wang et al., 2020) . The exact mechanism of action of HQ and HCQ is unknown although these drugs increase the pH of endosomes that the virus uses for cell entry and can interfere with the glycosylation of the cellular receptor of SARS-CoV, angiotensin-converting enzyme 2 (ACE2) and associated gangliosides (Devaux et al., 2020) . Other mechanisms have also been proposed (Quiros Roldan et al., 2020) . CQ has a half-life of 20-60 days and can accumulate at higher levels in metabolically active tissues (Wong et al., 2020) . Similarly, a macrolide antibiotic AZ can synergise with HCQ to block viral entry into cells and decrease viral replication (Andreani et al., 2020) . Despite this, the effectiveness of HCQ in treating COVID-19 has been highly contentious (Kupferschmidt and Cohen, 2020; Lenzer, 2020) . An initial non-randomised trial showed remarkable efficacy in combination with AZ in clearing SARs-Cov2 (Gautret et al., 2020) . Other studies have produced conflicting results (Chen et al., 2020a; Chen et al., 2020b; Gao et al., 2020; Geleris et al., 2020; Magagnoli et al., 2020; Mahevas et al., 2020; Molina et al., 2020; Rosenberg et al., 2020; Tang et al., 2020; Yu et al., 2020) . Recently, the Henry Ford Covid-19 Task Force found that when controlled for COVID-19 risk factors, HCQ alone and in combination with AZ was associated with a reduction in mortality (Arshad et al., 2020) . However, adverse cardiovascular effects may predispose certain patient groups to ventricular arrhythmias (Giudicessi et al., 2020; Ray et al., 2012) . The US Federal Drug Administration (FDA) and the World Health Organisation (WHO) halted HCQ trials due to a lack of efficacy in reducing death rates of already hospitalised patients (https://covid19treatment guidelines.nih.gov/therapeutic-options-under-investigation). However, concerns about side-effects were then influenced by the withdrawal of papers (Mehra et al., 2020a; Mehra et al., 2020b) . The FDA has granted emergency use authorisation for HCQ in the treatment of a limited number of hospitalised cases but has cautioned against its use outside of a clinical trial or hospital setting. In this context, aside from the treatment of malaria, HCQ has also been used widely over the past decade at lower concentrations to treat auto-immune diseases such as systemic lupus erythematosus and rheumatoid arthritis where it can reduce inflammation (Thome et al., 2014; Thome et al., 2013) . Given this situation, HCQ is still being used by many countries and thousands of patients globally to treat There are more than 200 trials presently underway around the world on its impact either as a prophylactic or for treatment for COVID-19. The efficacy may vary with the dose used in the various studies. One recent study from the multi-center COVID-19 RISK and Treatments (CORIST) Collaboration, using 400-600 mg of HCQ day found that treatment was associated with reduced mortality (Di Castelnuovo et al., 2020) . Another recent study from Belgium using similar doses also reported reduced mortality (Catteau et al., 2020) . The doses and onset of treatment in these studies differed from the UK RECOVERY trial where 1600 mg on the first day and 800 mg on days 2-9 were given (Horby et al., 2020) . Overall, there is an interest whether HCQ might be more effective if used early in infection, before the need for hospitalisation. A large COPCOV trial on the role of HCQ as a prophylactic for COVID-19 health workers was recently re-approved by the regulatory authorities in the United Kingdom (https://www.tropmedres.ac/covid-19/copcov). Funded by the COVID-19 Bill & Melinda Gates Foundation, Wellcome and Mastercard Therapeutics Accelerator grant, the COPCOV study will enroll over 40,000 frontline health care workers who have close contact with patients to determine whether chloroquine or hydroxychloroquine are effective in preventing Many countries continue to use HCQ to treat COVID-19 health workers who are suspected or confirmed of infection (Nina and Dash, 2020) . HCQ and CQ have been reported to have mixed and contradictory effects on the immune system. One report showed that CQ enhances human CD8+ T cell responses (Accapezzato et al., 2005) , while another showed that HCQ inhibits CD4+ T-cell activation (Wu et al., 2017) . HCQ is well known to inhibit autophagy (Bonam et al., 2020) , may reduce the efficacy of antigen-presentation (Thome et al., 2014; Ziegler and Unanue, 1982) and decrease the production of proinflammatory Th17relatedccytokines (Wozniacka et al., 2008) . Another report showed that HCQ enhanced the function of suppressive regulatory T cells (Tregs) (Thome et al., 2013) while others have reported that HCQ increases the presence of Tregs and plasmacytoid dendritic cells together with a decrease in activated CD4+ T-cells and plasmacytoid dendritic cells in patients infected with the human immunodeficiency virus (HIV-1) (Piconi et al., 2011) . In this context, the past 15 years have witnessed a revolution in the application of immunotherapy for the treatment of cancer. Immune checkpoint blockade (ICB) uses monoclonal antibodies that block the binding of inhibitory receptors (IRs) on T cells to their natural ligands often expressed on cancer cells. The blockade of cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD-1) or the PD-1 ligand, PD-L1 have achieved survival rates of 20-30% in treating cancers such as non-small cell lung carcinoma (NSCLC), melanoma, kidney, and bladder . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 30, . . https://doi.org/10.1101 cancer (Page et al., 2013; Sharma et al., 2011) . The tumor microenvironment (TME) can alter the make-up and function of TILs (Balkwill and Mantovani, 2001) . With the advent of the COVID-19 crisis, an increasing number of cancer patients on ICB are being infected with SARS-CoV2. This raises an important question as to the best therapeutic approach for COVID-19 patients, one that limits viral spread, while allowing for the benefit of checkpoint immunotherapy in promoting reactivity against tumor neo-antigens. This has been further complicated by reports that HCQ can reverse the drug sequestration in lysosomes (Li et al., 2018) and enhance chemo-sensitization in cancer patients (Li et al., 2018) . Phase II trial studies showed that HCQ is effective in treating patients with recurrent oligometastatic prostate cancer by promoting cell death in cancer cells. In breast cancer, autophagy has been reported to be tumour-suppressive (Cianfanelli et al., 2015; Cicchini et al., 2014) , while in other cases, can promote tumors (Karsli-Uzunbas et al., 2014; Yang et al., 2014) . In a similar vein, AZ can inhibit primary antibody responses, and recall responses on bacterial infections (Fernandez et al., 2004) . Although these previous studies emphasized effects on the gut microbiota (Routy et al., 2018; Vetizou et al., 2015) , it is noteworthy that AZT and ciprofloxacin (CPX) also act as lipophilic weak bases where they affect intracellular organelles similar to CQ and HCQ (Poschet et al., 2020) . Given the urgent clinical context, there is a pressing need to assess the effects of both HCQ and AZ on the immune checkpoint response against cancer. In this study, we show that HCQ and AZ, alone or in combination, impaired the ability of anti-PD-1 therapy to promote the elimination of the B16 melanoma. We further show that HCQ or HCQ/AZ combined therapy selectively inhibits the appearance of self-renewing CD8+ PD-1+TCF1+ TILs and CD8+PD-1+TOX+ effector T-cells. We also identified TCF-1 expression as a marker in patient samples for the action of COVID-19 and HCQ. Our study indicates that HCQ negatively affects anti-PD-1 immune checkpoint blockade in the promotion of tumor rejection. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 30, . . https://doi.org/10.1101 To address this issue, we assessed whether HCQ or AZ was beneficial, or harmful to tumor immunotherapy. Mice were implanted with B16-PD-L1 melanoma cells intradermally into C57B6/J mice 4 days before the injection of anti-PD-1 alone (200ug/mouse), or in conjunction with HCQ or AZ at days 4, 7, 11 and 14. Tumors were harvested on day 17. The dosing with HCQ and AZ was the same as used in clinical trials to treat SARS-CoV-2 infection (Gautret et al., 2020) . Fig. 1A shows that the growth curve in which tumour volume increased from 100mm 3 on day 10 to 610mm 3 from day 16 (n=14). Anti-PD-1 reduced the growth of tumour as early as day 12 (i.e. 100 vs 210 mm 3 ) until day 16 (i.e. 220 vs 610mm 3 ). Importantly, HCQ impaired the anti-PD-1 reduction in tumor growth as seen on day 16 (i.e. 425mm 3 for HCQ/anti-PD-1 vs. 210mm 3 for anti-PD-1). Injection of AZ alone showed a trend in reducing the beneficial effect of anti-PD-1 (375mm 3 vs 210mm 3 for anti-PD-1), although this did not achieve statistical significance. The combination of HCQ and AZ reversed the benefits of anti-PD-1 to the same extent as HCQ alone (430mm 3 vs 220mm 3 for anti-PD-1) (also see spider graphs in Figs. S1,2). Interestingly, neither HCQ nor AZ affected tumor growth in the absence of anti-PD-1 (Fig. 1B) . Fig. 1C . Neither drug treatment resulted in major loss of mouse weight (Fig. 1D) , while scatter plot analysis showed a correlation between tumour volume and weight in reducing tumor size in response to anti-PD-1, HCQ and HCQ/AZ (p=0.025) (Fig. S3 ). HCQ did not directly affect the growth of in vitro cultured B16 cells (Fig. S4) . These data showed that HCQ and HCQ/AZ impaired the ability of anti-PD-1 to promote tumor regression. We next examined the composition of TILs in tumors where anti-PD-1 showed a trend in increasing CD45+ tumour infiltrating lymphocytes (TILs) (Fig. 1E) . Neither HCQ nor AZ affected this overall increase. Conversely, anti-PD-1 showed a trend in reducing the percentage of CD45-cells, although again, this was not statistically significant (Fig. 1F) . In examining specific subsets, we showed that anti-PD-1 caused a significant increase in the overall presence of CD8+ TCRβ+ TILs, which was unaffected by HCQ or AZ (Fig. 1G) . The same treatment showed a trend in decreasing the percentage of CD4+ T-cells and CD4+ FoxP3+ TILs. Neither AZ, nor HCQ affected this trend. Within the CD8 TIL compartment, anti-PD-1 therapy increased the representation of CD8+CD44+PD-1+ cells (Fig. 1H) , while the presence of HCQ and AZ interfered with the ability of anti-PD-1 to achieve statistical significance relative to untreated mice. By contrast, no differences were noted in CD8+ central memory TILs (Fig. 1I ) or CD8+ effector memory T-cells (Fig. 1J) , B cells (Fig. 1K ). . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 30, . . https://doi.org/10.1101 In terms of myeloid cells, cancer-driven emergency myelopoiesis give rises to myeloid-derived suppressor cells (MDSCs) that express PD-1 (Rudd, 2020; Strauss et al., 2020) . Anti-PD-1 therapy showed a trend in decreasing the presence of suppressive M-MDSCs (Fig. 1L ) and PMN-MDSCs ( Fig. 1M ) as well as in increasing the ratio of M-MDSCs to PMN-MDSCs (Fig. 1N) , as reported (Strauss et al., 2020) . HCQ nor AZ had a statistically irrelevant effect on this trend. Similarly, anti-PD-1 showed a trend in increasing the presence of cDC TILs which was unaffected by HCQ or HCQ/AZ (Fig. 1O) . No obvious effect of therapies on the presence of DC1 (Fig. 1P ) or DC2 and 3 TILs was observed (Fig. 1Q ). We next examined the surface expression of PD-1, PD-L1 and CD80 receptors on different TILs ( Fig. 2) . Anti-PD-1 treatment showed a trend in increasing PD-1 expression on PMN-MDSCs ( Fig. 2A) . Neither HCQ nor AZ monotherapy in combination with anti-PD-1 affected this trend, although the combination of HCQ/AZ reduced expression to control levels. No effect on the expression of PD-L1 or CD80 was observed. Anti-PD-1 increased the percentage of PD-1 expressing PMN-MDSCs which was unaffected by co-injection of HCQ or AZ in mice (Fig. 2B) . The percentage of PMN-MDSCs TILs expressing PD-L1 was unaffected by anti-PD-1 or anti-PD-1 with HCQ or AZ (Fig. 2B) . Similarly, no effect was observed on the expression of class 2 major histocompatibility antigens (MHC) on B-cells ( Fig. 2C) or cDCs (Fig. 2D) . In contrast, anti-PD-1 increased the expression of PD-L1 on the surface of CD45-CD31-, SSC+ and FSC+ B16 tumor cells and this effect was inhibited by the co-expression of HCQ and HCQ/AZ (Fig. 2E) . Similarly, when cultured in vitro, anti-PD-1 increased the expression of PD-L1 on B16 cells (Fig. 2F ). Co-incubation with different concentrations of HCQ and HCQ/AZ inhibited the increase. These data showed that HCQ and HCQ/AZ could act in in vitro and in vivo to reduce an increase in PD-L1 expression on tumor cells that was induced by anti-PD1. No effect on the expression of other key ligands on myeloid, B-cells or on the B16 melanoma cells was observed. Likewise, we did not observe changes in the presence of IgM or IgG antibodies against PD-L1 in mice treated with anti-PD1, alone, or in combination with HCQ/AZ (Fig. S4) . Index value was calculated by normalizing B16-IgG and B16-IgM MFI of the sample to the average of tumor-free mice ( Fig. S4D-F) . We also did not observe effects on the composition of thymic subsets (Fig. S5) . Lastly, as a control, we monitored the localisation of HCQ in cellular compartments using autofluorescence (Sauer et al., 2019) . Autofluorescence was observed in the CD45-fraction which was mostly comprised of B16 cancer cells (Fig. 2G ) as well as in the CD8+ and CD4+ TIL population (Figs. 2H and I, respectively). . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 30, . . https://doi.org/10.1101 Given the effect of HCQ and AZ on CD8+ TILs, we next used viSNE and Cytobank analysis to interrogate the composition of this subset (Fig. 3) . viSNE can visually define groupings of immune cells by simultaneously combining multiple markers (Amir el et al., 2013) . The CD8 compartment could be separated into different areas by staining with anti-PD-1 and anti-TCF-1 (Fig. 3A) . One island of CD8+ cells showed moderate to high levels of PD-1 and CD44 expression (island (i)), while a separate grouping of cells expressed primarily TCF-1 but no PD-1 (island (ii)). A grouping of cells between islands (i) and (ii) showed moderate levels of PD-1 and TCF-1 co-expression (island (iii)). TCF-1 defines progenitor CD8+ T-cells (Raghu et al., 2019; Weber et al., 2011) which give rise to effector CD8+ T-cells (Page et al., 2013; Rudd, 2020) . We first assessed the presence of TCF-1+PD-1-and TCF-1+PD-1+ progenitor TILs in response to anti-PD-1 therapy, either alone or in conjunction with HCQ or AZ. Anti-PD-1 did not alter the presence of CD8+TCF-1+PD-1-cells relative to the overall CD8+ TIL population ( Fig. 3B ) or relative to the tumor volume (Fig. 3C) . HCQ and HCQ/AZ also had no effect on the presence of this subset of TILs. By contrast, anti-PD-1 consistently increased the presence of CD8+TCF1+PD-1+ as measured relative to tumor volume (Fig. 3D) . Significantly, HCQ and HCQ/AZ inhibited this event. Similarly, anti-PD-1 therapy increased the presence of a subset of TCF1+PD-1+TILs expressing the receptor CD44 ( Fig. 3E ). CD44 is a marker for antigen (Ag)-experienced, effector and memory T cells (Baaten et al., 2012) . In this instance, we also compared the effects of HCQ and HCQ/AZ from tumors of responsive and non-responsive mice. Mice which were responsive to the effects of HCQ and HCQ/AZ had larger tumors (i.e. big), unaffected by anti-PD-1. By contrast, mice which were non-responsive to the effects of HCQ and HCQ/AZ had smaller tumors (i.e. small) similar in size to those seen with anti-PD-1 therapy alone. Tumors from responsive mice showed a clear inhibition of the presence of CD8+TCF-1+PD-1+ progenitors, while tumors where from HCQ non-responsive mice showed an increase in the presence of progenitors similar to that seen with anti-PD-1 therapy. In terms of receptor expression, dot plot profiles showed that anti-PD-1 increased the MFI for TCF-1 expression on CD8+CD44+TCF1+ TILs (Fig. 3F) . Co-therapy with HCQ and HCQ/AZ had marginal but significant effects on preventing anti-PD-1 from achieving statistical significance relative to untreated control mice. These data showed that anti-PD-1 increased both the numbers of CD8+CD44+TCF1+ cells as well as the level of TCF-1 expression on these cells and that these effects were inhibited by both HCQ and HCQ/AZ. Less obvious effects of HCQ on anti-PD-1 induced increases in PD-1 (Fig. 3G) , the transcription factor T-bet ( Fig. 3H ) or the activation marker Ki67 were seen (Fig. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 30, 2020. . https://doi.org/10.1101/2020.09.29.20193110 doi: medRxiv preprint 3I). Interestingly, HCQ and HCQ/AZ increased the expression CD8 when treatment was combined with anti-PD-1 (Fig. 3J) . Overall, these data showed that HCQ has a specific effect in inhibiting the ability of anti-PD-1 immunotherapy to increase the generation of CD8+PD-1+TCF1+ and CD8+CD44+ PD-1+TCF1+ progenitor TILs relative to tumor volume. We next assessed the effects of HCQ and AZ on the generation of CD8+ effector TILs, which do not express TCF-1, but are derived from progenitors (Miller et al., 2019) . viSNE analysis showed the patterns of cells stained with anti-CD8, CD44, PD-1, TOX and TIM-3 ( Fig. 4A) . 5 different clusters could be identified based on levels of receptor expression (Fig. 4B ). This included TCF1-CD8+ cells with low-intermediate levels of PD1, TOX, TIM-3 (i.e. cluster 2: PD1+TOX+TIM-3+) as well as cells with higher levels of PD1, TOX, TIM-3 expression (i.e. clusters 3-5). Cells with the higher levels of PD-1 and TIM-3 expression classically correspond to exhausted T-cells (Jin et al., 2010; Wherry, 2011) . The status of cells with lower levels of these inhibitory receptors is unclear, but most likely corresponds to activated functional CD8+ T-cells, which will eventually become exhausted, if exposed to repeated, ongoing antigenic stimulation. From this, we observed that anti-PD-1 induced a clear increase in TCF1-CD8+ cluster 2 expressing low levels of PD1, TOX, TIM-3 (i.e. PD1+TOX+TIM-3+) (upper table and middle panel). Significantly, HCQ and HCQ/AZ inhibited the increase in the presence of this population of cells. By contrast, no effect was seen on the presence of this population in anti-PD-1 untreated mice (lower panel). Dot plot analysis confirmed that anti-PD-1 increased the ratio of CD8+CD44+PD1+TOX+TIM-3 low TILs (cluster 2) relative to tumor volume and that this increase was blocked by the presence of HCQ or HCQ/AZ (Fig. 4C) . These data showed that HCQ inhibited the expansion of CD8+ TILs induced by anti-PD-1 therapy, expressing low levels of the activation markers PD-1 and TIM-3, either due to the inhibition of the presence of TCF-1+PD-1+ progenitor T-cells and/or due to direct effects on the PD1+TOX+TIM-3+ cells themselves. At the same time, anti-PD-1 decreased the presence of CD8+CD44+ T-cells with higher levels of PD-1, TIM-3 and TOX-3 corresponding to clusters 3-5 (Fig. 4B) . In the case of clusters 4 and 5, the decrease was statistically significant. HCQ and HCQ/AZ treatment inhibited the anti-PD-1 induced reduction in the presence of exhausted T-cells as most clearly seen in cluster 4. Unfortunately, as in other studies (Wei et al., 2017) , due to small numbers of recovered TILs, it was not possible to assess directly whether these subsets of cells were functionally non-responsive or dysfunctional. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 30, 2020. . Importantly, an analysis of the cluster 2 (effector-like) to cluster 4 (terminal exhaustion) frequency ratio relative to tumor weight further showed that shift to cluster 2 in response to anti-PD-1 and its inhibition by HCQ/AZ was correlated with the loss of tumor control (Fig. 4D) . Lastly, we observed that anti-PD-1 showed a trend in increasing the percentage of CD8+CD44+ T-cells expressing GZMB and this trend was unaffected by HCQ or HCQ/AZ (Fig. 4E) . Anti-PD-1 also showed a trend in increasing the percentage of CD8+CD44+ T-cells expressing IFNγ1 which was unaffected by HCQ or HCQ/AZ (Fig. 4F) . Lastly, as was seen in the progenitor population, HCQ and HCQ/AZ had an usual effect on the expression of CD8 where it cooperated with anti-PD-1 to significantly increase the MFI of CD8 expression on the CD8+ effector-like (cluster 2) T-cells (Fig. 4G) . Overall, these results showed that HCQ and HCQ/AZ interfered with the ability of anti-PD-1 to increase CD8+effector-like TILs while at the same time prevented CD8+ TILs from acquiring a phenotype indicative of T-cell exhaustion. A general ability of HCQ to inhibit T-cell activation would be consistent with this phenotype were it both inhibits the generation of functional CD8+ effectors while also preventing their progression into a more exhausted phenotype. Aside from the immune system, we also examined the vasculature in mice (Fig. 5) . To evaluate potential alterations in the morphological features of blood vessels, we examined retinal and brain cortical vasculature in treated mice. Whole mounts of retinal vasculature showed no changes in retinal vessel morphology or branching between groups. (Fig. 5A) . Further, an examination of the pial cerebral vessels, cortical vessels, vessel bifurcation and length showed no differences (Fig. 5B) . Images of retinal and brain cortical vasculature are shown in Fig. 5C . These data indicate that neither anti-PD-1 nor HCQ or AZ had any detectable effects on the vasculature in mice. Furthermore, we found no effect of treatments on the overall branching structure in the cortical vasculature. These results show that the topological structure of the retinal and brain cortical vascular networks was not affected by the treatments. Given the continuing widespread global use of HCQ in treating patients who are infected with SAR-CoV-2, we attempted to identify a phenotypic marker in human peripheral blood for the effects of COVID-19 and HCQ on the immune system (Fig. 6) . Peripheral T-cells from the blood of cancer and non-cancer COVID-19 patients treated with HCQ were analyzed. viSNE analysis identified three separate clusters of CD8+ human T-cells, one island (i) staining with PD-1 (upper panel), Tim-3 (upper . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 30, 2020. . https://doi.org/10.1101/2020.09.29.20193110 doi: medRxiv preprint middle) and GZMB (lower panel) (Fig. 6A, upper circles) , and a two other distinct islands (lower circles ii and iii) which stained clearly with anti-TCF-1 (lower middle panel). Images are a compilation of data from >20 patients. TCF-1+ clusters (ii) and (iii) could be distinguished on the basis of the expression of Ki67 and CCR7 which preferentially stained island (iii) (Fig. 6B) . Ki67 is an activation marker while CCR7 mediates the homing of T cells to secondary lymphoid organs via high endothelial venules (HEV) and can act as a marker for memory T-cells (Sharma et al., 2015) CD8+ T-cells from the blood of COVID-19 patients relative to healthy donors showed an increase in the staining with anti-PD-1, TIM-3 and GZMB, concurrent with a reduction in the staining of both TCF-1+ islands (Fig. 6B) . By contrast, HCQ treatment increased the presence of TCF-1 and CD8+TCF-1+Ki67-CCR7+/-cells in island (ii), both in the samples from patients with the COVID-19. HCQ treatment had no effect in the CD8+TCF-1+Ki67+CCR7+ (iii) subset. The same pattern was seen in COVID-19 patients with cancer, and in the one sample we were able to obtain from a patient treated with COVID-19/cancer/HCQ. All cancer patients had either pancreatic or oesophageal cancer who had been treated with chemotherapy. These data showed that HCQ could prevent the loss of TCF-1+ progenitors in blood with a less activate or differentiated phenotype. SARs-CoV-2 infection decreased the overall expression of TCF-1 staining both in terms of the percentage of cells and the intensity of staining (MFI (Fig. 6C ) and in the ratio of TCF-1 to PD-1 MFIs (Fig. 6D) . HCQ showed a trend in reversing this decrease and in the ratio of TCF-1 to PD-1. Overall, our findings showed that the expression of TCF-1 could be used as a marker for the both SARs CoV2 and treatment with HCQ. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 30, 2020. . https://doi.org/10.1101/2020.09.29.20193110 doi: medRxiv preprint Globally, HCQ continues to be used to treat thousands of COVID-19 patients with new and ongoing clinical trials underway to establish its efficacy. This situation makes an inquiry into the effects on HCQ and AZ on cancer immunotherapy an immediate pressing issue. In this study, we show that HCQ alone, or in combination with AZ, at the doses used to treat COVID-19 patients, reverses the ability of anti-PD-1 therapy to control the growth of cancer. This reversal was accompanied by a selective interference with CD8+TCF1+ progenitor and CD8+PD-1+ effector TIL expansion or infiltration of tumors. Further, we show that TCF-1 expression serves as a marker in peripheral blood CD8+ Tcells for the response of the immune system to SARs-CoV-2 and HCQ therapy. (Rudd, 1999; Wilkinson et al., 2004) . Differences in signaling have also been noted between CD4 and CD8+ T-cells where IL-2 induces quantitatively stronger proliferation in CD8 + T cells compared to CD4 + T cells (Gesbert et al., 2005) due to greater IL-2Rβ chain expression (Smith et al., 2017) . We also failed to find differences in antibody titres in mice or in levels of IgG1/G2 antibodies against PD-L1. This susceptibility of CD8+ T-cells to inhibition by HCQ in the context of anti-PD-1 immunotherapy may also involve factors in the tumor microenvironment that affected immune activation and function. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 30, . . https://doi.org/10.1101 As reported by others (Siddiqui et al., 2019; Wei et al., 2017) , we observed that anti-PD-1 immunotherapy increased the presence of PD-1+ TCF-1+ progenitors and PD-1+CD44+TCF-1-effector TILs. These progenitors in turn are needed to produce more differentiated PD-1+TCF1-cells in immunotherapy (Siddiqui et al., 2019) . Importantly, HCQ and HCQ/AZ inhibited the ability of anti-PD-1 therapy to increase in the presence of these PD-1+TCF-1+ progenitors. Consistent with this, HCQ also inhibited the ability of anti-PD-1 to expand CD8+CD44+ TILs. These cells generally expressed low- (Routy et al., 2018; Vetizou et al., 2015) . These previous studies emphasized effects on the gut microbiota (Routy et al., 2018; Vetizou et al., 2015) . The differences in these findings may relate to the dose of antibiotics used or the coverage of bacterial inhibition by AZT versus ciprofloxacin (CPX) and other antibiotics. It might also relate to the nature of gut biota in mice which is likely to vary in different containment facilities. In terms of the expression of specific molecules, HCQ had variable effects on markers indicative of activation. For example, while HCQ inhibited the expression of TCF1 TILs, it did not reproducibly affect the expression of the activation marker Ki67 in progenitor T-cells. One surprising observation was . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted September 30, 2020. . https://doi.org/10.1101/2020.09.29.20193110 doi: medRxiv preprint the potent effect of HCQ in increasing CD8 expression on progenitor and effector T-cells. It is paradoxical given our previous findings showing that CD8 (and CD4) bind to p56 lck to initiate a proteintyrosine phosphorylation cascade in T-cells that is needed for T-cell activation (Barber et al., 1989; Rudd et al., 1988) . Further, the down-regulation of CD8-lck has been suggested as a mechanism for peripheral tolerance (Schonrich et al., 1991; Zhang et al., 1995) , while subpopulations with low CD8 have been reported during chronic diseases (Grisotto et al., 2001) and in acute immune responses to pathogens (Walker et al., 1995) . HCQ and AZ increased CD8 expression in combination with anti-PD-1. No effect was seen in the absence of anti-PD-1 indicating that altered signaling linked to anti-PD-1 blockade synergizes with HCQ and AZ to promote increased CD8 expression. It was observed on progenitors and effector T-cells. The functional consequences of this increased expression are unclear, although an increased number of CD8 molecules engaged by MHC during antigen-presentation would be expected to increase the activation and proliferation of T-cells (Rudd, 1999) . Alternatively, depending on the relative stoichiometry and localisation of expression, an excess of CD8 receptors might lead to few receptors bound to p56 lck and hence, reduced activation. CD4 sequestration of p56 lck complex may also prohibit the induction of activation signals through the TCR-CD3 complex (Haughn et al., 1992 ). Many questions remain related to HCQ, COVID-19 and tumor immunology. Whether the inhibitory effects on anti-PD-1 immune therapy may be offset by its reported benefit in sensitizing chemotherapy remains to be determined. For example, HCQ has been reported to enhance the anticancer activity of the histone deacetylase inhibitor, vorinostat (VOR), in pre-clinical models and early phase clinical studies of metastatic colorectal cancer (mCRC) (Patel et al., 2016) . Lastly, the inhibitory effects on anti-PD-1 therapy in this study used HCQ doses that have been used in the treatment of COVID-19 patients (Chen et al., 2020a; Chen et al., 2020b; Gao et al., 2020; Gautret et al., 2020; Geleris et al., 2020; Magagnoli et al., 2020; Mahevas et al., 2020; Molina et al., 2020; Rosenberg et al., 2020; Tang et al., 2020; Yu et al., 2020) . It remains unclear whether lower concentrations of drug will . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted September 30, . . https://doi.org/10.1101 have the same effect, although the effectiveness of lower concentrations in treating auto-immune diseases such as SLE is in part likely due to its immune-suppressive properties (Thome et al., 2014; Thome et al., 2013) . The utility of HCQ may vary dependent on the dose of the drug and the nature of cancer therapy. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted September 30, . . https://doi.org/10.1101 Board #2021-6881. The peripheral blood lymphocytes were isolated at the MUHC-RI by MUHC staff using the density gradient centrifugation method. Cells were washed 3 times in PBS and fixed in 3.5% paraformaldehyde at room temperature for 30 minutes for the inactivation of the SARS coronavirus as described (Kariwa et al., 2004) . COVID-19 patients were treated with 400mg HCQ twice a day (BID) on day 1 followed by 200 po BID for the next 5 days, reassessed based on progression (BID = 2x daily). Patients were monitored for hemolytic anemia with CBC every 2 days. Testing for G6PD levels was overlayed on a ficoll layer for lymphocyte collection. Lymphocytes were then stained for viability followed by surface staining with FACS antibodies before fixation. Intracellular staining was performed using the FoxP3/Transcription Factor Staining Buffer Set (ebiosciences, San Diego, CA, USA). For PBL isolation . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 30, 2020. . https://doi.org/10.1101/2020.09.29.20193110 doi: medRxiv preprint from mice, blood was collected in EDTA-coated tubes and peripheral blood mononuclear cells (PBM were isolated by Ficoll (Corning, NY, USA). After 3 washes, PBMCs were stained for FACS. For the preparation of thymocytes, thymi were collected from all mice immediately after sacrifice, dissociated passed through 70 µm cell strainers into RPMI medium. Following 3 washes, cells were stained for viability and fixed in 2% PFA until further analysis. Tumour infiltrating lymphocyte collection. Briefly, tumours were dissected and placed in RPMI m on ice. Tumours were minced with razor blades into pieces and then digested in the presence of libe and DNase at 37 o C for 30 min, with manual shaking every 5 min. The mixture was then strained thro µm cell strainers, and collected cells were rinsed twice in 2 % FCS-PBS and overlayed over on a fill for lymphocyte. After ficoll separation, TILs were collected and subjected to viability staining, then fix with 2% PFA. were nd then ibodies . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 30, . . https://doi.org/10.1101 Human PBMC isolation and FACs staining. Blood was collected from healthy donor, cancer or CO CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 30, 2020 . . https://doi.org/10.1101 Eyes were prefixed in 4% PFA for 15 min at room temperature. Dissected retinas were blocked overnight at 4°C in blocking solution (0.1Tris-HCl, 150 mM NaCl, 0.2% Perkin Elmer blocking reagent and, and 0.5% Triton-X). Retinas were then incubated with IsolectinB4 and immunostained with anti-Smooth Muscle Actin-FITC (Sigma). Quantification of retinal vasculature branch points was done using the angiogenesis analyzer tool for ImageJ. Statistics. All data are expressed as mean ± SEM. A t-test was used when only two groups were compared, and a one-way ANOVA was used when more than two groups were compared. A two-way ANOVA was used for multiple comparison procedures that involved two independent variables. Dunett or Bonferroni tests were used for posthoc analyses. A difference in mean values between groups was significant when p ≤ 0.05 *; p ≤ 0.01 ** and p ≤ 0.001 ***. Supplemental Information can be found online at xxxxxx is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 30, 2020 . . https://doi.org/10.1101 arranging for ethical approval for their use. C.E.R. wrote the manuscript with assistance from J.K, F.S., M.I.,R.K., C.M. and A.K. The authors declare no competing interests. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 30, . . https://doi.org/10.1101 Ziegler, H.K., and Unanue, E.R. (1982) . Decrease in macrophage antigen catabolism caused by ammonia and chloroquine is associated with inhibition of antigen presentation to T cells. Proc Natl Acad Sci U S A 79, 175-178. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 30, 2020. Chloroquine enhances human CD8+ T cell responses against soluble antigens in vivo viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia Lag-3, Tim-3, and TIGIT: Co-inhibitory Receptors with Specialized Functions in Immune Regulation In vitro testing of combined hydroxychloride and azithromycin as a treatment for SARs-CoV-2 shows synergistic effect Task Force