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NK cells are endowed with tumor killing ability, nevertheless most cancers impair NK cell functionality, and cell-based therapies have limited efficacy in solid tumors. How cancers render NK cell dysfunctional is unclear, and overcoming resistance is an important immune-therapeutic aim. Here, we identify autophagy as a central regulator of NK cell anti-tumor function. Analysis of differentially expressed genes in tumor-infiltrating versus non-tumor NK cells from our previously published scRNA-seq data of advanced human prostate cancer shows deregulation of the autophagic pathway in tumor-infiltrating NK cells. We confirm this by flow cytometry in patients and in diverse cancer models in mice. We further demonstrate that exposure of NK cells to cancer deregulates the autophagic process, decreases mitochondrial polarization and impairs effector functions. Mechanistically, CCAAT enhancer binding protein beta (C/EBPβ), downstream of CXCL12-CXCR4 interaction, acts as regulator of NK cell metabolism. Accordingly, inhibition of CXCR4 and C/EBPβ restores NK cell fitness. Finally, genetic and pharmacological activation of autophagy improves NK cell effector and cytotoxic functions, which enables tumour control by NK and CAR-NK cells. In conclusion, our study identifies autophagy as an intracellular checkpoint in NK cells and introduces autophagy regulation as an approach to strengthen NK-cell-based immunotherapies., Competing Interests: Competing interests: T. Tonn is named as an inventor on patents in the field of cancer immunotherapy. The remaining authors declare no competing interests. Ethics statement: This study adheres to the principles outlined in the Declaration of Helsinki and the ethical standards set by our Institution. All human participants provided informed consent, and animal experiments were conducted with approval from the Institutional Animal Care. We promote diversity, equity, and inclusion in research, and we have disclosed any potential conflicts of interest., (© 2024. The Author(s).)

Esophageal cancer is a highly lethal malignancy, representing 5% of all cancer-related deaths. The two main subtypes are esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). While most research has focused on ESCC, few studies have analyzed EAC for transcriptional signatures linked to diagnosis or prognosis. In this study, we utilized single-cell RNA sequencing and bulk RNA sequencing to identify specific immune cell types that contribute to anti-tumor responses, as well as differentially expressed genes (DEGs). We have characterized transcriptional signatures, validated against a wide cohort of TCGA patients, that are capable of predicting clinical outcomes and the prognosis of EAC post-surgery with efficacy comparable to the currently accepted prognostic factors. In conclusion, our findings provide insights into the immune landscape and therapeutic targets of EAC, proposing novel immunological biomarkers for predicting prognosis, aiding in patient stratification for post-surgical outcomes, follow-up, and personalized adjuvant therapy decisions.

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) with post-transplant cyclophosphamide is a curative treatment for many hematological malignancies, yet a majority of patients still suffers from recurrent infections. Post-transplant infusion of memory T-cells could potentially enhance immunological protection without increasing the risk of eliciting acute graft-versus-host disease, which is mainly induced by naïve T-cells. Here, we performed longitudinal analysis of the lymphocyte compartment in 19 patients who underwent haplo-HSCT previously enrolled in a phase II prospective clinical trial (www.clinicaltrials.gov as #NCT04687982), in which they received post-transplant CD45RA-depleted donor lymphocyte infusions (DLI). T-cell receptor sequencing analysis showed that, surprisingly, CD45RA-depleted DLI do not increase T-cell clonal diversity, but lead to prominent expansion of a selected number of infused memory T-cell clones, suggesting recruitment of these cells in the immune response. Pathogen-specific memory T-cells, including cytomegalovirus (CMV)-specific cells, were engrafted and were able to persist for at least 1 month. Deep immunophenotyping revealed strong polyfunctional effector CMV-specific T-cell responses in the majority of patients, with their expansion correlating with the frequency of CMV-specific cells in the donor. These findings provide a rationale behind the suggested improved protection against viral infections in patients receiving CD45RA-depleted DLI., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Circular RNAs (circRNAs) are a class of non-coding RNAs increasingly emerging as crucial actors in the pathogenesis of human diseases, including autoimmune and neurological disorders as multiple sclerosis (MS). Despite several efforts, the mechanisms regulating circRNAs expression are still largely unknown and the circRNA profile and regulation in MS-relevant cell models has not been completely investigated. In this work, we aimed at exploring the global landscape of circRNA expression in MS patients, also evaluating a possible correlation with their genetic and epigenetic background., Methods: We performed RNA-seq experiments on circRNA-enriched samples, derived from peripheral blood mononuclear cells (PBMCs) of 10 MS patients and 10 matched controls and performed differential circRNA expression. The genetic background was evaluated using array genotyping, and an expression quantitative trait loci (eQTL) analysis was carried out., Results: Expression analysis revealed 166 differentially expressed circRNAs in MS patients, 125 of which are downregulated. One of the top dysregulated circRNAs, hsa_circ_0007990, derives from the PGAP3 gene, encoding a protein relevant for the control of autoimmune responses. The downregulation of this circRNA was confirmed in two independent replication cohorts, suggesting its implementation as a possible RNA-based biomarker. The eQTL analysis evidenced a significant association between 89 MS-associated loci and the expression of at least one circRNA, suggesting that MS-associated variants could impact on disease pathogenesis by altering circRNA profiles. Finally, we found a significant correlation between exon methylation and circRNA expression levels, supporting the hypothesis that epigenetic features may play an important role in the definition of the cell circRNA pool., Conclusion: We described the circRNA expression profile of PBMCs in MS patients, suggesting that MS-associated variants may tune the expression levels of circRNAs acting as "circ-QTLs", and proposing a role for exon-based DNA methylation in regulating circRNA expression., Competing Interests: Declaration of Competing Interest The authors report no conflict of interest., (Copyright © 2022. Published by Elsevier B.V.)

Background & Aims: The landscape and function of the immune infiltrate of intrahepatic cholangiocarcinoma (iCCA), a rare, yet aggressive tumor of the biliary tract, remains poorly characterized, limiting development of successful immunotherapies. Herein, we aimed to define the molecular characteristics of tumor-infiltrating leukocytes with a special focus on CD4+ regulatory T cells (Tregs)., Methods: We used high-dimensional single-cell technologies to characterize the T-cell and myeloid compartments of iCCA tissues, comparing these with their tumor-free peritumoral and circulating counterparts. We further used genomics and cellular assays to define the iCCA-specific role of a novel transcription factor, mesenchyme homeobox 1 (MEOX1), in Treg biology., Results: We found poor infiltration of putative tumor-specific CD39+ CD8+ T cells accompanied by abundant infiltration of hyperactivated CD4+ Tregs. Single-cell RNA-sequencing identified an altered network of transcription factors in iCCA-infiltrating compared to peritumoral T cells, suggesting reduced effector functions by tumor-infiltrating CD8+ T cells and enhanced immunosuppression by CD4+ Tregs. Specifically, we found that expression of MEOX1 was highly enriched in tumor-infiltrating Tregs, and demonstrated that MEOX1 overexpression is sufficient to reprogram circulating Tregs to acquire the transcriptional and epigenetic landscape of tumor-infiltrating Tregs. Accordingly, enrichment of the MEOX1-dependent gene program in Tregs was strongly associated with poor prognosis in a large cohort of patients with iCCA., Conclusions: We observed abundant infiltration of hyperactivated CD4+ Tregs in iCCA tumors along with reduced CD8+ T-cell effector functions. Interfering with hyperactivated Tregs should be explored as an approach to enhance antitumor immunity in iCCA., Lay Summary: Immune cells have the potential to slow or halt the progression of tumors. However, some tumors, such as intrahepatic cholangiocarcinoma, are associated with very limited immune responses (and infiltration of cancer-targeting immune cells). Herein, we show that a specific population of regulatory T cells (a type of immune cell that actually suppresses the immune response) are hyperactivated in intrahepatic cholangiocarcinoma. Targeting these cells could enable cancer-targeting immune cells to act more effectively and should be looked at as a potential therapeutic approach to this aggressive cancer type., Competing Interests: Conflict of interest E.L. receives research grants from Bristol Myers Squibb and consulting fees from BD Biosciences. A.L. reports receiving consulting fees from Intercept Pharma, AlfaSigma, Takeda, AbbVie, Gilead, and MSD and travel expenses from Intercept Pharma, AlfaSigma and AbbVie. M.I. participates in advisory boards/consultancies for Gilead Sciences, Roche, Third Rock Ventures, Antios Therapeutics, Amgen, Asher Bio, Allovir, ENYO Pharma. The other authors have no competing interests. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2022 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Tumor-associated macrophages (TAMs) are correlated with the progression of prostatic adenocarcinoma (PCa). The mechanistic basis of this correlation and therapeutic strategies to target TAMs in PCa remain poorly defined. Here, single-cell RNA sequencing was used to profile the transcriptional landscape of TAMs in human PCa, leading to identification of a subset of macrophages characterized by dysregulation in transcriptional pathways associated with lipid metabolism. This subset of TAMs correlates positively with PCa progression and shorter disease-free survival and is characterized by an accumulation of lipids that is dependent on Marco. Mechanistically, cancer cell-derived IL-1β enhances Marco expression on macrophages, and reciprocally, cancer cell migration is promoted by CCL6 released by lipid-loaded TAMs. Moreover, administration of a high-fat diet to tumor-bearing mice raises the abundance of lipid-loaded TAMs. Finally, targeting lipid accumulation by Marco blockade hinders tumor growth and invasiveness and improves the efficacy of chemotherapy in models of PCa, pointing to combinatorial strategies that may influence patient outcomes., Competing Interests: Disclosures: R.A. DePinho reported being a Founder and Advisor for Tvardi Therapeutics, Asylia Therapeutics, Nirogy Therapeutics, Stellanova Therapeutics, and Sporos Bioventures. The focus of these companies is not directly related to the content of this manuscript. S. Gordon reported personal fees from Verseau, Myeloid Therapeutics, and Alnylam outside the submitted work. No other disclosures were reported., (© 2021 Masetti et al.)

Gut Microbiota (GM) dysbiosis associates with Atherosclerotic Cardiovascular Diseases (ACVD), but whether this also holds true in subjects without clinically manifest ACVD represents a challenge of personalized prevention. We connected exposure to diet (self-reported by food diaries) and markers of Subclinical Carotid Atherosclerosis (SCA) with individual taxonomic and functional GM profiles (from fecal metagenomic DNA) of 345 subjects without previous clinically manifest ACVD. Subjects without SCA reported consuming higher amounts of cereals, starchy vegetables, milky products, yoghurts and bakery products versus those with SCA (who reported to consume more mechanically separated meats). The variety of dietary sources significantly overlapped with the separations in GM composition between subjects without SCA and those with SCA (RV coefficient between nutrients quantities and microbial relative abundances at genus level = 0.65, p -value = 0.047). Additionally, specific bacterial species ( Faecalibacterium prausnitzii in the absence of SCA and Escherichia coli in the presence of SCA) are directly related to over-representation of metagenomic pathways linked to different dietary sources (sulfur oxidation and starch degradation in absence of SCA, and metabolism of amino acids, syntheses of palmitate, choline, carnitines and Trimethylamine n -oxide in presence of SCA). These findings might contribute to hypothesize future strategies of personalized dietary intervention for primary CVD prevention setting.

It has long been known that in vitro polarized macrophages differ in morphology. Stemming from a conventional immunohistology observation, we set out to test the hypothesis that morphology of tumor-associated macrophages (TAMs) in colorectal liver metastasis (CLM) represents a correlate of functional diversity with prognostic significance. Density and morphological metrics of TAMs were measured and correlated with clinicopathological variables. While density of TAMs did not correlate with survival of CLM patients, the cell area identified small (S-TAM) and large (L-TAM) macrophages that were associated with 5-yr disease-free survival rates of 27.8% and 0.2%, respectively (P < 0.0001). RNA sequencing of morphologically distinct macrophages identified LXR/RXR as the most enriched pathway in large macrophages, with up-regulation of genes involved in cholesterol metabolism, scavenger receptors, MERTK, and complement. In single-cell analysis of mononuclear phagocytes from CLM tissues, S-TAM and L-TAM signatures were differentially enriched in individual clusters. These results suggest that morphometric characterization can serve as a simple readout of TAM diversity with strong prognostic significance., Competing Interests: Disclosures: A. Mantovani reported personal fees from Ventana, Pierre Fabre, Verily, AbbVie, Astra Zeneca, Verseau Therapeutics, Compugen, Myeloid Therapeutics, Third Rock Venture, Imcheck Therapeutics, Ellipses, Novartis, Roche, Macrophage Pharma, Biovelocita, Merck, and Principia; grants from Novartis; and "other" from Cedarlane Laboratories Ltd, Hycult Biotechnology, eBioscience, BioLegend, ABCAM Plc, Novus Biologicals, Enzo Life (ex-Alexis Corp.), and Affymetrix outside the submitted work. He is the inventor of patents related to PTX3 and other innate immunity molecules and receives royalties for reagents related to innate immunity. No other disclosures were reported., (© 2020 Donadon et al.)

Background: Inflammation is a key component of cardiac disease, with macrophages and T lymphocytes mediating essential roles in the progression to heart failure. Nonetheless, little insight exists on other immune subsets involved in the cardiotoxic response., Methods: Here, we used single-cell RNA sequencing to map the cardiac immune composition in the standard murine nonischemic, pressure-overload heart failure model. By focusing our analysis on CD45 + cells, we obtained a higher resolution identification of the immune cell subsets in the heart, at early and late stages of disease and in controls. We then integrated our findings using multiparameter flow cytometry, immunohistochemistry, and tissue clarification immunofluorescence in mouse and human., Results: We found that most major immune cell subpopulations, including macrophages, B cells, T cells and regulatory T cells, dendritic cells, Natural Killer cells, neutrophils, and mast cells are present in both healthy and diseased hearts. Most cell subsets are found within the myocardium, whereas mast cells are found also in the epicardium. Upon induction of pressure overload, immune activation occurs across the entire range of immune cell types. Activation led to upregulation of key subset-specific molecules, such as oncostatin M in proinflammatory macrophages and PD-1 in regulatory T cells, that may help explain clinical findings such as the refractivity of patients with heart failure to anti-tumor necrosis factor therapy and cardiac toxicity during anti-PD-1 cancer immunotherapy, respectively., Conclusions: Despite the absence of infectious agents or an autoimmune trigger, induction of disease leads to immune activation that involves far more cell types than previously thought, including neutrophils, B cells, Natural Killer cells, and mast cells. This opens up the field of cardioimmunology to further investigation by using toolkits that have already been developed to study the aforementioned immune subsets. The subset-specific molecules that mediate their activation may thus become useful targets for the diagnostics or therapy of heart failure.

Reticulated platelets (RPs) are larger, hyperreactive platelets that contain significantly more ribonucleic acid (RNA) compared with mature platelets (MPs). High levels of RPs in peripheral blood are predictors of an insufficient response to dual antiplatelet therapy in cardiovascular patients and of adverse cardiovascular events. However, the mechanisms underlying these correlations remain widely unknown and the biology of RPs has not been investigated yet. Here, we compared for the first time the transcriptomic profiles of RPs and MPs isolated from peripheral blood of healthy donors. Total RNA sequencing revealed 1,744 differentially expressed genes (670 downregulated, 1,074 upregulated) in RPs compared with MPs. In particular, transcripts for the collagen receptor GP6 , thromboxane receptor A2 ( TBXA2R ), thrombin receptor PAR4 ( F2RL3 ), and adenosine triphosphate receptors P2RX1 , ORAI2 , and STIM1 (both involved in calcium signaling) were significantly upregulated in RPs, whereas several RNA regulators as the ribonuclease PARN , the RISC-component TNRC6A , and the splicing factor LUC7L3 were downregulated in RPs. Gene ontology analysis revealed an enrichment of relevant biological categories in RPs including platelet activation and blood coagulation. Gene Set Enrichment Analysis showed an overrepresentation of several platelet activation pathways like thrombin, thromboxane, and glycoprotein IIb/IIIa signaling in RPs. Small-RNA sequencing reported 9 micro-RNAs significantly downregulated in RPs with targets involved in platelet reactivity. Our data show for the first time an enrichment of several prothrombotic transcripts in RPs providing a first biological explanation for their hyperreactive phenotype., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

Background & Aims: Mesenchymal stem cells (MSCs) are pluripotent cells that can promote expansion of immune regulatory cells and might be developed for the treatment of immune disorders, including inflammatory bowel diseases. MSCs were reported to reduce colitis in mice; we investigated whether MSC localization to the intestine and production of paracrine factors, including tumor necrosis factor-induced protein 6 (TSG6), were required for these effects., Methods: MSCs were isolated from bone marrow (BM-MSCs) of 4- to 6-week-old C57BL/6, C57BL/6-green fluorescent protein, or Balb/c Tsg6-/- male mice. Colitis was induced by ad libitum administration of dextran sulfate sodium for 10 days; after 5 days the mice were given intraperitoneal injections of BM-MSCs or saline (controls). Blood samples and intestinal tissues were collected 24, 48, 96, and 120 hours later; histologic and flow cytometry analyses were performed., Results: Injection of BM-MSCs reduced colitis in mice, increasing body weight and reducing markers of intestinal inflammation, compared with control mice. However, fewer than 1% of MSCs reached the inflamed colon. Most of the BM-MSCs formed aggregates in the peritoneal cavity. The aggregates contained macrophages and B and T cells, and produced immune-regulatory molecules including FOXP3, interleukin (IL)10, transforming growth factor-β, arginase type II, chemokine (C-C motif) ligand 22 (CCL22), heme oxygenase-1, and TSG6. Serum from mice given BM-MSCs, compared with mice given saline, had increased levels of TSG6. Injection of TSG6 reduced the severity of colitis in mice, along with the numbers of CD45+ cells, neutrophils and metalloproteinase activity in the mucosa, while increasing the percentage of Foxp3CD45+ cells. TSG6 injection also promoted the expansion of regulatory macrophages that expressed IL10 and inducible nitric oxide synthase, and reduced serum levels of interferon-γ, IL6, and tumor necrosis factor. Tsg6-/- MSCs did not suppress the mucosal inflammatory response in mice with colitis., Conclusions: BM-MSCs injected into mice with colitis do not localize to the intestine but instead form aggregates in the peritoneum where they produce immunoregulatory molecules, including TSG6, that reduce intestinal inflammation. TSG6 is sufficient to reduce intestinal inflammation in mice with colitis., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Objective: Inflammation plays crucial roles in the pathogenesis of several chronic inflammatory disorders, including Crohn's disease (CD) and UC, the two major forms of IBD. The urokinase plasminogen activator receptor (uPAR) exerts pleiotropic functions over the course of both physiological and pathological processes. uPAR not only has a key role in fibrinolysis but also modulates the development of protective immunity. Additionally, uPAR supports extracellular matrix degradation and regulates cell migration, adhesion and proliferation, thus influencing the development of inflammatory and immune responses. This study aimed to evaluate the role of uPAR in the pathogenesis of IBD., Design: The functional role of uPAR was assessed in established experimental models of colitis. uPAR deficiency effects on cytokine release, polarisation and bacterial phagocytosis were analysed in colonic macrophages. uPAR expression was analysed in surgical specimens collected from normal subjects and patients with IBD., Results: In mice, uPAR expression is positively regulated as colitis progresses. uPAR-KO mice displayed severe inflammation compared with wild-type littermates, as indicated by clinical assessment, endoscopy and colon histology. The absence of uPAR led to an increased production of inflammatory cytokines by macrophages that showed an M1 polarisation and impaired phagocytosis. In human IBD, CD68(+) macrophages derived from the inflamed mucosa expressed low levels of uPAR., Conclusions: These findings point to uPAR as an essential component of intestinal macrophage functions and unravel a new potential target to control mucosal inflammation in IBD., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Agrin, an extracellular matrix protein belonging to the heterogeneous family of heparan sulfate proteoglycans (HSPGs), is expressed by cells of the hematopoietic system but its role in leukocyte biology is not yet clear. Here we demonstrate that agrin has a crucial, nonredundant role in myeloid cell development and functions. We have identified lineage-specific alterations that affect maturation, survival and properties of agrin-deficient monocytic cells, and occur at stages later than stem cell precursors. Our data indicate that the cell-autonomous signals delivered by agrin are sensed by macrophages through the α-DC (DG) receptor and lead to the activation of signaling pathways resulting in rearrangements of the actin cytoskeleton during the phagocytic synapse formation and phosphorylation of extracellular signal-regulated kinases (Erk 1/2). Altogether, these data identify agrin as a novel player of innate immunity.

Hematopoiesis is the process leading to the sustained production of blood cells by hematopoietic stem cells (HSCs). Growth, survival, and differentiation of HSCs occur in specialized microenvironments called "hematopoietic niches," through molecular cues that are only partially understood. Here we show that agrin, a proteoglycan involved in the neuromuscular junction, is a critical niche-derived signal that controls survival and proliferation of HSCs. Agrin is expressed by multipotent nonhematopoietic mesenchymal stem cells (MSCs) and by differentiated osteoblasts lining the endosteal bone surface, whereas Lin(-)Sca1(+)c-Kit(+) (LSK) cells express the α-dystroglycan receptor for agrin. In vitro, agrin-deficient MSCs were less efficient in supporting proliferation of mouse Lin(-)c-Kit(+) cells, suggesting that agrin plays a role in the hematopoietic cell development. These results were indeed confirmed in vivo through the analysis of agrin knockout mice (Musk-L;Agrn(-/-)). Agrin-deficient mice displayed in vivo apoptosis of CD34(+)CD135(-) LSK cells and impaired hematopoiesis, both of which were reverted by an agrin-sufficient stroma. These data unveil a crucial role of agrin in the hematopoietic niches and in the cross-talk between stromal and hematopoietic stem cells.

In addition to its role as neurotransmitter, serotonin (5-HT) is an important modulator of inflammation and immunity. Here, we report novel findings suggesting a 5-HT involvement in T cell migration. In particular, we show that 5-HT tunes the responsiveness of human T lymphocytes to the broadly expressed chemokine CXCL12 in transwell migration assays. By real-time PCR, western blot analysis and electrophysiological patch clamp experiments, we demonstrate that the type 3 5-HT receptor (5-HT(3)) is functionally expressed in human primary T cells. In addition, specific 5-HT(3) receptor agonists selectively decrease T cell migration towards gradients of CXCL12 but not of inflammatory chemokines, such as CCL2 and CCL5. In transmigration experiments, 5-HT(3) receptor stimulation reverts the inhibitory effect of endothelial-bound CXCL12 on T cell migration. Our data suggest that the reduced T cell responsiveness to CXCL12 induced by 5-HT may occur to facilitate T cell extravasation and migration into inflamed tissues.

The finding that in addition to CD4 molecule HIV-1 uses, CCR5 or CXCR4 receptors to enter target cells prompted the research to identify polymorphisms in coreceptor genes affecting disease progression. In this study we analyzed the prevalence of CCR5-delta32, CCR2-641 and SDF1-3'A alleles in a highly selected group of 42 Long-Term Nonprogressors (LTNPs) compared to 112 subjects with a typical course of HIV-1 infection (TPs) and 117 healthy controls (HCs). In addition, we correlated CCR5, CCR2 and SDF-1 genotypes with molecular indexes of HIV-1 replication, cell-free RNA and both unspliced (US) and multiply spliced (MS) intracellular transcripts, to investigate the role of the mutant alleles in determining a long-term nonprogressive course of HIV-1 disease. Our results indicate a significantly higher prevalence of CCR5-delta32 allele in LTNPs compared to TPs (p=0.0434), while the proportions of CCR2-64I and SDF1-3'A alleles were comparable between the two groups. However, SDF-1 wild type LTNP subjects showed significantly lower levels of HIV-1 genomic RNA, US and MS transcripts than SDF1-3'A heterozygous ones (p=0.0021, 0.016, 0.0031, respectively), whereas both CCR5 and CCR2 wild type individuals had similar rates of viral replication compared to CCR5-delta32 and CCR2-64I heterozygous ones. CCR5, CCR2 and SDF-1 combined genotypes were also studied and this analysis did not identify a specific protective cluster of alleles in LTNPs. Taken together, our results indicate that genetic background involving CCR5, CCR2 and SDF-1 alleles may play a limited role in the natural history of HIV-1 infection.