A CD4+ T cell reference map delineates subtype-specific adaptation during acute and chronic viral infections

Paving the way for T cell-based immunotherapies in chronic hepatitis E

T cell exhaustion and loss of memory potential occur during many chronic viral infections and cancer. We investigated when during chronic viral infection virus-specific CD8 T cells lose the potential to form memory. Virus-specific CD8 T cells from established chronic infection were unable to become memory CD8 T cells if removed from infection. However, at earlier stages of chronic infection, these virus-specific CD8 T cells retained the potential to partially or fully revert to a memory differentiation program after transfer to infection-free mice. Conversely, effector CD8 T cells primed during acute infection were not protected from exhaustion if transferred to a chronic infection. We also tested whether memory and exhausted CD8 T cells arose from different subpopulations of effector CD8 T cells and found that only the KLRG1(lo) memory precursor subset gave rise to exhausted CD8 T cells. Together, these studies demonstrate that CD8 T cell exhaustion is a progressive developmental process. Early during chronic infection, the fate of virus-specific CD8 T cells remains plastic, while later, exhausted CD8 T cells become fixed in their differentiation state. Moreover, exhausted CD8 T cells arise from the memory precursor and not the terminally differentiated subset of effector CD8 T cells. These studies have implications for our understanding of senescence versus exhaustion and for therapeutic interventions during chronic infection.

Limited efficiency of endogenous interleukin-7 levels in T cell reconstitution during HIV-1 infection: will exogenous interleukin-7 therapy work?

The molecular basis of the failed immune response in chronic HBV: Therapeutic implications

Targeting Innate Immunity: A New Step in the Development of Combination Therapy for Chronic Hepatitis B

The range of polyclonal CD4 T cell receptor (TCR) affinities that participate during the response to viral infection is currently unknown. Utilizing a highly sensitive two-dimensional micropipette adhesion frequency assay (2D), we determined the TCR affinity and frequency of CD4 T cells that expand and survive throughout multiple phases of the chronic and acute lymphocytic choriomeningitis virus (LCMV) T cell response. We discovered consistently more (4-5 fold) CD4 T cells specific for the IAb GP66-77 LCMV epitope than detected with pMHC II tetramer. The 2D identified lower affinity cells participate in the anti-viral response and are apparent at all time points of acute and chronic infection. Average TCR affinities measured at peak and later stages of the chronic response remained constant and equivalent to corresponding acute response measurements. However, programmed death 1 (PD-1) expressing CD4+ T cells late in chronic infection are functionally unresponsive despite static affinities. In contrast, memory cells in the acute response have a higher functional avidity compared to peak effectors (produce IFNγ in response to lower amount of peptide antigen) yet T cell memory is not restricted to select TCR affinities. Our findings suggest long term antigen exposure and functional enhancement/impairment exert no selective pressure in the survival or deletion of specific CD4 TCR affinities and TCR affinity alone does not always predict biological response.

In the first phase of the viral life cycle, the virus enters cells using a specific cell surface receptor. Many viruses use multiple receptors: some of which are unique to a certain cell type, whereas others are found in many cell types. After the virus enters into cells, various cellular proteins may interact with it; some support virus replication, while others inhibit it. Once virus succeeds to establish its life cycle in the target cell, the progeny viruses disseminate within the tissues or systemically via viremia. The intrinsic pro- or anti-viral cellular machinery differs among cell types. Thus, depending on the receptors used, the viral cell tropism is determined, resulting in the characteristic distribution of virus-infected cells/tissues and the disease outcome in vivo. How viral cell tropism, determined by the receptor used, can affect the disease outcome in acute and chronic virus infection is a major subject under extensive investigation in Virology. To understand the mechanism which causes human diseases by viruses, that is, viral pathogenesis, we have studied various aspects of virus infection at a cell/tissue level or using animal models. In this context, the recent development of reverse genetics allows us to visualize virus-infected cells/tissues or even the virus itself. By applying such manipulated viruses to animal models, it is also possible to analyze the dynamics of virus infection in vivo. In this Research Topic, we selected on studies connecting virus receptor usage and the pathogenesis of various viruses causing acute or chronic infection. Eventually, it could expand to cover the receptor-pathogenesis relationship in various acute and chronic virus infection. This research topic comprises an original research article on HIV-1, two opinions articles on the hepatitis C virus (HCV) and norovirus, while the remaining review articles on HTVL-1, measles virus (MV), mouse hepatitis virus (MHV), influenza virus, HCV, and enterovirus (EV) provide overviews on various aspects of viral pathogenesis. In all these review/opinion articles, at least one comprehensive table or figure is incorporated so that readers who are unfamiliar with these viruses can get a message at a glance. With regards to the cell tropism of HIV-1, Terahara et al. (2012) presented his recent study using CCR5-tropic and CXCR4-tropic HIV-1 with distinct fluorescent reporter. These HIV-1s allowed us to detect HIV-infected cells at a different stage of infection and to evaluate the level of virus replication in CD4+ T cells with distinct differentiation phenotype including CCR5+ memory. In contrast, a receptor for HTLV-1 and related pathogenesis is still intriguing issue, which is described by Hoshino (2012) in his extensive review. The two reviews on the MV (Kato et al., 2012; Takeda et al., 2012) were published at a very appropriate time as a third receptor for MV entry into epithelial cells, nectin 4, had just been discovered (Muhlebach et al., 2011; Noyce et al., 2011). Here, Kato et al. (2012) focused on the receptor usage of MV in vivo which may influences the disease outcome using monkey models, while Takeda et al. (2012) discussed about the dual-tropic nature of MV using SLAM and nectin 4 expressed in immune cells and epithelial cells, respectively. Ito et al. (2012) addressed the importance of B cells as a reservoir for persistent HCV infection. In two reviews on HCV, Moriishi and Matsuura (2012) overviewed a current research focus on lipid components for the HCV pathogenesis, while Shoji et al. (2012) discussed about glucose metabolic disorders associated with HCV infection. Nishimura and Shimizu (2012) and Yamayoshi et al. (2012), both of whom successfully identified two novel receptors for EV, overviewed the current knowledge about receptor usage and various diseases associated with EV infection. For the coronavirus, Taguchi and Hirai-Yuki (2012) overviewed studies on the receptor and related cellular factors for MHV, which may contribute to the mouse susceptibility to MHV infection. As regards to the virus recognizing sugar moieties, Ramos and Fernandez-Sesma (2012) provided insights about the interaction of influenza A virus with sialic acid receptors on immune cells with special reference to the innate immune response. Shirato (2012) described about the norovirus with distinct genotypes which recognize a specific structure of sugar chain. We will learn by these articles the fact that to identify a receptor is the first important step to know a virus, but many questions remain in order to fully understand human diseases caused by viruses. I would like to express my cordial thanks to all the contributors for this topic. I hope readers find the content interesting, but most importantly, that the information will prove very useful for future research.

In chronic infections and cancer, exhausted CD8 T cells exhibit heterogeneous subpopulations. TCF1+PD-1+ progenitor exhausted CD8 T cells (Tpex) can self-renew and give rise to Tim-3+PD-1+ terminally differentiated CD8 T cells that retain their effector functions. Tpex cells are thus essential to maintaining a pool of antigen-specific CD8 T cells during persistent antigenic stimulation, and only they respond to PD-1-targeted therapy. Despite their potential as a crucial therapeutic target for immune interventions, the mechanisms controlling the maintenance of virus-specific Tpex cells remain to be determined. We observed approximately 10-fold fewer Tpex cells in the spleens of mice chronically infected with lymphocytic choriomeningitis virus (LCMV) one-year post-infection (p.i.) than at three months p.i. Similar to memory CD8 T cells, Tpex cells have been found to undergo self-renewal in the lymphoid organs, prominently the bone marrow, during chronic LCMV infection. Furthermore, ex vivo treatment with IL-15 preferentially induced the proliferation of Tpex cells rather than the terminally differentiated subsets. Interestingly, single-cell RNA sequencing analysis of LCMV-specific exhausted CD8 T cells after ex vivo IL-15 treatment compared with those before treatment revealed increased expression of ribosome-related genes and decreased expression of genes associated with the TCR signaling pathway and apoptosis in both Tpex and Ttex subsets. The exogenous administration of IL-15 to chronically LCMV-infected mice also significantly increased self-renewal of Tpex cells in the spleen and bone marrow. In addition, we assessed the responsiveness of CD8 tumor-infiltrating lymphocytes (TILs) from renal cell carcinoma patients to IL-15. Similar to the data we obtained from chronic viral infection in mice, the expansion of the Tpex subset of PD-1+ CD8 TILs upon ex vivo IL-15 treatment was significantly higher than that of the terminally differentiated subset. These results show that IL-15 could promote self-renewal of Tpex cells, which has important therapeutic implications.

During immune responses against chronic viral infection or cancer, expression of the immune-inhibitory receptor Programmed cell death-1 (PD-1) on T cells results in cellular exhaustion, characterized by poor cellular immune functions ultimately leading to a failure to respond appropriately to antigen. Intriguingly, exhausted PD-1hi cells from a chronic infection remain exhausted even after removal from chronic antigen exposure, indicating an active epigenetic mechanism is employed to inhibit PD-1 expression. This study elucidates the role of the histone H3K4me1/me2 demethylase LSD1 in mediating epigenetic changes at the PD-1 locus which lead to gene silencing. Blimp-1, a known inhibitor of PD-1 that associates with epigenetic modifiers, was bound to the locus in CD8 T cells during both acute and chronic modes of infection. However, LSD1 was only recruited during acute infection concurrent with PD-1 silencing. Accordingly, the active histone modifications H3K4me1 and me2, which are removed by LSD1, were absent after acute but not chronic infection. PD-1 expression on T cells was enhanced and prolonged during acute infections in an LSD1 conditional knockout mouse. Expansion of these studies to an in vivo melanoma model also showed that loss of LSD1 resulted in higher expression of PD-1 on tumor-infiltrating lymphocytes, and a corresponding failure to clear tumor cells. Together these data highlight a distinct role for LSD1 as a defining factor mediating the silencing of PD-1 expression following acute immune activation. This study elucidates a key epigenetic mechanism governing the switch in cell fate between acute effector or exhausted T cell, and highlights the overall role that epigenetics play in modulating immune responses.

During many chronic infections, the responding CD8 T cells become exhausted as they progressively lose their ability to elaborate key effector functions. Unlike prototypic memory CD8 cells, which rapidly synthesize IFN-gamma following activation, severely exhausted T cells fail to produce this effector molecule. Nevertheless, the ontogeny of exhausted CD8 T cells, as well as the underlying mechanisms that account for their functional inactivation, remains ill defined. We have used cytokine reporter mice, which mark the transcription of IFN-gamma mRNA by the expression of Thy1.1, to decipher how activation events during the early stages of a chronic infection dictate the development of exhaustion. We show that virus-specific CD8 T cells clearly respond during the early stages of chronic lymphocytic choriomeningitis virus infection, and that this early T cell response is more pronounced than that initially observed in acutely infected hosts. Thus, exhausted CD8 T cells appear to emerge from populations of potently activated precursors. Unlike acute infections, which result in massive expansion of the responding T cells, there is a rapid attenuation of further expansion during chronic infections. The exhausted T cells that subsequently emerge in chronically infected hosts are incapable of producing the IFN-gamma protein. Surprisingly, high levels of the IFN-gamma transcript are still present in exhausted cells, demonstrating that ablation of IFN-gamma production by exhausted cells is not due to transcriptional silencing. Thus, posttranscription regulatory mechanisms likely disable this effector module.

During infection, antigen-specific T cells undergo tightly regulated developmental transitions controlled by transcriptional and post-transcriptional regulation of gene expression. We found that the microRNA miR-31 was strongly induced by activation of the T cell antigen receptor (TCR) in a pathway involving calcium and activation of the transcription factor NFAT. During chronic infection with lymphocytic choriomeningitis virus (LCMV) clone 13, miR-31-deficent mice recovered from clinical disease, while wild-type mice continued to show signs of disease. This disease phenotype was explained by the presence of larger numbers of cytokine-secreting LCMV-specific CD8+ T cells in miR-31-deficent mice than in wild-type mice. Mechanistically, miR-31 increased the sensitivity of T cells to type I interferons, which interfered with effector T cell function and increased the expression of several proteins related to T cell dysfunction during chronic infection. These studies identify miR-31 as an important regulator of T cell exhaustion in chronic infection.

The innovation of histocompatibility locus antigen (HLA) class I tetramers has revolutionized our understanding of virus-specific T cells (1). To generate this class of reagent, HLA class I molecules are made in Escherichia coli and then refolded with epitope peptides. A peptide tag added at the carboxy terminus of the heavy chain allows the complex to be biotinylated enzymatically, so that 4 HLA molecules bind 1 fluorochrome-marked streptavidin molecule. Although monomeric HLA binding is of too low an affinity to be useful in T-cell recognition, these labeled tetrameric complexes bind stably to antigen-specific CD8+ T cells, because of the avidity they gain by forming tetramers. Tetramer–T cell receptor (TCR) complexes are internalized after binding, which also preserves the staining of the target CD8+ T cells (2). One surprise to come out of this work concerns the deduced prevalence of virus-specific T cells. More than 1% of blood CD8+ T cells stain for single epitopes from persistently infecting viruses such as Epstein-Barr virus (3), human T-cell lymphotropic virus type I (4, 5), or HIV (6). These numbers are more than 10 times greater than those calculated in limiting dilution assays (LDAs), yet there is no evidence that use of tetramers leads to an overestimate of the number of antigen-specific T cells. In acute viral infections, the number of specific CD8+ T cells is even greater. In acute infectious mononucleosis, up to 44% of blood CD8+ T cells can react with a single Epstein-Barr virus epitope (3). Similar numbers are found in mice infected with lymphocytic choriomeningitis virus (LCMV) (7), influenza virus (8), and Listeria monocytogenes (9). These huge expansions are short-lived — most of the T cells die by apoptosis, which may explain the discrepancy between the direct counts of specific T-cell subpopulations and the values measured by the LDA. In the LDA, single clones have to survive, divide, and differentiate before they can be detected, so this assay may measure long-term memory T cells, whereas tetramers stain both memory cells and an expanded effector population that can no longer divide (10). It has been questioned whether all of the CD8+ T cells that proliferate in response to acute infections actually function in immune responses (11). Here too, HLA class I tetramers have provided useful information. Once T cells expressing specific receptors have been stained using these tetramers, their function can be assessed by sorting stained cells or by costaining with antibodies to cytokines, chemokines, or perforin. Despite clues that in most instances such cells are active, there are reports of impaired function. Mice infected with high-titer, aggressive LCMV have expansions of tetramer-staining cells that seem functionless (12). In addition, dysfunctional tumor-specific T cells were found in a patient with melanoma (13), and LCMV-infected mice that had been depleted of CD4+ T cells expressed CD8+ T cells that stained with an appropriate tetramer but appeared functionless (11). In HIV infection, CD4+ T-cell function is impaired early, raising the question of whether all of the CD8+ T cells seen in HIV-infected individuals are active against the virus. There is evidence that these cells function, but the issue remains of how CD4+ T cells influence the CD8+ T-cell response. This uncertainty demonstrates the need to develop HLA class II tetramers that could be used to explore the role of CD4+ T cells in infections, autoimmunity, and atopic diseases. Several groups have tried to develop such reagents, but all have met with technical difficulties. Soluble α and β chains from H-2 IEk have been made in E. coli, and then refolded in the presence of excess peptide and biotinylated (14). Monomeric, dimeric, trimeric, and tetrameric complexes formed could be used to study T-cell activation, but yields were low and the method has been difficult to reproduce with other class II alleles. Successful class II tetramer formation requires interaction of 3 components — α and β chains and the peptide — making it more complex than the corresponding process for class I molecules, which can occur in 2 steps. We have attempted to refold HLA DR1 made by E. coli without success, despite having added leucine zippers to link the peptide to the β chain (A. Kelleher, unpublished results). However, E. coli expression has been used successfully by another group (L. Stern, personal communication). Crawford et al. (15) developed a second approach that uses recombinant baculovirus or direct transfection to express class II molecules in insect cells, taking advantage of the ability of these cells to express HLA class II molecules at high levels in the absence of antigen processing. These authors linked a number of peptide epitopes to the amino terminus of the β chain using a short flexible linker to produce H-2 IEk and H-2 IAk, from which they prepared biotinylated tetramers. These tetramers stained T-cell hybridomas; the intensity of their staining correlated with their affinity for the TCR and the level of TCR expression. Antigen-specific T cells were detectable in TCR transgenic mice. HLA DR4 tetramers were also made (J. Kappler, personal communication). We have prepared HLA DR1 coupled to an HIV gag peptide in this way (A. Kelleher, unpublished results). A drawback to this approach is that the peptide has to be engineered into the construct, which is cumbersome, but in this issue of the JCI, Novak et al. (16) describe production of HLA DR4 tetramers that contain an influenza virus epitope. DR4 was expressed in empty form in insect cells, with the 2 chains linked at their carboxy termini by a leucine zipper. Empty DR4 was purified and then soaked in the required epitope peptide to give a stable molecule that could be biotinylated and used to stain influenza virus–specific CD4+ T cells. This staining confirms the specificity of the reagent and affords Novak and colleagues the first glimpse of a human antiviral CD4+ T-cell response. They could detect no staining of T cells in blood from immune humans, but stimulation in vitro with influenza antigen induced CD4+ T cells that stained with the tetramer. By costaining these cultures with 5-carboxyfluorescein diacetate succinimidyl ester, which dilutes exponentially as the cells divide, Novak et al. could calculate the number of divisions that the cells completed — up to 10 — within 7 days of antigen stimulation. These data allow the number of precursors in the original blood sample to be determined, avoiding the considerable difficulties associated with the LDA. Precursor frequency determined by these calculations agrees with estimates from the LDA (< 1 in 104). The number of virus-specific CD4+ T cells appears to be lower than that of the CD8+ T cells cited above, but the latter were measured during persisting infections. Influenza virus is eliminated after each attack; specific CD8+ T cells can be found in the blood of healthy adults but at similar low levels (1 in 104). It remains to be seen whether the CD4+ T-cell response to a persisting or acute virus is smaller than the massive CD8+ T-cell response. Many questions about the human CD4+ T cell response to natural antigens can now be explored in detail.

Hepatitis C virus (HCV) causes persistent infection in most patients. To clarify the mechanisms underlying establishment of this persistent infection, nucleotide sequences of the E1/E2 region were characterized in 5 patients with acute and chronic HCV infection. We used direct DNA sequencing methods to identify the major sequence of HCV in each patient. Each HCV genome displayed a high frequency of nucleotide sequence variation in the hypervariable region (HVR) of E2. However, patient-specific conserved nucleotide sequences were identified in the E1/E2 region during the course of infection and conserved the higher-order protein structure. In the acute phase HCV infection, amino acid substitution in HVR-1 as the monthly rate of amino acids substitution per site (%) between each point exceeded 10.2%. In the chronic phase HCV infection, a significantly lower rate of amino acid substitution was observed in patients. The host immune responses to HVR-1 of each HCV isolates from all clinical courses were characterized using synthetic peptides and ELISA. One chronic patient serum (genotype 1b) did not react at all to its own HVR-1 peptides, however another patient (genotype 2b) reacted to all clinical course. These results indicated that HVR-1 might not always exhibit neutralizing epitopes of HCV infection. The sequence variation in HVR-1 may instead indicate the existence of various clones in acute phase infection and the adaption of these clones is thought to have caused persistent and chronic infection in each patient.

Background: T cell exhaustion is a hyporesponsive state commonly found in cancer and is characterized by overexpression of inhibitory receptors and secretion of immunosuppressive cytokines. Immune checkpoint inhibitors (ICIs) target inhibitory receptors such as PD-1 and CTLA-4 to reinvigorate exhausted T cells and promote anti-tumor responses. While ICIs have improved clinical outcomes, no biomarker to date reliably determines the extent of T cell exhaustion. Since robust T cell receptor (TCR) signaling is critical for effective antitumor immunity, we hypothesized that interrogating the phospho-signaling states within the T cells along with their functional states will enhance our understanding of T cell exhaustion and yield potential biomarkers. Methods: In this study, we developed a validated mass cytometry panel of 24 markers to quantify phosphorylation states of 8 intracellular kinases involved in the TCR signaling within multiple CD8+ T cell subtypes. To identify potential differences between the local and peripheral immunological responses, we profiled CD8+ T cells derived from four sites (tumor, tumor draining lymph node, spleen and peripheral blood) in mice bearing MC38-induced flank tumors. We performed a clustering analysis of the dataset in aggregate using FlowSOM and visualized the results with UMAP, a dimensionality reduction algorithm, to compare the phospho-profiles of CD8+ T cell subtypes in all four sites. Results: Bulk analysis of CD8+ T cells demonstrates site-specific concordance between phosphoprotein markers and T cell functional markers (i.e. Ki67 and Granzyme B) with the highest expression in tumor followed by tumor draining lymph node, spleen and peripheral blood. Based on the results from the clustering analysis, site-specific breakdown of CD8+ T cells revealed high prevalence of effector, memory, and exhausted memory T cells in tumors compared to other sites where naïve T cells predominated. Despite these variations in CD8+ T cell immunophenotypic distribution, TCR signaling profiles correlated strongly with specific T cell subtypes across all sites. Conclusion: These data demonstrate that subtype-specific TCR signaling is preserved systemically and that phospho-immune subtyping of CD8+ T cells in the peripheral blood may be used to identify T cell exhaustion states that are found in the tumor microenvironment. Further studies will be conducted to investigate the changes in the TCR phospho-profiles in response to ICI treatment and their associated functions. Citation Format: Zaw Phyo, Rohan Verma, Won J. Ho, Elizabeth M. Jaffee. Site-specific profiling of TCR signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4475.

The infection from Epstein-Barr virus (EBV) or virus of infectious mononucleosis, together with other herpes viruses’ infections, represents a prototype of persistent viral infections characterized by the property of the latency. Although the reactivations of the latent infection are associated with the resumption of the viral replication and eventually with the “shedding”, it is still not clear if this virus can determine chronic infectious diseases, more or less evolutive. These diseases could include some pathological conditions actually defined as “idiopathic”and characterized by the “viral persistence” as the more credible pathogenetic factor. Among the so-called idiopathic syndromes, the “chronic fatigue syndrome” (CFS) aroused a great interest around the eighties of the last century when, just for its relationship with EBV, it was called “chronic mononucleosis” or “chronic EBV infection”.Today CFS, as defined in 1994 by the CDC of Atlanta (USA), really represents a multifactorial syndrome characterized by a chronic course, where reactivation and remission phases alternate, and by a good prognosis. The etiopathogenetic role of EBV is demonstrated only in a well-examined subgroup of patients, while in most of the remaining cases this role should be played by other infectious agents - able to remain in a latent or persistent way in the host – or even by not infectious agents (toxic, neuroendocrine, methabolic, etc.). However, the pathogenetic substrate of the different etiologic forms seems to be the same, much probably represented by the oxidative damage due to the release of pro-inflammatory cytokines as a response to the triggering event (infectious or not infectious).Anyway, recently the scientists turned their’s attention to the genetic predisposition of the subjects affected by the syndrome, so that in the last years the genetic studies, together with those of molecular biology, received a great impulse. Thanks to both these studies it was possibile to confirm the etiologic links between the syndrome and EBV or other herpesviruses or other persistent infectious agents.The mechanisms of EBV latency have been carefully examined both because they represent the virus strategy to elude the response of the immune system of the host, and because they are correlated with those oncologic conditions associated to the viral persistence, particularly lymphomas and lymphoproliferative disorders. Just these malignancies, for which a pathogenetic role of EBV is clearly documented, should represent the main clinical expression of a first group of chronic EBV infections characterized by a natural history where the neoplastic event aroused from the viral persistence in the resting B cells for all the life, from the genetic predisposition of the host and from the oncogenic potentialities of the virus that chronically persists and incurs reactivations.Really, these oncological diseases should be considered more complications than chronic forms of the illness, as well as other malignancies for which a viral – or even infectious - etiology is well recognized. The chronic diseases, in fact, should be linked in a pathogenetic and temporal way to the acute infection, from whom start the natural history of the following disease. So, as for the chronic liver diseases from HBV and HCV, it was conied the acronym of CAEBV (Chronic Active EBV infection), distinguishing within these pathologies the more severe forms (SCAEBV) mostly reported in Far East and among children or adolescents. Probably only these forms have to be considered expressions of a chronic EBV infection “sensu scrictu”, together with those forms of CFS where the etiopathogenetic and temporal link with the acute EBV infection is well documented. As for CFS, also for CAEBV the criteria for a case definition were defined, even on the basis of serological and virological findings. However, the lymphoproliferative disorders are excluded from these forms and mantain their nosographic (e.g. T or B cell or NK type lymphomas) and pathogenetic collocation, even when they occur within chronic forms of EBV infection. In the pathogenesis, near to the programs of latency of the virus, the genetic and environmental factors, independent from the real natural history of EBV infection, play a crucial role.Finally, it was realized a review of cases - not much numerous in literature – of chronic EBV infection associated to chronic liver and neurological diseases, where the modern techniques of molecular biology should be useful to obtain a more exact etiologic definition, not always possibile to reach in the past.The wide variety of clinical forms associated to the EBV chronic infection makes difficult the finding of a univocal pathogenetic link. There is no doubt, however, that a careful examination of the different clinical forms described in this review should be useful to open new horizons to the study of the persistent viral infections and the still not well cleared pathologies that they can induce in the human host.