Primary immunodeficiencies and the control of Epstein Barr virus infection

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1 Ann. N.Y. Acad. Sci. ISSN ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Issue: The Year in Immunology Primary immunodeficiencies and the control of Epstein Barr virus infection Umaimainthan Palendira 1,2 and Alan B. Rickinson 3 1 Centenary Institute, Newtown, New South Wales, Australia. 2 Discipline of Medicine, Sydney Medical School, University of Sydney, NSW, Australia. 3 Cancer Sciences and Centre for Human Virology, University of Birmingham, Birmingham, United Kingdom Address for correspondence: Umaimainthan Palendira, Centenary Institute, Locked Bag No. 6, Newtown, NSW 2042, Australia. m.palendira@centenary.org.au Human primary immunodeficiency (PID) states, where mutations in single immune system genes predispose individuals to certain infectious agents and not others, are experiments of nature that hold important lessons for the immunologist. The number of genetically defined PIDs is rising rapidly, as is the opportunity to learn from them. Epstein Barr virus (EBV), a human herpesvirus, has long been of interest because of its complex interaction with the immune system. Thus, it causes both infectious mononucleosis (IM), an immunopathologic disease associated with exaggerated host responses, and at least one malignancy, EBV-positive lymphoproliferative disease, when those responses are impaired. Here, we describe the full range of PIDs currently linked with an increased risk of EBVassociated disease. These provide examples where IM-like immunopathology is fatally exaggerated, and others where responses impaired at the stage of induction, expansion, or effector function predispose to malignancy. Current evidence from this rapidly moving field supports the view that lesions in both natural killer cell and T cell function can lead to EBV pathology. Keywords: primary immunodeficiencies; Epstein Barr virus; genetic defects; cell-mediated immunity Introduction The multiplicity of hematopoietic cell types and effector pathways constituting the human immune system bears witness to the multiple challenges that infectious agents have posed throughout the evolution of our species and its antecedents. Any one type of pathogen is likely to alert several response pathways, and so understanding the relative importance of those pathways to overall control of that infection is a complex task. In this regard, the study of patients with primary immunodeficiencies (PIDs) can be hugely instructive. 1 3 Such individuals, carrying mutations in single immune system genes, have to deal with the vast range of naturally acquired infections but will present in the clinic only with those against which their defenses are especially compromised. The human herpesviruses are particularly interesting litmus tests of immune competence. All are potentially pathogenic but, having coevolved with our species and its antecedents over millions of years, have arrived at a virus host balance that is critically dependent upon host control. Most of these agents are widespread in the population, often being acquired silently or with mild symptoms in childhood and then carried for life as asymptomatic latent infections. PID patients are therefore likely to be exposed to these viruses relatively early in life and will have to deal with them both as a primary infection and as a persistent challenge. Here, we focus on one particular herpesvirus, Epstein Barr virus (EBV), which largely conforms to the abovementioned pattern of asymptomatic infection in the immunocompetent host, yet is etiologically linked to a range of nonmalignant and malignant diseases. 4,5 How the human immune system normallycontrolsebvandtowhatextentaberrantcontrols contribute to disease pathogenesis remain to be fully determined. 6,7 A brief survey of the biology and immunology of EBV infection, as currently inferred doi: /nyas Ann. N.Y. Acad. Sci (2015) C 2015 The Authors Annals of the New York Academy of Sciences This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

2 Palendira & Rickinson Primary immunodeficiencies and Epstein Barr virus from studies in the immunocompetent host and in immunosuppressed patients, is given below as a prelude to the evidence emerging from PID states. EBV infection in the immunocompetent host EBV is a gamma-1 herpesvirus, a genus whose members are distinguished by their restriction to primate hosts, their persistence in the B lymphoid system, and their ability to drive growth through coordinate expression of a unique set of latent cycle genes. 4,8 10 As illustrated in Figure 1, the virus is acquired orally and replicates as a lytic infection within the oropharynx, leading to high levels of infectious virus shedding in throat washings. Little is known about these very early events; however, lytic replication is thought to occur in oropharyngeal epithelium and possibly also locally infiltrating s. Thereafter, from the evidence of tonsillar tissues from infectious mononucleosis (IM) patients undergoing primary EBV infection, 11 the virus appears to spread into the system by transient activation of a latent growth-transforming infection. Many of these expanding cells express the full panel of virus latent proteins and, by implication, resemble the lymphoblastoid cell lines (LCLs) that arise when the virus transforms normal Bcellsin vitro. However, in at least some cells, the growth-transforming program is suppressed and those cells, now carrying the virus genome as a truly latent (antigen-negative) infection, enter the recirculating memory pool (see Fig. 1 legend for details and Refs ). This pool then appears to be stably maintained for life, with occasional cells reactivating from latency into lytic infection at oropharyngeal sites, thereby initiating new transforming infections as well as seeding secondary foci of virus replication at oropharyngeal surfaces that lead to low-level virus shedding. 4,6,9 Beneath this broad framework, the detailed virological and immunological events involved in asymptomatic infection are still poorly understood. Much is inferred from studies on patients, mainly young adults, in whom an atypically delayed primary infection is clinically manifest as IM; even in these circumstances, the characteristic disease symptoms (fever, lymphadenopathy, and + T lymphocytosis) only develop some 4 6 weeks after virus acquisition, 15 and little is known about this long prodromal phase. Much of the + Tcell expansion seen in acute IM blood appears to be virus specific and mainly directed against immediate early and some early proteins of the lytic cycle, with smaller responses against the latent proteins. 6,16 Coincident with the resolution of symptoms, these responses contract to smaller virus-specific memory populations that persist for life, at levels not obviously different from those found in long-term viruscarrierswithnohistoryofim. 16,17 Thereisalso a parallel, but much smaller, CD4 + Tcellresponse to both lytic and latent cycle antigens in IM, from which CD4 + T cell memory populations are likewise derived. 18,19 In addition, recent work has shown that while total natural killer () cell numbers are not obviously increased in the blood in acute phase, this masks a significant expansion of activated cells with a phenotype intermediate between the less mature CD56 bright,kir subset normally dominant in lymphoid tissues, and the more mature CD56 dim, KIR + subset normally dominant in blood. 20 IM is increasingly considered to be an immunopathologic disease whose febrile symptoms and malaise are caused by proinflammatory cytokines released from hyperactivated T cells. It is not clear whether the immune responses seen in IM simply exaggerate those occurring during asymptomatic primary infection, or if the two situations are also qualitatively distinct. Recent data, albeit in a humanized mouse model, suggest that in asymptomatic primary infection, cells act to limit the initial replication of the virus, thereby reducing the peak antigen load and avoiding antigen-driven overexpansion of the + T cell response that drives the disease process. 21 Such a role for cells would accord to earlier in vitro work demonstrating that an infected cell s entry into the lytic cycle is accompanied by increased sensitivity to cell recognition. 22 Furthermore, other innate effectors may also be involved at such early times. In that context, although there have been no studies of invariant natural killer T (it) cell activation in acute IM, there is evidence that it cells are able to recognize and kill s immediately after EBV infection in vitro 23 and, when adoptively transferred in humanized mouse models in vivo, cancontrol the outgrowth of EBV-positive tumor cell lines Multiple arms of the cell-mediated response could therefore be employed at one point or another in the control of EBV, and the relationship between them remains to be determined. Ann. N.Y. Acad. Sci (2015) C 2015 The Authors Annals of the New York Academy of Sciences 23

3 Primary immunodeficiencies and Epstein Barr virus Palendira & Rickinson Primary infection (IM) Persistent infection Oropharynx Tonsil Tonsil Oropharynx L3 L3 Epithelial cells Lyt Lyt Lyt Lyt L3 L3 L3 CD4 CD4 L0 Virus reservoir L0 CD4 Latent responses Reactivation signal L0 Lyt CD4 Lyt Epithelial cells Lytic responses Latent responses Lytic responses CD4 CD4 Blood CD4 L0 L0 CD4 Blood CD4 Lytic primary responses Latent primary responses Latent memory responses Lytic memory responses Figure 1. A schematic diagram to illustrate the main features of EBV infection in the immunocompetent host, as currently understood. Orally acquired EBV replicates locally as a lytic infection (Lyt), probably in oral epithelial cells (and possibly locally infiltrating s), leading to high levels of viral shedding detectable in throat washings. On the basis of studies of tonsillar tissues from IM patients, the virus then spreads through initiating a latent growth-transforming infection (Latency III, L3) of s that expand through clonal proliferation; this appears to involve the expression of the full array of EBV latent proteins, just as when EBV infects s in vitro to produce EBV-transformed LCLs. Thereafter, in at least some cells, the virus downregulates the expression of the latent proteins; the cells move out of cycle and then carry the virus genome as a true antigen-negative latent infection (Latency 0, L0). Such cells are found exclusively within a memory reservoir that preferentially recirculates between the peripheral blood and oropharyngeal lymphoid tissues. How the virus selectively populates the memory (but not naive) pool in vivo is not clear, though various models have been proposed, including the possibility that virus-infected naive s are driven through a germinal center reaction, thus exploiting the physiologic route into memory. 4,9,12 14 Thereafter, during long-term virus carriage, some latently infected s will reactivate into the lytic cycle (possibly as a result of cognate antigen-driven differentiation into plasma cells) and produce infectious virions. These may either initiate new growth-transforming infections in neighboring s or seed secondary foci of lytic replication at oropharyngeal sites, thereby leading to low-level virus shedding in throat washings. Host immune responses have the potential to interfere with these events at many points. During primary infection (as seen in IM), cells are activated, and there is a rapid expansion of + T cells against the lytic cycle and to some extent latent cycle antigens (both of which are more apparent in the blood than the tonsil), while there is a much smaller expansion of latent and lytic antigen-specific CD4 + T cells. As the primary infection resolves, both virus load in the blood and (eventually) virus shedding in the throat fall to much lower levels; similarly, virus-specific T cell responses contract to smaller long-term populations that persist for life both in the blood and, at higher frequencies, in tonsillar tissues. During virus persistence, latent antigen-specific memory T cells may control new transforming infections, while lytic antigen-specific memory T cells may control new foci of virus replication in the oropharynx. Black arrows on the diagram represent movement of infected cells, dotted arrows represent movement of virus particles, and red arrows represent the targeting of and T cell responses. EBV infection in the immunosuppressed host Further evidence that cell-mediated surveillance is important in maintaining the EBV host balance comes from the study of immunosuppressed solid organ and stem cell transplant recipients. In the first-year posttransplant when the immunosuppressive regime is most intense, such patients show increased latent viral loads in the blood and are at risk of EBV-driven B lymphoproliferative disease (B-LPD). This disease can present initially as an IM-like lymphadenopathy but develops into oligoclonal or monoclonal foci of B lymphoproliferation. 24 Ann. N.Y. Acad. Sci (2015) C 2015 The Authors Annals of the New York Academy of Sciences

4 Palendira & Rickinson Primary immunodeficiencies and Epstein Barr virus Here, as in in vitro transformed LCLs, most cells express the full spectrum of latent cycle proteins, while a few are switching from the transformed state into the lytic cycle. 27,28 Essentially, the same disease was also often seen in human immunodeficiency virus (HIV)-infected patients who, before the era of highly active retroviral therapy, progressed to late-stage acquired immune deficiency syndrome (AIDS) with profound CD4 + T cell lymphopenia and associated + T cell exhaustion. 29 In both the posttransplant and late-stage AIDS contexts, it is tempting to attribute the appearance of EBVdriven B-LPD entirely to a loss of effective T cell surveillance. Certainly, the LPD lesions themselves can be effectively targeted by adoptive transfer of human leukocyte antigen (HLA)-matched T cell preparations enriched for EBV-specific (mainly ) reactivities by LCL stimulation in vitro. 30,31 However, the circumstances that precede B-LPD development, that is, the tipping of the virus host balance toward the high virus loads, may not be entirely a consequence of impaired T cell controls. Thus, there is evidence that T cell suppressive drugs, such as cyclosporine, and also uncontrolled HIV infection can both impair some aspects of cell function As fully described elsewhere, EBV is etiologically linked to a number of other malignancies. 4,5,29 For the purposes of this review, we mention only those tumors where there is either clear evidence or reasonable suspicion that tumor risk is elevated by immune impairment, that is, tumors whose appearance might therefore be taken as a sign of impaired EBV control in specific PID settings. In that context, like EBV-driven B-LPD, an EBV-positive smooth muscle cell tumor (leiomyosarcoma) has only been observed in highly immunocompromised transplant and late-stage AIDS patients. 35 Three other tumors, Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and diffuse large lymphoma (DLBCL) (the latter often recorded as non-hodgkin lymphoma), can occur in either EBV-positive or EBV-negative forms and are not as directly linked to profound immune impairment. However, all three occur at increased incidence in AIDS patients, as do the Hodgkin and diffuse large lymphomas in two further groups: solid-organ transplant patients on long-term immune suppression, and elderly patients in the general population who may suffer age-related immune senescence. 8,29,36 Primary immune deficiency states and EBV-associated disease The explosion in human genome sequencing means that there are now over 200 genetically defined PIDs affecting one or other aspect of hematopoietic cell development and/or immune effector function. 37 Here, we focus on those with evidence of EBVassociated disease. To give structure to the review, we have arranged those conditions into four groups, based upon their overall susceptibility to infectious agents and/or lymphoma; we would emphasize that the boundaries between these groups are not set in stone, and may indeed change in the future. As each group is introduced in the coming text, the essential characteristics of the constituent PIDs will be summarized in Tables 1 4. Table 1. PIDs selectively susceptible to EBV Disorder Affected gene Inheritance Associated immune defects EBV-associated disease Other pathogenic infections XLP Impaired lysis, targets Most with severe IM, fatal HLH None SH2D1A it Absent X-linked T Impaired recognition B Hypogamma XIAP Normal Some with severe IM, HLH None BIRC4 it Reduced numbers X-linked T Apoptosis prone B Hypogamma in some cases HLH, hemophagocytic lymphohistiocytosis. Ann. N.Y. Acad. Sci (2015) C 2015 The Authors Annals of the New York Academy of Sciences 25

5 Primary immunodeficiencies and Epstein Barr virus Palendira & Rickinson Immunocompetent XLP Lysis NTBA NTBA SAP + Lysis pmhc-tcr Impaired lysis NTBA NTBA SHP-1 SHP-1 Lysis pmhc-tcr target CD48 2B4 SAP SHP-1 Non- target target CD48 2B4 Non- target CD48 NTBA NTBA Lysis 2B4 SAP SAP + Activatory receptors Lysis CD48 Impaired lysis NTBA NTBA 2B4 SHIP-1 SHIP-1 Activatory receptors Lysis SHIP-1 Figure 2. A schematic diagram of the molecular pathogenesis of XLP. In immunocompetent hosts, the + T cell or cell interaction with targets relies on positive signals delivered through the signaling lymphocyte activation molecule family of receptors (SLAMFRs). In this regard, NTBA NTBA interactions and 2B4 CD48 interactions deliver a positive signal to T cells or cells via the adaptor protein SAP. The presence of SAP also prevents the binding of negative regulators, such as SHP-1 or SHIP-1, to SLAMFR. The ensuing positive signals activate + T cells and cells to lyse targets. By contrast, + Tcellor cell recognition of non- targets, such as dendritic cells and epithelial cells, is independent of the SLAMFR interaction. In XLP patients, when + T cells and cells attempt to engage targets, the absence of SAP allows the recruitment of negative regulators such as SHP-1 ( + T cells) and SHIP-1 ( cells) to the SLAMFRs, resulting in impaired target lysis. 159 However, the effector functions of SAP-deficient + T cells and cells against non- targets remain unaffected. Group 1: PIDs selectively susceptible to EBV X-linked lymphoproliferative syndrome X-linked lymphoproliferative syndrome (XLP) is a rare X-linked immune deficiency first recognized through young boys presenting with an extreme, often fatal, form of IM following primary EBV infection (Fig. 2) The acute disease was characterized by massive sustained expansions of EBVinfected s, + T cells, and cells in blood and tissues, and a resultant cytokine storm that led on to macrophage activation and hemophagocytic lymphohistiocytosis (HLH). Surprisingly, other virus infections appeared to be handled normally, making XLP the paradigmatic example of genetic susceptibility to a single infectious agent. However, not all boys with the XLP trait presented in this way; others had a history of hypogammaglobulinemia or lymphoma that was independent of, and often preceded, EBV infection. 38 Reconciling these disparate presentations only became possible once the genetic basis of XLP was discovered. The affected gene SH2D1A encodes an adaptor protein, the signaling lymphocyte activation molecule (SLAM)-associated protein (SAP), which is expressed in T cells, cells, and it cells. SAP binds to the cytoplasmic tails of the SLAM family of receptors, namely, SLAM (CD150), 2B4 (CD244), NTBA, CD229, 4, and CRACC. These receptors are expressed on a variety of hematopoietic cells and, with one exception (2B4), mediate cell cell communication through homodimeric interactions; 2B4 has a heterologous ligand, CD48, that is highly expressed on EBV-infected s. Loss-of-function mutations in SAP lead to the complex immunodeficient phenotype seen in XLP. Affected boys lack it cells, have specific defects in and T cell function, and cannot generate classical T cell dependent memory. 42 While the absence of it cells has fueled speculation of a role for these cells in EBV control, greater insight came with the findings that cells, 43,44 and subsequently EBV-specific + T cells, 45,46 from XLP patients failed to form immune synapses with B lymphocytes; in contrast, recognition of other target cell types was unaffected. As illustrated in Figure 2 and described in the legend, recognition by cells and T cells requires positive 26 Ann. N.Y. Acad. Sci (2015) C 2015 The Authors Annals of the New York Academy of Sciences

6 Palendira & Rickinson Primary immunodeficiencies and Epstein Barr virus signaling through two SLAM receptors, 2B4 and NTBA; in the absence of SAP, the 2B4 CD48 and NTBA NTBA interactions still occur but now appear to deliver a negative signal. These functional defects in and T communication help to explain several aspects of the XLP patients varied clinical presentation, in particular their unique susceptibility to EBV. Thus, the virus colonizes its host specifically via infection, a cell type that in XLP patients neither cells nor virus-specific + T cells can efficiently recognize. These effectors therefore cannot contain the infection nor, in the case of + T cells, can they receive the restimulation-induced cell death signals through which highly expanded T cell responses are normally curtailed. 47 In the absence of this autoregulatory route, lymphoproliferation is further amplified, generating the cytokine storm that presages HLH. Nevertheless, some XLP patients survive the initial onslaught of EBV infection and go on to maintain a stable EBV viral load in the blood. Remarkably, several such patients showed evidence of somatic reversion to SAP positivity in a small fraction of circulating lymphocytes, almost exclusively in the + T cell compartment. These reverted cells had not only regained effective recognition but also many appeared to be EBV specific, suggesting that selective pressure from EBV infection had expanded a rare population of spontaneous revertants to detectable levels. 48 Clearer evidence that the + Tcellresponse to EBV is primarily driven by virus infection in the compartment, and not, for example, by replication in oropharyngeal epithelial cells, came from a study of XLP carrier mothers. In such individuals, random X-chromosome inactivation means that 50% of T cells are SAP positive, and 50% SAP negative. While T cell memory to two non-b tropic viruses (influenza and the cytomegalovirus (CMV)) was present in both subsets, EBV-specific + T cells against both lytic and latent cycle antigens were almost all found in the SAP-positive population. 45 X-linked inhibitor of apoptosis protein deficiency A second immunodeficiency sharing some characteristicswithxlpstemsfrommutationofan adjacent gene on chromosome Xq25, BIRC4, which encodes the X-linked inhibitor of apoptosis (IAP) protein, XIAP. Indeed, the trait was first identified by screening 15 families in which boys presented with XLP-like symptoms; three of these families were found to have a BIRC4 rather than SH2D1A gene mutation, with seven of 12 affected boys having recurrent HLH possibly linked to EBV infection. However, subsequent surveys of HLH in genetically identified XIAP patients have not found such a strong link with EBV, and it is likely that the initial focus on XLP-like patients may have contributed to a high degree of EBV penetrance in the original report. Clearly, XIAP and XLP share important clinical features, notably presentation either as HLH and hypogammaglobulinemia, but are different in other respects; in particular, XIAP patients sometimes present with chronic colitis but not with lymphoma. The relationship between the two conditions therefore remains a subject of debate How loss of XIAP protein function leads to the clinical phenotype is unclear. The protein is one of eight members of the IAP family and acts mainly as a potent inhibitor of caspases 3, 7, and 9; however, it is also involved in a variety of other signaling pathways, including nuclear factorkappa B (NF- B) and c-jun N-terminal kinase activation. 53 Interestingly, the reduced it cell numbers first noticed in XIAP 51 appear to be restricted to EBV-positive patients, 54 implying that EBV infection itself may drive activation-induced it cell death. By contrast, cell numbers and 2B4-dependent effector function appear to be normal in XIAP patients, as are circulating T cell numbers despite the fact that T cells are more sensitive to apoptosis-inducing signals in vitro. Arecent study of one XIAP kindred (albeit one also carrying a rare CD40 ligand polymorphism) has identified individuals with persistent lymphocytosis, and unusually large expansion of EBV-specific CD4 + and + T cells, in the blood many years after symptomatic primary EBV infection. 55 This is at least consistent with the idea that hyperexpansion of the EBV-induced T cell response could underlie at least some cases of XIAP-associated HLH. Group 2: PIDs with broader virus susceptibility but frequent EBV disease CD27 deficiency CD27 deficiency, a PID arising in almost all cases from homozygous CD27 mutation, was Ann. N.Y. Acad. Sci (2015) C 2015 The Authors Annals of the New York Academy of Sciences 27

7 Primary immunodeficiencies and Epstein Barr virus Palendira & Rickinson Table 2. PIDs with broader virus susceptibility but frequent EBV disease Disorder Affected gene Inheritance Associated immune defects EBV-associated disease CD27 def CD27 Autosomal recessive it T Normal Low-normal Normal 12/17 with severe IM or chronic EBV; 9/17 with EBV + tumors: B-LPD, HL, DLBCL B Hypogamma in some cases XMEN Impaired lysis 4/7 with EBV + tumors: B-LPD, BL, HL, NHL MAGT1 it? X-Linked T Impaired lysis B Normal Other pathogenic infections Influenza, VZV, CMV Viral pneumonia, M. contagiosum, VZV, HSV ITK def Normal 8/9 with EBV + tumors: B-LPD, HL VZV, CMV ITK it Reduced numbers Autosomal recessive T Progressive CD4 lymphopenia B Progressive hypogamma Coronin 1A def CORO1A it Normal Near absent 4/6 with EBV + tumors: B-LPD, HL, DLBCL Autosomal recessive T T lymphopenia B Hypogamma CD16 def FCGR3A it Impaired lysis Normal 1/3 with severe IM, 1/3 with EBV + tumor: B-LPD Autosomal recessive T Normal B Normal HSV, HPV HPV, VZV MCM4 def Impaired maturation 1/13 with EBV + tumor: B-LPD HSV, VZV MCM4 it Normal Autosomal recessive T Normal B Normal VZV, varicella-zoster virus; CMV, cytomegalovirus; NHL, non-hodgkin lymphoma; HSV, herpes simplex virus. recently recognized through its unexpectedly high incidence of EBV-associated pathology. Among 17 affected individuals identified to date, 12 had either suffered symptomatic primary EBV infection/lymphadenopathy early in life or had presented with chronic EBV viremia. Importantly, nine of the above 17 (not always those individuals with prior EBV problems) eventually developed malignancy, six involving EBV-positive LPD/lymphomas, and three Hodgkin lymphomas that seem likely to have been EBV positive though their virus status was not recorded. Clearly, however, CD27 deficiency is not entirely EBV selective in its effects since some patients also had a history of severe infections with influenza virus or with herpesviruses such as varicella-zoster virus (VZV) or CMV CD27, a costimulatory molecule belonging to the tumor necrosis factor receptor (TNFR) family, is constitutively expressed as a transmembrane homodimer on all memory s, on all T cells except for a terminally differentiated (CD57 + ) subset, and on CD56 bright cells. Its ligand, CD70, is transiently expressed on activated dendritic cells (DCs), as well as on s and T cells following antigen receptor signaling. 59 In + T cells, signaling through CD27 has been shown to induce expression of IL-2 and the prosurvival protein Bcl-XL, consistent with its importance for the 28 Ann. N.Y. Acad. Sci (2015) C 2015 The Authors Annals of the New York Academy of Sciences

8 Palendira & Rickinson Primary immunodeficiencies and Epstein Barr virus survival of virus-specific + T cells in mouse models. 60 In addition, mouse models have also shown that in CD4 + T cells, CD27 CD70 signaling suppresses Th17 differentiation and promotes an IFN- secreting Th1 phenotype. 61 Perhaps surprisingly, CD27-deficient patients showed very little disturbance of lymphocyte populations in the blood, with normal numbers and normal distribution of most B and T cell subsets (though + effector memory T cells are sometimes expanded), and normal numbers of cells, it cells, and T cells. Studies to date on immune functions have shown, at most, only subtle impairments cell cytotoxicity was reduced in some but not all patients, 57 while one report speaks about hypogammaglobulinemia along with reduced T cell dependent responses in vitro. 56 However, since no EBV-seronegative individuals have yet been identified so far, it is unclear whether these are mild primary features of CD27 deficiency per se or secondary effects of EBV infection. Importantly, the two patients analyzed for EBV-specific T cell responses to date both had normal levels of EBV-specific + T cells and these cells appeared to be functional in cytokine- and degranulation-based assays. 57 Why CD27-deficient patients have such preferential susceptibility to EBV is as yet unclear. Some subtle impairment of T cell, or CD56 bright cell, function remains a possibility. It is worth noting that, in vitro, EBV infection upregulates the CD27 ligand CD70 to high levels on the surface. 62 Thus, CD27 CD70 interactions may be particularly important in determining their susceptibility to virus-specific T cell or cell surveillance. This latter possibility is interesting given that another PID, affecting a different TNFR family member OX40, was identified through the patient s unusual susceptibility to the Kaposi sarcoma-associated herpesvirus (KSHV). That susceptibility may reflect the importance of OX40 OX40 ligand interactions in KSHV surveillance since the ligand is highly upregulated on virus-infected endothelial cells. 63 MAGT-1 deficiency A second PID recently identified through its impairment of EBV control is caused by loss of the X-chromosome encoded Mg 2+ ion transporter MAGT-1, hence its definition as X-linked immunodeficiency with Mg 2+ defect, EBV infection, and neoplasia (XMEN). Of the seven patients reported, all had high EBV genome loads in the blood and four had developed one of the following EBV-positive lymphomas: classical B-LPD, Burkitt lymphoma, Hodgkin lymphoma, and non- Hodgkin lymphoma. Again, the immunodeficiency is not selective for EBV. These patients also have a history of recurrent respiratory infections, viral pneumonia, and severe poxvirus (molluscum contagiosum) or herpesvirus (VZV and HSV) infections This implies a wider impairment of viral immune surveillance, which subsequent studies have addressed with interesting results. MAGT1, one of 20 mammalian Mg 2+ transporters, is expressed in many cell lineages but its immunological importance was only realized with discovery of the XMEN syndrome. In T cells, MAGT1 mediates the Mg 2+ influx induced by T cell receptor (TCR) stimulation and, through downstream Mg 2+ pathway signaling, also optimizes Ca 2+ influx. MAGT1-deficient patients have low CD4 + T cell numbers, likely due to poor thymic output, whereas cell and + T cell (including EBV-specific + T cell) numbers appear normal. However, both and + T cell cytotoxicity was partly impaired. As illustrated in Figure 3, this led to the finding that free Mg ions regulate these cells expression of G2D, a membrane protein that engages its ligand, G2DL, on the target cell surface. As the name implies, G2D was first identified as an activating receptor but it also now appears to be required for optimal cytotoxicity by G2D-expressing + T cells. Thus, the cytotoxic function of - and EBV-specific T cells from XMEN patients in vitro could be fully restored by Mg 2+ supplementation of the medium, coincident with upregulation of G2D expression. Furthermore, an Mg 2+ -supplemented diet was also able to reduce EBV genome loads in vivo, strongly suggesting a role for one or both of these effectors in maintaining the EBV host balance. 66 It is likely that and/or T cell control of other viruses is similarly impaired. ITK deficiency Homozygous mutations in the interleukin-2 inducible T cell kinase gene (ITK) underpin another PID, again first recognized through its high prevalence of EBV-associated disease but not strictly EBV specific in its effects. ITK is one of the Ann. N.Y. Acad. Sci (2015) C 2015 The Authors Annals of the New York Academy of Sciences 29

9 Primary immunodeficiencies and Epstein Barr virus Palendira & Rickinson Immunocompetent XMEN disease G2D Lysis Impaired lysis MAGT1 G2DL G2DL Mg 2+ EBV + target EBV + target pmhc-tcr Reduced Mg 2+ uptake MAGT1 Activatory receptor G2D Lysis G2DL G2DL Impaired lysis Figure 3. A schematic illustration of the molecular pathogenesis of XMEN disease. In mammalian cells, there are several different Mg 2+ transporters involved in the regulation of Mg 2+ influx. One such transporter, MAGT1 is an important regulator of intracellular free Mg 2+ in immune cells. In T cells, optimal activation following TCR stimulation depends on Mg 2+ influx. In addition, the expression of the activatory receptor G2D on T cells and cells is also dependent on free Mg 2+. In immunocompetent individuals, a functional MAGT1 receptor therefore facilitates optimal T cell and cell activation upon recognition of virally infected targets. In patients with XMEN disease, the defective MAGT1 receptor results in reduced Mg 2+ uptake in T and cells, leading to reduced G2D expression and impaired target cell lysis. five members of the Tec family that function as nonreceptor protein tyrosine kinases. 67 Its expression is restricted to T cells, cells, it cells, and mast cells. In T cells, it is induced upon TCR activation or IL-2 stimulation and is thought to play a critical role in downstream signaling. Altogether nine patients from six kindred have been identified to date, with inactivating mutations affecting different domains of the protein. The condition is characterized by a progressive loss of CD4 + T cells, particularly naive CD4 + T cells, and a progressive hypogammaglobulinemia. Most of the patients have normal levels of + T cells and cells, whereas circulating it cell numbers are heavily reduced The recent identification of an EBV-naive patient has confirmed that these features are primary consequences of ITK deficiency and not EBV induced. 71 Remarkably, the first eight reported ITK-deficient patients from five unrelated kindred had all developed EBV-positive malignancies, either B-LPD or Hodgkin lymphoma. 72 Note that several of these cases were discovered, following the initial report of EBV disease in two ITK-deficient sisters, 70 by screening for ITK mutations in patients who had a history of EBV-associated tumors; hence, the seemingly very high penetrance of EBV disease in ITK deficiency may be an overestimate. Nevertheless, the recently identified EBV-naive patient subsequently acquired EBV and developed a smooth muscle cell tumor. 71 Several instances of severe VZV and CMV infections were also recorded among the abovementioned patients and so the overall picture of viral susceptibility associated with ITK mutation remains unclear. The incidence of EBV-associated disease is nevertheless interesting and further work is needed to determine to what extent it deficiency and/or impaired T or cell function is responsible. Coronin 1A deficiency Recent work has identified three kindred with a combined immune deficiency linked to homozygous or heterozygous mutations in the gene encoding the coronin family member coronin 1A. This protein, widely expressed in hematopoietic 30 Ann. N.Y. Acad. Sci (2015) C 2015 The Authors Annals of the New York Academy of Sciences

10 Palendira & Rickinson Primary immunodeficiencies and Epstein Barr virus Immunocompetent CD16 mutation Activatory receptors Activatory receptors CD16 CD2 EBV + EBV + target target cell cell CD2 X mcd16 Lysis Impaired lysis Figure 4. A schematic view of the immune defect associated with CD16 deficiency. In immunocompetent individuals, cell lysis of target cells depends upon a favorable balance between activatory and inhibitory receptors. CD16 is one such activatory receptor that can deliver activating signals to cells when it engages its ligand, CD2, expressed on target cells. Missense mutations in FCGR3A, leading to expression of a mutant CD16 (mcd16), abrogate the CD16 CD2 interaction and impair spontaneous cell lysis of virally infected targets cells. cells, directly binds filamentous (F) actin and regulates actin polymerization through interaction with the actin-related protein (ARP) 2/3 complex. 73 Murine studies first showed that coronin 1A deletion caused peripheral T lymphopenia, perhaps by affecting thymic emigration 74 or TCR signaling in naive T cells, 73 and led to impaired T-dependent antibody responses; by contrast, cell and naive development were unaffected. Subsequent screening of patients with a T-deficient, B- and -sufficient phenotype indeed identified individuals with coronin 1A mutations; where studied, it cell numbers were also grossly reduced in these patients. 75 All six affected individuals identified to date showed a general predisposition to various infections but with a high penetrance of EBV-associated disease. A first case, with heterozygous gene mutations and no detectable coronin 1A protein, had a history of respiratory infections, oral thrush, and inability to control a live attenuated VZV vaccine before stem cell transplantation at 4 years of age. 76 In another kindred, where a homozygous missense coronin 1A mutation led to barely detectable protein expression, three affected siblings again had a history of recurrent respiratory infections but interestingly all three later developed EBV-positive LPD. 77 Finally, in a third kindred, two siblings with heterozygous mutations suffered multiple cutaneous infections with HSV, human papilloma viruses (HPVs), and/or Mycobacterium leprae in their first decade before, in one case, developing Hodgkin lymphoma and then a diffuse large lymphoma, both tumors being confirmed as EBV positive. 78 CD16 impairment Homozygous missense mutations in FCGR3A, the gene encoding the Fc receptor CD16 expressed on cells, have been identified in at least three patients from unrelated families Importantly, these patients have normal numbers of cells, T cells, and s but, while T and functions appear unaffected, and key aspects of cell function are impaired. Normally, cells recognize target cells for destruction when signals received through their activatory receptors outweigh those received through inhibitory receptors. One such activatory receptor is CD16 expressed by mature cells; its best known function is to engage the immunoglobulin G (IgG) Fc domain of IgG-opsonized target cells and induce antibody-dependent cellular cytotoxicity (ADCC). However, CD16 can also promote conventional lysis through a coactivating interaction with CD2 on the target cell surface. Interestingly, the FCGR3A mutations described above leave ADCC function intact but, as illustrated in Figure 4, abrogate the CD16 CD2 interaction and impair spontaneous cell cytotoxicity. This -selective PID Ann. N.Y. Acad. Sci (2015) C 2015 The Authors Annals of the New York Academy of Sciences 31

11 Primary immunodeficiencies and Epstein Barr virus Palendira & Rickinson therefore offers the clearest litmus test of the importance of cell mediated killing in viral defense. All three patients suffered from a number of viral infections, including HPV and VZV; once again, however, there was a significant incidence of EBV-associated disease. One patient suffered a prolonged IM-like illness that lasted nearly 10 months, while another developed recurrent EBV-associated B-LPD. Group 3: PIDs generally susceptible to viral and nonviral infections Activating mutations of the PI3K pathway Recent work has identified a new class of PIDs, marked by recurrent bacterial and viral infections and caused by hyperactivation of the phosphatidylinositol-3-oh kinase (PI3K) pathway. The class I PI3Ks, responsible for phosphorylation of the inositol ring of phosphatidylinositol Table 3. PIDs generally susceptible to viral and nonviral infections Disorder Affected gene Inheritance Associated immune defects EBV-associated disease Hyperactive PI3K P110 /p85a it Normal numbers? 2/30 with EBV + tumors: HL, DLBCL Dominant active T CD4 lymphopenia, exhaustion B Hypogamma in some cases STK4 def STK4/MST1 it Normal numbers? 3/8 with EBV + tumors: B-LPD, HL Autosomal recessive T Naive T cell deficiency B Low-normal numbers ZAP70 def ZAP-70 it Normal numbers Normal numbers 1/19 with EBV + tumor: DLBCL Autosomal recessive T CD4 reduced B Normalnumbers CTPS1 def CTPS1 it Normal numbers Absent 8/8 with recurrent IM, 3/8 with EBV + tumor: B-LPD Autosomal recessive T CD4 lymphopenia B Reduced memory NHEJ def Low-normal (1) 2/6 with EBV + 1. Artemis 2. DNA Lig IV Hypomorphic it T? Reduced numbers tumors: B-LPD (2) 2/17 with EBV + tumors: DLBCL B Reducednumbers Gata 2 def GATA2 Autosomal dominant it T Near absent Normal CD4 lymphopenia in some 2/57 with EBV + tumors: smooth muscle, mesenchymal cases B Near absent Chediak-Higashi Impaired lysis Some cases of accelerated LYST it? disease/hlh? Autosomal recessive T Normal (?) B Normal Other pathogenic infections Respiratory bacteria, VZV, HSV, CMV Bacterial, fungal, HPV, VZV, CMV Bacterial, fungal, HSV, HPV, M. contagiosum Bacterial, HPV, VZV, and other viruses Bacterial, viral Bacterial, VZV, CMV, HSV, HPV Respiratory bacteria 32 Ann. N.Y. Acad. Sci (2015) C 2015 The Authors Annals of the New York Academy of Sciences

12 Palendira & Rickinson Primary immunodeficiencies and Epstein Barr virus membrane lipids, consist of a catalytic subunit (p110, p110, or p110 ) and a regulatory subunit (p85, p85, orp55 ). 82 The p110 / p85 heterodimer in particular plays a key role in both TCR and B-cell receptor (BCR) signaling, and mutations affecting both subunits have been described. Thus, two studies have reported a combined total of 26 PID patients from 14 kindred with p110 mutation; all cases had gain-of-function mutations that resulted in hyperactivation of the PI3K Akt mtor pathway. 83,84 Similar hyperactivation can also result from heterozygous splice site mutations leading to a truncated p85 subunit that has lost regulatory function In each case, the main immune defects were a progressive loss of CD4 + (particularly naive CD4 + ) T cells, defective T cell dependent antibody responses, a reduction in class-switched memory s, and accumulation of immature transitional s. cell numbers were normal in most cases but, from evidence in mouse models, 88 their function may be compromised. All patients with PI3K mutations suffered recurrent respiratory bacterial infections and increased susceptibility to several viral infections; details varied between reports, to some extent reflecting the depth of analysis. In one study, five of the 17 patients with p110 mutations had a history of severe herpesvirus infections, 83 whereas two of nine patients in a parallel study developed EBV-positive tumors, one a classical Hodgkin lymphoma and the other a diffuse large lymphoma; furthermore, all nine had high EBV viral loads, and some also high CMV loads, in the blood. 84 Also one of the four patients with p85 mutation had high EBV and CMV viral loads in the blood. 87 When analyzed, most patients were able to generate large numbers of EBV-specific + T cells. However, like the population as a whole, these cells were skewed toward terminally differentiated phenotype, possibly indicative of chronic antigen challenge and/or functional impairment. 84 STK4 deficiency A similar broad susceptibility to infections is seen in another recently defined PID caused by homozygous mutations in the gene encoding the ubiquitously expressed serine threonine kinase 4 (STK4), which is also known as mammalian sterile 20-like protein (MST1). 89,90 SKT4 s role in immune cells is poorly defined, but its ability to phosphorylate members of the FOXO transcription factor family can have multiple downstream effects. For example, in T cells, FOXO1 regulates the expression of the IL-7 receptor and of the lymphoid homing molecules CCR7 and CD62L. Loss of STK4 has all the hallmarks of a severe T cell deficiency. The main immune defects include a progressive loss of naive T cells, restricted TCR diversity, and CD4 + Tcell lymphopenia. + T cell and numbers are normal in some cases but low in others, whereas cell numbers are normal. To date, eight STK4-deficient patients have been identified from four kindred. All suffered recurrent infections in early life with bacteria, fungi, and virusessuchashpv,hsv,vzv,andebv.specifically, three of four children from two unrelated families had high EBV loads in blood and, of these, one went on to develop EBV-positive Hodgkin lymphoma. 90 In two further reports, one of three affected siblings and a single child in another family both developed EBV-positive LPD ZAP70 deficiency Another severe T cell deficiency is caused by mutations affecting ZAP70, a nonreceptor tyrosine kinase that is a key component of the TCR signal transduction pathway. Upon TCR stimulation, ZAP70 is recruited to the CD3 chain where, after its phosphorylation by Lck, it phosphorylates a number of downstream targets to initiate the signaling cascade. ZAP70 mutation is characterized by a profound T cell deficiency with near absence of + T cells and impaired TCR signaling in the remaining CD4 + T cells, whereas the and cell compartments seem unaffected. 92 So far, at least 19 ZAP70-deficient patients have been identified. Although their clinical presentations have been heterogeneous, generally, they show increased susceptibility to recurrent bacterial, fungal, or viral infections in the first 2 years of life and a failure to thrive. Of the viral infections, HSV, molluscum contagiosum, and HPV are the most pathogenic. The EBV status of many patients was often not determined. However, one infant with normal numbers of s and CD4 + T cells, but a near absence of + T cells, developed an aggressive EBV-positive diffuse large lymphoma. 93 CTPS1 deficiency A recent study has identified homozygous mutations in the gene encoding cytidine 5 triphosphate Ann. N.Y. Acad. Sci (2015) C 2015 The Authors Annals of the New York Academy of Sciences 33

13 Primary immunodeficiencies and Epstein Barr virus Palendira & Rickinson synthase 1 (CTPS1) as responsible for another combined deficiency of adaptive immunity. CTPS1 is strongly and specifically activated in T cells following TCR engagement and appears to be the main source of cytidine 5 triphosphate supporting deoxyribonucleic acid (DNA) synthesis for TCR-induced proliferation. Thus, T cells from CTSP1-deficient patients retained the immediate signaling events after TCR triggering in vitro but could not sustain the subsequent proliferative burst. proliferation following BCR stimulation in vitro was also impaired, whereas IL-2 induced cell responses were much less affected. Some, but not all affected individuals had T cell,, and, to some extent, cell lymphopenia, most obviously during active infections, while further analysis in one case failed to detect any it cells. 94 Eight CTSP1-deficient patients in this first report suffered recurrent encapsulated bacterial infections as well as multiple viral infections, with EBV and VZV particularly prominent. Thus, four of the eight patients studied had a history of a severe IM-like syndrome within the first year of life, while another three developed EBV-positive B-LPD lesions in the central nervous system, a classic site for uncontrolled EBV transforming events. 94 The susceptibility to herpesvirus, especially EBV, infections would accord with failure of the virus-induced primary T cell burst. Nonhomologous DNA end-joining deficiencies The process of nonhomologous DNA end joining (NHEJ) is generally important for DNA doublestrand break repair in mammalian cells, but also crucial for the generation of BCR and TCR diversity in lymphocytes through V(D)J recombination. Genetic defects in several components of the NHEJ machinery cause severe combined immunodeficiency in humans, abrogating all B and T lymphopoiesis and necessitating hematopoietic stem cell transplantation (HSCT). 95 However, hypomorphic mutations in two of these components, the Artemis protein and DNA ligase IV, have beenfoundallowinglimitedbandtlymphocyte development. All six reported cases of hypomorphic Artemis gene (DCLRE1C) mutation were susceptible to multiple infectious agents early in life However, two of four affected individuals in the original report eventually died of an EBV-positive LPD, one of which developed spontaneously and the other as a post-hsct lesion. 96 At least 17 cases of hypomorphic DNA ligase IV mutation have been identified, again with multiple recurrent infections. Three of these cases developed an EBV-positive diffuse large lymphoma. 99,107 In one instance, the disease arose in one of two sisters, both heterozygous for a null and a hypomorphic DNA ligase IV (LIG4) allele and showing reduced T cells, almost absent s, but normal cell numbers and an expanded T cell population. 99 GATA2 deficiency The hematopoietic transcription factor GATA2 is a member of a family of zinc finger transcription factors that regulate gene expression during hemopoiesis. 108 It appears to be more important for non-t cell lineages and indeed is highly expressed in CD56 bright cells. 109 Heterozygous mutations of GATA2 are now recognized to underlie a complex range of immune-deficient phenotypes, typically characterized by marked reductions in cell, monocyte,, and DC numbers in blood; half of those surveyed to date also had reduced CD4 + Tcell numbers. With respect to cells, the CD56 bright subset is particularly affected but, in some patients, CD56 dim cells are also low and their function is impaired. 109,110 Interestingly, what is now recognized as the index case of GATA2 deficiency was originally reported as an cell defect linked to severe VZV, CMV, and HSV infections. 111 However, a recent survey of 57 patients found generalized susceptibility to multiple viruses, also including HPV, as well as to mycobacterial and fungal infections. Interestingly, only a small minority of patients had high EBV viral load in the blood, but two individuals developed unusual EBV-positive malignancies: one was a smooth muscle cell tumor and the other an uncharacterized tumor of mesenchymal origin. 110 Given the complexity of the GATA2-deficient phenotype, it is difficult to interpret these findings in terms of EBV control. The relative rarity of high EBV blood loads, and the absence of EBV-positive LPD, may simply reflect the paucity of mature s; indeed, the frequency of EBV infection in these patients is still poorly documented. A limited reservoir may reduce the chance of EBV establishing its conventional latency; however, in these circumstances, the absence of effective cell control over replicative infection may allow the virus to access atypical cell lineages, such as smooth muscle cells, in which it cannot replicate but is potentially oncogenic. 34 Ann. N.Y. Acad. Sci (2015) C 2015 The Authors Annals of the New York Academy of Sciences