Pathways Case Record: EBV-associated Hemophagocytic Lymphohistiocytosis: A Case of Unchecked Inflammation

A 71-year-old man with a past medical history of bipolar depression, hypothyroidism, atrial fibrillation, and type 2 diabetes was admitted to another hospital with weakness and loss of consciousness. A few weeks before hospitalization, he began to experience gait changes, intermittent fevers, and fatigue. Upon presentation at the hospital, he was in septic shock, hypotensive, and had atrial fibrillation with rapid ventricular response. He was treated with fluid resuscitation, stress-dose steroids, and vasopressors. Further evaluation was notable for high liver function tests, elevated ferritin, and enlarged lymph nodes. Additionally, he was positive for Epstein-Barr virus (EBV), but EBV IgG was negative, suggesting acute EBV infection. He was then transferred to Massachusetts General Hospital for suspected hemophagocytic lymphohistiocytosis (HLH).

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Upon transfer to Mass General, the patient's evaluation was notable for pancytopenia (reduced red blood cells, white blood cells, and platelets), persistently abnormal liver function tests, high ferritin, and positive EBV PCR testing. Lumbar puncture revealed increased cell count, predominantly immune cells, and EBV in the cerebral spinal fluid, suggesting EBV meningoencephalitis. He was briefly treated with antiviral medications. His EBV titer declined, and his liver function tests returned to normal. He developed an ulcer that grew multiple microbes that was treated with antibacterial and antifungal medications.

The Pathways Service in the Department of Medicine at Mass General was consulted and focused on two main questions:

1. Does our patient have HLH?
2. Why did this patient develop inflammation on the spectrum of HLH?

Background and Diagnosis

The pathophysiology of HLH is not completely understood. Presentation of an overactivation immune system response (excessive inflammation, tissue destruction, and often end-organ dysfunction) in the setting of immune dysregulation is a hallmark of this serious inflammatory disease (Biol Blood Marrow Transplant). The immune system is regulated by normal downregulation by macrophages and lymphocytes. In HLH, however, these normal counter-regulatory mechanisms may be dysfunctional for various reasons. As a result, there is inappropriate, sustained overactivation of cytotoxic CD8+ T cells with excessive cytokine release, representing unchecked inflammation. Classically, HLH has been categorized into primary and secondary forms. Primary HLH is typically present in young children and caused by key gene mutations resulting in impaired cytotoxic T lymphocytes (CTL) and natural killer (NK) cell activity. Secondary HLH is primarily in adults and is an induced impairment of CTL and NK cell activity (Blood). In either case, defects in CTL/NK cell function lead to compensatory up-regulation of other inflammatory pathways that drive HLH pathogenesis.

There are two different sets of diagnostic criteria for adult patients with suspected HLH. The first is the identification of a heterozygous mutation that is known to be associated with HLH, in the appropriate clinical presentation (Pediatr Blood Cancer). Alternatively, a patient may meet diagnostic criteria based on clinical presentation if they have 5 of the following 9 findings: [1] fever, [2] enlarged spleen, [3] low blood counts, [4] high triglycerides or deficiency of fibrinogen in the blood, [5] phagocytosis of blood cells in bone marrow, spleen, lymph node, or liver, [6] low or absent natural killer (NK) cells, [7] high ferritin, [8] elevated soluble CD25 (soluble IL-2 receptor alpha [sIL-2R]), and [9] elevated CXCL9. While our patient did not meet formal diagnostic criteria, he did show four of these findings.

A bone marrow biopsy in our patient may have provided the evidence to confirm HLH diagnosis. Often, patients with suspected HLH are too critically ill to defer treatment while waiting for diagnostic test results and/or too unstable to tolerate a bone marrow biopsy. Therefore, there are modified criteria that are often relied upon to evaluate a patient for HLH and more promptly initiate necessary therapies. Based on the presence of fever, splenomegaly, hepatitis, and increased ferritin, our patient met this less specific diagnostic threshold.

Factors that predispose individuals to HLH include genetic and acquired susceptibilities. Many genetic mutations linked to HLH are involved in cell-mediated cytotoxicity and/or lymphocyte activation and survival (J Allergy Clin Immunol). Genetic defects are often considered to be deleterious mutations that produce a defective protein product unable to perform its function (for example, PRF1 mutations can cause defective perforin functioning resulting in no cytolytic activity). There may also be partial loss-of-function mutations that produce slightly defective protein products (Blood Res). There are 25 documented genes known to predispose patients to HLH. These genes can largely be broken down into three categories: [1] genes involved in degranulation and cytotoxic function (e.g., PRF1), [2] genes involved in lymphocyte activation (e.g., IL2R, IL7R, CD70), and [3] genes involved in apoptosis (e.g., caspases).

In addition to genetic contributions, acquired susceptibilities, such as hematologic malignancies, rheumatologic disorders, or HIV infection, may predispose a patient to develop HLH. Largely, these predisposing factors cause immune system suppression (Best Pract Res Clin Rheumatol). In patients with underlying immune deficiencies, the inability to control acute infections prompts overly aggressive compensatory immune system activation and therefore contributes to HLH development. A common example of such a trigger is a viral infection. The most common example is EBV, as seen in our patient. An underlying genetic mutation predisposing a patient to HLH is often coupled with the viral-mediated development of HLH (Best Pract Res Clin Rheumatol). It is reasonable to suspect that a loss-of-function mutation in one of this patient's key anti-viral genes may play a role in his presentation.

In patients with HLH, the combination of immune activation in the setting of underlying immune dysregulation results in abnormally sustained inflammation. While there may be an underlying trigger (e.g., viral infection), the abnormal immune response is largely responsible for the clinical presentation of HLH. In acute EBV infection, the virus infects B cells, which function as antigen presenting cells and in turn activate cytotoxic T lymphocyte (CTL) cells. This overactive immune response is driven by cytotoxic T cells, rather than macrophages, in HLH. For example, a patient may have a genetic mutation (e.g., PERF1 deficiency) that impairs one's ability to kill cells that have been infected by the virus, resulting in sustained antigen presentation to CD8 T cells and activation of the immune system. Additionally, the overly activated CD8 T cells stimulate antigen presenting cells and macrophages, resulting in the release of excessive amounts of cytokines, namely IL-1β and IL-18, resulting in a 'cytokine storm' that causes tissue damage, end-organ failure, and high mortality. As such, this disease process is marked by pathologic immune amplification loops that propagate unchecked inflammation (Best Pract Res Clin Rheumatol).

To understand why our patient improved, it is important to consider the natural immune response to a viral infection. When a virus is introduced, it is quickly phagocytized and fragments of its protein components are presented by MHC class I and II complexes to subsequently activate antigen-specific T and B lymphocytes. While CTLs immediately go into the bloodstream to kill virally-infected cells, B cells undergo affinity maturation to produce more specific antibodies against this viral antigen, resulting in an early CTL response and delayed antibody and B cell response. Typically, this earlier CTL response results in the curbing of exponential viral growth and systemic inflammation until the antibody response can effectively clear circulating virions (Nat Rev Immunol). If this CTL response is delayed or ineffective, however, the virus grows and divides exponentially, causing systemic inflammation and potentially HLH. When the IgM response eventually becomes activated, the virus will be controlled, and inflammation will resolve (Biol Blood Marrow Transplant). For this reason, we believe a loss-of-function mutation in a key CTL gene could cause HLH which would eventually resolve in our patient.

Summary and Future Steps

While it is important to note that EBV is one of the most common causes of HLH and acts through a variety of mechanisms including bone marrow exhaustion, viral dysregulation of cytokine balance, suppression of NK cell cytolytic activity, and evasion of immune recognition, it does not explain why such a common infection can cause such a severe, HLH-like presentation in an elderly gentleman (Front Immunol).

To examine whether this patient has a loss-of-function mutation in one of these genes, genetic testing of a panel of 25 HLH-associated genes should be performed (Blood). Additionally, whole exome sequencing may provide more insights into potential mutations of T cell function genes not included in the group of known HLH-associated genes.

To elucidate the cellular dynamics, we propose tracking EBV reactive T cells, anti-EBV IgM titers, and EBV viral load longitudinally across our patient's hospitalization.

Lastly, to better understand lymphocyte responses developed at different stages of the infection, we propose to run flow cytometry to identify naive, activated, effector, and memory B and T cell subtypes from the patient's blood longitudinally. These studies will provide us with key insights into the pathophysiology of EBV-related immune dysregulation and potentially identify therapeutic targets for the future.

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