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Prepublished online June 17, 2004; doi:10.1182/blood-2004-04-1431

Unusual Immunophenotype of CD8+ T-cells in Familial Hemophagocytic Lymphohistiocytosis Nitin J Karandikar, Steven H Kroft, Subramanian Yegappan, Beverly B Rogers, Victor M Aquino, Kyung-Mi Lee, Vinay Kumar, F J Guenaga, Elaine S Jaffe, Daniel C Douek and Robert W McKenna

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Nitin J. Karandikar , Steven H. Kroft , Subramanian Yegappan , Beverly B. Rogers , Victor M. Aquino2, Kyung-Mi Lee3,4, Vinay Kumar3, F. Javier Guenaga5, Elaine S. Jaffe6, Daniel C. Douek5 and Robert W. McKenna1 From the Departments of 1Pathology and 2Pediatrics, The University of Texas Southwestern Medical Center at Dallas, TX 75390, USA; 3Department of Pathology, The University of Chicago, Chicago, IL, USA; 4Korea University College of Medicine, Seoul, Korea 136-705; and 5The Vaccine Research Center, National Institute of Allergy and Infectious Diseases and 6

Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. Short Title: Unusual CTL Immunophenotype in FHL

Address correspondence to: Nitin J. Karandikar, MD, PhD Departments of Pathology and Neurology The University of Texas Southwestern Medical Center 5323 Harry Hines Blvd. Dallas, TX 75390-9072 Phone: 214-648-1416 Fax: 214-648-4070 e-mail: [email protected] Total Text Word Count: 1,199 Abstract Word Count: 146 Scientific Heading: Clinical Observations, Interventions, and Therapeutic Trials NJK is supported by USPHS NIH grant AI49990 and the UT Southwestern President’s Research Council Distinguished Young Investigator Award.


Copyright (c) 2004 American Society of Hematology

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ABSTRACT Familial hemophagocytic lymphohistiocytosis (FHL) is an inherited, fatal disorder of infancy.

We report here a 17-day old female infant who presented with high fever,

hepatosplenomegaly, hypertriglyceridemia, hypofibrinogenemia, thrombocytopenia and liver failure. Leukocytosis was detected with circulating “atypical” lymphoid cells. Flow cytometric studies revealed expanded subpopulations of CD8+ T-cells with unusual immunophenotypic features, including a subset that lacked CD5 expression.

A liver biopsy showed

hemophagocytic lymphohistiocytosis with exuberant infiltrates of CD8+ T-cells that lacked perforin. Mutational studies revealed a 666C A (H222Q) missense mutation in the perforin gene. T-cell receptor studies on flow-sorted T-cell subpopulations revealed no evidence of monoclonality. Analysis of T-cell receptor excision circle levels indicated long proliferative history in the aberrant CD8+ T-cell subsets. This case provides an instructive example of uncontrolled reactive proliferation of CD8+ T-cells in FHL, resulting in atypical morphology and unusual immunophenotypic features that may suggest malignancy in other clinical settings.


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hemophagocytic lymphohistiocytosis (HLH), is a disorder of infancy that usually presents with acute illness characterized by fever, hepatosplenomegaly, cytopenias, hyperlipidemia, coagulopathy and hemophagocytosis


. Most patients with FHL have deficient NK and

cytotoxic T-cell function associated with T-cell activation, impaired apoptosis, increased cytokine secretion and activation of mononuclear phagocytes


. An inherited mutation of

the perforin gene underlies the pathophysiology of FHL in a subset of patients


patients may bear mutations in different cytotoxicity-associated genes


, while other



expression of perforin is associated with increased activation and expansion of cytotoxic T cells


. The morphologic and immunophenotypic features of these T-cells have not been


Here, we report the case of a 17-day old girl with liver failure who was

suspected to have leukemia due to the presence of circulating atypical lymphoid cells. A liver biopsy revealed HLH associated with perforin deficiency.

The case provides an

instructive example of the morphologic, immunophenotypic and molecular attributes of Tcells that have undergone dysregulated activation and proliferation in the setting of FHL.


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MATERIALS AND METHODS Patient Presentation, Clinical Course and Laboratory Studies. A 17-day old female presented with high fever, hepatosplenomegaly, hematuria and progressive liver failure. An older sibling had died following a similar presentation. There was no evidence of bacterial or viral infection [including cytomegalovirus (CMV) or EpsteinBarr virus (EBV)]. There was mild anemia (Hemoglobin 11.7 g/dl), thrombocytopenia (21 × 109/L) and a leukocytosis (26.9 × 109/L) composed of atypical lymphoid cells (54% of leukocyte differential).

Blood was submitted for flow cytometric studies to evaluate for

leukemia. The patient had hypertriglyceridemia (153 mg/dl) and hypofibrinogenemia (114 seconds.

Liver biopsy findings were consistent with FHL (see Results). The patient suffered from post-biopsy blood loss and expired. Flow Cytometric Evaluation. Blood leukocytes were processed and analyzed as described previously




following markers were evaluated: CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD14, CD15, CD16, CD19, CD20, CD22, CD25, CD33, CD34, CD36, CD38, CD45, CD45RA, CD45RO, CD56, CD57, CD61, CD64, CD79a, T-cell receptor (TCR)-

, TCR- ,

kappa light chain, lambda light chain, myeloperoxidase, TdT. Flow Sorting. Leukocytes were stained for CD5, CD8 and CD4 and sorted into the indicated subsets using the BD FACSVantage SE sorter



Post-sort purity was >95% for each

subpopulation. TCR- Gene Rearrangement Studies. DNA was extracted from blood leukocytes or flow-sorted T-cells and used for TCRPCR

. 2.0 µl of the PCR product was added to 1.0 µl of ROX500 molecular markers and


24 µl of de-ionized formamide, denatured at 95oC for 5 minutes and immediately cooled on


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ice, followed by analysis on an ABI310 Genetic Analyzer using GeneScan software (Applied Biosystems). Perforin Gene Mutational Analysis. Sequencing of the perforin (PRF1) gene was performed for mutational analysis 4. T-cell receptor excision circle (TREC) levels. TREC levels were evaluated using quantitative real time PCR assays 14 albumin gene being used as a control for quantification .



with the

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RESULTS AND DISCUSSION Morphology and Flow Cytometry The peripheral blood contained numerous medium-sized to large lymphoid cells showing abundant basophilic cytoplasm with intense peripheral basophilia, oval to irregular nuclear contours, slightly dispersed chromatin and occasional nucleoli (Figure 1, Panel A). Flow cytometry (Figure 1, Panel B) revealed that 78% of events were mature, surface CD3+ T lymphocytes with a predominance of CD8+ T-cells (CD4:CD8 ratio = 0.35:1). CD4+ T-cells were composed of a CD45RO CD45RO+ subset (memory/effector).

subset (presumably naïve) and an expanded

CD8+ T-cells were composed of three major sub-

populations (blue, black and red in Figure 1, Panel B) with a CD2+, CD7+, CD45bright+, CD25predominantly–, CD56predominantly–, TCR-


and TdT– immunophenotype.




were also present and were distinct from these populations. The smallest subpopulation (~2.5% of total events; blue) was composed of small cells (forward light scatter characteristics) and exhibited a CD4–, HLA-DR–, CD45RO–, CD45RAbright+, CD5bright+ phenotype and most likely represented normal, naïve CD8+ T-cells. The other two subpopulations showed partial dim CD4 and slightly bright CD8 expression and were predominantly CD57–, uniformly CD45RO+ and CD45RAdim+to-, bearing some similarity to a previously described case


. The population indicated in black (~43% of total

events) showed variable cell size with dim CD5, variable HLA-DR and bright CD38 expression. The population indicated in red (~12% of total events) was composed of small to medium-sized HLA-DR+ cells that showed complete lack of CD5 expression. Thus, the CD8+ T-cells consisted of a small proportion of naïve T-cells and two unusual expanded subpopulations with an “effector/memory phenotype” and under-expression of CD5. Liver Biopsy The liver biopsy showed an exuberant lymphocytic infiltrate within portal triads, with occasional foci within lobules. The infiltrates were composed predominantly of CD3+ T-cells (Figure 2), including mainly CD8+ and few CD4+ cells with a high degree of pleomorphism, 6

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enlarged nuclei, eosinophilic nucleoli and abundant mitoses. Scattered macrophages were observed within sinusoids with occasional phagocytosis of neutrophils.

Kupffer cell

hyperplasia was noted on CD4 and CD68 staining. The infiltrating cells showed positive staining for granzyme B and T cell intracellular antigen-1 (TIA-1).

However, they were

negative for perforin. Scattered cells showed weak diffuse cytoplasmic staining, which was distinct from the perforin staining pattern seen in positive controls 7. These findings are 7 consistent with a diagnosis of FHL arising from a perforin mutation . Interestingly, CD5

staining revealed few positive cells, likely corresponding to the CD4+ T-cells. Thus, the infiltrating CD8+ T-cells in the liver corresponded to the CD5dim+ or CD5– subpopulations identified in blood. Clonality, Proliferation History and Mutational Analysis To evaluate for potential monoclonality in the T-cells, we flow-sorted four subpopulations (Figure 1, Panel B) and performed PCR reactions for TCR- rearrangement. All sorted populations and bulk blood leukocytes showed oligoclonal or polyclonal electrophoretograms (Figure 1, Panel C). Thus, despite the aberrant immunophenotype and atypical morphology, there was no evidence of a monoclonal/neoplastic lymphoproliferative disorder. This suggested a dysregulated reactive proliferation of CD8+ T-cells in the setting of perforin deficiency, leading to expansions of phenotypically unusual subsets. To test this hypothesis, we assessed proliferative history by measuring TRECs, which are episomal DNA fragments (excised from the TCR genes during thymic development) that are mitotically diluted during proliferation


. As predicted, the highest TREC levels were

observed in naïve CD8+ T-cells (Population II). Importantly, there were no detectable TRECs in the two aberrant subpopulations (III and IV), confirming their prolonged proliferative history (Figure 1, Panel C). To clarify the underlying genotype, we performed mutational analysis of the perforin gene


and discovered a homozygous missense point mutation in exon 3 [666C A


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(H222Q)]. This point mutation was recently described in a study of North American families with HLH 16. This case is an example of FHL associated with perforin deficiency and dysregulated proliferation of CD8+ T-cells, leading to atypical morphology and unusual immunophenotype. In other clinical settings (for example, in an adult patient), these findings would be suspicious for a clonal/malignant T-lineage proliferation.

However, there was no evidence of

monoclonality in any subpopulation, indicating that this was a reactive proliferation, probably driven by antigenic stimulation of CD8+ T-cells with impaired cytotoxic and apoptotic functions 3,9,17. This case study provides several valuable clinical lessons. First, it depicts the wide spectrum of morphologic and immunophenotypic variability in reactive (non-malignant) Tcells. Second, it provides a detailed description of the morphology and immunophenotype of FHL-associated, dysregulated CD8+ T-cells. In future studies, it may be worthwhile testing whether this phenotype is predictive of impaired cytotoxicity in the setting of HLH, thereby making it an indicator of focused diagnostic tests. Finally, this case re-emphasizes the need for a multi-modal approach to the diagnosis of hematolymphoid disorders, where data from morphology, immunophenotyping and molecular/genetic tests contribute to the diagnostic process.


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ACKNOWLEDGEMENTS The authors would like to thank Sterling Ortega and Bonnie Darnell for technical assistance.


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Janka GE, Schneider EM. Modern management of children with haemophagocytic

lymphohistiocytosis. Br J Haematol. 2004;124:4-14 2. Arico M, Allen M, Brusa S, Clementi R, Pende D, Maccario R, Moretta L, Danesino C. Haemophagocytic lymphohistiocytosis: proposal of a diagnostic algorithm based on perforin expression. Br J Haematol. 2002;119:180-188 3.

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Figure 1.

Blood Findings: Morphologic, Flow Cytometric and Molecular Studies.

Panel A depicts the morphologic appearance of the “atypical” lymphoid cells in the peripheral blood of the patient (1000X). Panel B demonstrates six representative dot plots from the flow cytometric studies.

The population in green (Population I) represents CD4+ T

lymphocytes. The three subpopulations of CD8+ T lymphocytes (Populations II, III and IV) are represented in blue, black and red, respectively. The regions drawn around populations in the sixth dot plot approximate the electronic gates used for flow-sorting the corresponding subsets. Panel C shows the results from the TCR- PCR reactions performed on genomic DNA extracted from either bulk blood leukocytes or sorted populations of T-cells (as indicated). The bottom row indicates a positive control PCR reaction (monoclonal TCR). TREC levels (per cell) from the corresponding populations are also indicated in the same panel to the right of each tracing.

Figure 2. Liver Biopsy. Panel A demonstrates the H&E-stained liver section. Panels B-K represent immunohistochemical staining for CD20, CD3, CD2, CD4, CD8, CD5, CD68, TIA1, granzyme B and perforin. Panel L shows positive perforin staining on a control section (spleen). All images are at 500X magnification. The infiltrate is composed predominantly of + CD8 T-cells with lack of perforin expression (FHL) and lack of CD5 expression, similar to

flow cytometric findings.


Karandikar, et al; Figure 1

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Karandikar, et al; Figure 2

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