Persistent interferon production by double negative T cells and collapsing focal segmental glomerulosclerosis

Abstract

Collapsing glomerulopathy has multiple associations including viral infections, medications like bisphosphonates and interferon, autoimmune diseases and genetic predisposition. We report a case of collapsing focal segmental glomerulosclerosis associated with persistently high levels of interferon gamma produced by T cell receptor (TCR) αβ (+), CD4-CD8- (double negative) T lymphocytes that progressed despite treatment and improvement of other cytokine levels. Double negative T cells are elevated and activated in autoimmune lymphoproliferative syndrome. Production of elevated interferon gamma levels from double negative T cells in autoimmune lymphoproliferative syndrome despite treatment provides insight to the pathophysiology of collapsing glomerulopathy, guiding future research for collapsing glomerulopathy.

Introduction

Collapsing glomerulopathy or focal segmental gomerulosclerosis (FSGS) can be idiopathic or caused by viral infections like human immune deficiency virus (HIV), parvovirus B19, select medications, apolipoprotein L1 (APOL1) genetic predisposition, autoimmune conditions like lupus, and hemophagocytic syndrome.[1] Collapsing FSGS is rapidly progressive with abrupt onset of kidney failure and nephrotic-range proteinuria. [2] The pathophysiology is incompletely understood and there is limited effective treatment. [2] We report a case of collapsing FSGS with autoimmune lymphoproliferative syndrome (ALPS) associated with high levels of interferon gamma (INF Ƴ) production from T cell receptor αβ CD4-CD8- double negative T cells, that failed to improve with treatment. ALPS, also known as Canale-Smith syndrome is a rare inherited disorder of disrupted lymphocyte homeostasis resulting in chronic lymphoproliferation of the T cells that have not undergone programmed cell death. [3, 4] This syndrome is usually characterized by increased double negative T cells.[4]

Case presentation

A 29-year-old African- American female presented with one-year history of lymphadenopathy with findings of atypical lymphoid proliferation on biopsy which at the time was attributed to dental infection and cellulitis. On presentation, she had high-grade fever of 105.1-degree Fahrenheit, night sweats, weight loss, fatigue, blood pressure of 108/61 mmHg and pulse of 148 per minutes. Physical examination was notable for splenomegaly. Serum creatinine was elevated at 1.9 mg/dL from a baseline of 0.7 mg/dL which later peaked at 4.2 mg/dL. One isomorphic red cell and 12 white blood cells/high power field were noted on urinalysis. 24-hour urine protein was measured at 5.6 grams and serum albumin was 2.7 g/L. Subsequent, kidney biopsy demonstrated several glomeruli with prominent podocyte capping with collapse of the underlying tuft, prominent podocyte droplets, detached podocytes in the urinary space and segmental mesangial hypercellularity suggestive of collapsing focal segmental glomerulosclerosis. Diffuse interstitial lymphoplasmacytic inflammation, tubular injury and focal intraepithelial inflammation were also noted. Crescents, thrombi and necrosis were not seen. Immunofluorescence revealed fine granular capillary wall and mesangial staining for IgG (1+), IgA (1+), IgM (1+), kappa and lambda light chain. Stains for C3 and C1q were negative. Electron microscopy showed diffuse foot process effacement without any electron dense deposits. (Figure 1)

Figure 1: Kidney biopsy showing collapsing Focal Segmental Glomerulosclerosis (FSGS).

Light Microscopy: H&E (upper left corner) and periodic acid schiff (upper right corner) with glomeruli showing proliferation of epithelial cells with collapse of the underlying glomerular tuft (red arrowheads) and detached podocytes in the urinary space (black arrowhead). Segmental mesangial hypercellularity (yellow arrowhead), diffuse interstitial lymphoplasmacytic inflammation (block arrow) and tubular injury and focal intraepithelial inflammation (black star) are also noted.

Electron Microscopy (Lower left corner): Diffuse podocyte foot process effacement (arrowheads) with focal microvillous transformation, glomerular basement membranes (white star) thickening and numerous endothelial tubuloreticular inclusions (blue block arrowhead) are noted.

Immunofluorescence (Lower right panel): There is 1+ staining for IgG, IgA, IgM, kappa and lambda light chains in the capillary wall and mesangium.

Anemia and mild thrombocytopenia were noted. (Table 1) Triglycerides were elevated at 503 mg/dL. Ferritin level peaked at 244,808 ng/mL (36-195). Interleukin (IL)-2 level peaked at 13,597 pg/mL (532-1891), INF Ƴ level at 480 pg/mL and IL-18 level at 535001 pg/ mL. T cell receptor αβ double negative T cell lymphocytes comprised of 2.5% of T cells and 2% of all cells on flow cytometry analysis, thus meeting criteria for diagnosis of ALPS (Figure 2A). There were high levels of tumor necrosis factor (TNF), IL-10 and IL-17 which were produced by double negative T cells, CD4+ and CD 8+ T cells (Figure 2B).

Table 1:

Laboratory characteristics at presentation

General lab	Patient’s results (Reference range)	Immunology lab	Patient’s results (Reference range)
White blood cell count (WBC)	6700 (4.5-11 cu/mm)	Interleukin-2 (IL-2)	13,597 (532-1891 pg/mL)
Lymphocytes	7 (24-44%)	Interleukin −6 (IL-6)	11.39 (0.31-5.0 pg/mL)
Hematocrit	23.4 (36-46%)	Interleukin-10 (IL-10)	227 (<6pg/mL)
Hemoglobin	7.9 (12-15 g/dL)	Interleukin-18 (IL-18)	535,001 (89-540 pg/mL)
Platelet count	109,000 (150,000-350,000 /cu mm)	Interferon gamma (INF Ƴ)	480 (<5 pg/mL)
Serum creatinine	1.9 −4.2 (0.5-1.2 mg/dL) Baseline 0.7	Absolute CD4+ Lymphocytes	1,829 (458 - 1,344 /cu mm)
Blood urea nitrogen	27 (7-22 mg/dL)	CD3+ Lymphocytes	90.6 (51 - 91 %)
Albumin	2.7 (3.5-5.3 g/dL)	CD16+56+ Lymphocytes	4.6 (7 - 31 %)
Triglycerides	503 (0-150 mg/dL)	CD19+ Lymphocytes	4.4 (6 - 19 %)
Ferritin	244,808 (36-195 ng/mL)	CD4/CD8 Ratio	0.53
Erythrocyte sedimentation rate (ESR)	117 (4-25 mm/h)	CD4+ Lymphocytes	33.8 (32 - 68 %)
Lactate dehydrogenase (LDH)	784 (122-220 U/L)	CD8+ Lymphocytes	63.7 (10 - 36 %)
Kappa light chain	151.2 (3.3-19.4 mg/L)	Complement C3	100.2 (81.0-157.0 mg/dL)
Lambda light chain	118.9 (5.7-26.3 mg/L)	Complement C4	10.9 (13.0-39.0 mg/dL)

ESR : Erythrocyte Sedimentation Rate; LDH: Lactate dehydrogenase

Fig. 2A. Flow cytometry demonstrating double negative T cells in healthy subject and patient with ALPS.

Percentage and absolute cell number of DN T cells increased in peripheral blood of ALPS patient, which decreased after dialysis treatment

Fig. 2B. Flow cytometry demonstrating cytokine levels in healthy subject and ALPS patients.

Double Negative T cells in peripheral blood of ALPS patient produces high level of cytokines (IFN-γ, IL-10, TNF or IL-17), which decreased after dialysis treatment, except the level of IFN-γ by DN T cells

Extensive infectious work up was completed with results negative for bacterial and fungal blood cultures, Epstein-Barr virus viral load, cytomegalovirus IgM, parvovirus IgM, coxsackie, HIV, human herpes virus (HHV)-8, HTLV-1, hepatitis B and C, syphilis, tuberculosis, Toxoplasma, Legionella, Histoplasma, Coccidiodes, Bartonella, Tularemia and Brucella. Autoimmune work up was unremarkable for antinuclear antibody (ANA), anti-double stranded DNA, anti-cardiolipin antibody, anti-mitochondrial antibody, rheumatoid factor and cyclic citrullinated peptide. Light chain kappa to lambda ratio was 1.27. Serum protein electrophoresis was negative for monoclonal protein. Peripheral blood smear, lymph node and bone marrow biopsies were unrevealing for hematopoietic malignancy and hemophagocytosis. Whole exome sequencing did not reveal mutations in genes associated with primary hemophagocytic lympho- histiocytosis (HLH) or the presence of apolipoprotein L1 (APOL1) risk alleles.

The patient subsequently developed hemorrhagic pericardial effusion with tamponade requiring pericardial drains and window, and large pleural effusions requiring chest tubes. Renal replacement therapy was initiated. She was started on cyclosporine, anakinra (IL-1 receptor antagonist), prednisone and subsequently underwent splenectomy. After the initiation of immunosuppression, there was improvement in fever and IL-2 levels decreased to 4476 pg/mL. Double Negative T cells and CD4+ counts decreased post treatment (Figure 2A). However, high levels of CD 8+ cells persisted post treatment along with high level of TNF produced by CD 8+ cells (Figure 2A and 2B).

Renal biopsy was repeated one month after treatment initiation with re-demonstration of collapsing glomerulopathy. Hence, prednisone was continued at 60 mg daily with gradual taper after 4-6 weeks. As illustrated in the figure 2, improvement in clinical course correlated with decrease in the double negative T cells and cytokines produced by double negative T cell, CD4+ and CD8+ cells after treatment except for INF Ƴ in double negative T cells and TNF by CD 8+ T cells. INF Ƴ in normal double positive T cell also improved with treatment unlike double negative T cells. Her clinical course consisted of progression to end stage kidney disease (ESKD). Hemodialysis was continued and she remained on immunosuppressive medications for ALPS.

Discussion

Collapsing glomerulopathy is an uncommon variant of FSGS defined by segmental or global wrinkling of the glomerular basement membranes associated with podocyte injury that leads to podocyte dedifferentiation, apoptosis, and proliferation, due to dysregulation of cell cycle–related proteins [5, 6] It is associated with rapid progression to end stage renal disease and poor response to treatment. [7] The pathologic features include visceral epithelial cell hypertrophy and hyperplasia, tubular microcysts, tubular epithelial degenerative and regenerative features and interstitial edema.[8] Various hypotheses for pathogenesis for collapsing glomerulopathy have been proposed without identification of definite trigger for epithelial cell proliferation.[6] Cytokines produced by the inflammatory cells not only causes glomerular and podocyte injury but also causes tubular cell damage. [6] Among the broad spectra of clinical associations and etiologic factors reported, our patient had collapsing glomerulopathy with high levels of cytokines, especially INF γ produced by double negative T cells associated with ALPS.

ALPS is clinically characterized by lymphadenopathy, splenomegaly and autoimmune cytopenias. [9] Around 80% of patients with ALPS have long history of tender lymphadenopathy. [9] The hallmark of ALPS is chronic lymphoproliferation with increased number of double negative T cells. [9] Double negative T cells constitute 1% in normal individuals but can reach up to 40% in patients with ALPS.[10, 4, 9] Double negative T cells could be either previously activated mature T cells that have lost CD8+ or CD4+ coreceptor or expression, or a special minor cell lineage selectively expanded owing to defect in Fas signaling.[4] Double negative T cells produce high levels of IL-10. [11] The diagnosis of ALPS is based on clinical observation and laboratory abnormalities. [9] The characteristics abnormalities that facilitate diagnosis of ALPS include presence of αβ double negative T cells, elevated levels of the cytokine IL- 10, IL-18, vitamin B12 and defective Fas-mediated apoptosis on in vitro assay.[10, 9] In our patient, compared to healthy population, the level of INF γ, TNF α, IL-10 and IL-17 produced by all T cells including CD4+, CD8+ and double negative T cells were increased. INF γ by double negative T cells and TNF by CD 8+ cells remain elevated despite treatment.

Steroids are the first line treatment for ALPS. [9] Mycophenolate mofetil and sirolimus are used as steroid sparing agents. [9] Our patient was treated with steroids followed by cyclosporine. Due to the intractable high-grade fever, anakinra was also used. Even though splenectomy is generally not recommended in ALPS, [9] given the severity of the inflammatory response, she underwent splenectomy as well.

A prominent feature in this patient were the increased double negative T cells producing high INF Ƴ levels that failed to improve with treatment and correlated with lack of renal recovery. Therapeutic interferon treatments (INF α, -β, or -γ) have been reported to cause collapsing FSGS. [12] This case illustrates the pathogenesis of collapsing glomerulopathy in relation to levels of cytokines pre and post treatment. Collapsing glomerulopathy carries a poor prognosis and rapid progression to ESKD. In this case, dysregulated cytokine production with persistently elevated interferon gamma levels produced by double negative T cells has potentially mediated the development of collapsing lesion and poor response to therapy.