Chronic Borrelia burgdorferi infection triggers NKT lymphomagenesis

TO THE EDITOR:

Borrelia burgdorferi sensu lato complex (Bb sl) is a group of Borrelia species that is responsible for Lyme disease transmitted to humans by Ixodes tick bites. Lyme borreliosis is an emerging zoonosis and the most important vector-borne disease of the northern hemisphere, with 30 000 to 300 000 cases reported each year in the United States¹  and 65 000 cases per year in Europe.²  The association between chronic bacterial infections and lymphomas has been suggested for a long time; however, with the exception of a few pathologies (ie, gastric B-cell lymphomas and Helicobacter pylori infection³ ), clear demonstrations of causative links are missing. Although various Borrelia strains have been associated with primary cutaneous lymphomas, the results have been almost exclusively reported as case studies or small retrospective series of cutaneous lymphomas^4-7  and remain controversial.^8,9 Bb sl DNA has been detected in 10% to 42% of patients with cutaneous mucosa-associated lymphoid tissue B-cell lymphomas, in 15% to 26% of cutaneous follicular and diffuse large B-cell lymphomas,^5,6  and in 18% of mycosis fungoides (MFs) diagnosed in some European countries.^7,10  Taken together, there is insufficient evidence to show a definitive association between Bb sl and lymphomas. Therefore, the development of animal models to show the direct link between Borrelia-driven infection and lymphomagenesis is required.

To explore the role of Bb sl in T-cell lymphomagenesis, we used p53^−/− mice that develop, in addition to the well-known immature thymic T-cell lymphomas,^11,12  spontaneous peripheral T-cell lymphomas (PTCLs) originating from natural killer T (NKT) cells.¹³  All animal studies and procedures were performed in accordance with European Union guidelines and were approved by the Animal Ethics Evaluation Committee.

p53^−/− mice were injected with live Borrelia afzelii IBS39 (n = 34) or with control medium (Barbour–Stoenner–Kelly medium; n = 39) intradermally to mimic tick bites. Of the 34 mice injected with B afzelii, 29 tested positive for anti-Borrelia immunoglobulin G (IgG) 15 and 30 days postinjection, confirming the infection. The median survival of 158 days was not significantly different from uninfected p53^−/− mice (median survival = 168 days) (Figure 1A). Among Borrelia-infected mice, almost 50% developed CD19⁻, CD3⁺, Thy1⁺ PTCL compared with only 32% in the uninfected group (Figure 1B; supplemental Figure 1, available on the Blood Web site). Macroscopically, these PTCLs were characterized by hepatomegaly and splenomegaly (Figure 1C), with a 58-fold and fourfold increase in lymphocytes in liver and spleen, respectively, compared with healthy mice (Figure 1D). The architecture of spleen and liver from PTCL mice was nodular and diffuse, with massive cell infiltration leading to effacement of the normal structure (Figure 1E). The PTCL infiltrate in the liver was massively perivascular but also intrasinusoidal, the later being where normal NKT cells are preferentially found in healthy liver (Figure 1E). Because Borrelia-expressed glycolipids enable direct NKT cell activation and expansion,¹⁴  we studied the NKT cell origin of these PTCLs using α-Galactosylceramide (αGalCer)-loaded CD1d tetramer. A set of PTCLs was characterized by positive CD1d tetramer staining, defining NKT lymphomas (NKTLs), whereas another set of PTCLs was negative, indicating conventional PTCL (Figure 1F; supplemental Figure 1; supplemental Table 1). As shown in Figure 1G, Borrelia infection significantly increased the incidence of NKTL (94% in the Borrelia-infected group vs 61% in the uninfected group). The NKT cell origin of these PTCLs was further validated by expression of the invariant Vα14-Jα18 rearrangement of the T-cell receptor α (TCRα) chain and by the rearrangement of the TCRβ chain with only Vβ7, Vβ8.2, and Vβ8.3 rearrangements (supplemental Table 1). In the Borrelia-infected group, NKTLs were clonal, with a clone-productive frequency of the TCRβ chain rearrangement between 84.9% and 99.8% (supplemental Table 1).

To further understand the role of chronic infection by Bb sl in NKTL development, we studied the incidence of NKTL under different experimental conditions, as well as the persistence of anti-Borrelia IgG, because these antibodies play an important role in immune responses against spirochetes.¹⁵ p53^−/− mice infected with live B afzelii produced high levels of anti-Borrelia IgG that persisted for >120 days (Figure 2A), and they developed significantly more NKTLs compared with mice injected with Barbour–Stoenner–Kelly medium (Figure 2B). Conversely, mice injected with heat-killed (HK) Borrelia or Borrelia-infected mice treated with antibiotics to eradicate bacteria¹⁶  did not produce long-term persisting anti-Borrelia IgG (Figure 2A) and did not show any significant increase in NKTL frequency (Figure 2B). These results demonstrated the importance of live Borrelia persistence (ie, chronic infection) in NKTL development. NKTL showed downregulated NK1.1 and increased PD-1 expression, 2 characteristics of chronically activated NKT lymphocytes (Figure 2C). The role of chronic TCR stimulation in NKT lymphomagenesis was further demonstrated by the increase in NKTLs after chronic injections with αGalCer (Figure 2D).

Normal NKT cells persist in vivo in the absence of TCR/CD1d interactions.¹⁷  Conversely, during chronic infections, the survival of antigen-specific memory T cells relies on the persistence of cognate antigens through repeated TCR engagements.¹⁸  To investigate the importance of CD1d-mediated chronic TCR engagement in NKTL survival, we transferred NKTL cells into CD1d^−/− recipient mice. All wild-type (WT) recipients succumbed to lymphoma, whereas all CD1d^−/− mice remained healthy (Figure 2E). Liver cellularity increased significantly in WT recipient mice; NKTL cells represented 92% to 95% of total liver cells, whereas they were undetectable in CD1d^−/− recipient mice (Figure 2F). Such dependence to CD1d/TCR interaction strongly suggests that NKTL development is driven by chronic TCR stimulation.

In summary, we described for the first time that chronic Bb sl infection increases the incidence of PTCL originating from NKT cells in p53^−/− mice. These results reinforce previous data demonstrating the role of TCR in NKT lymphomagenesis.¹³  In addition to transforming viruses, such as Epstein-Barr virus¹⁹  and human T-cell leukemia virus, type 1,²⁰  which are implicated in several non-Hodgkin lymphoma subtypes, chronic immune stimulation by pathogens, such as H pylori³  and hepatitis C virus,²¹  has been reported to trigger lymphomagenesis. This study constitutes a novel illustration of the association among bacterial infections, chronic antigen receptor stimulation, and lymphoma development, as well as a formal demonstration that Bb sl infection drives T-cell lymphomagenesis. Whether Bb infection also triggers NKTL in humans needs to be further investigated, because our p53^−/− model may not recapitulate the pathophysiological genetic events of mycosis fungoides or other PTCLs associated with B burgdorferi infection. However, recent data from whole-exome sequencing have identified TP53 mutations and copy number alterations as the most prevalent genetic abnormalities in cutaneous T-cell lymphoma,^22,23  suggesting that TP53 alterations might be the genetic driver events associated with chronic TCR stimulation in cutaneous T-cell lymphomagenesis associated with Borrelia infection. In addition, PLZF, a key transcription factor in NKT cell differentiation, is overexpressed in some human T-cell lymphomas/leukemias, such as MF and Sézary syndrome (SS),^24,25  suggesting that at least some SSs and/or MFs may be NKTLs.