Malignant T Cell Activation by a Bacillus Species Isolated from Cutaneous T-Cell Lymphoma Lesions

Abstract

Cutaneous T-cell lymphoma (CTCL) is a life-debilitating malignancy of lymphocytes homing to the skin. Although CTCL is thought to arise from a combination of genetic, epigenetic, and environmental factors, specific triggers are unclear. The skin is colonized by a unique microbiota and is heavily influenced by its interactions. We hypothesized that adaptive immune responses to skin commensals lead to clonal T-cell proliferation and transformation in the appropriate genetic background. We therefore collected lesional and nonlesional skin microbiota from patients with CTCL to study T cell interactions using skin T cell explants and peripheral, skin-homing CD4⁺ T cells. By various methods, we identified Bacillus safensis in CTCL lesions, a rare human commensal in healthy skin, and showed that it can induce malignant T cell activation and cytokine secretion. Taken together, our data suggest microbial triggers in the skin microbiota of patients with CTCL as potential instigators of tumorigenesis.

Keywords: ASV, amplicon sequence variant; CLA, cutaneous lymphocyte‒associated antigen; CTCL, cutaneous T-cell lymphoma; MF, mycosis fungoides; STAT3, signal transducer and activator of transcription 3; rRNA, ribosomal RNA.

Figure 1

Characterization of skin microbiome of patients with CTCL using 16S rRNA sequencing. (a) Pie charts depicting the genera that are at least 1% of total genera in the dataset. A total of 15 genera met this criterion and are shown, representing 78.68% of overall 16S rRNA V1‒V3 reads at the genus level. Genera are shown as averages. Skin swabs from the arms of healthy subjects, n = 4; lesional/nonlesional skin swabs of the arms of subjects with CTCL, n = 4, swabs, n = 5. Skin swabs from the legs of healthy subjects, n = 5, swabs, n = 5; lesional/nonlesional skin swabs of the legs of subjects with CTCL, n = 6, swabs, n = 12. Swabs from the feet of healthy subjects, n = 4, swabs, n = 4; Lesional/nonlesional skin swabs of the feet of subjects with CTCL, n = 2, swabs, n = 5. All nonlesional and lesional samples represent paired CTCL samples. f indicates family, and g indicates genus. (b) Total number of Bacillus genera ASV counts in MF samples (n = 48) compared with those in HD skin swabs (n = 40) and SLE skin swabs (n = 76); P = 7.504e–008 healthy versus MF; P = 9.629e–009 MF versus SLE. (c) B. safensis ASV unrarefied counts from healthy (n = 40), MF (n = 48), and SLE skin swabs (n = 76); P = 0.0605 healthy versus MF; P = 0.0076 MF versus SLE. (d) Pie charts representing the percentage of subjects positive for B. safensis ASVs, defined as having a 16S rRNA V1‒V3 sequence homology (>99%) to CTCL. Nonlesion/lesion indicate CTCL skin swabs from nonlesional or lesional sites; control swabs indicate air swabs taken before sampling skin sites. ASV, amplicon sequence variant; CTCL, cutaneous T-cell lymphoma; HD, healthy donor; MF, mycosis fungoides; rRNA, ribosomal RNA; SLE, systemic lupus erythematosus. P-values were calculated using the unpaired two-tailed Student’s t-test. Significance levels are indicated by asterisks: ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001.

Figure 2

Alpha and beta diversity of CTCL and HD skin microbiomes as well as culture isolates from lesional and nonlesional CTCL skin. (a) Alpha diversity as measured by Shannon‒Weiner diversity index between skin swabs of arms of healthy subjects, n = 4, swab n = 4; lesional/nonlesional skin swabs of arms from subjects with CTCL, n = 4, swabs, n = 5. Skin swabs of legs from healthy subjects, n = 5, swabs, n = 5; lesional/nonlesional skin swabs of legs from subjects with CTCL, n = 6, swabs, n = 12. Skin swabs of feet from healthy subjects, n = 4, swabs, n = 4; lesional/nonlesional skin swabs of feet from subjects with CTCL, n = 2, swabs, n = 5. All nonlesional and lesional samples represent paired CTCL samples. No statistical difference in Shannon diversity was found. (b) Beta diversity as measured by principal-coordinate analysis of unweighted UniFrac distances. The blue sphere indicates healthy, the red sphere indicates nonlesional CTCL, and the orange square indicates lesional CTCL. No statistical difference was found between CTCL and HD unweighted UniFrac with the exception of the foot (PERMANOVA 999 permutations P = 0.034). (c) Alpha rarefaction curve showing all samples from the arm, leg, and foot to a sequencing depth of 10,000 reads. (d) Relative abundance of rarefied counts of Staphylococcus aureus ASV in healthy (n = 3) and MF (n = 20) skin swabs color coded by subject. (e) Single colony culturing from lesional and nonlesional swabs of patients with CTCL. Lesional and nonlesional sites were sterilely swabbed and then cultured in two different growth media as described in Materials and Methods. Approximately 1 of every 100 colonies per plate or quarter plate was picked and then sequenced. The table shows an overview of bacterial strains per individual, all strains with at least one single colony per swab. All results represent the colonies identified under aerobic growth. Anaerobic media did not show any growth in patient samples or controls. Patient MF07 did not provide samples for culturing. ASV, amplicon sequence variant; CTCL, cutaneous T-cell lymphoma; HD, healthy donor; MF, mycosis fungoides.

Figure 3

Alignments and rarefied counts of ASVs from Bacillus safensis. (a, b) The 16S rRNA V1‒V3 ASVs were aligned using Clustal omega to corresponding 16S rRNA V1‒V3 of a B. safensis culture isolate from a patient with CTCL (MF05) and a representative B. safensis strain (FO-36b 16S rRNA NR_041794.1). (a) Percent identity matrix showing aligned sequence percent identity relative to each other. (b) Aligned nucleotide sequences in the same order as in a. B. safensis strain FO-36b is listed in this figure as NR_041794.1. (c) Number of ASV counts from a present in patients with CTCL (four lesional and two nonlesional hits as indicated) compared with that of absent counts in healthy donors (n = 5). ASV, amplicon sequence variant; CTCL, cutaneous T-cell lymphoma; HD, healthy donor; MF, mycosis fungoides; rRNA, ribosomal RNA.

Figure 4

Evolutionary relationships of Bacillus species. The phylogenetic relationship of the Bacillus species identified in this study and related types of strains as well as the identified staphylococci and other skin commensals was examined. The evolutionary history was inferred using the Minimum Evolution method (Rzhetsky and Nei, 1992). The optimal tree with the sum of branch length = 0.35505038 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) are shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and are in the units of the number of base substitutions per site.

Figure 5

Isolation of CTCL T cells from skin biopsies and immunoblotting for phosphorylated STAT3. (a) A representative image from a Petri culture dish showing malignant T cells isolated from patient skin biopsy that are growing on collagen-coated grids. (b, c) Western blotting shows higher phosphorylation of STAT3 in cutaneous lesional T cells than in cutaneous nonlesional T cells after loading equal amounts of total protein per lane. The blot is representative of two replicates. CTCL, cutaneous T-cell lymphoma; phospho, phosphorylated; pSTAT3, phosphorylated signal transducer and activator of transcription 3; STAT3, signal transducer and activator of transcription. P-values were calculated using the unpaired two-tailed Student’s t-test. Significance levels are indicated by asterisks: ∗P < 0.05.

Figure 6

In vitro T cell proliferation and cytokine studies using patient-isolated cutaneous T cells. (a) Proliferative responses of human malignant T cells isolated from skin biopsies (MF05) to bacteria isolated from lesional compared with that from nonlesional (control) regions (2 inches next to each matched lesion). Y-axis indicates proliferation as RLUs using a nonradioactive ATP release assay. (b, c) Cytokine concentrations (in pg/ml) from the supernatant of the cutaneous T cells stimulated for 72 hours with bacteria as indicated, represented as (b) Z-score and (c) individual graphs. Functional phenotypes were characterized by cytokine secretion of IL-21, GM-CSF, IFN-γ, TNF-α, IL-17A, IL-4, IL-5, IL-13, and IL-10, respectively. Data points represent duplicates. P-values were calculated using the unpaired two-tailed Student’s t-test. Significance levels are indicated by asterisks: ∗P < 0.05; ∗∗P < 0.01. ATP, adenosine triphosphate; MF, mycosis fungoides; RLU, reactive light unit.

Figure 7

In vitro T cell cytokine studies using patient-isolated cutaneous T cells. (a) Proliferative responses of human malignant T cells isolated from skin biopsies (MF06) to bacteria isolated from lesional compared with that from nonlesional (control) regions (2 inches next to each matched lesion). Y-axis indicates proliferation as RLUs using a nonradioactive ATP release assay. (b) Individual cytokine graphs with cytokine concentrations (in pg/ml) from the supernatant of the cutaneous T cells stimulated for 72 hours with heat-killed bacteria as indicated. Functional phenotypes were characterized by cytokine secretion of IL-21, GM-CSF, IFN-γ, TNF-α, IL-17A, IL-4, IL-5, IL-13, and IL-10, respectively. Data points represent duplicates. P-values were calculated using the unpaired two-tailed Student’s t-test. Significance levels are indicated by asterisks: ∗P < 0.05; ∗∗P < 0.01. ATP, adenosine triphosphate; MF, mycosis fungoides; RLU, reactive light unit.

Figure 8

In vitro T cell proliferation and cytokine studies using patient-isolated peripheral blood T cells. (a) Human CD4⁺ T cells isolated from patient peripheral blood (MF06) selected for skin-homing markers CCR4 and CLA show proliferation to Bacillus safensis compared with unstimulated T cells. Y-axis indicates proliferation as RLUs using a nonradioactive ATP release assay. (b, c) Cytokine concentrations (in pg/ml) from the supernatant of the cutaneous T cells stimulated for 72 hours with bacteria as indicated, represented as (b) Z-score and (c) individual graphs. Functional phenotypes were characterized by cytokine secretion of IL-21, GM-CSF, IFN-γ, TNF-α, IL-17A, IL-4, IL-5, IL-13, and IL-10, respectively. Data points represent duplicates. P-values were calculated using the unpaired two-tailed Student’s t-test. Significance levels are indicated by asterisks: ∗P < 0.05; ∗∗P < 0.01. ATP, adenosine triphosphate; CLA, cutaneous lymphocyte‒associated antigen; MF, mycosis fungoides; RLU, reactive light unit.

Figure 9

Photographs, histology, and FISH of skin lesions from patients with CTCL sampled in this study. (a) Representative lesional photos (upper and lower panel) from one representative patient (MF04). The region marked in blue represents the area that was swabbed. (b) Representative H&E sections of three patients at the time of diagnosis (MF05, MF06 and MF01). Bar = 100 μm. (b) The images show (i) the histology from a biopsy of the right lateral thigh with an atypical T cell infiltrate with exocytosis involving the epidermis, (ii) the histology from a biopsy of the right lateral periorbital region with features of folliculotropic mycosis fungoides (patient MF06), and (iii) the histology from a biopsy of the left buttock region with infiltration of hair follicles by atypical intrafollicular and perifollicular lymphocytes (together with additional clinical data supporting clonal T-cell receptor gene rearrangement; patient MF01). (c) FISH of skin biopsies from patients with MF and psoriasis. Cutaneous lesional biopsies from three patients with MF (MF05, MF06, and MF07) stain positive for a gyrB-specific 16S FISH probe (green) as well as for the eubacterial probe EUB338 (red). A merge of gyrB and eubacterial staining is shown in yellow. Cutaneous biopsies from psoriasis lesions were negative for gyrB (shown is one representative of two). Magnification for MF05, MF06, psoriasis = 40×; magnification for MF07 = 60×. Bar = 20 μm. CTCL, cutaneous T-cell lymphoma; MF, mycosis fungoides.

Figure 10

Hypothesized involvement of Bacillus safensis and related strains in CTCL tumorigenesis. (a, b) The hypothesized mechanistic role for B. safensis in the pathogenesis of MF—the most common form of CTCL—is schematically depicted in the left panel a and illustrated in the skin in the right panel b. B. safensis bacteria are indicated as blue rods, and Staphylococcus aureus bacteria are indicated as yellow circles. Cutaneous bacteria, B. safensis in this study, are taken up by professional antigen-presenting cells (APCs) such as Langerhans cells (LCs) that migrate to the local lymph node in order to present antigens to circulating T cells. Next, activated T cells are recruited to the skin via the skin homing markers CLA and CCR4. The direct antigen contact in the epidermis potentially triggers T cell proliferation and cytokine secretion activating STAT3 phosphorylation, thereby inducing a progressive inflammatory response that supports the malignant transformation of cutaneous T cells. Additional inflammatory events mediated by other microbiota (such as staphylococci, indicated by S. aureus) likely fuel this process, resulting in the formation of Pautrier’s microabscesses and ultimately promoting the progression of the T cell malignancy. Graphs were created with BioRender.com. APC, antigen-presenting cell; CLA, cutaneous lymphocyte‒associated antigen; CTCL, cutaneous T-cell lymphoma; LC, Langerhans cell; MF, mycosis fungoides; STAT3, signal transducer and activator of transcription 3.