Th22 cells increase in poor prognosis multiple myeloma and promote tumor cell growth and survival

Figures & data

Table 1. Characteristics of the patients

									Testing of IL-22^+ frequency
Patients	Age (yr)	Sex	% PC in BM aspirate	% PC in BM biopsy	Ig type	DS	ISS	Therapy	PB	BM
MGUS
#426	44	M	4	10	IgGk	n.a.	n.a.	none	+	+
#0037	67	F	4	8	IgGλ	n.a.	n.a.	none	-	+
#0091	72	M	2	4	IgGλ	n.a.	n.a.	none	+	-
#0083	75	M	2	9	IgGk+λ	IA	n.a.	none	-	+
#0245	78	M	9	9	IgAκ	n.a.	n.a.	none	-	+
SMM
#337	67	M	14	27	IgGλ	IA	I	none	+	+
#411	80	M	10	42	IgAk	IA	II	none	+	+
#0059	73	M	35	37	IgGκ	IA	I	none	+	+
#307	58	M	6	13	IgGλ	IA	I	none	+	+
#571	37	F	17	n.a.	IgGκ	IA		none	+	-
#0681	77	M	4	12	IgMk+Gλ	n.a.	n.a.	none	+	+
#0182	70	M	33	38	IgGκ	IA	I	none	+	-
#0200	62	F	26	40	IgAk	IB		none	+	-
#340	68	F	16	24	IgGκ	IA	I	none	+	-
MM Diagnosis
#0017	55	M	52	46	IgGκ	IIIA	I	none	+	-
#345	73	F	42	37	IgGκ	IIIA	I	none	-	+
#0075	40	F	8	n.e.	IgGλ	IIIA	I	none	+	+
#375	70	M	20	35	IgGλ	IIIA	I	none	+	+
#622	65	M	n.e.	42	IgGκ	IIIA	I	none	+	+
#245	74	M	2	n.e.	IgAk	IA	I	none	+	+
#0284	34	M	32	50	IgGκ	IIA	I	none	+	+
#0079	64	M	72	55	IgAκ	IIIA	II	none	+	+
#647	66	M	47	68	IgAκ	IA	II	none	+	+
#126	84	M	n.e.	18	IgGκ	IIIA	II	none	+	+
#646	59	F	40	<10	IgGk	IIA	II	none	+	+
#462	40	M	82	>90	IgGk	IIIA	II	none	+	-
#0575	81	F	40	n.e.	IgGλ	IIIA	II	none	+	+
#0603	70	F	40	n.av.	IgGλ	IIA	II	none	+	-
#25	67	F	80	52	IgGκ	IIIA	III	none	-	+
#356	40	M	78	95	IgGλ	IIIA	III	none	+	+
#609	63	M	33	33	IgAκ	IIIB	III	none	+	+
#785	59	M	35	70	IgGλ	IIIB	III	none	+	+
#157	58	M	3	53	IgGλ	IIIB	III	none	+	+
#608	74	M	24	47	IgAκ/IgGκ	IIIA	III	none	-	+
#279	78	F	39	36	IgGκ	IIIA	III	none	+	-
#304	77	F	71	67	IgGλ	IIA	III	none	+	-
#0773	58	M	81	62	IgGκ	IIIA	III	none	+	+
Relapsed/Refractory
#177	66	M	17	58	λ	IIA	III	Table 2	+	+
#0121	81	F	24	48	K	IIIA	n.av.	Table 2	+	+
#374	64	F	22	30	IgGk	IIA	I	Table 2	+	+
#0046	74	M	71	n.av.	IgGλ	IIIA	I	Table 2	+	+
#412	69	F	39	41	IgAk	IIIA	n.av.	Table 2	-	+
#71	67	F	44	48	IgGλ	IIIA		Table 2	+	+
#623	74	M	8	n.av.	IgAk	IIIA	III	Table 2	-	+
#0449	73	F	42	37	IgGk	IIIA	I	Table 2	+	+
#0618	65	M	10	24	IgAk	IA	I	Table 2	+	+
#0088	67	F	6	16	IgGλ	IIIA	I	Table 2	+	+
#0151	44	F	5	n.av.	IgGk	IIIA	II	Table 2	-	+
#0251	61	F	1	0	IgGk	IIIB	III	Table 2	+	+
#0024	63	M	12	27	IgGλ	IIIB	III	Table 2	-	+
#0071	72	M	4	7	IgGk	IIIA	III	Table 2	-	+

BM, bone marrow; ±, conducted/not conducted; DS, Durie–Salmon staging system at diagnosis; ISS, International Staging System at diagnosis; n.a., not applicable; n.e., not evaluable; n.av., not available; PCs, plasma cells; PB, peripheral blood.

Table 2. Previous therapies of relapsed/refractory patients

MM relapsed/refractory	Relapse #	Therapies	Time from last therapy
#177	1	Btz based + ASCT × 2	8 months
#0121	1	Btz - MP	3 years + 7 months
#374	1	Ln based + ASCT × 2	1 year + 7 months
#0046	3	ASCT × 2/ Btz ± MP	10 months
#412	3	MPT/Ln-Dx	6 months
#71	1	VAD + ASCT × 2	11 years
#623	1	Btz based	1 year + 6 months
#0449	1	Btz based ASCT × 2	9 months
#0618	1	Thal based + ASCT × 2 + RT	4 years
#0088	1	VAD + ASCT × 1	7 years
#0151	3	BtzASCT + alloSCT/Btz/Ln/CT	2 years in therapy
#0251	1	Btz based + ASCT × 2	2 years
#0024	2	VAD + ASCT × 2/Thal/alloSCT	7 years
#0071	1	Thal based + ASCT × 2 + Thal	2 years

Abbreviations: alloSCT, allogenic stem cells transplantation; ASCT, autologous stem cells transplantation; Btz, bortezomib; CT, polychemotherapy; Dx, dexamethasone; Ln, lenalidomide; MP, melphalan + prednisone; MPT, melphalan + prednisone + thalidomide; RT, radiotherapy; Thal, thalidomide; VAD, vincristine + adriamycin + dexamethasone.

Figure 1 (see previous page). IL-22⁺IL-17⁻IL-13⁺ CD3⁺ T cells are increased in the PB and BM of MM patients with poor prognosis. Tukey plots of cumulative results (A, B, D, and E) and dot-plots of representative data from cytokine-ICS analyses (A [left], B [left], C, and F). (A) Analysis for IL-22 expression was conducted on CD3⁺ cells (left, representative of PBMCs of patient #356). Percentage of IL-22⁺ T cells in PB of healthy donors (n = 15), MGUS+SMM (n = 11), MM at diagnosis (n = 20), and relapsed/refractory MM (n = 9). (B) Analysis for IL-22 expression was conducted on CD3⁺ cells (left, representative of BMMCs of patient #356). Percentage of IL-22⁺ T cells in the BM of healthy donors (n = 4), MGUS+SMM (n = 9), MM at diagnosis (n = 18), and relapsed/refractory MM (n = 14). (C) Representative ICS of PBMCs and BMMCs of patient #177. Top: IL-22 and IL-17 expression. Bottom: IL-22 and IL-13 expression. Gate of IL-22⁺IL-17⁻ (R1) cells was used for analysis of IL-22⁺IL-17⁻IL-13⁺ cells (R2). (D) Percentage of IL-22⁺IL-17⁻IL-13⁺ T cells in PB of healthy donors (n = 15), MGUS+SMM (n = 11), MM at diagnosis divided into stage I+II (n = 13) and stage III (n = 7) and relapsed/refractory MM (n = 9). (E) Percentage of IL-22⁺IL-17^-IL-13⁺ T cells in BM aspirates of healthy donors (n = 4), MGUS+SMM (n = 9), MM at diagnosis divided into stage I+II (n = 11) and stage III (n = 7) and relapsed/refractory MM (n = 14). Responses significantly different by the Mann–Whitney U test are indicated as: *, P < 0.05 and **, 0.001 < P < 0.01. (F) Representative ICS for IL-22 and TNFα expression in PBMCs (left) and BMMCs (right) of patient #177.

Figure 2. Distribution of CD4⁺CCR6⁺ T-cell clones based on the profile of IL-22 and IL-17 secretion. Cake graphs of percentages of IL-22⁺IL-17⁻ (red slices), IL-22⁺IL-17⁺ (light blue slices), IL-22⁻IL-17⁺ (green slices), and IL-22⁻IL-17⁻ (violet slices) clones obtained from the (A) BM of MM patients (B) and PB of MM patients, and (C) healthy donors. Clonal efficiencies for cloning conducted after in vitro expansion were (A) 48.2% and 55.6% for BM of patients #356 and #121, respectively and (B) 25.4% and 21.1% for PB of the same patients, respectively. (C) Clonal efficiencies for PB of healthy donors were 45.8% and 76.9% for BC01 and BC60, respectively. Clonal efficiencies for cloning conducted immediately after sorting were 4%, 11.9%, 2.3%, and 7.6% for BM of patients #609, #177, #0046, and #374, respectively (A). For patient characteristics, see Table 1.

Figure 3. Chemokine receptor and cytokine profile of Th22 clones from the BM of MM patients. (A) Surface expression of CCR6, CXCR4, CCR4, and CCR10. (Left) Representative histograms: isotype control staining (solid gray), chemokine receptor-specific staining (black line). (Right) RFI (i.e., fold increase in mean fluorescence intensity relative to isotype control) calculated for each clone. (B, D, and F) Cumulative cytokine secretion by clones from different MM patients represented as Whisker plots. (B) Th22 clones (n = 4). (D) Th22-Th1 clones (n = 8). (F) Th22-Th2 clones (n = 10). (C-E-G) (Left and middle) Analysis of CXCR3 and CRTh2 expression in one representative Th22 (C), Th22-Th1 (E), and Th22-Th2 (G) clone, respectively. Dot plots (left and middle upper) and histograms (middle lower) from representative clones are shown. (Right) Cumulative RFI of paired CXCR3 and CRTh2 staining calculated for each clone.

Figure 4. IL-22RA1 is expressed in a fraction of MM cell lines and primary tumors. (A) mRNA expression of IL-22RA1 in MM cell lines. COLO205 cells were positive controls. Epstein–Barr virus-transformed B lymphoblastoid cell lines (LCLs) were negative controls. (B) mRNA expression of IL-22RA1 in primary MM cells of a cohort of 414 newly diagnosed patients described in ref. 34. The expression pattern for the probe set 220056_at is shown. Continued line: median. Dotted line: 2× standard deviation. (C) IL-22RA1 protein expression in BM biopsies from MM patients #121 (negative) (left) and #177 (positive) (right). Images were acquired at a ×40 magnification.

Figure 5 (see previous page). IL-22 induces MM cell activation, proliferation, and resistance to drug-induced cell death. (A) Representative staining for phosphorylated STAT-1 and STAT-3 (n = 6). MM1S are representative of IL–22RA1-positive cells, KMS34 for low/absent IL-22RA1 expression. IL-6 and IFNγ were positive controls for phosphorylated STAT-3 and STAT-1, respectively; unstimulated cells (gray line) and cytokine-stimulated cells (black line). (B) Cumulative percentage of induction of STAT-3 phosphorylation by recombinant IL-22 (left) and supernatants of Th22 clones (i.e., C37 and 374-19) (right) in the presence of anti-IL-22 or isotype control antibodies (Abs). Recombinant IL-22 was used at 20 ng/mL. IL-22 measured in the supernatants was 20 and 26.5 ng/mL for clone C37 and clone 374-19, respectively. (C and D) ³H-thymidine incorporation of MM cells cultured in the absence (nil) or in the presence of IL-22 (50 ng/mL) or IL-6 (50 ng/mL) in IL–22RA1-positive (C) and -negative (D) cells. Representative data from 1 of 4 independent experiments are depicted for each cell line. (E) Percentages of dying MM cells after (i) 24-h incubation with Dx in the absence (nil) or presence of IL-22 (20 ng/mL) or IL-6 (20 ng/mL) and in the presence of anti-IL-22 or isotype control Abs (2.5 μg/mL); (ii) 48-h incubation with Ln; and (iii) 24-h incubation with Ln followed by 24-h incubation with both drugs in the absence or the presence of IL-22 or IL-6 and in the presence of anti-IL-22 or isotype control Abs (n = 2). (F) WB of phosphorylated (p) p38, p38, pERK1/2, ERK1/2, pAKT, and AKT in MM1S untreated or treated with IL-22 or IL-6, as indicated. (G) pp38 data were quantified by densitometric analysis, normalized using p38, and divided by the value of untreated cells (Arbitrary Units, A.U.). (H) WB analysis of Mcl^-1, Bcl^-2, Bcl^-X_L, and cleaved PARP and caspase-3 in MM1S untreated or treated with IL-22 or IL-6 and in the absence or in the presence of Dx, as indicated (n = 2). A.U. were calculated as in (G), using β-tubulin (βTUB) for normalization, and are reported at the bottom of the lanes. IL-6 was used throughout as the positive control. Significant values calculated with the Mann–Whitney U test (C and D) or unpaired, one-tailed Student's t-test (E) are indicated as: *, P < 0.05; **, 0.001 < P < 0.01; and ***, P < 0.001.

Figure 6. IL-13 induces BM-MSC activation and adhesion molecule upregulation, enhances IL-6 production and IL–13-treated BM-MSCs favor MM cell proliferation. (A) Representative staining for phosphorylated STAT-6 (n = 3). TNFα was used as negative control. Unstimulated cells (gray line) and cytokine-stimulated cells (black line). (B) Cumulative percentage of induction of STAT-6 phosphorylation by recombinant IL-13 (left) and the supernatants of Th22 clones (i.e., A15 and 05.08) (right) in the presence of anti-IL-13 or isotype control Abs. Recombinant IL-13 was used at 10 ng/mL. Clone A15 supernatant (IL-13 = 90 ng/mL) was used diluted (1:10) and clone 05.08 supernatant (IL-13 = 15 ng/mL) was used undiluted. (C) Representative VCAM-1 and ICAM-1 expression on untreated (nil) or IL–13-treated BM-MSCs (n = 2). TNFα and IFNγ were positive controls. (D, left) Representative IL-6 secretion by untreated (-IL-13, white bars) or treated (+IL-13, gray bars) BM-MSCs in the absence or in the presence of MM1S cells (n = 3). (D, right) Effect of addition of anti-IL-13 or isotype control Abs on IL-6 secretion by BM-MSCs. (E) Representative MM1S cell proliferation in the presence of untreated (-IL-13, white bars) or IL-13 (+IL-13, gray bars)-treated BM-MSCs (n = 3). IL-13 in (D) and (E) was used at 50 ng/mL. Responses significantly different are indicated as: *, P < 0.05 and ***, P < 0.001 (determined by paired, one-tailed Student's t-test).