T-cell lymphoproliferative disorders are associated with a rich infiltrate of monocyte-derived cells. Normal skin and biopsies obtained from CTCL patients with mycosis fungoides (A) or lymph node biopsies from patients with PTCL (B) were stained for monocyte-derived macrophages (CD68) or dendritic cells (CD1a, CD11c, S-100). Representative examples (CTCL, n = 10; PTCL, n = 12) are shown (×200).

Malignant T cells produce IL-10 and STAT-3 is phosphorylated within the tumor microenvironment. (A) PBMCs obtained from CTCL patients were cultured in the presence of PMA, ionomycin, and brefeldin A. Malignant T cells were identified as before and intracellular staining for IL-10 was performed. A representative example is shown (n = 2). (B) Plasma was obtained from normal donors (n = 24) and T-cell NHL patients (n = 25) and IL-10 levels were determined (mean ± 95% confidence interval is shown; P = .04). (C) Immunohistochemical analysis of pSTAT3 expression in CTCL biopsy specimens was performed (n = 10). Three representative examples are shown (×200).

CCL5, produced by malignant T cells, attracts monocytes to the tumor microenvironment. (A) MyLa-conditioned media were used to attract monocytes in a 3-hour chemotaxis assay. An isotype control or neutralizing anti-CCL5 antibody was included in the assay, and spontaneous monocyte migration in response to media alone was subtracted from the data shown. Data shown are representative of at least 3 similarly performed experiments (mean ± SD). (B) Skin biopsies from CTCL patients (n = 10) were stained for CCL5. Two representative examples are shown, including an intraepidermal nest of malignant T cells (ie, Pautrier microabscess, indicated by [→]). (C) CCL5 was measured in plasma obtained from both normal donors (n = 24), and from patients with CTCL (n = 23) or PTCL (n = 29). Mean values ± 95% confidence intervals are shown; P < .001.

Monocytes promote survival of malignant T cells. (A-E) Cells were stained with annexin V and 7-AAD and viability of Sézary cells (SCs) was reported. PBMCs were either mock or CD14 depleted (A-C) and cultured for approximately 7 days before staining. Malignant T cells were identified by staining for the appropriate T-cell receptor (TCR) Vβ chain. For samples in which TCR Vβ use was unknown, malignant CD4⁺ T cells were identified by the aberrant down-regulation of CD7. The data shown in panel B represent the mean ± SD of 4 independent samples. (C) CD14-depleted samples were supplemented with either purified autologous monocytes (first 3 samples shown, indicated by “mono”) or with supernatants (ie, monocyte-conditioned media; last 7 samples shown, indicated by “Mono-CM”) derived from cultured patient-derived monocytes or with monocyte-conditioned media (mono-CM) derived from normal donor monocytes, as indicated. (D) SCs derived from PBMCs or skin from the same patient were cultured in serum-free media supplemented with autologous mono-CM and viability of CD4⁺CD7⁻ SCs was determined by annexin V and 7-AAD staining. (E) Total PBMCs (n = 6) or skin-infiltrating lymphocytes (n = 4) were cultured in serum-free (SF) conditions (■) or in SF media supplemented with 40% mono-CM (□) and viability of CD4⁺CD7⁻ malignant T cells was determined, as described for panel D.

Monocytes prevent chemotherapy-induced cell death and promote tumor engraftment in a human xenograft model. (A) HuT 78 cells were cultured in serum-free media alone, in B cell–conditioned media, T cell–conditioned media, patient mono-CM (Mp), or mono-CM obtained from 3 normal donors (Mnd), and thymidine incorporation was determined 72 hours later (mean ± SD). (B) HuT 78 cells in SF or mono-CM were stained with annexin V/PI and viability was determined 48 hours later. (C) HuT 78 cells were cultured in media alone or in media supplemented with mono-CM, and doxorubicin was added at the concentration (80 ng/mL) that inhibited thymidine incorporation at 72 hours by approximately 50%. Data shown are representative of at least 3 independently performed experiments (mean ± SD). (D) MyLa cells, alone or combined with purified monocytes (ratio 1:1), were injected subcutaneously into the flanks of NOD-SCID mice (each group n = 10). Tumors were considered established when the average tumor diameter reached at least 3 mm. Data shown are representative of 4 similarly performed experiments. (E) Tumors were harvested from both groups of mice and immunohistochemistry was performed for markers (CD11c, CD68) expressed by monocyte-derived cells (×200).

Malignant T cells inhibit DC maturation in an IL-10–dependent fashion. (A) Immature DCs (iDCs) were generated from monocytes and LPS (1 μg/mL) was added for the last 24 hours of culture to generate mature DCs (mDCs). A group of DCs was cocultured with HuT 78 cells 24 hours before LPS maturation. CD11c⁺ DCs were purified and iDCs, mDCs, or DCs that had been cocultured with HuT 78 cells before LPS maturation were used to stimulate proliferation of purified CD4⁺ T cells at the T-cell/DC ratios shown. (B-C) DCs were generated, matured with LPS, and used as stimulators in an allo-mixed lymphocyte reaction at a T-cell/DC ratio of 100:1. As in panel A, before maturation, groups of DCs were cocultured with either HuT 78 (B) or SU-DHL-1 (C) cells in the presence of an isotype control or neutralizing IL-10 monoclonal antibody (2 μg/mL), as indicated. Data are mean ± SD. (D) DCs were generated and matured with LPS in the presence or absence of HuT 78 or SU-DHL-1 cells, as indicated. An isotype control or neutralizing IL-10 antibody was included. Cells were stained with an isotype control (shaded) or anti-CD83 (solid line). Only CD11c⁺ cells were gated and included in the analysis. Mean fluorescent intensity (MFI) for CD83 is shown in each histogram. Data shown (A-D) are representative of at least 3 similarly performed experiments. (E) Benign dermatitis (n = 10) and CTCL (n = 10) skin biopsies were immunohistochemically stained for both CD11c and CD83. Four representative examples of each are shown.