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The field of flow cytometry has witnessed rapid technological advancements in the last few decades. While the founding principles of fluorescent detection on cells (or particles) within a uniform fluid stream remains largely unchanged, the availability more sensitive cytometers with the ability to multiplex more and more florescent signals has resulted in very complex high-order assays. This results in the co-use of fluorophores with increased levels of emission overlap and/or spillover spreading than in years past and thus requires careful and well thought out planning for flow cytometry assay development. As an example, we present the development of a large 18-color (20 parameter) flow cytometry assay designed to take an in depth analysis of effector lymphocyte phenotypes, with careful attention to assay controls and panel design.

Tumor-induced immune suppression involves the accumulation of suppressive infiltrates in the tumor microenvironment such as regulatory T-cells (Tregs). Previous studies demonstrated that NK-dependant increases in CCL22 secretion selectively recruit Tregs toward murine lungs bearing Lewis Lung Carcinoma (LLC). To extend the in vitro studies, the present studies utilized in vivo depletion of NK cells to ascertain the contribution of NK-derived CCL22 toward total CCL22 and subsequent Treg levels in both normal and LLC-bearing lungs. However, NK depletion had the unexpected effect of increasing both CCL22 secretion and Treg levels in the lungs of NK-depleted LLC-bearing mice. This was concurrent with an increase in tumor burden. Flow cytometry and a series of both immunomagnetic and FACS isolations were used to identify the CCL22-producing cellular fractions in LLC-bearing lungs. A novel CD11b(+)CD11c(+) cell population was identified that accumulates in large numbers in NK-depleted LLC-bearing lung tissue. These CD11b(+)CD11c(+) cells secreted large amounts of CCL22 that may overcompensate for the loss of NK-derived CCL22 in the lungs of NK-depleted LLC-bearing mice. Taken together, these data suggest that NK cells play both a positive and negative role in the regulation of CCL22 secretion and, in turn, the recruitment of Tregs toward LLC-bearing lungs.

Tumor-induced immune suppression involves the accumulation of immune-suppressive infiltrates in the microenvironment. This study demonstrates increased numbers of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) in the lungs of C57BL/6 mice bearing a metastatic Lewis lung carcinoma (LLC) variant. These Tregs suppressed the proliferation of endogenous CD4(+)CD25(-) cells and expressed higher levels of the chemokine receptor CCR4 than other types of T cells. LLC-bearing lungs secreted elevated levels of the CCR4-associated chemokine CCL22 compared with normal lungs. However, CCL22 was not secreted by LLC or normal epithelial controls, suggesting that CCL22 is secreted by a nonepithelial component of the microenvironment. Migration assays revealed that medium conditioned by LLC-bearing lungs selectively recruited Tregs at higher frequencies than did medium conditioned by normal lungs. Neutralization of CCL22 significantly reduced this selective recruitment toward both conditioned media. A series of immunomagnetic isolations, FACS, and flow cytometric analyses were used to isolate different cellular fractions from both normal and LLC-bearing lungs. When isolated, only the NK-containing fractions secreted CCL22, and the same fraction isolated from LLC-bearing lungs secreted higher levels. Depletion of NK cells from both normal and LLC-bearing lung tissue significantly reduced CCL22 secretion, suggesting that a large portion of secreted CCL22 is NK cell dependent. Flow cytometric analysis of the lung NK compartments revealed no significant increase in NK cell numbers across LLC-bearing lung tissue as a whole as compared with normal tissue. However, immunofluorescent staining revealed an increased frequency of NK cells at the tumor periphery that were closely associated with the elevated FoxP3(+) infiltrate.