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To extravasate into normal and neoplastic tissue, lymphocytes must migrate through the subendothelial basement membrane and underlying interstitium, structures rich in extracellular matrix (ECM). We have performed a time-course study of the development of motility in ECM by murine lymphocytes during in vitro exposure to high titers of IL-2 (1000 Cetus units/ml). This protocol generates immunotherapeutic lymphocyte populations expressing lymphokine-activated killer activity. Spontaneous motility was measured in three-dimensional gels of type I (interstitial) collagen or Matrigel, a model basement membrane. A newly developed assay permitted not only the measurement of distance traveled by the leading cell front, but also the separation of lymphocytes on the basis of three types of behavior. The motile fraction consisted of lymphocytes that penetrated beneath the ECM gel surface during an 18-h migration period. There were also two nonmotile fractions: the nonadherent fraction, which failed to bind to the gel surface; and the adherent fraction, which bound but did not penetrate during the assay period. During a 3- to 5-day exposure to high titer IL-2, both adherence and motility increased significantly. In type I collagen, cells of the NK lineage developed greater surface adherence and less motility than cells of the T lineage. The surface-adherent fraction expressed higher lymphokine-activated killer and NK activity than did the nonadherent or motile fractions. Under prolonged IL-2 stimulation (7 to 12 days), there was a decline in the percentage of cells exhibiting motility in both types of ECM, and an increase in the percentage of surface-adherent cells. The findings indicate that the behavior of an IL-2-stimulated lymphocyte population in ECM is profoundly influenced by the duration of IL-2 exposure. Furthermore, lack of lymphocyte motility may reflect two different behaviors, nonadherence and adherence without motility. The nonadherent and surface-adherent populations may differ in phenotypic distribution and function. The motility system described in this report will be useful in separating and studying the mechanisms that produce lymphocyte adherence and motility, and in understanding the in vivo implications of these behaviors.

The migration of lymphocytes through extracellular matrix (ECM) is an essential feature of the infiltration process. In the course of their extravasation into poorly perfused neoplastic lesions, lymphocytes often encounter regions of acidified ECM. This study was designed to determine whether lymphocyte adherence and motility in ECM are influenced by ambient pH. Murine splenic lymphocytes, activated by culture with high-titer IL-2, were allowed to migrate into three-dimensional gels of Type I collagen, a major component of interstitial stroma, or into Matrigel, a basement membrane model. After 18 hr at pH 7.1, the leading cell front traveled a mean distance of approx 475 microM into Type I collagen gel. Approx 50% of the cells remained nonadherent, 25% adhered to the gel surface, and 25% were motile (penetrated beneath the surface). At pH 6.7, the leading-front distance increased significantly, by a factor of 1.4X, but there was little change in the proportion of cells exhibiting nonadherence, surface adherence, or motility. The relative motilities of CD3+ and AsGM1+ subsets were also unaltered. It therefore appears that acidification of collagen matrix increases the locomotory activity of motile lymphocytes, but causes little recruitment of nonmotile lymphocytes into the motile pool. Similar results were obtained in experiments with Matrigel. The increased motility observed at pH 6.7 did not reflect breakdown or relaxation of matrix lattices, as measured by the passive diffusion of latex beads of defined diameter. Preincubation of lymphocytes at pH 6.7 did not alter their subsequent motility in pH 7.1 gels. The findings establish ambient pH as a microenvironmental variable which can influence lymphocyte migration through ECM. The weak acidity characteristic of certain tumor microenvironments may be a factor which encourages lymphocyte infiltration through tissue matrix. Treatments which alter intratumor pH could potentially be used to manipulate the infiltration process for immunotherapeutic benefit.

The movement of lymphocytes through extracellular matrix (ECM) is an essential component of normal traffic and infiltration into inflammatory sites. This review surveys current knowledge of the mechanisms of lymphocyte migration through ECM, most of which was derived from work with in vitro models of basement membranes, interstitial stroma, or their constituent components. Normal lymphocyte motility is an extremely plastic property. Naive lymphocytes tend to be unresponsive to ECM components and many chemoattractants, but when exposed to antigens, artificial mitogens and certain lymphokines, they rapidly acquire locomotory capacity, which is expressed as increased polarity, adhesiveness, invasiveness and chemotactic response. Acquisition of locomotory capacity is associated with the G0/G1 transition, and activation of protein kinase C appears to be a key event. Preliminary evidence indicates that mitogenesis and differentiation to the memory phenotype trigger a long-lasting, possibly permanent elevation of locomotory response to ECM. Receptors for fibronectin, laminin and collagens I and IV have been implicated as mediators of lymphocyte motility, but these receptors have not been characterized in detail. Heparanases facilitate T cell movement through the basement membrane, but the role of proteases has not yet been defined. Major gaps remain in our understanding of the connection between in vitro models and specific stages of the infiltration process in vivo and of motility regulation at the molecular level.

Murine splenic lymphocytes nonspecifically stimulated with recombinant human interleukin-2 in vitro were fractionated according to their ability to migrate through type I collagen gel during a 24-hour period. After labeling with 111In and adoptive transfer into hosts of subcutaneous mammary tumors, motile fractions exhibited approximately twofold greater tumor localization and approximately twofold lower lung localization than nonmotile fractions. The results suggest that lymphocyte properties associated with motility through extracellular matrix also influence patterns of lymphocyte localization in vitro. It should be possible to identify these properties by comparative analysis of motile and nonmotile fractions obtained from various matrices.

The motility of murine splenic lymphocytes stimulated nonspecifically by recombinant interleukin 2 (RIL-2) was studied in a three-dimensional collagen-gel system. Nonadherent BALB/c splenic lymphocytes were cultured in medium containing Cetus RIL-2 (700 to 1000 units/ml) or excipient control. They were then allowed to locomote randomly for 16 to 18 h into slabs of type I rat tail collagen gel. The gels were digested with collagenase, and total lymphocyte populations and motile subpopulations were collected and compared with respect to their lymphokine-activated killer activity (measured as 4-h cytotoxicity against the natural killer-resistant mammary adenocarcinoma line 410.4), their natural killer activity (measured as 4-h cytotoxicity versus lymphoma YAC-1), and their subset distribution (defined by immunofluorescence). Some of the slabs were not digested but fixed for measurement of leading-front distance. RIL-2-stimulated lymphocyte populations displayed greater motility than unstimulated populations; the mean leading front distance was 2.4 times greater, and the percentage of cells exhibiting motility was approximately doubled. The most motile RIL-2-stimulated cells, however, were not the most tumoricidal. Motile subpopulations displayed approximately 25 to 60% lower lymphokine-activated killer activity than did the total populations from which they were derived. Natural killer activity followed a similar pattern. Motile subpopulations contained a lower proportion of asialo-GM1+ and T-null cells than did total populations and a higher proportion of L3T4+ cells. Chemokinetic stimulation with alpha-interferon increased overall motility, but the lymphokine-activated killer activity of the motile subpopulation was still lower than that of the total population. Lymphocyte motility is important in the infiltration of tumors and other inflammatory lesions. The results indicate that the most tumoricidal lymphocytes in RIL-2-stimulated populations may not be the best tumor infiltrators, and that the tumoricidal activity of circulating lymphocytes may be a misleading indicator of the effectiveness of immunotherapy.

Hoechst 33342 (HO 33342) is a fluorescent dye which binds specifically to DNA and can be used to label lymphocytes for in vivo migration studies. Lymphocytes were treated with varying concentrations of HO 33342 and assayed in vitro for effects on viability, mitogen-stimulated proliferation, and motility. In vivo traffic studies were performed to determine a dye concentration with minimal toxicity for lymphocytes, but sufficient fluorescence for detection of cells in frozen sections. The concentration reported to yield quantitative staining of nuclear DNA (10.7 microM, or 6 micrograms/ml) reduced motility and proliferative response, and resulted in an altered lymphocyte migration pattern compared to untreated lymphocytes. A concentration of 0.25 microM, however, produced no toxicity in the in vitro assays, and an in vivo migration pattern similar to that of untreated cells; lymphocytes stained with 0.25 microM HO 33342 for 30 min were readily observable in histological sections. This study indicates that the concentration of HO 33342 optimal for DNA staining may exert deleterious effects on in vivo lymphocyte traffic studies, and that far lower dye concentrations are more suitable for such studies.

The relationship between the basal (spontaneous) motility of murine lymphocytes and their position in the cell cycle was examined in a new collagen gel motility assay system. Concanavalin A-stimulated or control lymphocytes were allowed to locomote into slabs of type I collagen gel. The assay configuration permitted extraction of both total populations and locomotory subpopulations as viable, single-cell suspensions suitable for phenotypic and cell analysis. Concanavalin A stimulation resulted in a significant increase in the mean distance traveled by the leading cell front in 4 hr, from 23 microns (controls) to 67 microns. The estimated percentage of motile cells increased from 0.9 to 2.8%. Similar increases were observed after 18 hr of locomotion. The SIg+, Thy-1+, L3T4+, and Ly-2+ subsets exhibited equivalent increases in motility. Total populations and locomotory subpopulations were allowed to incorporate 5-bromo-2'-deoxyuridine, and their cell cycle profiles were compared by dual parameter anti-5-bromo-2'-deoxyuridine, propidium iodide fluorescence analysis. Total population and locomotory subpopulations did not differ significantly with respect to the ratio G0/G1:S, indicating that lymphocytes in these two phases exhibited approximately equal motility. Cells in late S and G2 + M were significantly less motile; locomotory subpopulations contained 60 to 75% fewer G2 + M cells than the total populations from which they were derived. Taken together, the results indicate that the concanavalin A-induced increase in motility commences before S phase and that motility diminishes shortly before or during G2 + M.

The composition of tumor infiltrating lymphocyte populations is often reported to be different from that of the lymphocyte pool of peripheral blood. This suggests that infiltration-regulating mechanisms reside in or near the tumor microenvironment. Available evidence indicates that these mechanisms exert their effects on lymphocyte traffic. Two models of regulation are proposed. In the selective immigration model, different lymphocyte types display different tendencies to extravasate into the tumor. Selective immigration could reflect heterogeneity of such lymphocyte properties as binding to vascular endothelium, response to chemotactic factors, or spontaneous locomotion. In the other model, selective entrapment, different types of lymphocytes exit from the tumor microenvironment at different rates. Entrapment could be regulated by selectively acting adhesive, locomotion-inhibiting, or negatively chemotactic factors. Available information supporting each model is presented. The composition of infiltrate may also be influenced by the composition of the circulating lymphocyte pool. Evidence is presented that this pool in turn may be influenced by tumor-induced systemic changes in lymphocyte traffic. Deliberate manipulation of lymphocyte traffic should improve the effectiveness of immunotherapeutic regimes. One promising method of traffic manipulation is the modulation of spontaneous motility. A collagen gel assay for spontaneous lymphocyte motility is described.