In this study, we analyzed fresh peripheral blood samples and characterized the expression pattern of T cell markers in patients with lymphedema as a consequence of surgical cancer treatment. The expression of PD-1, Tim-3, and Lag3 and the frequency of Treg on CD4 + T cells were significantly upregulated in patients with lymphedema, compared with those in HC. On the other hand, we could not find any relationship between exhaustion markers on CD8 + T cells and lymphedema. Notably, for both CD4 + and CD8 + T cells, the proportions of the naïve phenotype subsets were decreased in patients with lymphedema patients, while the fraction of Tscm in the naïve cell-like phenotype was increased, suggesting that the Tscm compartment was retained under the concurrent shrinkage of naïve-like cell pools. To our knowledge, this is the first report of detailed observations of peripheral T cells in patients with lymphedema. Investigation of functional T cell landscapes in lymphedema is crucial for a deeper understanding of the immune status underlying this unique medical complication. Moreover, these findings might influence on prudent decision of cancer treatment, particularly in the case of the lymph node dissection.
The PD-1 regulatory pathway plays indispensable roles in downregulating the immune response, and in promoting tolerance to self-antigens by suppressing T cell activation through B7-CD28 co-stimulatory molecules that deliver critical inhibitory signals . In the context of autoimmune diseases, PD-1 is known as a protective molecule that acts by inducing apoptosis in activated effector T cells and reducing the apoptosis of regulatory T cells . Tim-3, a member of the T cell Ig and mucin domain-containing molecule superfamily, is a key regulatory molecule for Th1 response. Initial studies on Tim-3 demonstrated its upregulation in activated Th1 cells [24–26]. The expression of Tim-3 in peripheral T cells is associated with the regulation of autoimmune encephalomyelitis (EAE) and rheumatoid arthritis [27, 28]. Lag-3 exhibits high affinity to major histocompatibility complex class II (MHC II) and regulates the proliferation, activation, and function of T cells [29, 30]. In this study, the expression of PD-1, Tim-3, and Lag3 on CD4 + T cells was significantly upregulated in patients with lymphedema, compared with that in HC. Importantly, these molecules are also known as markers of Treg subsets that possess enhanced immunosuppressive function [31, 32]. Previous studies on the immunopathology of lymphedema have shown that CD4 + T cells play a role in aggravating tissue fibrosis and lymphatic dysfunction [8–11]. Upregulated exhaustion markers on CD4 + T cell populations in lymphedema might reflect not only chronic consumption of effector CD4 + T cells, but also the counterbalancing enhancement of Treg function to inhibit the progress of tissue inflammation and fibrosis. This hypothesis is supported by our observations in the present study that the proportions of Treg II and Treg III, but not those of Treg I, were more significantly elevated in patients in lymphedema patients than in HC.
Treg cells compete for the T cell growth factor IL-2 via expression of high-affinity IL-2 receptor complexes and exert direct suppressive activity by secreting immunosuppressive cytokines such as TGF-β and IL-10 . Treg Ⅰ cells are able to proliferate themselves upon T cell receptor stimulation and can convert to Treg Ⅱ cells . The Treg Ⅱ subset is functionally important in regard to its potent suppressive function, which is caused due to its high expression of CTLA-4 and CD25 and its higher sensitivity to IL-2 than that of other Treg subpopulations . Treg Ⅲ secretes a high amount of effector cytokines (IL-2, IL-17, and IFN-γ) without suppressive activity [19, 34]. Treg Ⅲ cells may be a heterogeneous subset between Treg cells and effector T cells. After dissection of the lymph node in an animal model, Treg infiltration is increased in lymphedematous tissues . The accumulation of Treg in the skin subsequently impairs bacterial phagocytosis because of the development of a Treg-mediated immunosuppressive environment . This observation suggests that, in the presence of chronic lymphedema, circulating Treg I may be prone to conversion into Treg Ⅱ, which can swiftly downregulate local inflammation.
Naïve T cells are subjected to an abnormal drive for differentiation under the pressure of IL-6 and TNF . Given that age strongly affects the number of circulating naïve T cells in healthy individuals, the proportions of naïve fraction observed in patients need to be age-matched [36, 37]. Irrespective of age, the reduction of naïve T cells was reported in patients with active rheumatoid arthritis  and autoimmune colitis , implying that such abnormalities may be common to immune-mediated inflammatory diseases. In this study, the number of naïve T cells was more significantly decreased in patients with lymphedema than in age-matched HC. The progressive depletion of the naïve pool induces homeostatic proliferation, where the naïve T cells turn over and differentiate into memory T cells . Naïve T cell numbers and clonal diversity represent the potential of the adaptive immune system to sense and respond to foreign pathogens and to the mutant proteins expressed by malignant cells . Loss of naïve T cells could leave many individuals vulnerable to infection.
Tscm cells exhibit enhanced proliferative capacity compared with that of CM phenotype T cells, the potential to differentiate into all other classically defined T cell memory subsets, and the ability to retain their phenotype following proliferation in vitro and in vivo model [40, 41]. Tscm cells are increased in chronic disease states. Specifically, Tscm cells are enhanced in individuals with type 1 diabetes , uveitis , systemic lupus erythematosus , cardiovascular disease , and rheumatoid arthritis . Tscm frequency might correlate with disease activity and might indicate immune therapies . In the present study, the proportion of the Tscm population was retained even though the number of naïve phenotype cells was significantly decreased in CD4 + and CD8 + T cells. Such a Tscm pool might function as a reservoir of effector T cells relevant to chronic inflammation induced by environmental factors in lymphedema.
All patients in this study had history of surgical resection of cancer without relapse. A dramatic reduction in PD-1 expression and peripheral frequencies of Treg to the normal level was observed within weeks after surgical resection of the primary cancer [47, 48]. It is plausible that the expression patterns of T cells in the present study are contributed to by lymphedema, and not by the history of primary cancer.
Circulating T cell proportion in patients with lymphedema was investigated using several expression markers in the present study. It is well known that patients with lymphedema often experience cellulitis without definite skin injury. The pathogenesis and progression of the symptoms are very rapid, and redness and local fever quickly spread throughout the whole extremity within an hour, which completely differs from usual cellulitis without lymphedema . Stewart and Treves reported that angiosarcomas associated with lymphedema develop after 9 years (range 8–24) from the onset of lymphedema . The median survival has been quoted as 24 months, with an overall 5-year survival rate of approximately 10% . Only early radical surgical removal, including amputation or disarticulation of the affected limb, or wide excision at an early stage offers the greatest chance of long-term survival. Frequent incidences of cellulitis and angiosarcoma cause heavy deterioration in the quality of life of patients with lymphedema. Because we believed that these pathological changes were associated with T cell modulation, we investigated circulating T cells using several expression markers in patients with lymphedema. We found that the exhaustion markers of CD4 + T cells and Treg were upregulated, and the naïve phenotype on CD4 + and CD8 + T cells was decreased in patients with lymphedema patients. These findings may be the result of the immune dysfunction that accompanies lymphedema.
Our study has some limitations. First, a small number of patients is always a cause of bias in this type of study. Second, we lack data on the similar profiling of T cells in age-matched patients with cancer but without lymphedema; therefore, we could not exclude the effects of pre-existing cancer from those of the lymphedema itself. In addition, the relationships between clinical conditions and therapeutic strategies need to be elucidated. A large-scale study in the future is warranted to assess the function of T cells in lymphedema more clearly.
In conclusion, the expression of PD-1, Tim-3, and Lag-3 in peripheral CD4 + T cells, and the relative proportions of Foxp3-positive Treg cells were upregulated in patients with lymphedema, compared with those in HC. The skewed number of naïve phenotype T cells and the increased frequency of Tscm both in naïve-like CD4 + and CD8 + T cells subsets were characteristic of the patients with lymphedema in our study. These distorted profiles of circulating T cells may be related to the frequent cellulitis and rare cases of angiosarcoma seen in patients with lymphedema. Our results may assist in designing new approaches for cancer treatment with the help of translational immunology.