New insights on the role of CD8⁺CD57⁺ T-cells in cancer

Abstract

Incomplete differentiation of CD8+ cytotoxic T-lymphocytes (CTLs) in the tumor microenvironment is associated with cancer progression. We describe a new type of tumor-infiltrating CD8+CD57+ T cell in cancer with hybrid phenotypic and functional properties of both an early effector-memory cell and a terminally-differentiated effector cell. These cells behave as incompletely-differentiated CTLs.

Melanomas are considered to be one of the most immunogenic cancers and are frequently infiltrated with CD8⁺ and CD4⁺ T lymphocytes specific for major tumor antigens. CD8⁺ cytotoxic T lymphocytes (CTL) mediate antigen-specific lysis of tumor cells through cytolytic granule proteins, such as granzymes, granulysin, and perforin. Previously, other investigators have shown a lack of complete CTL differentiation in solid tumors.¹ However, the exact stage of differentiation affected was unclear. We recently reported in Clinical Cancer Research a novel subset of CD8⁺ tumor-infiltrating lymphocytes (TILs) that co-expressed early effector-memory markers (CD27, CD28) and a marker for end-stage, senescent T cells (CD57).²

CD28 and CD27 are markers of early CD8⁺ effector-memory T (T_EM) cells. Based on studies on virus-specific T cells in humans, it was postulated that CD8⁺ T_EM cells differentiate in a linear pathway from CD28⁺CD27⁺ (early-differentiated) to CD28^-CD27⁺ (intermediate-differentiated) to CD28^-CD27^- (late-differentiated).³ As CD8⁺ T_EM transitions to end-stage effector (T_EFF), the loss of CD28 and gain of CD57 is an immunological characteristic of humans and non-human primates, but not of mice.³ CD57 is a marker on highly differentiated CD8⁺CD27^-CD28^- T cells needed to control CMV and other endemic viruses in humans.^3,4 CD57 was also proposed as a marker of end-stage, senescent CD8⁺ T cells in HIV patients exhibiting highly cytotoxic potential.^3,5 However, in HIV progressors, a failure to coordinately downregulate CD27 and upregulate CD57 resulted in an accumulation of an unusual subset of HIV-specific CD8⁺CD27⁺CD57⁺ cells.⁴ CD8⁺CD27⁺CD57⁺ T lymphocytes have also been observed in the peripheral blood of melanoma patients after vaccination with gp100 tumor antigen.⁶

We set out to determine the role of CD8⁺CD57⁺ T cells in the melanoma. By performing flow cytometry staining on TIL isolated from 44 metastatic tumors, we found that the CD8⁺CD57⁺ subset was 16.2 ± 3.5% of the total tumor-infiltrating CD8⁺ T cells. The vast majority of these CD8⁺ T cells were also “locked” in the T_EM stage and, on average, > 20% of the CD8⁺CD57⁺ subset co-expressed CD27 and CD28. These T cells were GB^hi but Perf^lo/-, and they recognized melanoma tumor antigens, MART-1 and gp100 in HLA-A2⁺ patients. In contrast, only a few (< 5%) of the CD8⁺CD57⁺ T cells in the PBMC of the same melanoma patients co-expressed CD27 and CD28, but they were GB^hi and Perf^hi. We also found a similar population in pleural effusions from metastatic breast cancer. Notably, this TIL subset was different from the Foxp3⁺ CD8⁺ early effector TILs, which did not express CD57, as reported earlier.⁷

Whether CD8⁺CD57⁺ T cells are really “senescent” has become a controversial issue.³ We addressed this issue in the CD8⁺CD27⁺CD57⁺ TIL subset and found that they indeed proliferated in response to IL-2. We then purified this subset from IL-2-cultured TILs by cell sorting. Interestingly, we found that the CD27⁺CD57⁺ TILs were able to proliferate and produce high levels of IFN-γ upon TCR stimulation, which was inconsistent with CD57 as a general marker for T-cell senescence. We also found that PD-1 expression was higher in the CD27⁺CD57⁺ subset compared with the CD27⁺CD57^- subset. Since CD8⁺ T cells naturally express PD-1 as a result of TCR activation, where it plays a regulatory mechanism to prevent over-reactivity,³ it is possible that the CD8⁺CD27⁺CD57⁺ subset are more highly activated T cells in the tumor microenvironment.

Building on our observation that CD8⁺CD57⁺ TILs were non-senescent, we hypothesized that these T cells were in a transition state and could be induced to differentiate further to cytotoxic end-stage effectors. Indeed, we found that the sorted CD8⁺CD27⁺CD57⁺ TILs, which were Perf^lo and poorly cytotoxic, could differentiate into a Perf^hi, highly cytotoxic CD27^-CD57⁺ or CD27^-CD57^- subset after TCR stimulation (Fig. 1). IL-2 treatment alone induced a minor fraction of CD27⁺ precursor cells to become CD27⁺CD57⁺, which suggested that IL-2 was sufficient to expand the CD27⁺CD57⁺ subset from CD27⁺ precursors (Fig. 1). TCR-stimulation induced the differentiation of sorted CD8⁺CD27⁺CD57^- TILs into a Perf^hi, CD27^-CD57^- subset, but the cells differentiating from the CD27⁺CD57⁺ subset on average acquire higher perforin levels and killing function (Fig 1). We also found that TGF-β1, produced by many melanomas, arrested the differentiation and cytotoxic activities of both the CD27⁺CD57^- and CD27⁺CD57⁺ subsets at the CD27⁺ stage. Taken together, these findings may help explain why metastatic melanomas are often infiltrated with only early T_EM CD8⁺ T cells that have limited ability to kill tumor cells. Thus, TGF-β1 may be a key suppressor of CTL differentiation in the tumor microenvironment. However, other factors such as PGE₂, indoleamine 2,3-diooxygenase (IDO), IL-10, or inhibitory signaling through PD-1, may also play a role.⁸

Figure 1. Differentiation pathway of the tumor-infiltrating CD8⁺ T cells in metastatic cancer. In situations where CD8⁺ T cells encounter persistent, chronic antigenic stimulation such as metastatic cancer or uncontrolled chronic viral infections, CD8⁺ effector-memory T (T_EM) cells fail to coordinate downregulation of CD27 with upregulation of an end-stage CTL marker, CD57 and acquire a more cytolytic phenotype. Thus T_EM fail to transition from a granzyme B (GB⁺) perforin^lo (Perf^lo) cells into Perf^hi, highly cytotoxic end-stage CTL. This resulted in accumulation of CD8⁺ T cells at a transitional stage where markers for early T_EM (CD27, CD28) are co-expressed with CD57, even though the cells remain Perf^lo. We also found that TGF-β1, an immunosuppressive cytokine frequently found in the microenvironment of metastatic cancer, could also contribute to the arrested differentiation and accumulation of CD27⁺CD57^- precursor T cells and CD27⁺CD57⁺ T cells. We found that PD-1 was expressed more in the CD27⁺CD57⁺ T cells, which implied that they may exhibit a higher level of effector activity. When these tumor-infiltrating lymphocytes (TIL) are expanded with IL-2, a minor fraction (~30%) of CD27⁺CD57^- subset differentiated into CD27⁺CD57⁺ T cells. After TCR stimulation ex vivo, the CD27⁺CD28⁺CD57^- subset directly differentiated to become CD27^-CD57^- T cells. On the other hand, CD27⁺CD57⁺ differentiated to become CD27^-CD57⁺, and, in some patients, CD27^-CD57^-. These phenotypic changes were accompanied by increased perforin expression and acquisition of potent cytotoxicity against tumor cells. We think that CD57 is not a marker for T-cell senescence, but rather marks highly differentiated T cells that are in the process of transitioning into a truly end-stage, effector CTL. Currently it is not known which set of markers define truly senescent, end-stage, highly cytolytic CTL. We also propose that ultimately, both the CD8⁺CD57⁺ and CD8⁺CD57^- subsets may ultimately differentiate into a subset of CD8⁺CD27^-CD28^-CD57^- cells that may be more NK-like, expressing higher levels of CD122, KLRG1, NKG2D and other NK receptors.

Another interesting observation we made was that both the CD8⁺CD27⁺CD57⁺ and CD8⁺CD27⁺CD57^- T cells seemed to ultimately differentiate into CD27^-CD57^- (also CD28^-) cells subset with high perforin and killing activity. Are these the true end-stage or terminally-differentiated state of CTL that actually may be more NK-like, by expressing CD122, other NK receptors (NKG2D and high KIR levels)? These cells have been described before, but their origin is unknown.⁹

In conclusion, we characterized a novel subset of TILs in metastatic melanoma and breast cancer that seems to be locked in a transitional state between early T_EM and fully-differentiated CTL. Our study also underscores the need for a greater understanding of the state of CTL differentiation and activity in patients in relation to tumor control. Overall, there has been an over-emphasis on the role of cytokine release by CD8⁺ T cells (e.g., IFNγ), which has generally not correlated with clinical efficacy during cancer immunotherapy. A case in point is the recent HIV vaccine trials where effective immunization correlated more with a gain of antigen-specific CTL phenotype and killing function rather than IFN-γ production.¹⁰

Glossary

Abbreviations:

CTL

cytotoxic T lymphocytes

TCR

T-cell receptor

TIL

tumor-infiltrating lymphocytes

ACT

adoptive T-cell therapy

PBMC

peripheral blood mononuclear cells

Perf

perforin

GB

Granzyme B

HNK-1

human natural killer-1

T_N

naïve T cells

T_EM

effector-memory T cells

T_TDE

terminally-differentiated effector cells