Metastatic Melanoma in the Older Patient

Immunologic Insights and Treatment Outcomes

Upendra P Hegde; Nitya Chakraborty; Bijay Mukherji; Jane M Grant Kels

Disclosures

Expert Rev Pharmacoeconomics Outcomes Res. 2011;11(2):185-193. 

In This Article

Ongoing Laboratory Studies & Preliminary Results

Our early laboratory studies of the aging immune system are based on our human anti-melanoma in vitro model examining both effector T lymphocytes and their regulation. We are quantifying CTL responses to a self and non-self peptide antigen (Mart-1 and influenza peptide, respectively) pulsed through an autologous peripheral blood monocyte-derived mature dendritic cell (mDC) serving as an APC using tetramer technology and flow cytometry. The functional studies used in this in vitro model include the ELISA technique to examine the profile of generated cytokines (Figure 2). These studies have demonstrated activation and expansion, as well as functional activity, of effector lymphocytes when presented with melanoma-specific antigens by mDC APCs, from both young and elderly patients. We have also examined the proliferation of CD4+ T lymphocytes in response to plate-bound anti-CD3 antibody and assessed inhibitory effects in this system of natural Treg lymphocytes from young and elderly patients, as well as from young normal donors on proliferation of these CD4 lymphocytes (Figure 3). The results of this assay suggest that marked inhibitory effects of natural Treg lymphocytes are seen in younger patients' CD4+ T lymphocytes compared with those of elderly patients. Our ability to engineer T lymphocytes with an avid anti-melanoma T-cell receptor has also helped us to distinguish T-cell defects inherent to the cell membrane, T-cell receptor or intracellular signaling pathways [CHAKRABORTY NG, HEGDE UP, MUKHERJI BJ, UNPUBLISHED DATA].

Figure 2.

Activation and expansion of melanoma-associated antigen MART-1-specific CD8+ cytotoxic T lymphocytes from HLA A2+ normal donors and older patients with melanoma when stimulated in vitro . (A) Basic CTL expansion/activation protocol in which M1 peptide is presented by mDC acting as an antigen-presenting cell to the CTL in the presence of cytokines IL-2 and IL-15. (B) Expansion of M1-specific CTL upon antigen presentation by antigen-presenting cell in culture analyzed by flow cytometry in both older melanoma patients (top panel) and normal donor (bottom panel). (C) Functional assay describing IFN-γ release by the M1-specific CTL; comparable levels of IFN-γ are released by CTL when M1 antigen is presented in vitro by antigen-presenting cell both from older melanoma patients (top panel) and normal donors (bottom panel). Appropriate negative control shows no cytokine release. Similarly, no expansion of CTL is observed when CTLs are not stimulated (negative control). T2 is a HLA-A2-positive antigen-presenting cell line, M1 is a MART-1 27–35 antigen and M3 is a control peptide. (D) In vitro cytotoxicity of a target cell line T2 pulsed with M1 peptide by the in vitro expanded human M1-specific CTL (effector cells). The x-axis represents an increasing ratio of effector cells (M1-specific CD8+ lymphocytes) to the target cells (M1-pulsed T2 cells) from 2.5:1, 5:1, 10:1, 20:1, 30:1 and 40:1. Increasing target cell lysis (y-axis) is seen with increasing ratio of effector to target cells (x-axis). The negative control in the assay is highlighted by the lack of lysis of target cells when effector cells are exposed to T2 cells not pulsed with M1 (solid squares) or T2 cells pulsed with control peptide (solid circles).
CTL: CD8+ T lymphocytes/effector cells; M1: Melanoma specific MART-1 peptide; mDC: Mature dendritic cell acting as an antigen-presenting cell; T: Tetramer.

Figure 3.

CD4+ T-lymphocyte proliferation in response to plate-bound anti-CD3 and its modulation by Tregs in elderly melanoma patients compared with their young counterparts with and without melanoma (CD4+ cell number represented on the y-axis). The first of the three histograms in each panel depicts CD4+ T lymphocytes stimulated by control antibody showing no expansion of T lymphocytes. The middle histogram depicts CD4+ T lymphocyte's proliferative response to stimulation by anti-CD3 antibody. Note that the CD4 lymphocyte expansion occurs in all three groups. The last histogram depicts the effects of autologous Tregs on expansion of CD4+ lymphocytes stimulated by plate-bound anti-CD3+ antibody. Note the lack of suppression of CD4+ lymphocytes proliferation in response to anti-CD3 antibody by Tregs in elderly patients with cutaneous melanoma (middle panel) compared with those in young normal donors and young patients with cutaneous melanoma whose CD4+ lymphocyte proliferation in response to anti-CD3 antibody is suppressed by autologous Tregs.
CPM: Count per minute; SD: Standard deviation; Treg: Regulatory T cell.

A summary of the preliminary results of our ongoing human research studies presents the following information:

  • Melanoma epitope-specific T-cell responses are preserved in some elderly patients diagnosed with CM, as well as in those with MM (Figure 2);

  • The T-lymphocyte proliferative response to anti-CD3 antibody is comparable to that seen in younger patients with and without CM (Figure 3);

  • The effects of natural Tregs is inhibitory on younger patients' lymphocytes although its effect in the older patients' lymphocytes is weaker (Figure 3);

  • The older patients' lymphocytes fail to demonstrate Th2 responses when polarized with Th2 conditions compared with their younger counterparts who show strong Th2 responses when polarized with Th2 conditions. Both groups of patients demonstrated preserved Th1 responses [Chakraborty NG, Hegde UP, Chhabra A et al., Unpublished Data];

  • Using engineered T-cell receptors in T lymphocytes, early results point to preserved T-cell responses, both at qualitative and quantitative levels, involving both CD4+ and CD8+ lymphocytes [Chakraborty NG, Hegde UP, Yadav M et al., Unpublished Data];

Based on our clinical observations in older patients with MM and their responses to treatment, as well as our immunologic insights, we propose the following conclusions:

  • The aging immune system does not imply a lack of anti-tumor immune response and, in fact, represents an imbalanced anti-tumor immunity;

  • A balance between regulation and effector T cells will determine the effectiveness of anti-tumor immunity. A weaker regulation would facilitate anti-melanoma responses, while a strong regulatory response could seriously impair the anti-melanoma immune response (Figure 1);

  • The favorable Th1 cytokine profile in some older patients is believed to facilitate active immunity against melanoma. The clinical relevance of this observation in 'fit' versus 'frail' elderly patients needs to be studied further;

  • Effective clinical responses to chemotherapy seen in some older patients with MM might represent activation of the unbalanced anti-melanoma immunity by tumor antigens generated during chemotherapy-mediated tumor cell death by partially effective chemotherapeutic agents;

  • The relatively weakened regulation might tip the immune response to an anti-melanoma response facilitated by skewed Th1 versus Th2 cytokines;

  • The clinical benefit of immune-based treatment of melanoma might be enhanced by the 'favorably' imbalanced immune system of the older patient. This will hopefully stimulate the future design of clinical trials to study the relevance of immune-based treatment in elderly patients with malignant melanoma.

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