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Concomitant Administration of Interleukin-2 During Therapeutic Vaccinations Against Cancer: The Good, the Bad, or the Evil?

Tumor Immunology Group, Danish Cancer Society, Copenhagen, Denmark

Julie Gehl, Poul Geertsen

Department of Oncology, Herlev University Hospital, Herlev, Denmark

Jürgen C. Becker

Department of Dermatology, University of Würzburg, Würzburg, Germany

To the Editor:

The article by Slingluff et al¹ report a phase II vaccination study in which melanoma patients were vaccinated with synthetic peptides in montanide ISA-51 and granulocyte-macrophage colony-stimulating factor (GM-CSF); combined with concurrent or delayed systemic administration of low-dose interleukin-2 (IL-2). In two groups, patients received either IL-2 beginning at day 7 (group 1), or a similar dose of IL-2 beginning at day 28 (group 2). Thus, the reported phase II trial was initiated with the aim of selecting whether upfront or delayed administration of IL-2 was more likely to enhance the efficacy of the vaccine. Importantly, evaluations of induced biologic responses were measured not only in peripheral blood lymphocytes (PBL), but also in the sentinel immunized node (SIN). The primary end points were in fact reactivity in SIN as measured by EliSpot assay. Secondary end points were antimelanoma and melanoma antigen–specific T-cell reactivity in blood, likewise measured by EliSpot assay and delayed-type hypersensitivity testing.

The major finding of the study was that reactivity was observed significantly less frequently in group 1 patients, who received IL-2 from day 7, compared with group 2 patients, receiving IL-2 from day 28. Thus, reactivity in PBL and SIN was 37% and 38%, respectively, in group 1 patients, compared with 53% and 83% in PBL and SIN, respectively, in group 2 patients.

The authors conclude from the study that "low-dose IL-2 at 3 MU/m2 is not a useful adjuvant for increasing T-cell responses to a peptide vaccine when administered on the schedule used in group 1."

We recently reported a significant decrease in CD8 T-cell reactivity in PBL throughout the course of low-dose IL-2 administration.² In a phase II clinical trial, melanoma patients were treated with electrochemotherapy and low-dose subcutaneous IL-2. Bleomycin was administered intratumorally, followed by short high-voltage pulses. Subcutaneous administration of IL-2 was given outside the electrochemotherapy area as a flat dose of 2 MU/d for 21 days. Up to three treatment cycles could be given, and weekly blood samples were collected for immunologic monitoring. Importantly, each treatment cycle was separated by a gap of 1 week devoid of treatment.

As in the study of Slingluff et al,¹ we conducted biologic monitoring taking advantage of the EliSpot technique, quantitating CD8 T-cell reactivity against the human leucocyte antigen A2 (HLA-A2) –restricted peptides Mart-1,³ tyrosinase-related protein 2 (TRP-2),⁴ survivin,^5,6 and gp100.⁷ Several of these antigens are targets for spontaneous reactivity in melanoma patients (Mart-1, gp100, and survivin). Consequently, monitoring of reactivity against these antigens offers the opportunity to reveal not only induction or increase of reactivity, but also any decrease or disappearance of reactivity during the course of treatment.

In all patients analyzed, we observed a reduction in peptide specific reactivity during administration of IL-2. Moreover, we observed a remarkable reappearance of T-cell reactivity when IL-2 administration was paused (eg, between the treatment cycles).

Clearly, this could represent a general phenomenon of migration out of circulation of CD8 cells during IL-2 administration. However, (1) CD4-CD8 ratios remained stable in each patient during treatment, and (2) the short in vitro culture before EliSpot analyses did not induce changes in CD4-CD8 ratios.

Both we and Slingluff et al took advantage of a short in vitro senzitation before EliSpot analysis, which is mandatory, as CTL frequencies in most cases are below the limit of detection if analyzed directly ex vivo. However, this implies that T cells may in fact die during culture (eg, due to peptide stimulation), and the threshold for stimulation/death of T cells could theoretically be altered during IL-2 administration. However, in our study of reactivity during electrohemo–IL-2 therapy, one of the patients could be analyzed by direct ex vivo EliSpot. Data from these experiments demonstrated the same pattern of decrease in reactivity during IL-2 administration, excluding that decrease in reactivity was due to death of CD8 cells during the in vitro culture.

Decrease in reactivity against the tumor-associated antigen (TAA)-derived peptides could represent a general decrease in CD8 T-cell reactivity. However, data from analyses of reactivity against the HLA-A2 restricted Epstein-Barr virus (EBV) peptide demonstrated that (1) the number of EBV-reactive CD8 T cells remained relatively stable during treatment, and (2) reactivity against EBV did not follow the pattern of decrease during IL-2 treatment and increase after the break.

These data strongly encouraged us to study in more detail the destiny of TAA-specific T cells during treatment. First, taking advantage of HLA tetramer complexes, we isolated Mart-1–specific T cells at different time points during treatment, and molecular fingerprinting by T-cell receptor (TCR) clonotype mapping allowed us to demonstrate that new Mart-1–specific T cells were induced during treatment. Thus, specific T-cell clonotypes disappearing from circulation during the course of IL-2 administration did not reappear after the pause. Rather, new Mart-1–specific T cells were introduced in each cycle.

Moreover, using similar techniques, we were able to demonstrate that interferon gamma (INF-) secreting TRP-2–specific T-cell clonotypes—disappearing from circulation during IL-2 administration—could subsequently be detected at the tumor site.

In terms of immunologic monitoring, our data point to at least two important features of antitumor T-cell responses. First, it is obvious that the information provided by our close weekly monitoring would not have been revealed by, for example, monthly or pre- and post-treatment EliSpot analyses. Second, it would not have been possible to draw any conclusions based on TAA EliSpots alone; only by scrutinizing CD4-CD8 ratios (before as well as subsequent to peptide stimulation) and EBV reactivity can careful conclusions be drawn. Subsequently, these conclusions could be strengthened by applying molecular tracking of specific T cells. The data raise the question as to whether analysis of T-cell reactivity in PBL is optimal, or even useful, when conducted alone.

We believe that our data suggest that IL-2 influences the capacity of CD8 T cells to home to the site of action. Moreover, considering the fact that virus-specific T-cell reactivity in PBL remained stable and that CD4-CD8 ratios were unchanged, nourishes the notion that this action of IL-2 is restricted to T cells, actively engaged with an ongoing immune attack. Most importantly, it is clear that the interpretation of our data would be completely different if the EliSpot data were interpreted alone, as this would favor an understanding in which administration of IL-2 diminished antitumor reactivity. Only when the compiled data are judged and combined does a completely different and more complex picture emerge.

Slingluff et al¹ conclude that low-dose IL-2 administered concurrently as in group 1 patients, is not a useful adjuvant for increasing T-cell responses to a vaccine. We are aware that data from our study were not available when Slingluff and colleges submitted their manuscript for publication. We do, however, believe that the stated conclusions should nevertheless be drawn with more caution. It is certainly admirable that the study includes analyses for reactivity in SIN as well as PBL; however, SIN may not be the only organ relevant for analyses of vaccination-induced responses. Thus, it is quite clear that T cells are guided in their migration pattern by expression of chemokine receptors; thus, (some) receptors associated with homing to skin, lymph nodes, and bone marrow have been characterized.⁸ Thus, there are numerous other (relevant) compartments besides PBL and SIN, which could comprise vaccination-induced T cells. To this end, it has been shown that tumor-specific memory T cells preferentially home to the bone marrow.⁹ Moreover, the authors state that the patients were clinically disease-free, for which reason it is unlikely that T-cell homing to the tumor site would significantly influence systemic frequencies of such T cells. Considering that the precursor frequencies are very low and that cancers can grow to a substantial size in the absence of any symptoms, this issue is more appropriately left as an unresolved question.

One of the secondary end points in the study of Slingluff et al is delayed-type hypersensitivity testing. This test has previously been demonstrated to correspond quite well with clinical responses.¹⁰ Unfortunately, no data are provided.

The study of Slingluff el al presents data from the monitoring of vaccinated melanoma patients administered different regimens of IL-2. Interestingly, both SIN and PBL were analyzed, leading to very interesting data. However, we feel strongly that the suggestions given with regard to the influence IL-2 may have on induction of antitumor T-cell reactivity in cancer patients are premature considering the presented data.

Authors' Disclosures of Potential Conflicts of Interest

The authors indicated no potential conflicts of interest.

REFERENCES

1. Slingluff CL Jr, Petroni GR, Yamshchikov GV, et al: Immunologic and clinical outcomes of vaccination with a multiepitope melanoma peptide vaccine plus low-dose interleukin-2 administered either concurrently or on a delayed schedule. J Clin Oncol 22:4474-4485, 2004[Abstract/Free Full Text]

2. Andersen MH, Gehl J, Reker S, et al: Dynamic changes of specific T cell responses to melanoma correlate with IL-2 administration. Semin Cancer Biol 13:449-459, 2003[CrossRef][Medline]

3. Kawakami Y, Eliyahu S, Sakaguchi K, et al: Identification of the immunodominant peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2- restricted tumor infiltrating lymphocytes. J Exp Med 180:347-352, 1994[Abstract/Free Full Text]

4. Wang RF, Appella E, Kawakami Y, et al: Identification of TRP-2 as a human tumor antigen recognized by cytotoxic T lymphocytes. J Exp Med 184:2207-2216, 1996[Abstract/Free Full Text]

5. Andersen MH, Ostergaard Pedersen L, Becker JC, et al: Identification of a cytotoxic T lymphocyte response to the apoptose inhibitor protein Survivin in cancer patients. Cancer Res 61:869-872, 2001

6. Otto K, Andersen MH, Eggert AA, et al: Therapy-induced T cell responses against the universal tumor antigen survivin. Vaccine 23:884-889, 2005[CrossRef][Medline]

7. Bakker AB, Schreurs MW, de Boer AJ, et al: Melanocyte lineage-specific antigen gp100 is recognized by melanoma-derived tumor-infiltrating lymphocytes. J Exp Med 179:1005-1009, 1994[Abstract/Free Full Text]

8. Moser B, Wolf M, Walz A, et al: Chemokines: Multiple levels of leukocyte migration control. Trends Immunol 25:75-84, 2004[CrossRef][Medline]

9. Letsch A, Keilholz U, Assfalg G, et al: Bone marrow contains melanoma-reactive CD8+ effector T cells and, compared with peripheral blood, enriched numbers of melanoma-reactive CD8+ memory T cells. Cancer Res 63:5582-5586, 2003[Abstract/Free Full Text]

10. Keilholz U, Weber J, Finke JH, et al: Immunologic monitoring of cancer vaccine therapy: Results of a workshop sponsored by the Society for Biological Therapy. J Immunother 25:97-138, 2002

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This Article Full Text (PDF) Erratum (v23,p6812) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Rights & Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Andersen, M. H. Articles by Becker, J. C. Search for Related Content PubMed PubMed Citation Articles by Andersen, M. H. Articles by Becker, J. C. Social Bookmarking                            What's this?