Abstract

Rituximab (Rtx) has shown significant therapeutic activity in follicular lymphoma (FL) patients, yet it’s exact mechanism of action has not been fully defined. Although killing of FL cells through complement dependent cytolysis, antibody dependent cellular cytotoxicity or direct induction of apoptosis may contribute to its effectiveness, these mechanisms are unlikely to be the only ones as;

  • the clinical and molecular responses to Rtx may continue for months after the last dose and;

  • the median duration of the second response to Rtx is longer than that of the first, findings that cannot be explained solely by the above mechanisms.

Rtx induced FL cell death likely results in the release of tumor antigens in a pro-inflammatory environment which we hypothesize may provoke a cell-mediated lymphoma specific immune response. Indeed, others have suggested that such a “vaccinal” effect may be an additional mechanism of action but to our knowledge there has been no direct evidence to support this in Rtx treated patients.

Methods: To provide support for this hypothesis we examined lymphoma idiotype (Id) specific T-cell responses in peripheral blood mononuclear cells (PBMC) from three patients with relapsed FL. PBMC were obtained prior to and 4–6 weeks after the last of 4 weekly doses (375mg/m2 q week) of Rtx. A lymph node biopsy was obtained prior to Rtx to generate the patient’s Id protein. Dendritic cells (DC) were generated from pre-Rtx PBMC and pulsed with;

  • no protein (control);

  • the patient’s Id protein (Id); or

  • an irrelevant (Irr) protein (Id from another patient).

For patient 1, pre- and post-Rtx PBMC were stimulated with the DC for 1 week, while for patients 2 and 3, PBMC were stimulated for 1 week then re-stimulated for another week with fresh DC. Effectors were then assayed in triplicate for IFN-γ producing cells by Elispot. A second independent experiment was conducted in patients 2 and 3 (ie. this study describes data from 3 patients, 5 separate experiments).

Results: Pre-Rtx: There was no consistent increase in the number of Id specific or Irr protein specific T-cells as compared to that of control T-cells. Post-Rtx: Whereas there was no consistent increase in the number of Irr protein specific T-cells as compared to that of control T-cells, in all three patients (including both replicates for patients 2 and 3) there was a consistent increase in the number of Id specific T-cells as compared to that of control T-cells. When composite data from all three patients were analyzed using a mixed ANOVA, the following p-values were obtained: Comparison Pre-Rtx Post-Rtx Control vs. Id 0.03 0.0003 Id vs. Irr 0.3 0.0005 Control vs. Irr 0.2 0.9

ComparisonPre-RtxPost-Rtx
Control vs. Id 0.03 0.0003 
Id vs. Irr 0.3 0.0005 
Control vs. Irr 0.2 0.9 
ComparisonPre-RtxPost-Rtx
Control vs. Id 0.03 0.0003 
Id vs. Irr 0.3 0.0005 
Control vs. Irr 0.2 0.9 

Conclusions: These data provide, to our knowledge, the first support for the hypothesis that Rtx treatment results in an increase in Id specific T-cell responses in FL patients. If indeed this is a mechanism of Rtx activity, then clinical strategies to augment this postulated vaccinal effect, such as anti-CTLA-4 antibodies or Id vaccination post Rtx, may further increase the clinical potential of this agent and change the way we develop combination therapies. Further study of immune responses in a larger number of FL patients treated with Rtx is warranted and ongoing.

Author notes

Disclosure:Employment: Daniel Gold is an employee of Favrille Inc. Consultancy: Steven Bernstein: Millenium, Genentech; Richard Fisher-Genentech, GSK, Millenium. Research Funding: Jonathan Friedberg - Genentech. Honoraria Information: Richard Fisher: Genentech, GSK, Millenium; Steven Bernstein: Millenium, Genentech. Membership Information: Richard Fisher: Genetech, Glaxo-Smith-Kline, Millenium; Steven Bernstein: Millenium, Genentech; Jonathan Friedberg: Genentech, Lily.