Immune and pathologic responses in patients with localized prostate cancer who received daratumumab (anti-CD38) or edicotinib (CSF-1R inhibitor).

BACKGROUND: The prostate tumor microenvironment (TME) is immunosuppressive, with few effector T cells and enrichment of inhibitory immune populations, leading to limited responses to treatments such as immune checkpoint therapies (ICTs). The immune composition of the prostate TME differs across soft tissue and bone, the most common site of treatment-refractory metastasis. Understanding immunosuppressive mechanisms specific to prostate TMEs will enable rational immunotherapy strategies to generate effective antitumor immune responses. Daratumumab (anti-CD38 antibody) and edicotinib (colony-stimulating factor-1 receptor (CSF-1R) inhibitor) may alter the balance within the prostate TME to promote antitumor immune responses. HYPOTHESIS: Daratumumab or edicotinib will be safe and will alter the immune TME, leading to antitumor responses in localized prostate cancer. PATIENTS AND METHODS: In this presurgical study, patients with localized prostate cancer received 4 weekly doses of daratumumab or 4 weeks of daily edicotinib prior to radical prostatectomy (RP). Treated and untreated control (Gleason score ≥8 in prostate biopsy) prostatectomy specimens and patient-matched pre- and post-treatment peripheral blood mononuclear cells (PBMCs) and bone marrow samples were evaluated. The primary endpoint was incidence of adverse events (AEs). The secondary endpoint was pathologic complete remission (pCR) rate. RESULTS: Twenty-five patients were treated (daratumumab, n=15; edicotinib, n=10). All patients underwent RP without delays. Grade 3 treatment-related AEs with daratumumab occurred in 3 patients (12%), and no ≥grade 3 treatment-related AEs occurred with edicotinib. No changes in serum prostate-specific antigen (PSA) levels or pCRs were observed. Daratumumab led to a decreased frequency of CD38+ T cells, natural killer cells, and myeloid cells in prostate tumors, bone marrow, and PBMCs. There were no consistent changes in CSF-1R+ immune cells in prostate, bone marrow, or PBMCs with edicotinib. Neither treatment induced T cell infiltration into the prostate TME. CONCLUSIONS: Daratumumab and edicotinib treatment was safe and well-tolerated in patients with localized prostate cancer but did not induce pCRs. Decreases in CD38+ immune cells were observed in prostate tumors, bone marrow, and PBMCs with daratumumab, but changes in CSF-1R+ immune cells were not consistently observed with edicotinib. Neither myeloid-targeted agent alone was sufficient to generate antitumor responses in prostate cancer; thus, combinations with agents to induce T cell infiltration (eg, ICTs) will be needed to overcome the immunosuppressive prostate TME.

Daratumumab Plus Atezolizumab in Previously Treated Advanced or Metastatic NSCLC: Brief Report on a Randomized, Open-Label, Phase 1b/2 Study (LUC2001 JNJ-54767414).

INTRODUCTION: The programmed death-ligand 1 inhibitor atezolizumab improves progression-free survival (PFS) and overall survival (OS) for patients with previously treated advanced NSCLC. Preclinical studies indicate that targeting CD38-positive cells with daratumumab may synergistically enhance atezolizumab's antitumor activity by increasing the effector T-cell activity. METHODS: This phase 1b-2 study included a safety run-in (one cycle of daratumumab plus atezolizumab) and randomized phases (daratumumab plus atezolizumab versus atezolizumab alone). The primary objective of the randomized phase was to compare overall response rates. The secondary objectives included evaluations of safety, clinical benefit rate (stable disease or better), PFS, OS, and pharmacokinetics. RESULTS: In total, 99 patients were enrolled (safety run-in, n = 7; randomized, n = 46 per arm). In the randomized phase, the overall response rate was 4.3% for daratumumab plus atezolizumab and 13.0% for atezolizumab alone (OR: 0.30; 95% confidence interval: 0.03-1.92). The respective clinical benefit rates were 52.2% and 43.5%. No improvements were observed in the median PFS or median OS for combination therapy. The study was terminated because of the limited efficacy of daratumumab plus atezolizumab. CONCLUSIONS: Daratumumab plus atezolizumab therapy did not improve efficacy versus atezolizumab monotherapy for patients with previously treated advanced NSCLC.

DNA vaccines targeting heavy chain C-terminal fragments of Clostridium botulinum neurotoxin serotypes A, B, and E induce potent humoral and cellular immunity and provide protection from lethal toxin challenge.

Botulinum neurotoxins (BoNTs) are deadly, toxic proteins produced by the bacterium Clostridium botulinum that can cause significant diseases in humans. The use of the toxic substances as potential bioweapons has raised concerns by the Centers for Disease Control and Prevention and the United States Military. Currently, there is no licensed vaccine to prevent botulinum intoxication. Here we present an immunogenicity study to evaluate the efficacy of novel monovalent vaccines and a trivalent cocktail DNA vaccine targeting the heavy chain C-terminal fragments of Clostridium botulinum neurotoxin serotypes A, B, and E. These synthetic DNA vaccines induced robust humoral and polyfunctional CD4(+) T-cell responses which fully protected animals against lethal challenge after just 2 immunizations. In addition, naïve animals administered immunized sera mixed with the lethal neurotoxin were 100% protected against intoxication. The data demonstrate the protective efficacy induced by a combinative synthetic DNA vaccine approach. This study has importance for the development of vaccines that provide protective immunity against C. botulinum neurotoxins and other toxins.

An Enhanced Synthetic Multiclade DNA Prime Induces Improved Cross-Clade-Reactive Functional Antibodies when Combined with an Adjuvanted Protein Boost in Nonhuman Primates.

UNLABELLED: The search for an efficacious human immunodeficiency virus type 1 (HIV-1) vaccine remains a pressing need. The moderate success of the RV144 Thai clinical vaccine trial suggested that vaccine-induced HIV-1-specific antibodies can reduce the risk of HIV-1 infection. We have made several improvements to the DNA platform and have previously shown that improved DNA vaccines alone are capable of inducing both binding and neutralizing antibodies in small-animal models. In this study, we explored how an improved DNA prime and recombinant protein boost would impact HIV-specific vaccine immunogenicity in rhesus macaques (RhM). After DNA immunization with either a single HIV Env consensus sequence or multiple constructs expressing HIV subtype-specific Env consensus sequences, we detected both CD4(+) and CD8(+) T-cell responses to all vaccine immunogens. These T-cell responses were further increased after protein boosting to levels exceeding those of DNA-only or protein-only immunization. In addition, we observed antibodies that exhibited robust cross-clade binding and neutralizing and antibody-dependent cellular cytotoxicity (ADCC) activity after immunization with the DNA prime-protein boost regimen, with the multiple-Env formulation inducing a more robust and broader response than the single-Env formulation. The magnitude and functionality of these responses emphasize the strong priming effect improved DNA immunogens can induce, which are further expanded upon protein boost. These results support further study of an improved synthetic DNA prime together with a protein boost for enhancing anti-HIV immune responses. IMPORTANCE: Even with effective antiretroviral drugs, HIV remains an enormous global health burden. Vaccine development has been problematic in part due to the high degree of diversity and poor immunogenicity of the HIV Env protein. Studies suggest that a relevant HIV vaccine will likely need to induce broad cellular and humoral responses from a simple vaccine regimen due to the resource-limited setting in which the HIV pandemic is most rampant. DNA vaccination lends itself well to increasing the amount of diversity included in a vaccine due to the ease of manufacturing multiple plasmids and formulating them as a single immunization. By increasing the number of Envs within a formulation, we were able to show an increased breadth of responses as well as improved functionality induced in a nonhuman primate model. This increased breadth could be built upon, leading to better coverage against circulating strains with broader vaccine-induced protection.

Co-Administration of Molecular Adjuvants Expressing NF-Kappa B Subunit p65/RelA or Type-1 Transactivator T-bet Enhance Antigen Specific DNA Vaccine-Induced Immunity.

DNA vaccine-induced immunity can be enhanced by the co-delivery of synthetic gene-encoding molecular adjuvants. Many of these adjuvants have included cytokines, chemokines or co-stimulatory molecules that have been demonstrated to enhance vaccine-induced immunity by increasing the magnitude or type of immune responses and/or protective efficacy. In this way, through the use of adjuvants, immune responses can be highly customizable and functionally tailored for optimal efficacy against pathogen specific (i.e., infectious agent) or non-pathogen (i.e., cancer) antigens. In the novel study presented here, we examined the use of cellular transcription factors as molecular adjuvants. Specifically the co-delivery of (a) RelA, a subunit of the NF-κB transcription complex or (b) T-bet, a Th1-specific T box transcription factor, along with a prototypical DNA vaccine expressing HIV-1 proteins was evaluated. As well, all of the vaccines and adjuvants were administered to mice using in vivo electroporation (EP), a technology demonstrated to dramatically increase plasmid DNA transfection and subsequent transgene expression with concomitant enhancement of vaccine induced immune responses. As such, this study demonstrated that co-delivery of either adjuvant resulted in enhanced T and B cell responses, specifically characterized by increased T cell numbers, IFN-γ production, as well as enhanced antibody responses. This study demonstrates the use of cellular transcription factors as adjuvants for enhancing DNA vaccine-induced immunity.

HIV-1 Env DNA vaccine plus protein boost delivered by EP expands B- and T-cell responses and neutralizing phenotype in vivo.

An effective HIV vaccine will most likely require the induction of strong T-cell responses, broadly neutralizing antibodies (bNAbs), and the elicitation of antibody-dependent cellular cytotoxicity (ADCC). Previously, we demonstrated the induction of strong HIV/SIV cellular immune responses in macaques and humans using synthetic consensus DNA immunogens delivered via adaptive electroporation (EP). However, the ability of this improved DNA approach to prime for relevant antibody responses has not been previously studied. Here, we investigate the immunogenicity of consensus DNA constructs encoding gp140 sequences from HIV-1 subtypes A, B, C and D in a DNA prime-protein boost vaccine regimen. Mice and guinea pigs were primed with single- and multi-clade DNA via EP and boosted with recombinant gp120 protein. Sera were analyzed for gp120 binding and induction of neutralizing antibody activity. Immunization with recombinant Env protein alone induced low-titer binding antibodies with limited neutralization breath. In contrast, the synthetic DNA prime-protein boost protocol induced significantly higher antibody binding titers. Furthermore, sera from DNA prime-protein boost groups were able to neutralize a broader range of viruses in a panel of tier 1 clade B viruses as well as multiple tier 1 clade A and clade C viruses. Further investigation of synthetic DNA prime plus adaptive EP plus protein boost appears warranted.

Innate lymphoid cells promote anatomical containment of lymphoid-resident commensal bacteria.

The mammalian intestinal tract is colonized by trillions of beneficial commensal bacteria that are anatomically restricted to specific niches. However, the mechanisms that regulate anatomical containment remain unclear. Here, we show that interleukin-22 (IL-22)-producing innate lymphoid cells (ILCs) are present in intestinal tissues of healthy mammals. Depletion of ILCs resulted in peripheral dissemination of commensal bacteria and systemic inflammation, which was prevented by administration of IL-22. Disseminating bacteria were identified as Alcaligenes species originating from host lymphoid tissues. Alcaligenes was sufficient to promote systemic inflammation after ILC depletion in mice, and Alcaligenes-specific systemic immune responses were associated with Crohn's disease and progressive hepatitis C virus infection in patients. Collectively, these data indicate that ILCs regulate selective containment of lymphoid-resident bacteria to prevent systemic inflammation associated with chronic diseases.

Intradermal DNA vaccination enhanced by low-current electroporation improves antigen expression and induces robust cellular and humoral immune responses.

DNA represents an ideal vaccine platform for HIV and many infectious diseases because of its safety, stability, and ease of manufacture. However, the immunogenicity of DNA vaccines has traditionally been low compared with viral vectors, recombinant protein, and live attenuated vaccines. The immunogenicity of DNA vaccines has been significantly enhanced by delivery with in vivo electroporation. Further improvements now allow electroporation to be performed in the dermis, which could potentially improve patient tolerability and may further enhance immunogenicity. In this study we examined how the current of intradermal vaccination impacts antigen expression, inflammation, and the induction of both humoral and cellular immunity in guinea pigs and nonhuman primates. We observed that a lower (0.1 A) current reduced inflammation and improved antigen expression compared with a 0.2 A current. The improved antigen expression resulted in a trend toward higher cellular immune responses but no impact on HIV- and influenza-specific binding titers. This study highlights the need for optimization of electroporation conditions in vivo in order to balance enhanced plasmid transfection with a loss of expression due to tissue inflammation and necrosis. These results suggest that a lower, 0.1-A current may not only improve patient tolerability but also improve immunogenicity.

An optimized SIV DNA vaccine can serve as a boost for Ad5 and provide partial protection from a high-dose SIVmac251 challenge.

One limitation in the development of an improved cellular response needed for an effective HIV-vaccine is the inability to induce robust effector T-cells capable of suppressing a heterologous challenge. To improve cellular immune responses, we examined the ability of an optimized DNA vaccine to boost the cellular immune responses induced by a highly immunogenic Ad5 prime. Five Chinese rhesus macaques received pVax encoding consensus (con) gag/pol/env intramuscularly (IM) with electroporation followed by the Merck Ad5 gag/pol/nef vaccine. A second group of five animals were vaccinated with Merck Ad5 gag/pol/nef followed by pVax gag/pol/env. One year following vaccination, Ad5-prime DNA-boosted monkeys and four unvaccinated controls received an intrarectal challenge with 1000 ID50 SIV(mac)251. The quality and magnitude of the T-cell response was analyzed by ELISpot and polyfunctional flow cytometry. We observed that an Ad5-prime DNA-boost resulted in significantly elevated SIV-specific T-cell responses even compared with animals receiving a DNA-prime Ad5-boost. Ad5 prime DNA boosted animals were capable of suppressing a pathogenic SIV(mac)251 challenge. Peak control correlated with the expansion of HLA-DR(+) CD8(+) T-cells two weeks post-infection. These data illustrate that high optimization of a DNA vaccine can drive of immune responses primed by a robust vector system. This previously unachievable feature of these newly optimized DNAs warrants future studies of this strategy that may circumvent issues of serology associated with viral vector prime-boost systems.

Clinical applications of DNA vaccines: current progress.

It was discovered almost 20 years ago that plasmid DNA, when injected into the skin or muscle of mice, could induce immune responses to encoded antigens. Since that time, there has since been much progress in understanding the basic biology behind this deceptively simple vaccine platform and much technological advancement to enhance immune potency. Among these advancements are improved formulations and improved physical methods of delivery, which increase the uptake of vaccine plasmids by cells; optimization of vaccine vectors and encoded antigens; and the development of novel formulations and adjuvants to augment and direct the host immune response. The ability of the current, or second-generation, DNA vaccines to induce more-potent cellular and humoral responses opens up this platform to be examined in both preventative and therapeutic arenas. This review focuses on these advances and discusses both preventive and immunotherapeutic clinical applications.

Selected approaches for increasing HIV DNA vaccine immunogenicity in vivo.

The safety, stability, and ability for repeat homologous vaccination makes the DNA vaccine platform an excellent candidate for an effective HIV-1 vaccine. However, the immunogenicity of early DNA vaccines did not translate from small animal models into larger non-human primates and was markedly lower than viral vectors. In addition to improvements to the DNA vector itself, delivery with electroporation, the inclusion of molecular adjuvants, and heterologous prime-boost strategies have dramatically improved the immunogenicity of DNA vaccines for HIV and currently makes them a leading platform with many areas warranting further research and clinical development.

Perforin and IL-2 upregulation define qualitative differences among highly functional virus-specific human CD8 T cells.

The prevailing paradigm of T lymphocyte control of viral replication is that the protective capacity of virus-specific CD8(+) T cells is directly proportional to the number of functions they can perform, with IL-2 production capacity considered critical. Having recently defined rapid perforin upregulation as a novel effector function of antigen-specific CD8(+) T cells, here we sought to determine whether new perforin production is a component of polyfunctional CD8(+) T cell responses that contributes to the control of several human viral infections: cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza (flu), and adenovirus (Ad). We stimulated normal human donor PBMC with synthetic peptides whose amino acid sequences correspond to defined CTL epitopes in the aforementioned viruses, and then used polychromatic flow cytometry to measure the functional capacity and the phenotype of the responding CD8(+) T cells. While EBV and flu-specific CD8(+) T cells rarely upregulate perforin, CMV-specific cells often do and Ad stimulates an exceptionally strong perforin response. The differential propensity of CD8(+) T cells to produce either IL-2 or perforin is in part related to levels of CD28 and the transcription factor T-bet, as CD8(+) T cells that rapidly upregulate perforin harbor high levels of T-bet and those producing IL-2 express high amounts of CD28. Thus, "polyfunctional" profiling of antigen-specific CD8(+) T cells must not be limited to simply the number of functions the cell can perform, or one particular memory phenotype, but should actually define which combinations of memory markers and functions are relevant in each pathogenic context.

Adenovirus-specific human T cells are pervasive, polyfunctional, and cross-reactive.

Pre-existing immunity to adenovirus (Ad) reduces the efficacy of Ad-based vaccines. The goal of this study was to define the prevalence, magnitude, functionality and phenotype of Ad-specific human T cells directly ex vivo. To study the magnitude of T-cell responses to Ad, we developed a highly reproducible whole Ad vector stimulation assay for use with polychromatic flow cytometry. Ad-specific CD4(+) and CD8(+) T-cells were detected in all 17 human subjects tested and were capable of proliferating upon restimulation. Ad5-specific CD4(+) T cells were primarily monofunctional CD4(+) T cells that produced IL-2, IFN-gamma or TNFalpha and expressed the memory markers CD27 and CD45RO. In contrast, Ad5-specific CD8(+) T cells were more polyfunctional, expressing effector-like combinations of IFN-gamma, MIP1alpha and perforin, and generally lacked CD27 and CD45RO expression. Ad-specific CD4(+) and CD8(+) T-cell responses against chimpanzee-derived AdC6 and AdC7 were found in all subjects, indicating the commonality of cross-serotype reactivity of Ad-specific T cells. This cross-reactivity is due in part to extensive CD4(+) and CD8(+) T-cell recognition of hexon regions conserved between multiple Ad serotypes. The prevalence, cross-reactivity and effector-like functions of Ad-specific T cells in humans may affect the efficacy of Ad vector-based vaccines by eliminating vector infected cells even when rare serotype Ad vectors are employed.

Vaccination with Ad5 vectors expands Ad5-specific CD8 T cells without altering memory phenotype or functionality.

BACKGROUND: Adenoviral (Ad) vaccine vectors represent both a vehicle to present a novel antigen to the immune system as well as restimulation of immune responses against the Ad vector itself. To what degree Ad-specific CD8(+) T cells are restimulated by Ad vector vaccination is unclear, although such knowledge would be important as vector-specific CD8(+) T cell expansion could potentially further limit Ad vaccine efficacy beyond Ad-specific neutralizing antibody alone. METHODOLOGY/PRINCIPAL FINDINGS: Here we addressed this issue by measuring human Adenovirus serotype 5 (Ad5)-specific CD8(+) T cells in recipients of the Merck Ad5 HIV-1 vaccine vector before, during, and after vaccination by multicolor flow cytometry. Ad5-specific CD8(+) T-cells were detectable in 95% of subjects prior to vaccination, and displayed primarily an effector-type functional profile and phenotype. Peripheral blood Ad5-specific CD8(+) T-cell numbers expanded after Ad5-HIV vaccination in all subjects, but differential expansion kinetics were noted in some baseline Ad5-neutralizing antibody (Ad5 nAb) seronegative subjects compared to baseline Ad5 nAb seropositive subjects. However, in neither group did vaccination alter polyfunctionality, mucosal targeting marker expression, or memory phenotype of Ad5-specific CD8(+) T-cells. CONCLUSIONS: These data indicate that repeat Ad5-vector administration in humans expands Ad5-specific CD8(+) T-cells without overtly affecting their functional capacity or phenotypic properties. This is a secondary analysis of samples collected during the 016 trial. Results of the Merck 016 trial safety and immunogenicity have been previously published in the journal of clinical infectious diseases [1]. TRIAL REGISTRATION: ClinicalTrials.gov NCT00849680[http://www.clinicaltrials.gov/show/NCT00849680].

Adenovirus-specific immunity after immunization with an Ad5 HIV-1 vaccine candidate in humans.

The immunologic basis for the potential enhanced HIV-1 acquisition in adenovirus serotype 5 (Ad5)-seropositive individuals who received the Merck recombinant Ad5 HIV-1 vaccine in the STEP study remains unclear. Here we show that baseline Ad5-specific neutralizing antibodies are not correlated with Ad5-specific T lymphocyte responses and that Ad5-seropositive subjects do not develop higher vector-specific cellular immune responses as compared with Ad5-seronegative subjects after vaccination. These findings challenge the hypothesis that activated Ad5-specific T lymphocytes were the cause of the potential enhanced HIV-1 susceptibility in the STEP study.

Baseline Ad5 serostatus does not predict Ad5 HIV vaccine-induced expansion of adenovirus-specific CD4+ T cells.

The mechanisms underlying possible increased HIV-1 acquisition in adenovirus 5 (Ad5)-seropositive subjects vaccinated with Ad5-HIV-1 vectors in the Merck STEP trial remain unclear. We find that Ad5 serostatus does not predict Ad5-specific CD4(+) T cell frequency, and we did not observe durable significant differences in Ad5-specific CD4(+) T cells between Ad5-seropositive and Ad5-seronegative subjects after vaccination. These findings indicate no causative role for Ad5-specific CD4(+) T cells in increasing HIV-1 susceptibility in the STEP trial.

AAV-1-mediated gene transfer to skeletal muscle in humans results in dose-dependent activation of capsid-specific T cells.

In a clinical trial for adeno-associated virus serotype 1 (AAV-1)-mediated gene transfer to muscle for lipoprotein lipase (LPL) deficiency, 1 subject from the high-dose cohort experienced a transient increase in the muscle enzyme creatine phosphokinase (CPK) 4 weeks after gene transfer. Simultaneously, after an initial downward trend consistent with expression of LPL, plasma triglyceride levels returned to baseline. We characterized B- and T-cell responses to the vector and the transgene product in the subjects enrolled in this study. IFN-gamma enzyme-linked immunosorbent spot (ELISpot) and intracellular cytokine staining assays performed on peripheral blood mononuclear cells (PBMCs) from the subject who experienced the CPK elevation showed the activation of capsid-specific CD4(+) and CD8(+) T cells. Four of 8 subjects had detectable T-cell responses to capsid with dose-dependent kinetics of appearance. Subjects with detectable T-cell responses to capsid also had higher anti-AAV-1 IgG3 antibody titer. No subject developed B- or T-cell responses to the LPL transgene product. These findings suggest that T-cell responses directed to the AAV-1 capsid are dose-dependent. Whether they also limit the duration of expression of the transgene at higher doses is unclear, and will require additional analyses at later time points.

Clinical activity and immune modulation in cancer patients treated with CP-870,893, a novel CD40 agonist monoclonal antibody.

PURPOSE: The cell-surface molecule CD40 activates antigen-presenting cells and enhances immune responses. CD40 is also expressed by solid tumors, but its engagement results in apoptosis. CP-870,893, a fully human and selective CD40 agonist monoclonal antibody (mAb), was tested for safety in a phase I dose-escalation study. PATIENTS AND METHODS: Patients with advanced solid tumors received single doses of CP-870,893 intravenously. The primary objective was to determine safety and the maximum-tolerated dose (MTD). Secondary objectives included assessment of immune modulation and tumor response. RESULTS: Twenty-nine patients received CP-870,893 in doses from 0.01 to 0.3 mg/kg. Dose-limiting toxicity was observed in two of seven patients at the 0.3 mg/kg dose level (venous thromboembolism and grade 3 headache). MTD was estimated as 0.2 mg/kg. The most common adverse event was cytokine release syndrome (grade 1 to 2) which included chills, rigors, and fever. Transient laboratory abnormalities affecting lymphocytes, monocytes, platelets, D-dimer and liver function tests were observed 24 to 48 hours after infusion. Four patients with melanoma (14% of all patients and 27% of melanoma patients) had objective partial responses at restaging (day 43). CP-870,893 infusion resulted in transient depletion of CD19+ B cells in blood (93% depletion at the MTD for < 1 week). Among B cells remaining in blood, we found a dose-related upregulation of costimulatory molecules after treatment. CONCLUSION: The CD40 agonist mAb CP-870,893 was well tolerated and biologically active, and was associated with antitumor activity. Further studies of repeated doses of CP-870,893 alone and in combination with other antineoplastic agents are warranted.

Exercise and lymphocyte activation following chemotherapy for breast cancer.

PURPOSE: To determine whether exercise training would increase lymphocyte activation in patients with breast cancer following chemotherapy. Activation was determined by the presence of CD4(+)CD69(+) T-helper lymphocytes, mitogen-induced proliferation, and levels of cytokines produced by mitogen-stimulated lymphocytes and in the patients' plasma. METHODS: Patients with breast cancer (N = 28) who participated in a 6-month exercise program were compared with patients (N = 21) who did not exercise. Following chemotherapy, and 3 and 6 months later, patients underwent fitness evaluations and had blood drawn. The exercise program consisted of resistance training and aerobic activity at 60-75% functional capacity three times a week with a personal trainer. Immunochemistry and flow cytometry were used to measure the number of CD4(+)CD69(+) blood lymphocytes. Whole blood was stimulated with concanavalin A (ConA), phytohemagglutin (PHA), or pokeweed mitogen (PWM) to determine proliferation potential. Enzyme-linked immunosorbent assays (ELISA) were used to determine the concentration of interferon-gamma (IFN-gamma) and interleukin-6 (IL-6) in the culture medium of mitogen-stimulated lymphocytes as well as the plasma concentrations of IL-6, soluble IL-6 receptor, soluble gp130, and IFN-gamma. Analysis of groups across time was done using the Wilcoxon signed rank test, and comparisons of groups were done using the Mann-Whitney U test. RESULTS: The exercising patients showed increases in maximal oxygen uptake and upper body strength. This group also showed a greater percentage of CD4(+)CD69(+) cells and a greater level of tritiated thymidine incorporation (DNA synthesis) when stimulated with ConA, PHA, and PWM at the end of the intervention. Plasma and mitogen-stimulated IL-6 and IFN-gamma production were similar in both groups. CONCLUSION: Exercise may improve immune function by increasing lymphocyte activation in patients with breast cancer following treatment.