Antiviral responses following L-leucyl-L-leucine methyl esther (LLME)-treated lymphocyte infusions: graft-versus-infection without graft-versus-host disease

Abstract

Although allogeneic hematopoietic progenitor cell transplant (HPCT) is curative therapy for many disorders, it is associated with significant morbidity and mortality, which can be related to graft-versus-host disease (GVHD) and the immunosuppressive measures required for its prevention and/or treatment. Whether the immunosuppression is pharmacologic or secondary to graft manipulation, the graft recipient is left at increased risk of the threatening opportunistic infection. Refractory viral diseases in the immunocompromised host have been treated by infusion of virus-specific lymphotyces and by unmanipulated donor lymphocyte infusion (DLI) therapy. L-leucyl-L-leucine methyl ester (LLME) is a compound that induces programmed cell death of natural killer (NK) cells, monocytes, granulocytes, most CD8(+) T cells, and a small fraction of CD4(+) T cells. We have undertaken a study of the use of LLME-treated DLI following T cell-depleted allogeneic HPCT, specifically to aid with immune reconstitution. In this ongoing clinical trial, we have demonstrated the rapid emergence of virus-specific responses following LLME DLI with minimal associated GVHD. This paper examines the pace of immune recovery and the rapid development of antiviral responses in 6 patients who developed viral infections during the time period immediately preceding or coincident with the administration of the LLME DLI.

Figure 1. LLME Depletion

Flow cytometry of product prior to LLME treatment shows the presence of NK (CD56 or 16 +/CD3−) cells, and CD3+/CD8+ cells, and CD3+/CD4+ cells. However, following LLME treatment, cells which contain perforin, namely the NK cells and a portion of CD8+ cells, are markedly depleted. The CD4 cell population is only minimally depleted in contrast (a). Following treatment with LLME, products showed adequate depletions of NK and CD8+ cells with a median 95.2% (range 79.9–100) depletion of NK cells, 76.1% (range 35.1–92.8) CD3+/CD8+ cells, but only 31.0% (0 – 88.8) CD3+/CD4+ cells (b).

Figure 1. LLME Depletion

Flow cytometry of product prior to LLME treatment shows the presence of NK (CD56 or 16 +/CD3−) cells, and CD3+/CD8+ cells, and CD3+/CD4+ cells. However, following LLME treatment, cells which contain perforin, namely the NK cells and a portion of CD8+ cells, are markedly depleted. The CD4 cell population is only minimally depleted in contrast (a). Following treatment with LLME, products showed adequate depletions of NK and CD8+ cells with a median 95.2% (range 79.9–100) depletion of NK cells, 76.1% (range 35.1–92.8) CD3+/CD8+ cells, but only 31.0% (0 – 88.8) CD3+/CD4+ cells (b).

Figure 2. Reconstitution of Perforin Positive Cells

Following LLME DLI, a product containing perforin negative cells, patients were able to reconstitute both perforin negative and then perforin positive cells. For the sample patient seen here, recovery of CD3+/CD4+ cells was apparent by Day 37 after LLME DLI (Figure 2a). Earlier time points were measured (Figure 2b) showing recovery of the same populations of cells but at lower numbers. The recovery of NK cells heralded (third dot plot & third column) preceded the recovery of CD8 cells. Reconstitution of CD8 cells included both perforin positive and perforin negative cells and preceded the recovery of CD4 positive cells.

Figure 2. Reconstitution of Perforin Positive Cells

Following LLME DLI, a product containing perforin negative cells, patients were able to reconstitute both perforin negative and then perforin positive cells. For the sample patient seen here, recovery of CD3+/CD4+ cells was apparent by Day 37 after LLME DLI (Figure 2a). Earlier time points were measured (Figure 2b) showing recovery of the same populations of cells but at lower numbers. The recovery of NK cells heralded (third dot plot & third column) preceded the recovery of CD8 cells. Reconstitution of CD8 cells included both perforin positive and perforin negative cells and preceded the recovery of CD4 positive cells.

Figure 3. Immune Reconstitution Following LLME DLI

Patients developed rapid and sustained reconstitution of lymphocyte subsets following LLME DLI. As shown in this sample patient, this included CD3+/CD4+, CD3+/CD8+ and CD3−/CD56 or 16+ cells (a) as well as both memory and naïve CD4 cells (b). In one patient who had not converted to 100% donor cells at the time he received LLME DLI, the recovery of memory CD4 cells was more prominent (c)

Figure 3. Immune Reconstitution Following LLME DLI

Patients developed rapid and sustained reconstitution of lymphocyte subsets following LLME DLI. As shown in this sample patient, this included CD3+/CD4+, CD3+/CD8+ and CD3−/CD56 or 16+ cells (a) as well as both memory and naïve CD4 cells (b). In one patient who had not converted to 100% donor cells at the time he received LLME DLI, the recovery of memory CD4 cells was more prominent (c)

Figure 3. Immune Reconstitution Following LLME DLI

Patients developed rapid and sustained reconstitution of lymphocyte subsets following LLME DLI. As shown in this sample patient, this included CD3+/CD4+, CD3+/CD8+ and CD3−/CD56 or 16+ cells (a) as well as both memory and naïve CD4 cells (b). In one patient who had not converted to 100% donor cells at the time he received LLME DLI, the recovery of memory CD4 cells was more prominent (c)

Figure 4. Anti-Viral Responses

Sample data are shown for two patients. In panels a and b, one sees the data from a patient who developed adenovirus hepatitis. Shortly after receiving the LLME DLI, he began to clear the adenovirus with a 10fold drop in pcr titers by 11 days s/p DLI (113 days s/p HPCT), and complete resolution by 42 days s/p DLI (144 days s/p HPCT). This was associated with an adenovirus specific CD4 IFN-γ response that was evident at 147 and 175 days post DLI. Sample from a second patient, who had developed CMV infection are shown in panels c and d. Once again, the patient had rapid resolution of CMV pcr titers with a significant drop within six days and complete resolution by 20 days s/p DLI (68 days s/p HPCT). He was also found to have developed both a CMV-specific CD8 response (anti-A2 peptide) and CD4 response (CMV ag), but no response to adenovirus.

Figure 4. Anti-Viral Responses

Sample data are shown for two patients. In panels a and b, one sees the data from a patient who developed adenovirus hepatitis. Shortly after receiving the LLME DLI, he began to clear the adenovirus with a 10fold drop in pcr titers by 11 days s/p DLI (113 days s/p HPCT), and complete resolution by 42 days s/p DLI (144 days s/p HPCT). This was associated with an adenovirus specific CD4 IFN-γ response that was evident at 147 and 175 days post DLI. Sample from a second patient, who had developed CMV infection are shown in panels c and d. Once again, the patient had rapid resolution of CMV pcr titers with a significant drop within six days and complete resolution by 20 days s/p DLI (68 days s/p HPCT). He was also found to have developed both a CMV-specific CD8 response (anti-A2 peptide) and CD4 response (CMV ag), but no response to adenovirus.

Figure 4. Anti-Viral Responses

Sample data are shown for two patients. In panels a and b, one sees the data from a patient who developed adenovirus hepatitis. Shortly after receiving the LLME DLI, he began to clear the adenovirus with a 10fold drop in pcr titers by 11 days s/p DLI (113 days s/p HPCT), and complete resolution by 42 days s/p DLI (144 days s/p HPCT). This was associated with an adenovirus specific CD4 IFN-γ response that was evident at 147 and 175 days post DLI. Sample from a second patient, who had developed CMV infection are shown in panels c and d. Once again, the patient had rapid resolution of CMV pcr titers with a significant drop within six days and complete resolution by 20 days s/p DLI (68 days s/p HPCT). He was also found to have developed both a CMV-specific CD8 response (anti-A2 peptide) and CD4 response (CMV ag), but no response to adenovirus.

Figure 4. Anti-Viral Responses

Sample data are shown for two patients. In panels a and b, one sees the data from a patient who developed adenovirus hepatitis. Shortly after receiving the LLME DLI, he began to clear the adenovirus with a 10fold drop in pcr titers by 11 days s/p DLI (113 days s/p HPCT), and complete resolution by 42 days s/p DLI (144 days s/p HPCT). This was associated with an adenovirus specific CD4 IFN-γ response that was evident at 147 and 175 days post DLI. Sample from a second patient, who had developed CMV infection are shown in panels c and d. Once again, the patient had rapid resolution of CMV pcr titers with a significant drop within six days and complete resolution by 20 days s/p DLI (68 days s/p HPCT). He was also found to have developed both a CMV-specific CD8 response (anti-A2 peptide) and CD4 response (CMV ag), but no response to adenovirus.

Figure 5. V Beta Spectratyping

Representative histograms of Vbeta spectratype analysis of the CD4⁺ T cells enriched from the peripheral blood of one of the patents/pairs. The repertoire complexity of the donor sample before LLME treatment (A), after LLME treatment (B), and reconstituting in the patient post-LLME DLI (C) exhibit similar levels of complexity.